Patent Application
20050272055
The present invention relates to methods of treating lethal shock using compositions and/or anitisense to turn off the expression of genes that are up-regulated by exposure to toxic agents or by increasing the amount of proteins or their products when genes that produce those proteins are down regulated by exposure to toxic agents.
The threat of terrorist action using biological warfare (BW), chemical or infectious agents has occurred throughout the world. These acts of terrorism are unpredictable and counter efforts have been aimed at rapid, accurate diagnosis and speedy treatment. Determination of the exact toxin that a subject has been exposed to is critical to treatment.
Current methods for pathogen or toxin identification require specialized reagents that are structural-based probes. For bio-engineered toxic agents, those probes may prove to be ineffective. The increased sophistication available for design of potential biological weapons will require reliance on better approaches to adequately identify such threats. Simple identification of toxins or infectious agents may be complicated by the fact that genetic manipulations could (1) make BW agents unrecognizable by structural-based technologies, or (2) enhance their devastating effects, making them toxic at undetectable levels. Furthermore, small amounts of common bacterial products, such as protein A or endotoxin, have been shown to markedly potentate activities of biological warfare threat toxins. The difficulties of identifying toxins experienced in the past could lead to potentially disastrous delays in responding appropriately to the threat or to the possibility of inappropriate treatment based on inadequate information. Thus far, diagnoses could only be made based on symptoms, which may take 4-24 hours or more to appear, and by that time, the damage is irreversible and death may result.
Description of a Selected Group of Toxic Agents:
There are many toxic agents that are a threat to humans in situations of biological warfare. For example, SEB: Staphyloccocal enterotoxin B is a potent bacterial toxin known to cause lethal shock. The mode of exposure could be aerosol, food or water contamination. It interacts with the lymphoid cells, proximal tubule (PT) kidney and other cells initiating cascades of reactions ultimately leading to lethal shock. The initial symptoms for SEB-induced intoxication are vertigo, muscle weakness (vasoconstriction in the extremities) within 1-8 hrs of exposure to the toxin. The symptoms that follow are nausea, vomiting and diarrhea, along with hypotension and vasodilation of blood vessels in kidney and other organs (1-24 h). Respiratory distress and pathological hypotension eventually lead to irreversible shock and death at about 40-60 hrs post exposure, although very early incidents (ca. 6 h) have been observed. The mechanism of its action is not clear, nor is it understood how SEB is massively potententated by trace levels of contaminants such as Protein A or endotoxin. In short, there is no system available to determine host exposure or individual responses and the toxin is rapidly (30 min) removed from the blood stream to the kidney PT (75%), liver and spleen.
Anthrax is another highly toxic agent. Anthrax is a natural disease of herbivorous animals that can be transmitted to humans. The causative agent Bacillus anthracis, can form spores which are extremely hardy and can remain alive for a very long time. After inhalation of a heavy dose of anthrax spores, however, the onset of the disease may occur within a day and death may follow rapidly in a couple of days. The molecular changes caused by this agent in the host is totally unknown, therefore identifying genes altered by this agent is very crucial for rapid and effective detection and for designing better treatments for this deadly pathogen. Anthrax is known to cause lethal shock.
Brucella is a highly infectious bacteria that causes disabling symptomatology (fever, chills, fatigue) in humans. Bacteria can be acquired through inhalation, ingestion, or penetration of damaged skin. As facultative intracellular parasites of macrophages, they primarily localize in the reticuloendothelial system. Bacteremia and symptoms occur from several days to several weeks after infection, presumably as a result of amplification of bacterial numbers in spleen, liver and bone marrow. Host response involves both Th1 and Th2 immune mechanisms, but is generally tilted toward Th1. In murine models of brucellosis, both antibody and T cells transfer immunity. Brucella LPS is relatively nonpyrogenic compared to LPS from Enterobacteriaceae. This property may explain the relative paucity of immune and inflammatory response early in infection.
Brucella has also been found to induce a cytokyne storm in humans which causes illness. The present invention includes treating a patient with anticytokyne therapy to reverse the harmful effects of the cytokine storm.
Plague is still another threatening toxic agent to man. The Y. pestis is an organism that causes plague. Plague symptoms include fever, chills, headache, hemoplysis and toxemia. This eventually leads to respiratory failure and death. Until now, diagnosis has been made by symptom analysis. This means that the progress of the illness can go unchecked before treatment is sought and is therefore, unsuccessful. A faster test is needed for plague. Plague also causes shock.
Botulinum toxin is extremely potent neurotoxins produced by different strains of the bacterium Clostridium botulinum. There are seven serotypes of botulinum toxins, which share the same functional mechanism: they have an endopeptidase activity that cleaves a protein in synaptic vesicles thereby inhibiting release of acetycholine. The resulting block in neurotransmitter release causes general skeletal muscle paralysis with death occurring due to respiratory failure. Following inhalation or ingestion of botulinum toxin, symptoms may appear within 24 to 36 hours or may take several days to appear. This toxin causes weakness, dizziness, dry mouth and throat, blurred vision and diplopia, dysarthria, disphonia, dysphasia and respiratory failure. A faster test for exposure to the botulinum toxin is needed.
Cholera Toxin (CT) causes vomiting, headache, diarrhea resulting in death. Mortality is as high as 80%. Diagnosis is done by symptoms of diarrhea and dehydration. The Cholera Toxin is a very difficult toxin to spot in a blood sample. Therefore, a faster, non-symptom related test is needed to prevent death.
There is no easy or fast detection method to confirm the exposure to these and other toxic agents. The deadly symptoms of lethal shock appear before they are diagnosed so the important life-saving treatment is delayed which results in deaths that could be prevented if an earlier test were available. Current methods for pathogen identification using structural-based probes may not be useful for early diagnosis for the reasons stated above.
One of the most harmful symptoms that are related to exposure to most toxic agents is the appearance of lethal shock. It is important to note that treatment of lethal shock initiated by multiple causes, has been an intractable medical problem that has been studied for (at least) decades. Clinical trials of therapies aimed at blocking/sequestering inflammatory mediators and involving huge numbers of patients, have not shown statistically significant benefits relative to no treatment.
Therefore, an object of the present invention is to provide for a method of treating patents that have been exposured to toxic agents by measuring distinct patterns in the levels of expression of specific genes and treating the patient based on the distinct patterns.
It is a further object of the invention to select a panel of genes, the altered pattern of expression of which will provide a fingerprint that is indicative of exposure to a particular toxic agent. This panel of genes will also indicate whether an exposed individual will develop the symptom of lethal shock. This panel of genes can show the potential to reveal the severity of exposure and the individual susceptibility to the agent, and can provide indicators of course of impending illness for even unknown toxic agents that leads to enlightenment of how to treat an exposed patient.
A still further object of the invention is to provide a method of early treatment of subjects exposed to threat agents, with the intervention of drugs or with agents, such as antisense oligos, which turn off the expression of genes that react detrimentally to toxins or by the addition of turned off advantageous proteins, based on the newly found gene changes.
A still further object of the present invention is to provide a method of treatment that is dependant upon the time of exposure to a toxic agent, wherein a particular treatment is effective at a particular time period after exposure.
With the method of the present invention, the problems experienced in the past are solved. With the present invention both known and presently unknown or bio-engineered biological warfare (BW) agents can be identified based on early host functional responses to exposure and the patient that has been exposed may be treated. The present method also has the benefit of revealing the presence of low-level potentiating contaminants, such as LPS and Protein A which cause the toxins to have a more potent effect on an exposed subject. The present invention provides early information regarding individual exposure and susceptibility which is useful for determining proper treatment. This approach offers the benefits of immediate diagnosis, and the ability to identify those who have been exposed to toxic agents but have not yet developed signs or symptoms. This approach also offers a viable and successful treatment for lethal shock to prevent the symptoms from occurring.
The present invention solves the problems of the past with a method whereby an individual's exposure and his/her response to a toxic agent based on alterations in gene expression in their peripheral blood lymphoid cells (also referred to as human lymphoid cells) can be determined. These cells are readily available from personnel. These cells serve as a reservoir of historical information; although they may not, themselves, be the pathogenic target of a toxic agent, the toxic agents can indirectly activate lymphoid cells to produce a unique gene expression patterns typical of the impending illness. In addition to diagnostics, the gene expression profile potentially provides a regimen for specially designed, stage dependent, appropriate treatment.
The present invention is thus, directed to a method of treating a patient that has been exposed to a toxic agent based on amounts and time of protein/gene expression present in a sample of mammalian tissue or mammalian body fluids that has been exposed to a toxic agent. The present invention is particularly useful because it can provide an early treatment based on diagnosis of exposure to a toxic agent before the onslaught of any symptoms.
The present invention also permits a determination of time of exposure based on measurement of amounts of up regulation and/or down regulation of certain genes at particular intervals after exposure. By determining time of exposure, lethal shock can be prevented by the administering of protein products of genes that are down regulated or the administering of antisense in the case where genes are upregulated by the toxic agent.
a is a graph showing the expression pattern of Ferretin Heavy chain in kidney cells in response to LPS;
b is a graph showing comparison of Ferritin Gene Expression in human kidney cells in response to LPS and SEB;
a is a graph of expression of cathepsin L in Anthrax treated cells;
b is a graph of expression of HCI and EIF3 upon exposure to Anthrax;
b is a digital differential display gel profile showing comparison of changes in gene expression in response to SEB and Cholera Toxin; and
FIGS. 66A-E are digital images of various organs and tissues showing the results of SEB exposure in piglets;
FIGS. 68 A-F are digital images of microscopic findings of histological examination of selected tissues in SEB treated piglets;
FIGS. 69A-D are digital images of a periarteriolar lymphoid sheath in SEB treated piglets;
FIGS. 70 A-F are digital images of histological findings of SEB treated piglets;
Discussion of the Figures and Tables:
It has been found that the host gene expression patterns act as diagnostic markers. The present inventors have compiled a library of genes altered by different toxic agents. These libraries consist of hundreds of genes altered upon exposure to a particular agent. These discoveries and method of diagnosing exposure to a toxic agent are set forth in U.S. Pat. No. 6,316,197, incorporated herein in its entirety by reference. Excerpts from U.S. Pat. No. 6,316,197 are provided below for convenience.
A gene library has been generated for each biological warfare agent in the present invention. This list gives the name of the gene and the ratio or fold difference of genes from the control values. These libraries allow the determination of the gene changes induced by each agent. The genes that are 2 fold and higher in ratio are good candidates for marker genes for determining exposure to each specific agent.
The inventors have identified a list of more than 200 genes per agent that change upon exposure to a toxic agent. These genes are important for not only early detection before the symptoms appear but also provide therapeutic targets that can be used for treatment of patients.
The gene lists provided in the following tables for each agent, provide the first glimpse ever at observing the molecular changes induced in the host upon exposure to toxic agents. No one has looked at the molecular events in the host before in such a global way.
The library of genes is a useful tool for developing a diagnostic chip that will contain all the disclosed gene names on one slide. These DNA chips are useful for confirmation of gene expression patterns upon exposure to toxic agents. The specific genes that are altered upon exposure serve as diagnostic markers and help predict the course of illness. A DNA chip containing specific genes for each agent, all in the same chip, which is used for diagnostic purposes.
With blood samples from exposed individuals to any of the above mentioned toxic agents, RNA is isolated and hybridized to the chip by methods known in the art to determine the gene changes. We have developed an extensive database of these gene changes with all the mentioned agents that can be used to identify the type of exposure. Targeting these genes for therapeutic intervention at various stages of illness is the key to this invention.
Effect of SEB on the Expression of Different Genes: Table 1
RT-PCR was performed on RNA samples from human lymphoid cells treated with SEB for different time periods. Several changes in expression of genes were observed that were up regulated or down regulated in response to the toxin in a time dependent manner as summarized in Table 1.
Differential display was used to identify various genes that are altered upon SEB exposure to human lymphoid cells in vitro. The differential display (DD-PCR) procedure has been completed using all the possible anchored and arbitrary primer combination (220) that has covered the entire RNA population. We have identified more than 900 genes that are altered upon SEB exposure. See Tables 1a and 1b.
Description of Gene Changes Induced by each Threat Agent that can be used for Diagnostic Tests:
Gene lists were obtained after screening of several gene arrays. Each agent was exposed to the cells and RNA isolated for gene array experiments. The untreated and treated samples were then labeled with 33P and hybridized to the arrays. The signals were obtained by scanning in a BIORAD scanner and the intensities of each spot was normalized with the housekeeping genes. Global normalization was also performed after the 16 bit Tiff image was aligned to the grid for each spot.
Each table represents columns showing first the function of the gene, the name of the gene, and the numbers represent the fold change at indicated time points. Fold change, was calculated after normalization of signals and was obtained by dividing the treated number with the untreated control. The ratio obtained after this is designated as fold change.
Use of Gene Array for Identification of Altered Genes in the Host:
The inventors have used gene array, a powerful tool, for identification of altered genes in the host upon exposure to the toxic agents. Libraries of genes were generated for each agent. The gene names are listed with each agent separately. The gene names are listed with each agent separately. The results of Tables 2-9 were obtained using gene array. These genes are altered specifically by each pathogen in a human upon exposure.
Table 2: Gene Library from Brucella Exposure.
Human lymphoid cells were exposed to Brucella Melitensis in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Table 2 shows the differences in expression pattern of untreated and treated samples. Many genes are upregulated and many genes are downregulated. They act as marker genes to predict exposure to Brucella.
Table 3: Gene Library for Yersinia Exposure.
Human monocytes were exposed to Yersinia pestis in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Table 3 shows the differences in expression patterns of untreated and treated samples. Each of these genes that are up regulated or down regulated 2 fold and higher can act as marker genes for Plague (Yersinia) exposure and also be used as therapeutic targets.
Table 4: Gene Library for SEB Exposure.
Human lymphoid cells were exposed to SEB in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Table 4 shows the differences in expression patterns of untreated and treated samples. Each of these genes that are up regulated or down regulated 2 fold and higher can act as marker genes for SEB exposure. These genes can be also targeted for therapy.
Table 5: Gene Library for Anthrax Exposure in Vitro.
Human lymphoid cells were exposed to Anthrax spores in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Table 5 shows the differences in expression pattern of untreated and treated samples. Each of the genes that are up regulated or down regulated 2 fold and higher act as marker genes for Anthrax exposure. These specific genes can be targeted for therapy and gives us much more choices other than using CIPRO which is the most common antibiotic treatment available today.
Table 6: Gene Changes Induced by Anthrax in Vivo in Monkeys
In vivo monkeys were exposed to Anthrax spores, whole blood collected at different time periods (24hr, 48hr, 72hr), RNA isolated and hybridized to Gene Array blots. Table 6 shows the ratio of treated over control samples. Each of the genes that are up regulated or down regulated 2 fold and higher act as marker genes for Anthrax exposure. A pattern of gene expression is also seen during these time points. Some of the early genes are upregulated by 24h and they disappear by 72h. However, some of the damaging genes causing cell death appear at later time points and they stay up regulated. These genes act as diagnostic markers and therapeutic targets for exposure to each of these BW agents.
Table 7a-7d: Gene Library for Venezuelan Equine Encephalitis (VEE) Virus Exposure in Vitro.
Human lymphoid cells were exposed to VEE virus in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Tables 7a and 7b shows the differences in expression patterns of untreated and treated samples. Many genes are upregulated and many genes are downregulated, acting as marker genes to predict exposure to VEE virus. Table 7a shows Array I and Table 7b shows Array II. Table 7c is a table showing gene changes induced by VEE virus invitro in human lymphoid cells for a cancer array. Table 7d is a comprehensive table showing gene changes induced by VEE virus in vitro in human lymphoid cells.
Table 8: Gene Library for Dengue Virus Exposure in Vitro.
Human lymphoid cells were exposed to Dengue virus in vitro for different time periods, RNA isolated and gene screening performed using Gene Array blots. Table 8 is a comprehensive table that shows the differences in expression patterns of untreated and treated samples. Many genes are upregulated and many genes are downregulated acting as marker genes to predict exposure to Dengue virus. These genes can be targeted specifically to combat the disease progression.
Table 9: Baseline Gene List
Approximately 244 genes were selected that were never expressed in 24 untreated control human lymphoid samples. The expression level of these genes were below the background levels in all these 24 samples. However, upon treatment with various agents, the expression of these genes was significantly altered. This leads us to believe that these genes can be used as specific diagnostic markers to identify exposure to the biological threat agents that we have tested. These genes since are never expressed in unexposed individuals, upregulation of these particular genes will indicate exposure to some agent. Only after exposure to a bacterial pathogen, or virus or toxin will alter the expression of these genes, thus these sets of genes are very important for diagnostic tests. These genes are also useful for targeting them after exposure to these BW agents for effective treatment.
Genes Identified Using Differential Display PCR: A Few Genes that were Identified Using DD-PCR to be Altered by SEB Exposure Were Selected and Confirmed their Level of Expression Using RT-PCR.
Effect of SEB on the Expression of CTAP-III Gene:
The CTAP III gene was identified to be down regulated by SEB, which was confirmed by RT-PCR, and by Northern blot analysis.
Effect of SEB on the Expression of Proteoglycan Gene:
Primers were designed for proteoglycan V1 (Vimentin) gene and RT-PCR performed on RNA samples from different time periods of SEB exposure. There was a dramatic decrease in expression upon SEB exposure (
Effect of SEB on Gene Expression of GBP:
SEB exposure caused a significant increase in expression of this gene that is involved in Guanylate cyclase regulation (
Effect of SEB on Gene Expression of Hypoxia Inducible Factor (HIF-1):
The expression of the gene HIF-1 was also up regulated in response to SEB in a time dependant manner (
Effect of SEB on gene expression of IL6:
IL6 gene expression was significantly up regulated upon SEB exposure within 2 hrs of exposure (
Effect of SEB Exposure on Gene Expression of Ferritin Heavy Chain:
SEB exposure caused a decrease in the expression of Human Ferritin gene as shown in
Confirmation of Gene Changes in Monkey Blood Samples Exposed to SEB:
We verified these findings in lymphocytes of monkeys challenged with SEB. Using PCR primers designed for the selected genes, we have found unique patterns in alteration of gene expression as early as 30 minutes post-aerosol challenge. We tested three genes in lymphocytes from monkey blood samples after exposure to SEB (
The expression of IL6 and GBP was up regulated by 30 minutes of SEB challenge in monkey samples. This was a sub-lethal dose given to the monkeys so the expression of CTAP-III was also shown to be up regulated in these samples by 30 minutes of exposure. Similar results were obtained with human cells in vitro when exposed to SEB.
Summary of Changes: Table 2a summarizes all the changes that were observed that were induced by these toxins in human lymphoid cells.
Comparison of the Effects of SEB and LPS on Expression of CTAP-III
Equal amount of the RNA samples treated with SEB and LPS along with proper controls were reverse transcribed as described elsewhere and amplified using custom designed primers of CTAP-III. Equal volumes of samples were resolved on a 1% agarose gel, visualized by ethidium bromide staining and quantitated by NIH image program 1.61. #1, Control; #2-4 were treated with 100 ng/ml SEB or LPS for different time periods and were normalized with expression of β-actin. #2; 2 hrs, #3, 4 hrs; #4, 24 hrs. Both SEB and LPS toxins were capable of down regulating the CTAP-III gene while showing a similar activation pattern. Effect of LPS was prominent compared to SEB. Down regulation of the CTAP III gene was visible as early as 2 hrs (SEB 50% of control levels and LPS 45% of control levels). After 24hrs of treatment expression of the CTAP-III gene induced by SEB was about 33-45% of control levels while LPS was 25-35% (
Comparison of the Effect of SEB and LPS on the Expression of the IL-6 GENE
Equal amount of the RNA samples treated with SEB and LPS along with proper controls were reverse-transcribed as described elsewhere and amplified using custom designed primers of IL-6. Equal volumes of samples were run on 1% agarose gel in a gel loading buffer, subjected to electrophoresis at IOOV for 40 min., visualized by ethidium bromide staining and quantitated by the NIH image program 1.61. #1, Control; #2-4 were treated with 100 ng/ml SEB or LPS for different time periods and were normalized with β-actin. #2; 2 hrs, #3, 4 hrs; #4, 24 hrs. Both toxins up regulated the expression of the IL-6 gene in a time dependent manner while the effect of SEB in human lymphoid cells was more prominent. An up regulation was seen as early as 2 hrs by both toxins (SEB 52-57 fold, LPS 7-8 fold), and was still up regulated at 24 hrs (SEB 30-35 fold, LPS 10-12 fold). SEB had a pronounced effect on IL-6 gene expression but with LPS it was not very significant (
Comparison of the Effects of SEB and LPS on Expression of GBP-2
Equal amount of the RNA samples treated with SEB and LPS along with proper controls were reverse-transcribed as described elsewhere and amplified using custom designed primers of GBP-2. Equal volumes of samples were resolved on a 1% agarose gel, visualized by ethidium bromide staining and quantitated by the NIH image program 1.61. #1, Control; #2-3 were treated with 100 ng/ml SEB or LPS for different time periods and were normalized with P-actin. #2; 2 hrs, #3, 24 hrs. GBP was clearly up regulated by SEB by 2hrs (7-8 fold), and was seen even after 24 hrs (3-3.5 fold). LPS had no effect on the expression of GBP-2 (
Comparison of the Effects of SEB and LPS on Expression of HIF-1
The HIF-1 gene expression was up regulated by SEB in a time dependent manner reaching an optimum value by 24 hrs (2.5-3 fold). Expression pattern of the HIF-1 gene by LPS was different to that observed for SEB. There was no significant change observed even after 24 hrs (
Summary of Unique Changes Induced by SEB and LPS:
Table A summarizes the changes induced by SEB and LPS. The time dependent changes are also noted in this table.
Differential Gene Expression Patterns in Human Kidney Cells Induced by SEB
The RhoE gene was identified by differential display (DD)—polymerase chain reaction (PCR) as one of the genes that was down regulated by SEB in renal proximal tubule epithelial cells (RPTEC). Two- to eight-fold reduction in expression, depending on the length of cell exposure to SEB, was confirmed by reverse transcription (RT)—PCR with specific primers (
Comparison of Gene Expression Patterns Induced by LPS and SEB in Human Kidney Cells.
A) Genes encoding ferritin, Guanylate binding protein (GBP) and interleukin-6 (IL-6) were differentially expressed in RPTEC (renal proximal tuble epithelial cell) stimulated with LPS. The peak expression of ferritin and GBP occurred at approximately 6 h of exposure, while the IL-6 did not show significant levels of expression until 24 h of the toxin stimulation. None of these genes were known to be differentially expressed in cells stimulated with SEB, as compared to the control cells (
B) Genes encoding hypoxia-inducible factor-1 (HIF-1) and myosin heavy chain showed no significant differences in expression patterns in LPS-stimulated RPTEC. However, both of these genes were up regulated in SEB-stimulated cells, with peak expression of HIF-1 and myosin occurring at approximately 2 h (greater than two-fold increase over control) and 24 h (greater than 20-fold difference increase over control), respectively (
In
In
In
In
Summary of Gene Changes in Human Kidney Cells in Response to SEB:
Table B summarizes all the 32 genes that were altered in kidney cells in response to SEB exposure. There were 14 genes that were up regulated and 18 genes that were down regulated.
Effect of Drugs to Block SEB Induced Responses:
We have tested three different drugs and have found them to be effective blockers of SEB induced responses. P-38 inhibitor is an inhibitor of a kinase that is crucial for signal transduction of SEB in human lymphocytes. It is preferred to administer P-38 within 2 hours of exposure to SEB. HPA-Na is a heteropolyanion that is a free radical scavenger that is also very effective in blocking the SEB effects. It is preferred to administer HPA-Na within 2-3 hours of exposure to SEB
Effect of P-38 Inhibitor on SEB Induced Cellular Events:
The drug known as P-38 was obtained from Smith Klien Beecham, NJ. Human TNF-α can either be as a membrane associated (26 kDa) or secreted (17 kDa) form (Kriegler, et al., cell, 53, 45-53, 1988). TNF-α induced by SEB is in the secreted form. TNF-alpha induces hemorrhagic necrosis and regression of tumors in animals, is cytotoxic to transformed cells, and promotes immunity, inflammation, insulin resistance, hypertension, shock and some cases chronic diseases (Tracey, et al., Annu. Rev. Cell Biol., 9, 317-343, 1993; Sidhu, et al., Pharmacol. Ther., 57, 79-128, 1993). Ability of P-38 inhibitor to block the induction of TNF-alpha makes this a solid therapeutic target.
Cells of the immune system utilize surface molecules for selective trafficking and focused cellular responses to a variety of inflammatory stimuli (Hogg, et al., Curr. Opin. Immunol., 5, 383-589, 1993; Mackay, et al., Immunol. Today, 1, 99-104, 1993). CD69 is a surface molecule that is rapidly expressed in response to various interleukins such as IL-2, IL-13 and is closely linked to the activation to protein kinase C in human T lymphocytes (Bjorndahl, et al., J. Immunol., 1, 4094-4098, 1988; Cebrian, et al., Eur. J. Immunol., 19, 809-816, 1989; Hamann, et al., J. Immunol., 150, 4920-4928, 1993; Testi, et al., J. Immunol., 150, 4920-4924, 1989). Flow cytometry is used for assessing surface molecule expression on selected cell populations. Ability of P-38 kinase inhibitor SB-203580 to reduce the production of CD69 induced by SEB increases the importance of P-38 inhibitor as a therapeutic target.
Effect of P-38 Inhibitor on SEB Induced Responses:
Effect of p-38 Inhibitor on Cell Proliferation.
P-38 inhibitor was administered at a concentration of 10 uM. P-38 inhibitor was able to block the growth of T-cells as shown in
Effect of P-38 Inhibitor on the Induction of TNF-alpha
Human lymphoid cells were treated with P-38 inhibitor followed by SEB exposure.
Upon the treatment of SEB with P-38, the TNF-α gene expression in human lymphoid cells almost doubled compared to untreated samples. When treated with 10 μg of P-38 inhibitor, the previously observed induction of the TNF-α gene by SEB was brought down back to control levels (
Effect of p-38 Inhibitor on the Induction of CD-69
When human lymphoid cells were treated with 100 ng/ml of SEB, we observed a clear stimulation of CD69 production in human lymphoid cells (15-20 pg/ml over control levels). This induction with SEB was clearly blocked upon the treatment with 10 uM of P-38 inhibitor (
Effect of HPA-Na and PKC Inhibitors on SEB Induced Responses:
SEB is known to induce rapid proliferation of the T cells, we tested different concentrations of the drugs on this assay and showed a definite decrease in proliferation. (
Effect of HPA on Proliferation Assay Induced by SEB:
The drug HPA-Na was given to human lymhpoid cells in the amount shown in
The drug HPA-Na (a heteropolyanion which is a metal ion derivative of polyoxotungstate) was synthesized in the laboratory using methods outlined in Heteropoly and Isopoly Oxometalateds, Michael Thor Pope, Springer Verlag, Berlin, Germany 1983. This drug is water soluble and stable at room temperature. Its structure is shown in
Effect of Inhibitors of Protein Kinase C:
As shown in
Treatment of Toxin Induced Illness with Antisense:
A new technique for treating patients is to prevent expression of specific genes by administering antisense to the mRNA for that particular gene. For the situation described in this application, persons exposed to toxic agents, in addition to classical drugs that target specific metabolic pathways, can be treated with antisense to mRNA coding for specific genes that we have determined to be critical for toxicity induced by the specific toxic agent. An example is that staphylococcal enterotoxin B illness is characterized by rapid drop in blood pressure, likely due to loss of regulation of vascular tone especially in organs. We have identified several genes, with altered expression in response to SEB that are involved in various aspects of regulation of vascular tone (Table 1b;
First one would determine, based on gene array analysis or conventional structural-based probes, that the patient had been exposed to a toxic agent. The probes used were designed to identify the agent such as SEB toxin gene or Anthrax genes, or genes specific for the pathogen itself.) If gene array analysis had been performed, detection of expressed genes known to be critical for the progression of the intoxication would be apparent by comparing the expression patterns with the gene libraries set forth in this description.
Dose of antisense: Typically patients have been treated and tolerate a dose of 0.5- 3 mg/kg/day delivered by continuous intravenous infusion. Antisense is easily designed for any gene based on methods well known in the art. Saline is an example of a carrier used to deliver it intravenously. In most cases for the toxic agents, there is a critical time period of the illness that lasts for 2-4 days. Treatment with antisense therapy for this length of time would not present a problem. One study treated ovarian cancer patients for 21 days on/ 7 days off (Yuen, et al., Yuen AR, et al., Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer, Clin Cancer Res 1999 Nov., 5(11): 3357-63 (1999). Other methods of administration are also under study including intraperitoneal, intramuscular and oral administration.
Antisense (complementary base pairs to the desired sequence) is typically constructed beginning with the 3 base “start code” for a specific mRNA and proceeding with the nucleotide sequence of the mRNA for the gene in question. Using Blast and other Gene search engines, one continues down the sequence of the desired gene until one determines that the sequence targets only the mRNA for the desired gene. An example in our laboratory is that for liver-fatty acid binding protein (L-FABP), a 19 base oligonucleotide sequence was specific for L-FABP. Hammameih, FASEB J. in press. (Das et al., Clin. Cancer Res., 7:1706-1715, 2001). This antisense was able to block the effects of L-FABP in cancer cells.
In general, this approach is successful because the antisense fragment binds to the complementary region of the selected gene. At that point, several theories exist such as that RNases are activated due to the complementary oligonucleotide bound to the mRNA or that blocking the “start code”, along with binding of the complementary oligonucleotide to the selected gene, prevents mRNA synthesis. Never the less, extensive studies indicate that directed antisense blocks synthesis of the gene in question. Shi Q, et al., Constitutive and inducible interleukin 8 expression by hypoxia and acidosis renders human pancreatic cancer cells more tumorigenic and metastatic. Clin Cancer Res 5(11):3711-21 (1999); Cho-Chung YS, Antisense DNA-targeting protein kinase A-RIA subunit: a novel approach to cancer treatment, Front Biosci 4:D898-907 (1999); Tian XX et al, Altered expression of the suppressors PML and p53 in glioblastoma cells with the antisense-EGF-receptor. Br J Cancer 81(6):994-1001 (1999). Additionally, some of the genes (and their corresponding proteins) found to be altered in response to toxic agents have already been studied for other reasons and specific inhibitors exist to treat the toxic agent-induced illness. Respiratory distress induced by SEB is an example (see Table 1b). (Table 1b. is a table showing a list of genes that have been identified to be altered upon SEB exposure using DD-PCR.)
Although no one knew previously that these genes and their corresponding proteins were altered in response to SEB or other listed bio-threat agents, these mediators were well known to be involved in asthma-induced respiratory distress. As such, specific inhibitors have been and are being designed to target these products, such as antisense to specific genes or inhibiting agents of an enzyme or a signaling pathway.
Intravenous administration of antisense therapy is likely to be the most successful route since most of the action of toxic agents might be expected to be associated with lymphoid and endothelial cells. In addition, IV could be distributed to the kidney, liver and spleen.
For example,
The genes that are disclosed as upregulated can be found in public gene libraries. The preparation of antisense to these known genes is easily accomplished by known techniques to those of ordinary skill in the art. Likewise, the preparation of proteins for known genes is easily accomplished by known techniques to those of ordinary skill in the art.
We have shown that when SEB was given to pigs and the kidney was analyzed for levels of EPO, there was a downregulation of EPO upon SEB exposure (
Effect of Anthrax on Expression of Different Genes in Human Lymphoid Cells in Vitro:
Cells were exposed to anthrax spores for different time periods and RNA isolated from the cells. Primers were designed for each gene and RT-PCR performed on RNA samples from different time periods of Anthrax exposure. Gene expression of Ferritin heavy chain and GBP did not alter in response to Anthrax (
Genes identified from differential display in anthrax treated cells were also tested for the level of expression by RT-PCR.
Comparison of Gene Expression Pattern in SEB and Anthrax Treated Cells:
The expression of GBP was compared in SEB and anthrax treated cells. There was a significant difference in response in these two sets. SEB showed an up regulation of the gene however there was no change in expression of the gene in anthrax treated cells (
Expression of IL6 was compared in cells exposed to these two BW agents. IL6 showed a 50-fold increase by two hours of SEB exposure and it remained high even after 24 hrs. There was no change of IL6 expression in two hours of Anthrax exposure however there was only a two fold increase by 24 hrs (
Expression of HIF- 1 was up regulated in both the groups with SEB and Anthrax treated cells (
In
Differential Display Gel Profiles of Each BW Agent:
RNA was isolated from lymphoid cells after treatment with each agent. RNA was processed using differential display kits (obtained from Beckman-Coulter, Calif.) using 33P to label the PCR products and was resolved on a long-read gel. The gels were dried and exposed to X-ray films.
Cells were treated with SEB for 16 hrs and different AP (anchored primers) and ARPs (arbitary primers) primers were used for the DD-PCR reaction (
Cells were treated with anthrax spores for 12 hrs and RNA isolated and compared to the control at 12 hrs. The comparison of SEB and anthrax is shown in
Monocytes were exposed to Yersinia pestis for 30 mins. and were inactivated in gentamycin for two hours prior to RNA isolation. Combination of different APs and ARPs were used on these RNA samples in duplicate and resolved on a long gel. Bands that showed changes were cut out for further analysis (
Lymphoid cells were exposed to Cholera toxin for 12 hrs prior to RNA isolation. DD-PCR reaction was performed and resolved on a long gel. Bands of interest were isolated and purified for sequencing (
A prototype example is described using 2 shock-inducing toxins, staphylococcal enterotoxin B (SEB) and endotoxin, of which lipopolysaccharide (LPS) is the smallest active unit.
At the present time we have now found about 829 genes with altered expression, which have been observed upon SEB exposure to peripheral blood human lymphoid cells. Of these genes, the identity of 120 genes has been determined by comparing their sequences to known sequences in GENBANK databases. Those genes have never previously been associated with SEB-induced lethal shock.
We have also identified 85 genes appearing as bands on gel in anthrax exposure to peripheral blood human lymphoid cells and 28 bands on gel in Plague exposure to peripheral blood human lymphoid cells and about 30 bands on gel in Cholera exposure to peripheral blood human lymphoid cells, each band indicating a specific gene. See
Gene Changes for Anthrax n Monkey (see FIGS. 44-56):
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These peripheral blood human lymphoid cells can be obtained readily from patients and provide a reservoir of information due to their responses to toxins, infectious agents, etc.
We have catalogued patterns of responses for several toxins; the objective was to relate genes expressed in response to a biological warfare insult, to a map of responses predictive of physiological responses. Examples of maps of responses are shown in
One need not know the identity of the toxic agent to determine the likely progression of symptoms, based on markers/mediators induced. The advantages in screening for specific mRNA for diagnostic markers induced by BW agents is that it will provide a target for early detection of surrogate markers of impending illness. Having identified what genes are affected by the toxic agent, we are able to design strategies for treatment approaches to block their function and thus prevent the lethal shock or any other symptoms manifested by the agent.
Advantages of the Invention Over Current Processes:
Structural based probes may not identify biologically altered toxic agents and most certainly will not detect trace levels of potentiating agents which have the ability to dramatically enhance toxicity. Use of the present system in which host response to exposure is examined, not only takes into account bioengineered agents or contaminants, but also assists in designing appropriate treatment based on factors such as degree of exposure and the individual response to the toxic agent.
Problems that the Invention is Designed to Solve:
Identification of toxic agents that have the potential to be used in terrorist attacks or accidental exposures, have previously been based on structural characteristics of the known toxic agents. Because of the threat of biologically altered toxic agents or undetectable levels of trace potentiating contaminants, we have proceeded to develop alternate approaches which rely on an individual host's response and is independent of the need to determine which toxic agent is present. Instead, the type of impending illness (shock, neurological toxicity, etc) can be determined by analyzing gene expression patterns of the peripheral blood lymphoid cells from exposed individuals. In vivo, we have seen gene expression patterns that are indicative of shock as early as 30 min post-SEB exposure. For in vitro studies, we chose 2 hr post exposure as the first time period; we also examined 16hr, 24h and later time periods as well.
Predicting exposure of a person to these agents before the symptoms appear will be of great advantage for timely treatment which can decrease morbidity and mortality from exposure to toxic agents. As stated above, these genes can be places on a blot or a small DNA chip that can be used for screening blood cell samples for rapid detection.
Other Uses for the Invention:
In the studies carried out so far, SEB and LPS induced gene alterations were compared since both agents can lead to lethal shock. Exposures to SEB can be detected based on host response and tailored treatment designed. Septic shock, induced by LPS from gram negative bacteria, is a usual emergency room occurrence daily; perhaps >20% of all emergency room cases are related to septic shock. Over at least the past 30 years, the finest pharmaceutical companies in the world have vigorously pursued studies to identify intervention tactics for septic shock; successes have occurred mainly. for early stages of shock. We have now identified genes, never before associated with lethal shock, that directly influence vascular tone (possibly the most critical element of lethal shock). Targeting these genes provide new approaches to combat this deadly illness.
Novel Aspects of the Invention:
We have identified a panel of host genes altered in response to BW agents that can be used as diagnostic markers. This has not been previously described. The advantages in screening for specific mRNA markers induced by toxic agents is that it provides a target for early detection of surrogate markers of impending illness. Having identified what genes are affected by the toxins, we have designed strategies for treatment approaches to block their function and thus prevent the lethal shock.
Patterns of Mediator Production Reflect Exposure to a Specific Toxic Agent:
We had previously observed that various toxins produced a distinctive pattern in production of mediators of illness when using either cultures of human lymphoid cells or when using plasma and/or lymphoid cells from animal experiments. It is impractical to try to measure mediators produced because a) they appear, usually transiently, from minutes to hours or days and b) they are usually unstable. Therefore, we decided to create a library of responses to toxins using mRNA, which has none of the problems associated with the mediators, themselves.
Patterns of Gene Expression Reflect Exposure to a Specific Toxic Agent:
We found that each toxic agent alters gene expression in the host in a unique pattern. Lymphoid cells provide a readily accessible reservoir of information that can reveal direct or indirect responses to toxic agents. As prototype toxic agents in our initial studies, we assessed the biologic effects on lymphoid cells by certain toxins that induce lethal systemic shock in primates. Though different mechanisms staphylococcal enterotoxin B (SEB) induce production of a cascade mediators whose activities lead to shock. The release of endotoxin, of which lipopolysaccharide (LPS) is its smallest active unit, from the cell wall of gram-negative bacteria, and subsequent production of numerous host mediators, is the initiating event of septic shock (Pugin, J., C. C. Schurer-Maly, D. Leturcq, and et. al. 1993. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA. 90:2744-2748; Wright, S. D., R. A. Ramos, P. S. Tobias, and et. al. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 249:1431-1433.1990). In contrast, SEB acts as a super antigen, stimulating T cell proliferation (Jett, M., R. Neill, C. Welch, T. Boyle, E. Bernton, D. Hoover, G. Lowell, R. E. Hunt, S. Chatterjee, and P. Gemski. 1994. Identification of staphylococcal enterotoxin B sequences important for induction of lymphocyte proliferation by using synthetic peptide fragments of the toxin. Infect Immun. 62(8):3408-15.1994; Neill, R. J., M. Jett, R. Crane, J. Wootres, C. Welch, D. Hoover, and P. Gemski. 1996. Mitogenic activities of amino acid substitution mutants of staphylococcal enterotoxin B in human and mouse lymphocyte cultures. Infect Immun. 64(8):3007-15. 1996), inducing a number of cytokine genes and other mediators in lymphocytes and monocytes (Yan, A., G. Yang, and M. Jett. 1997, Cholera toxin induces TNF-a production by human monocytes via cAMP independent pathways. FASEB J. 10:2746.). In our laboratory we have shown that SEB induces high levels of CD69 (Yan,, 1997. Protein kinase C is involved in SEB induced TNF-α production. FASEB J. 10:1922) while LPS produces a minor change in this surface marker. In contrast, TNF-α production is rapidly elevated by LPS whereas SEB produces modest changes in its production (Yan). These changes which we have characterized are just a few of a battery of potential biomarkers indicative of patterns of impending illness. Production of a unique pattern of mediators of serious illness in response to toxic agents, is indicative of the type of illness or toxicity that will follow.
We have now proceeded to identify a spectrum of genes altered in response to toxic agents using the technique of differential display. Briefly, we have identified 829 altered genes in response to SEB; many of these genes differ from the genes activated by LPS. Furthermore, our studies with SEB have revealed completely new responses to the toxin that have never before been characterized and present new therapeutic approaches. We have further verified in monkeys challenged with SEB (compared with using each monkey as its own control in a saline sham), that the selected genes were altered as predicted in response to the toxin. These genes not only provide diagnostic capabilities for toxic agents, they indicate exposure dose, and also they also provide potential new targets for events that ultimately lead to SEB induced lethal shock. We have further characterized gene responses induced by several other biothreat agents and they also provide new targets for therapy.
Lymphoid Cells:
This approach centers on the fact that peripheral blood lymphoid cells can serve as a reservoir of historical information and can be readily obtained from an exposed individual. Furthermore, even though lymphocytes may not be the cells most affected by a biological, infectious or chemical agent, they tend to respond to BW agents by either direct or secondary stimulations. Indeed, certain tissues most affected by many toxic agents are inaccessible.
Our approach relies on determination of a battery of unique genes altered in response to each of the toxic agents. We have used staphylococcal enterotoxin B (SEB) as a prototype and have found 829 genes with significant alterations in expression upon exposure, in vitro, of human peripheral blood lymphocytes to the toxin (See
Global Library:
This invention provides for a library of gene responses to BW agents. These genes can be targeted for treatment regimes for these agents. We have provided a list of genes that are induced by Brucella, Plague, SEB, SE's Anthrax, VEE and Dengue. These agents fall into groups causing similar gene alterations for some agents, yet pinpointing unique responses with a battery of other genes. With SEB and LPS, IL-6, TNF-alpha and a few other mRNA changes, may not distinguish between the two shock-inducing toxins. In contrast 6 of the numerous genes exhaustively examined to date show unique alteration in response to SEB and not to LPS. Selected genes act as markers, in a time-dependent manner, predicting the pattern of illnesses before the actual symptoms appear. Identification of specific genes that are differentially expressed in response to BW agents has revealed molecular pathogenesis that will enable us to design intervention to prevent or ameliorate impending severe illness.
The host gene expression patterns act as diagnostic markers. We have generated a library of genes altered by each toxic agent. These libraries consist of hundreds of genes altered upon exposure to each agent. See Tables 2-9.
We have shown changes in gene expression in lymphoid cells induced by Brucella, Plague, SEB, Anthrax, VEE and Dengue. We have shown gene changes in monkeys exposed to Anthrax and SEB. We have shown changes in gene expression in kidney cells induced by SEB, and have confirmed the changes in monkey samples. We have compared the pattern in SEB with LPS induced changes in both the cell systems. We have also shown the effect of drugs to block the SEB induced effects in lymphoid cells.
Changes in Gene Expression Induced by SEB.
We decided to examine the changes in levels of gene expression induced by these toxins in order to move away from the inherent difficulties in quantitating cytokine changes and to try to identify new therapeutic targets. Using SEB as a prototype, we studied changes in expression of mRNA using selected RT-PCR primers and subsequently performed the technique, differential display (DD). Table 1 shows changes in expression patterns of numerous genes both up- and down-regulated. These genes have been isolated, cloned, sequenced and characterized.
Genes 1, 2 and 5, that have been positively identified by database comparisons, are genes coding for proteins, not previously implicated in SEB action on lymphoid cells. They have varying activities and functions; there is a common theme of association with adhesion molecule function. These proteins may provide clues for new approaches in the treatment of lethal shock.
Although some gene sequences are not identified, the diagnosis of toxin can be made based on the location of the gene on the gel as shown in
Discussion of the genes in Table 1b.
Gene #1—Connective Tissue Activating Protein III (CTAP-III)
A cDNA which codes for a protein released from activated platelets and represents an inactive precursor connective tissue activating protein III (CTAP-III) (85 amino acids) was down regulated. This inactive precursor chemokine has shown to be proteolitically cleaved by leukocytes and leukocyte derived proteases at the N-terminus (Harter et al., 1994). These proteases have been shown to proteolitically process the above inactive chemokine to a neutrophil activating chemokine near sites of inflammation and vascular lesions (Harter, et al., 1994). The activation of the neutrophil activating chemokine has shown to aggravate the course of thrombotic diseases and their sequelae, as in atherosclerosis, by inducing inflammation and tissue damage (Walz, et al., J. Exp. Med. 170(5), 1745-1750, 1989). Inflammation and tissue damage are two conditions that are widely associated with SEB exposure. Here we show a cDNA, which had a high identity to CTAP-III, which was down-regulated through DD-PCR, and the down regulation was confirmed through RT-PCR and northern hybridization (
Gene #2—Chondroitin Sulphate Proteoglycan Versican 1
A cDNA that was down regulated is known to code for a chondroitin sulphate proteoglycan versican V1 that belongs to a growing family of large aggregating proteoglycans (Doege, et al., J. Biol. Chem, 266, 894-902, 1991; Doege, et al., J. Biol. Chem, 262, 17757-17767, 1987). The side chains containing a few chondroitin sulphate chains of these proteins protects the endothelium from oxidant injury and direct cytotoxycity (Nakazona, et al., Proc. Natl. Acad. Sci. USA, 88, 10045-10048, 1991; Abrahamsson, et al., Circ. Res., 70, 264-271 1992; Redni, et al., biochem. J., 252, 515-519, 1988). It is known that the changes in heparan sulfate metabolism might lead to profound changes in the physiology of blood vessels and removed from the endothelium in the course of inflammation. This was present in all types of blood vessels, ranging from the large caliber aorta to smallest capillaries. A decrease in proteoglycan may contribute to the loss of barrier properties therefore reducing in the thickness of the blood vessels, which may contribute to low blood pressure conditions, which is common in patients exposed to SEB and are symptoms associated with SEB induced shock. It is the first time such a gene has been identified to explain the low blood pressure conditions associated with SEB.
Gene #3
A novel gene that appeared on the gel but did not match with any of the available sequences of GenBank.
Gene #4—Interleukin-6 (IL-6)
Expressing of high levels of interleukin-6 by SEB is well documented. Experiments done on peripheral blood mononuclear cells (PBMC), with SEB have indicated the detection of elevated levels of IL-6 within 48 hours (Sperber, et al., Clin Degn Lab Immunol., 4, 473-477, 1995). Other experiments done using nonlethal dose SEB studies on human primates have indicated significant increased levels of IL-2 and IL-6 after four hours of receiving non lethal doses of SEB (Kerakaumer, et al., Mil. Med., 9, 612-615, 1997). Our results agreed with the above results, as we also observed high levels of IL-6 production within two hours of SEB induced human lymphoid cells first by DD-PCR and second by RT-PCR (Fig.5). As IL-6 is a common cytokine induced by many toxins, it cannot be used to differentiate the effect of SEB from other toxins.
Gene #5—Myosin Class 1 (Myc-1)
A cDNA, which coded for myosin class 1 was clearly up-regulated through DD-PCR. This motor domain containing proteins have shown to lead to significant cardiac dysfunction (Colbert, et al., J. Clin. Invest., 100, 1958-1968, 1997) showed a two fold up-regulation through RT-PCR and may explain the cardiac discomfort observed in subjects who are already suffering from other diseases and elderly who have been exposed to SEB.
Gene #6—Hypoxia Inducible Factor 1 (HIF-1)
Upon stimulation by SEB a set of genes that are observed under reduced oxygen content were differentially expressed. A key step to hypoxia inducible activation is the formation of a heterodimeric complex of two helix loop helix PAS proteins (Wang, et al., Proc. Natl. Acad. Sci.USA, 92, 5510-5514, 1995). The helix loop helix transcriptional factor consists of a 120 kDa subunit complexed with a 90-94 kDa subunit induces respiratory distress. The up regulation of this cDNA, which codes for hypoxia inducible factor-I (HIF-1) detected through DD-PCR was confirmed by RT-PCR (
Gene #7, #9 and #10
Novel genes that appeared on the gels but did not match with any of the available sequences in Gen Bank.
Gene #8—Guanylate Binding Protein (GBP)
An up-regulated cDNA detected through DD-PCR is known to code for an interferon (IFN) induced 67 kDa guanylate binding protein-2, which has a wide variety of basic cellular functions such as protein synthesis, signal transduction, and intracellular protein transcription (Bourne, et al., Cell, 53, 669-671, 1988). Its ability to increase cyclase activity results in the production of high levels of NO, vasodilation and a threat to the endothelium. SEB induction of this gene suggests (
Confirmation of Gene Changes in Monkey Samples Exposed to SEB
We exposed several rhesus monkeys with a sublethal dose of SEB (12-24 ug/kg cumulative via aerosol) and the controls with a saline challenge, isolated blood cells and prepared RNA from them. RT-PCR was performed for three separate genes that were altered in response to SEB in human lymphocytes. IL6 showed an increase over the control monkey samples suggesting that this cytokine does play a crucial role in SEB induced toxicity (
Comparison of Changes in Gene Expression in SEB and LPS Induced Lymphoid Cells:
When genes identified by DDPCR were analyzed and compared in two different toxins, we found there were some differences in their expression patterns. As shown in
In an attempt to determine how the kidneys may be contributing to SEB-induced lethal shock, Gene changes observed in human kidney cells (renal proximal tubule epithelial cells-RPETC):
Expression pattern of RhoE in Human Lymphoid Cells
RhoE is a small G protein that lacks intrinsic GTPase activity (Foster, et al., 1996). This protein is involved in cell adhesion. As shown in
Comparison of Gene Changes Induced by SEB and LPS in Kidney Cells:
Genes such as GBP, IL6 and Ferritin were induced by LPS in the kidney cells (
Genes encoding HIF-1 and Myosin heavy chain were both up regulated in kidney cells but LPS did not show any change (
Primary Cell Cultures: Cell Isolation/Purification from Plasma of Healthy Human Donors.
Human lymphocytes and monocytes were prepared from leukopacks from noimal donors according to Jett et al 1994 using lymphocyte separation medium histopaque 1077. Lymphocytes and monocytes were purified and separated further by counterflow centrifugation-elutriation with PBS as the eluant. Jett et al 1994.
Differential Display:
The differential display approach was introduced in the past few years and has become a potent tool for identifying genes that are differentially expressed in various eukaryotic cells and organs or under altered conditions. Differential Display was used to obtain the results shown in tables 1a, 1b.
The cells (12.5 E6 monocytes plus 50E6 lymphocytes in plastic tissue culture flasks containing 175 cm2) were exposed to these toxins for various appropriate time periods (1 hr-24hrs) andmRNA was isolated. The technique of differential display involves isolation of undegraded mRNA free of genomic DNA. Reverse transcriptase (RT) is necessary for conversion of mRNA to single stranded cDNA by using a two base-anchored oligo-dT primer T12MA, T12MC, T12MG and T12MT where M is a mixture of dA, dC and dG obtained from Beckman Coulter, Calif. A fraction of this reaction mixture of the cDNA was amplified by PCR using appropriate primers and radio labeled dNTP. The PCR products were separated on a 6% Sequencing polyacrylamide gel, after developing the gel we looked for differences in the treated vs untreated lanes for presence/absence/intensity of bands as described previously. Both positive and negative controls were included to avoid false positives. In addition to samples with and without toxin, controls include +/− RT product, +/−primer, etc. Once the different bands are identified, they were cut out of the gel, eluted by soaking in PCR buffer at 37 C for 30 min and reamplified by a repeated PCR using the same primers pairs of AP and ARP to confirm the changes. The final confirmation was carried out on a Northern blot, where the MRNA samples were run on a gel and each of these bands labeled and used as a probe to see if the changes are reproducible. Once this is confirmed then the cDNAs was cloned into a vector. Cloning was performed in a TA-TOPO vector from Invitrogen according to their protocol and sequenced to identify the nature of the gene. The sequence was compared to the gene bank database to look for homology with other already identified genes or find out if they are unique in any way. RT-PCR was also performed to confirm the changes in gene expression by each agent.
This technique is highly sensitive and reproducible, and is a rapid method for identifying unique genes, quantitatively, which are altered upon treatment of cells with the compound of interest. This information provides a library of genes that are activated by toxins/agents producing serious illness, it will aid in identification of new treatment modalities. Thus this technique has enormous potential; identifying the changes occurring at the molecular level in a system has radically changed concepts in biomedical research by opening new avenues for diagnosis and therapy. We have already used this technique and have identified many genes altered in expression in our prototype studies with SEB.
Other techniques that have been used are Gene Microarray technique to identify the changes induced by these toxic agents.
Gene array:
This technique allows us to screen thousands of genes for their expression pattern in one experiment. The gene array blots were purchased from Clontech laboratories or were slides custom printed in house, the RNA samples were labeled with 33P and hybridized to the blots according to the manufacturer's instructions. For slides RNA was labeled with fluorescent dyes, hybridized to the slide and scanned in Axon scanner. The image of the blots was scanned in a BIORAD Multiflor scanner and the data was analyzed using various softwares. ATLAS software 2.0, Gene pix, Gene Spring was used to get numbers for each spot for control and treated samples. The numbers were normalized and then the ratio obtained by dividing the adjusted numbers of treated sample over the control. The tables presented here represent the fold change induced by each agent at various time points.
Using these techniques, we screened 7,000 genes at a time to yield information in a time efficient manner and to quickly build a gene library for each toxic agent.
Measurement of Gene Changes by Using DNA Chips:
This is an innovative approach of analyzing changes in gene expression in a sample for a large number of genes simultaneously. The development of recent technologies allows us to immobilize DNA to a solid surface such as glass and exposed to a set of labeled probes. The array is then exposed to fluorescent labeled sample RNA, hybridized and the positive signals analyzed.
Biorobotics machine can spot thousands of genes on 48 slides at a time in duplicate on glass microscope slides in an area of 2.5 cm by 0.75 cm with the use of this high speed arraying robotic machine. Because allele-specific probes for each mRNA are specifically chosen and synthesized in known locations on the arrays, the hybridization patterns and intensities can be interpreted in terms of the identity and the concentrations of various mRNAs simultaneously. Multiple spots for each cDNA can be used to better quantify the concentration of mRNA. Probes specific for each symptoms will be used such as genes for lethal shock, or genes for neurotoxic agents that will determine which agent was involved in causing the gene changes in the blood sample.
The genes listed for each agent have been selected to construct gene chip specific for each agent, the inventors also have combined all the gene list and has created a gene chip with all the genes presented here. These chips can be used routinely to screen several samples in a cost effective manner.
In this example, lymphoid cells are treated with pathogens/toxins: 2, 6, 16 hr exposure; RNA is isolated. Lymphoid cells are exposed to various BW agents for defined time periods and RNA free of genomic DNA is isolated using trizol method. Enough human lymphoid cells are started to isolate RNA at all the time points for each BW agent. This RNA is used for screening of changes in gene expression pattern by several methods.
In this example, DD-PCR, +/− SAGE or Gene Array is used to isolate altered genes, purify, and amplify. DD-PCR is performed using various combinations of anchored and arbitary primers to cover the entire cDNA population. The DD-PCR products are resolved on a sequencing gel and changes for each agent analyzed. An example of this is shown in Table 1a. (Table 1a is a table describing the number of genes altered with each primer combination using DD-PCR with SEB treated cells.) At each step proper negative (reaction minus RT products, etc) and positive controls (supplied RNA from manufacturer) are used and samples are handled in duplicates to avoid false signals. Genes are up- or down-regulated by each BW agent. Gene arrays from Genome Systems Inc. St. Louis, Mo., can be used to screen a whole library of 18,000 genes at a given time. To obtain more global changes SAGE can be used, a new technique for analyzing the whole cDNA more rapidly.
The techniques outlined in the Examples above are used to identify specific genes altered in response to the 6 listed BW agents. We have also verified the changes using dose and time course variations in direct analysis using standard PCR primers. Changes identified from all these techniques can be verified by northern blots to avoid false positives. Some of the BW agents used may require the longer (24 h) incubation times for gene changes to appear; also, secondary effects (because of other tissues being the BW target) may cause gene changes which would not be seen in the in vitro system. Potentially, some of those changes will still be picked up upon in vivo exposure to the BW agent.
Purify, sequence genes from Example 3, identify using GENBANK databases; catalogue the genes identified for each specific agent and select genes which will discriminate among a variety of B/W agents. Each gene is re-amplified and sequenced using either cycle sequencing kit (Amersham) or using the ABI kit. We have currently found that ⅔ of the genes give a positive match in the Genebank database. Any new genes that look important as a BW agent marker, are cloned into a bacterial plasmid; we can then screen a cDNA library and identify the gene. This will provide a selected a pattern or panel of genes for each BW agent.
After confirming the changes identified by DDPCR, and Gene array, specific oligos can be designed or cDNAs that will be used to verify responses to various agents in vitro and in vivo. These genes can be attached to a matrix (membrane or on glass surface) for establishing a diagnostic tool for rapid detection. Since these are known genes whose sequence information is already available in the Gene Bank, antisense oligos to these genes can be also designed for specific treatment.
RT-PCR and northern analyses to confirm these changes, and determine alterations at intermediate time periods. Develop a quantitative PCR for selected genes: Specific primers are designed for each gene identified and a northern blot analysis is performed for all the RNA samples. A standardize method is used to quantify our PCR results-using nonradioactive probes [biotin-labeled specific probes for a PCR ELISA]. All necessary controls are used for such a procedure.
Expose animals/non-human primates to the BW agent in question: Blood samples are taken from various animals exposed to respective BW agents at 0, 2, 16 h; the blood samples are collected, lymphoid cell fraction are isolated, RNA is extracted, quantitative PCR measurements based on the unique genes altered in response to each specific agent are performed. The selected genes are confirmed by simple RT-PCR methods, then if appropriate these samples are tested on DNA array matrices.
Homo sapiens Down Syndrome critical region,
Homo sapiens elf-1 related protein (ELFR) mRNA,
Homo sapiens mRNA for dual specificity
Homo sapiens regulator of G protein signaling 10
H. sapiens Wnt-13 Mrna
Homo sapiens N-terminal acetyltransferase complex
Homo sapiens ribosomal protein L26 (RPL26
Excised cDNA of differentially expressed genes by SEB were subjected to RT-PCR using custom designed primers. Equal quantities of expressed DNA were resolved on an agarose gel, quantified, normalized with actin and the expression was compared to control levels.
X represents no effect,
‘up’ and ‘down’ represents an up and down regulation of the gene by the respective toxin respectively and
N.D. represents the values not obtained at the respective time point.
Renal proximal tubule epithelial cells were incubated with or without 50 ng/ml SEB for 12 hours. Total mRNA was isolated and DD-PCR performed as described. The 32 differentially expressed genes are currently at various stages of isolation, purification, sequencing, and identification.
*AP - anchored primer
**ARP - arbitrary primer
14 up regulated
18 down regulated
Total of 85 bands have been identified to be altered by Anthrax in human lymphocytes using differential display. So far 10 bands have been sequenced, the rest are being sequenced currently.
Description of gene changes induced by each threat agent that can be used for diagnostic as well as therapeutic strategies:
Gene lists were obtained after screening of several gene arrays. Each agent was exposed to the cells and RNA isolated for gene array experiments. The untreated and treated samples were then labeled with 33P and hybridized to the arrays. The signals were obtained by scanning in a BIORAD scanner and the intensities of each spot was normalized with the housekeeping genes.
Gene Based Solution for Therapy:
The present invention uses gene expression patterns to identify genes that are turned on or off in response to exposure to a toxin agent. Some of the early genes have been used as diagnostic markers. With this understanding of the pathways involved in signaling of various biothreat agents, we have identified targets for therapeutic agents. The present invention is directed towards treatment of patients when exposed to various biological threat agents based on gene targets identified.
a. Major Gene Changes Induced by SEB Toxication:
Genes involved in various functions have been identified. These genes are regulated by exposure to a toxic agent and provide therapeutic potential for treatment of the disease caused by these agents by an understanding of the time of appearance of these gene changes and their function. For SEB, genes whose expression was downregulated after 24 hr of SEB lethal challenge are ABP (angiotesin-binding protein), AVRlA (arginine vasopressin receptor 1A), and VAP (vasopressin). Genes whose expression was upregulated after 24 hr of SEB lethal challenge are ANG2 (angiopoietin 2), Tie2 (it is receptor for ANG2), VEGF, (vascular endothelial growth factor), FLT1 (VEGF receptor), iNOS (its product is nitric oxide (NO), NO is a potent vascular dilator)). Several cytokines and cytokine regulated genes such as Interleukin-2, TNF-alpha, Interleukin-6, Guanylate binding protein, Interferon-gamma were also upregulated compared to saline treated pigs. It is important to know time zero of exposure to a toxic agent that induces cytokine release to calculate the appropriate anti-cytokine therapy.
In
For genes that are downregulated, increasing the proteins or their products helps in treatment of the disease.
1. ABP (angiotesin-binding protein): Involved in contractile responses of arteries and muscle cells to angiotensin II. Tissue angiotensin II is known in the regulation of inflammatory and fibrogenic components of repair in vascular and nonvascular sites of cardiac injury, the rat heart. This protein is involved in healing and downregulation of this gene is bad for the body (Sun et al, J Lab Clin Med. 2004 January;143(1):41-51).
2. Vasopressin: Vasopressin is a protein secreted by the kidney and can induce vasoconstriction. Vasopressin is emerging as a rational therapy for vasodilatory shock states. Unlike other vasoconstrictor agents, vasopressin also has vasodilatory properties. There are now multiple agents being developed for the treatment of heart failure designed to block many of the neurohormones that are increased in these patients. One of the hormones that is increased in chronic heart failure is vasopressin. Vasopressin reduces free water secretion and at high concentrations, causes vasoconstriction in the peripheral vasculature. Administering vasopressin to a patient that shows the symptom of down regulation of the gene for vasopressin is an effective treatment.
For genes that are upregulated, blocking these genes or gene products with antisense to these genes is beneficial for the treatment of the disease.
1. INOS: INOS's product is NO. NO is a potent vascular dilator.
Nitric oxide (NO), a potent vasodilator, plays a significant role in the vascular hyposensitivity to vasoconstrictors related to portal hypertension. Chronic NO inhibition ameliorates portal-systemic collaterals in portal hypertensive rats.
2. Angiogenic growth factors such as Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF) induce NO and require NO to elicit an effect.
3. 5HT2A: 5HT2A is also a potent vascular constrictor. 5HT2A can lead to the smooth muscle in the veins to constrict and thus lead to even further vascular and capillary damage
4. VEGF and Flt and their related genes are responsible for the vascular leakage by damaging endothelial cells.
1. Effect of antithrombin for treatment of lethal shock induced by SEB:
We have identified genes that are involved in coagulation and therefore antithrombin was tested for its effect to block lethal shock in our piglet animal model. Lethal shock is triggered by inflammatory mediators, vascular leakage and ischemia. We believe that antiihrombin can block these effects.
Antithrombin HI (AT III) is a serine protease inhibitor, which acts as a major inhibitor of thrombin. Apart from its role in homeostasis, AT III exerts anti-inflammatory properties and improves survival in animal sepsis models and disseminated intravascular coagulation (DIC). AT III reduces leukocyte-endothelial cell interaction, prevents microvascular leakage and ameliorate ischemia/reperfusion injury.
When antithrombin was administered after the symptoms (2 hrs after exposure to the toxin) appeared after exposure to lethal dose of SEB (a biological threat agent), the animals showed improved pathology when compared to the untreated controls. When antithrombin was given long after the symptoms appeared, that is 6 hrs after exposure and 24 hrs after exposure, the pigs still survived the lethal dose of the toxin suggesting therapeutic potential as a treatment regimen long after exposure. Antithrombin can be administered 2-24 hours after exposure and it is preferred to administer 2-12 hours after exposure.
Anti-Thrombin (lmg / animal-250-300 ug/ Kg) was administered in two ways:
This drug blocks the effects of a cytokine called TNF-a, tumor necrosis factor-alpha. Pentoxiflyline is a methylxanthine derivative that inhibits the production of TNF-a by endotoxin-stimulated monocytes/macrophages at the transcriptional level. It is effective in reducing TNF-a levels in mice with endotoxic shock. Pentoxifylin is an anticytokine.
Pentoxifylline (50 mg/animal, 12.5-16.5 mg/Kg body weight) was administered in two ways:
It is preferred to administer Pentoxifylin within 4 hours of exposure to a lethal shock inducing agent. When administered at 24h after SEB challenge, it had no effect. So early administration is the key for effective therapeutic window.
3. Tyrosine Kinase Inhibitors for Treatments of Lethal Shock:
There were several tyrosine kinases that were activated upon exposure to these toxic agents. We tested to see if inhibiting these kinases would have any effect on the symptoms induced by the toxin in the animals. These inhibitors (Herbimycin, Genistin) did not show any significant changes upon treatment compared to the untreated controls.
Herbimycin (250 ug/ animal—Herbimycin 62.5 ug/Kg, Genistin 50 ug/Kg) was administered:
Genistin (200 ug/animal) was administered:
Hetastarch 6% in 0.9% saline administered at 72 hours during stage of lethal shock failed to revive an SEB intoxicated Piglet.
5. Effect of Zofran for Treatment of Incapacitation:
Treatment for SEB-induced incapacitation: In a prior Non human primate (NHP) incapacitation study in which we examined the appearance of various inflammatory mediators in plasma, we observed elevated plasma serotonin (5-HT) levels, and we realized that many of the clinical signs could result from the elevated levels of that mediator (
We developed the piglet model to test 5-HT receptor blockers, because NHP are difficult to use for incapacitation studies, since they cannot be handled without anesthesia, and NHP hide signs of illness. Both Kytril and Zofran were effective as the 5-HT receptor blockers (Zofran was easier to use). We did not administer the drug until after the onset of vomiting and diarrhea. There were usually one or two more incidents of retching or diarrhea after administration of Zofran, then the animal would usually go to the food dish and begin to eat. (
When Zofran was administered at symptoms the animals recovered from emesis and there was a slight improvement in lowering the temperature at 72h post treatment. However there was slight improvement in lowering the temperature at 72 hours post treatment. However, there was no change in the blood pressure levels in treated and untreated animals.
Zophran® (1,2,3,9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)]4H-carbazol-4-one,monohychloride, dehydrate) is manufactured by Glaxo Wellcome, Inc., Research Triangle, North Carolina. We have shown that Zofran blocks the cytokine surge in these animals, no one before has shown effect of Zofran on SEB induced symptoms or on cytokine responses.
It is preferred to administer Zofran within 2 to 3 hours of exposure to a lethal shock inducing agent. It is preferred to administer Kytril within 2 hours of exposure to a lethal shock inducing agent.
As shown in
It has been found that different drugs administered at different times block edemas (
6. Use of EPO as a Treatment for Lethal Shock:
Erythropoietin, the principal growth factor of erythropoiesis, stimulates proliferation and differentiation of erythropoietic cells (Erslev, 1987) and amplifies the production of red blood cells by inhibiting the premature death (apoptosis) of their precursor cells (Koury and Bondurant, 1988).
Erythropoietin is the only know hematopoietic growth factor that acts like a hormone (Spivak, 1995). It is predominantly produced by the pertubular cortical fibroblast-like cells of the kidney. The site of its action is hematopoietic cells in the bone marrow. Expression of EPO is strictly tissue specific and in fact tissue hypoxia is the only physiological stimulus for EPO production (Spivak, 1995). A key element in this stimulation is a heterodimeric transcription factor called hypoxia inducible factor I (HIF-I), which upon activation binds to an enhancer element 3′ to the EPO gene (Wang and Semenza, 1995). For over a decade, treatment with recombinant erythropoietin was part of the therapy of renal diseases and chemotherapy-induced anemia (Krantz, 1995). We have examined the role of erynthropoietin in controlling the blood pressure in SEB induced cells in vivo.
No one has examined regulating the blood pressure in SEB induced lethal shock in vivo using erythropoetin or other proteins in its regulatory pathway. Our results suggested that kidney cells play a very important role in SEB induced lethal shock. A very preliminary finding is that the kidney from a piglet treated with SEB did not show detectable EPO gene expression while a control animal kidney expressed the EPO gene in abundance. We hypothesize that giving EPO to patients who have been exposed to SE toxins will be able to regulate the blood pressure. See
As shown in
Erythropoitin (500 U /Kg body weight) was administered in the following ways:
Gross Pathology:
A) Administered at 2 hr/ 12 hr/ 24hr: 2/3 piglets had moderate gross pathology, while 1/3 had similar pathology compared to SEB.
B) At 2 hrs post SEB—1/1 pig had moderate pathology—probably 10-20% improvement in pathology over SEB controls
C) at 12 hours post SEB—4/5 pigs had similar pathology to SEB control, while 1/5 pig showed a slight reduction in pathological symptoms
D) At symptoms (i.e., 3-4 hrs post SEB)—2/2 pigs—probably 10% improvement in pathology over SEB controls, but the animals died at 96 hours (Lethal shock).
It is preferred to administer EPO at 2-12 hours after exposure to a lethal shock inducing agent.
Some of the Promising Treatments/Prophylaxes Based on Gross Pathology Are:
Pentoxifylline (best therapeutic up to 4 hours)—No perirenal or mesenteric edema, though there is mesenteric lymphadenopathy (
Anti-thrombin—No generalized lymphadenopathy, but some perirenal and mesenteric edema observed (
Anti-translocating Peptide—definitely appears to be the best of the lot. Peptide was administered 2-5 mins prior to SEB intoxication.
Staphylococcal enterotoxin B (SEB) causes serious gastrointestinal illness, and intoxication with this superantigen can lead to lethal toxic shock. In order to overcome significant shortcomings of current rodent and non-human primate models, we developed a piglet model of lethal SEB intoxication. Fourteen-day-old Yorkshire piglets were given intravenous SEB, observed clinically and euthanized at 4, 6, 24, 48, 72 or 96 hours post treatment. Clinical signs were biphasic with pyrexia, vomiting and diarrhea within 4 hours, followed by terminal hypotension and shock by 96 hours. Widespread T-lymphocyte proliferation was apparent in most piglets by 24 hours and all piglets by 48 hours. By 72 hours lymphadenopathy had progressed to markedly enlarged, dark red lymph nodes characterized histologically by hemorrhage, edema, perivascular fibrin accumulation and widespread lympholysis. At 72 hours there was severe widespread edema, most prominent in the mesentery, between loops of spiral colon, and in retroperitoneal connective tissue. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and brain, circulating lymphoblasts and lymphocytic portal hepatitis. Study of this piglet model will further elucidate the pathogenesis of SEB intoxication and enable us to test new therapeutic regimes.
The Staphylococcal enterotoxins (SE) are a group of pyrogenic exoproteins produced by gram-positive Staphylococcus aureus. Exposure to SE has been shown to initiate a range of clinical abnormalities from gastrointestinal upset to lethal toxic shock syndrome (TSS). Once introduced into host tissues these proteins have the ability to elicit pathology in many different systems. Within 4 hours of ingestion SE symptoms can be documented and these include: vomiting, diarrhea, nausea, and abdominal pain (Jett M, Brinkley W, Neill R, Gemski P, Hunt R: Infect Immun 1990, 58:3494-3499). Normally enterotoxicosis abates within 24 hours with mild anorexia that persists for up to five days. Currently there are twelve serotypes of SE described, named sequentially by letter (Jarraud S, Peyrat M A, Lim A, Tristan A, Bes M, Mougel C, Etienne J, Vandenesch F, Bonneville M, Lina G: J Immunol 2001, 166:669-677). Staphylococcal enterotoxin B (SEB) is one of the most clinically significant and well-studied members of this family. SEB is known to induce typical food poisoning symptoms, such as fever, vomiting and diarrhea, is implicated as a potent inducer of TSS, and is a potential biological threat agent (Marrack P, Kappler J: Science 1990 Jun 1;248(4959):1066). Much of the lethal effects of SEB have been attributed to superantigenicity and subsequent T-cell proliferation with massive inflammatory cytokine release (Miethke T, Wahl, C.,et al.: Journal of Experimental Medicine 1992, 175:91-98; Johnson H M, Torres B A, Soos J M: Proc Soc Exp Biol Med 1996, 212:99-109).
Unlike traditional antigens, superantigens (SAgs) can stimulate up to 20% of the host's T-cell repertoire. This is accomplished by their unique ability to bypass conventional antigen processing and presentation. Extracellular SE successfully binds both MHC II on antigen presenting cells and the T-cell receptor; creating a functional immunological synapse Jardetzky T S, et al: Nature 1994, 368:711-718). Specifically, it has been shown that interactions with SAgs primarily involves the variable region of the TCR beta chain (Johnson H M, Torres B A, Soos J M: Proc Soc Exp Biol Med 1996, 212:99-109). Subsequent to proliferation, most T cells whose cognate antigen is not present will undergo clonal deletion, resulting in immunosupression. By contrast, in susceptible individuals activated T cells may continue to be stimulated and exacerbate autoimmune disease (Johnson H M, Russell J K, Pontzer C H: Faseb J. 1991, 5:2706-2712).
Of great interest is SEB's ability to interact with non-immunological tissue. In the gastrointestinal tract it has been shown that SEB posses the ability to bind and traverse protective intestinal epithelia (Hamad A R, Marrack P, Kappler J W: J Exp Med 1997, 185:1447-1454; McKay D M, Singh P K: J Immunol 1997, 159:2382-2390). After this process of transcytosis, SEB gains access to circulation and systemic tissue. In the kidney proximal tubule SEB has been shown to bind galactosylceramide. This binding has potential implication in the etiology of SEB-induced hypotension and renal failure (Chatterjee S, Khullar, M., and Shi, W. Y.: Glycobiology 1995, 5:327-333; Chatterjee S, Jett M: Mol Cell Biochem 1992, 113:25-31; Normann S J: Lab Invest 1971, 25:126-132). In in vitro systems SEB demonstrated marked effects on pulmonary arterial cells. Toxin exposure elicited barrier dysfunction which occurred in the absence of effector cells or their intermediate products (Campbell W N, Fitzpatrick M, Ding X, Jett M, Gemski P, Goldblum S E: Am J Physiol 1997, 273:L31-39).
Many in vivo systems for studying SEB have been and are currently being employed. However this area is deficient in an effective and economic animal model, which closely parallels human staphylococcal enterotoxicosis. The non-human primate model (Macaca mulatta) (Normann S J, Jaeger R F, Johnsey R T: Lab Invest 1969, 20:17-25; Stiles J W, Denniston J C: Lab Invest 1971, 25:617-625) has proven to diagram SEB disease progression, but is limited because of high cost, short supply, and complexity of animal care. Rabbit models have been developed to specifically map the lesion progression of toxic shock syndrome toxin-1 (TSST-1, another exotoxin produced by S. aureus) however high doses are required and they need to be introduced via continual peritoneal infusion. Multiple strains of the murine species have also been used as in vivo models for SEB. Results are often skewed and hard to interpret because mice are insensitive to the effects of SEB and traditional mouse models of SEB intoxication require either genetic manipulation (Anderson M R, Tary-Lehmann M: Clin Immunol 2001, 98:85-94; Yeung R S, et al. :Eur J Immunol 1996, 26:1074-1082; Chen J Y, Qiao Y, Komisar J L, Baze W B, Hsu I C, Tseng J: Infect Immun 1994, 62:4626-4631) or prior sensitization, with D-galatosamine, or endotoxin (Miethke T, Wahl, C., Heeg, K., Echtenacher, B., Krammer, P., and Wagner, H.: Journal of Experimental Medicine 1992, 175:91-98). Even, with co-administered D-gal, the clinical syndrome in mice does not mimic that seen in higher order mammals.
In the present study a lethal SEB model using 14-day-old Yorkshire piglets was assessed for diagnostic parameters and relevance to human disease progression. This model could provide a promising alternative to traditional in vivo models for SEB. Piglets are easy to obtain, cost efficient, and require minimal care compared to those of primates. This paper characterizes the clinical syndrome, histological lesions and post mortem findings of intravenous SEB-exposed (lethal dose) piglets at varying time points.
Materials and Methods
Animals:
All animal use was carried out in accordance with AR 70-18, paragraph 12.d., in compliance with the Animal Welfare Act, adhering to the principles enunciated in The Guide for the Care and Use of Laboratory Animals. Litters of ˜8, 12-day-old, male and female Yorkshire piglets were obtained from Archer Farms (Darlington, Md.) and housed in groups of ˜3 piglets (assigned by treatment) in metal runs lined by rubber mats. Piglets were maintained under controlled lighting (12-hour light-dark cycle), at a temperature of 85° F. and humidity of ˜60%. Animals were fed swine pre-starter complete feed (Hubbard Feeds, Mankato, Minn.). Piglets had continual access to feed, water and a 2-3 heat lamp sources at one end of the run. At ˜18-days of age, anesethetized piglets (isofluorane (3% initially, achieving maintenance at ˜1.5-2%) (Abbott Labs, North Chicago, Ill.) received a lethal dose of SEB (150 μg/kg) or an equivalent volume of saline, administered into the ear vein using a 22 g 3/4 inch catheter. At 4, 6, 24, 48, 72 or 96 hours post treatment, animals were anesthetized with isofluorane, terminal measurements and blood were obtained and the piglets were euthanized using Buthanasia-D (Bums Biotech, Omaha, Nebr.) administered via intracardiac injection.
Toxin Preparation:
SEB, lot 14-30, purified by the method of Schantz et al (Schantz EJ, et al.: Biochemistry 1965, 4:1011-1016), was stored as a dry powder in pre-measured vacuum ampules. A working stock solution was made by dissolving the SEB in sterile pyrogen-free water to achieve a concentration of 5 mg/ml and that solution was aliquoted and stored frozen. At the time of use, an appropriate aliquot was thawed and diluted with i.v. injectable saline to 300 μg/ml. LD˜95 was achieved using 150 μg/kg. Lethality was also observed at 50 μg/kg but not at 30 μg/kg.
Clinical Observations and Measurements:
Animals were monitored continuously for clinical signs for the first 18 hours post treatment and every 6 hours until euthanasia. Recorded clinical observations included clinical sign results for at least 3 piglets per time period and for 3 different experiments (
Gross and Microscopic Pathology:
After euthanasia a complete necropsy was performed as follows: 4 hours (1 piglet), 6 hours (1 piglet), 24 hours (5 piglets), 48 hours (5 piglets), 72 hours (7 piglets) and 96 hours (4 piglets). At least one saline control piglet was examined per litter, with a total of 7 saline controls. A full set of tissues from each animal was fixed in 10% neutral buffered formalin. Fixed tissues were routinely trimmed, embedded in paraffin, sectioned at 5-7 μm and stained with hematoxylin and eosin for microscopic examination. Tissues examined microscopically for this report were: thymus, stomach, jejunum, spiral colon, descending colon, liver, spleen, pancreas, kidney, adrenal gland, urinary bladder, multiple lymph nodes, lung, heart, and brain.
Gene Studies
Whole blood samples were collected into CPT™ Vacutainer™ tubes (BD, Franklin Lakes, N.J.) at various time points and processed in accordance with the manufacturer's specifications which allow for the enrichment of peripheral blood mononuclear cells (PBMC). Total RNA was subsequently isolated from PBMCs using TRIzol reagent (Life Technologies, Grand Island, N.Y.) following the manufacturer's protocol.
Preliminary gene array yielded data that implicated several gene profile changes post-SEB treatment (data not presented). Five representative genes were chosen and primer pairs to be used for PCR were designed based on known mRNA sequences (Genbank, PubMed) using Primer software3 or Genelooper 2.0 from Geneharbor.
Equal amounts of total RNA were reverse transcribed to cDNA using oligo (dT) and Superscript reverse transcriptase II (Invitrogen, Carlsbad, Calif.). The obtained cDNA was used as a template for PCR reactions using PCR master mixture (Roche, Indianapolis, Ind.). Each cDNA was subjected to 25-30 PCR cycles using a GeneAmp 9600 thermal cycler (Perkin Elmer, Norwalk, Conn.) with conditions that resulted in a single specific amplification product of the correct size. Amplification was empirically determined to be in the linear range. mRNA amounts were normalized relative to 18S rRNA. Reaction products (10 μl ) were visualized after electrophoresis on a 1% agarose gel using SYBR Green I (Kemtek, Rockville, Md.). Gels were digitized using a BioRad Molecular Imager FX (BioRad, Hercules, Calif.) and band intensities were used to calculate mRNA abundance.
Results
Clinical Signs
Administration of SEB either IV or intratracheal at 150 μg/kg was lethal (or deemed non-survivable by the attendant veterinarian) in 31/31 piglets. An IV dose of 50 μg/kg resulted in lethality while 30 μg/kg was not lethal. After administration of the SEB, pre-established behavioral characteristics were recorded for each animal as a function of time post exposure during the course of the experiment (continually for the first 6 h and intermittently during the rest of the experiment). Five descriptions of piglet behavior for each of 3 categories (healthy, incapacitation, prostration) were established based on observed behavior from other studies with piglets. The animals showed onset of typical incapacitation signs (transient vomiting [˜3-6 episodes], severe diarrhea, anorexia) at 0.8-1.5 h post exposure (
Gross Findings
Gross changes were progressive over time. No significant gross changes were present in the both piglets necropsied at 4 and 6 hours post SEB treatment or in any saline control animals. By 24 hours mildly enlarged mesenteric lymph nodes and mild splenomegaly were present in 2 of 5 animals. By 48 hours post SEB treatment in all animals there was consistent mild splenomegaly when compared to control animals (
Gross lesions were most remarkable at 72 and 96 hours post exposure. All animals necropsied at these time periods had severe mesenteric edema that was most prominent between loops of spiral colon, (
Microscopic Findings
Histologic examination of selected tissues confirmed gross observations and helped to further characterize changes. The general progression of histologic changes in the mesenteric lymph nodes was: mild lymphoid hyperplasia by 24 hours, progression to moderate lymphoid hyperplasia and congestion by 48 hours, and marked lymphoid necrosis with hemorrhage, edema and fibrin accumulation by 72 to 96 hours (
Lymphoid hyperplasia was also present in all spleens examined at 24 hours post treatment and later. This change was characterized by mild diffuse expansion of the periarteriolar lymphoid sheaths (PALS) (FIGS. 69A-B). The lymphocytes in the affected PALS were larger, with increased cytoplasm and a large irregularly round stippled nucleus and there were increased numbers of mitotic figures in these areas (FIGS. 69C-D).
Severe mesenteric edema between loops of spiral colon seen grossly at 48 and 96 hours (
Additional histologic findings included lymphoblastic perivascular infiltrates and mild portal lymphoplasmacytic hepatitis. Small perivascular lymphocytic cuffs were present in the lungs of most animals examined at 48 hours and later (5 of 6 and 48 hours, 7 of 7 at 72 hours and 3 of 4 at 96 hours) (
SEB-Induced Gene Changes
After initial survey using custom gene microarrays, five genes were selected for study at 2, 6, 24, 48, and 72 hours post SEB exposure using RT-PCR (
Discussion
We have developed a clinically relevant piglet model of lethal SEB intoxication that we propose is superior to the current monkey and rodent models. This model more realistically parallels SEB intoxication in people than described mouse models and piglets are easier to obtain, maintain and handle than the non-human primate model.
This piglet model exhibits a biphasic clinical response to SEB intoxication that is virtually identical to that described in people but is not described in mouse models. Although lethal SEB intoxication has been achieved in previously manipulated mouse models, none of these models exhibit the typical initial gastrointestinal signs described in humans. In addition, the small size of these animals (Mice) makes obtaining many clinical measurements such as repeat routine hematology, serum chemistries, blood pressure and body temperature difficult.
The monkey model of lethal SEB intoxication is more clinically relevant than mouse models. However, although rhesus monkeys show a subtle clinical biphasic response to SEB intoxication it is not as exuberant or easily detected and monitored as that seen in the piglet model (One author's personal observation, MJ). This is likely a result of the fact that the laboratory Rhesus monkey retains many behavioral characteristics of its wild counterpart, including remarkable masking of clinical disease, which increases survival under natural adverse conditions; this is in marked contrast to the domestic pig whose disposition has been markedly altered by selective breeding. In addition, working with non-human primates, especially rhesus macaques, comes with a unique set of limitations, most notably high expense, limited supply and biosafety concerns. The aggressive nature of these monkeys and complications associated with Herpes B positive colonies make heavy sedation or anesthesia necessary for many routine procedures. In contrast, the piglets used in this model are easy to obtain and relatively inexpensive. The social nature of these animals allows routine procedures to be preformed without anesthesia or sedation and with minimal stress to the animal and handler.
In addition, study of other porcine models of human disease indicate that this species shows strong similarities to humans with respect to vascular responsiveness (Feletou M, Teisseire B: Edited by Swindle M M, Moody D C, Phillips L D. Ames, Iowa State Universtiy Press, 1992, pp 74-95) and is a good model in which to study cardiovascular disease. In fact, Lee et al (Lee P K, Vercellotti G M, Deringer J R, Schlievert P M: J Infect Dis 1991, 164:711-719) used porcine aortic endothelial cells to demonstrate that TSST-1), has a direct toxic effect on endothelium. There is also a described swine model of septic shock that culminates in a hypotensive crisis (Hoban L D, et al.: Awake porcine model of interperitoneal sepsis. Edited by Swindle M M, Moody D C, Phillips L D. Ames, Iowa State Universtiy Press, 1992, pp 246-264) that is similar to that observed in this model.
We have shown that administration of intravenous SEB to piglets results in terminal hypotension and shock similar to that seen in toxic shock syndrome in people and SEB intoxication in the rhesus macaque. Postmortem findings in people, monkeys and piglets indicate that hypotension and shock in SEB intoxication is a result of leakage of fluid from vessels into extravascular spaces. Pulmonary edema is the most consistent and remarkable gross lesion associated with death in the primate model of intravascular SEB intoxication (Finegold M J: Lab Invest 1967, 16:912-924) and in people with toxic shock syndrome (Larkin S M,et al: Ann Intern Med 1982, 96:858-864). One major difference in this piglet model compared to the disease in humans is that terminal edema is predominantly focused on the abdomen rather than the thorax resulting in severe mesenteric and perirenal edema with comparatively minor edema at other sites. It is interesting to note, that other natural and experimental angiotoxic diseases in the pig result in vascular leakage with edema predominantly in the abdominal region. In edema disease, a well characterized porcine disease, direct endothelial binding of Shiga-like toxin type Hie (SLT-IIe) secreted by E. coli, results in marked spiral colon mesenteric edema similar to that seen in this SEB piglet model (Gelberg H B: Alimentary system. Thomson's Special Veterinary Pathology. Edited by McGavin M D, Carltom W W, Zachary J F. St. Louis, Mosby, 2001, pp 42-43). In another porcine model that displays classical signs of circulatory shock, edema of the gastric wall and gall bladder is a result of experimental intravenous administration of T-2 toxin, a mycotoxin secreted by Fusarium species thought to cause moldy corn disease in swine (Pang V F, Lorenzana RM, Beasley VR, Buck WB, Haschek WM: Fundam Appl Toxicol 1987, 8:298-309). The abdominally focused edema in pigs may constitute a species difference that should be considered, especially in research aimed at treating late stage hypotensive shock and pulmonary edema. However, we feel strongly that this model is still a valid model for pathogenesis studies and lethal SEB intoxication prophylactic, early and mid-stage treatment trials.
Another characteristic unique to swine is the unique porcine lymph node architecture. Porcine lymph nodes are essentially reversed from other mammalian lymph nodes in that lymphoid tissue is centrally located and surrounded by loose peripheral lymphoreticular tissue resembling the medullary sinuses in other species. Although porcine lymph nodes are morphologically different, the functional flow of lymph is essentially identical to other species (Landsverk T: Immune system. Textbook of Veterinary Histology. Edited by Dellmann D, Eurell J A. Baltimore, Williams & Wilkins, 1998, pp 137-142) and in the author's (YAV) opinion does not represent a significant species difference, except perhaps in interpretation of lesions by a swine-naive histopathologist.
Histological lesions in this piglet model are similar to those described in other animal models of SEB intoxication. Ulrich et al (Ulrich R G, et al.: Textbook of Military medicine. Part I Warfare, Weaponry, and the Casualty. Ed. by Sidell F R, Takafuji E T, Franz D R. Washington, Office of the Surgeon General, 1997, pp 621-630 )provides a detailed description of both pulmonary and non-pulmonary lesions associated with lethal aerosol SEB exposure in the rhesus macaque. This model also had wide spread T-lymphocyte hyperplasia with enlarged lymph nodes, expanded PALS and circulating lymphoblasts. In addition, lymphocytic portal infiltrates similar to those seen in this model where also reported in the exposed monkeys. Another report of lethal aerosol SEB exposed monkeys described pulmonary perivascular lymphocytic infiltrates similar to those seen in this study (Mattix M E, Hunt R E, Wilhelmsen C L, Johnson A J, Baze W B: Toxicol Pathol 1995, 23:262-268). Lymphoid hyperplasia followed by lympholysis in the spleen is described in an Actinomycin-D primed mouse model (Chen J Y, Qiao Y, Komisar J L, Baze W B, Hsu I C, Tseng J: Infect Immun 1994, 62:4626-4631.). A similar change was noted in a mouse model of aerosol SEB exposure (Vogel, Pa., personal communication). These findings are consistent with the immunological manesfestations of SAg exposure.
As in the mouse models marked lympholysis was apparent in most piglets at 72 and 96 hours post SEB administration. However, this change was limited to severely affected lymph nodes and was not apparent in the thymus or spleen. It is possible that the severe lymphoid depletion noted at autopsy of several lethal cases of human toxic shock syndrome (Larkin S M,et al: Ann Intern Med 1982, 96:858-864) was a sequela of massive lympholysis. As TSS is lethal only in a small percentage of cases it is interesting to hypothesize that this change may be associated with lethality.
In summary we have characterized the clinical syndrome and post mortem findings of a 14-day-old Yorkshire piglet model of lethal SEB intoxication. We propose that this model is superior to previously described models. It is our hope that study of this piglet model will further elucidate the pathogenesis of SEB intoxication and enable us to test new therapeutic regimes.
The febrile state of treated animals is of particular interest and raises many questions. Studies using SEA mutants suggest that the emetic and superantigenic activity of SEs may be separate32. Immediately following exposure, piglets presented with an emetic phase that was not associated with temperature increase. Marked temperature elevation was not recorded in animals until after the last emetic event. If superantigenic T cell stimulation and subsequent cytokine production was solely responsible, one would suspect that the timing of emesis and fever would closely overlap. These data support the previously discerned hypothesis that the gastrointestinal and pyrogenic effects of SE may in fact be of different mechanism.
The timing of clinical symptoms, vital measurements, and pathologic lesions appears to be in direct concert (
By increasing peripheral vasculature resistance, blood pressure can be returned to a level that ensures adequate tissue perfusion. In this study, V1a mRNA levels are increased notably at 24 h, a time when systolic blood pressure re-equilibrates, and these levels are further increased at 72 h at the onset of the hypotensive crisis.
The complex nature of SE pathophysiology has posed many questions and much of the host's response to these toxins has been explained in terms of their effect on the body's immune system. As we progress further in understanding the chronology and severity of lesions induced by SEB, it will be necessary to further investigate SEs interaction with non-immunological tissue. Most notably would be the correlation of SEs effect on endothelium and on epithelial tissues with the presence of irreversible shock.
In summary we have characterized the clinical syndrome and post mortem findings of a 14-day-old Yorkshire piglet model of lethal SEB intoxication and propose that this model is superior to previously described models. It is our hope that study of this piglet model-will further elucidate the pathogenesis of SEB intoxication and enable us to test new therapeutic regimes.
References
J Exp Med 1997, 185:1447-1454
66(1): 158-64
39. Teresa Krakauer* and Bradley G. Stiles Pentoxifylline Inhibits Superantigen-Induced Toxic Shock and Cytokine Release Clin Diagn Lab Immunol. 1999 July; 6(4): 594-8.
AU = upregulated at all time points;
AD = downregulated at all time points:
U = upregulated;
M = moderately upregulated
D = downregulated
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While the preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
This application is a CIP of U.S. Ser. No. 10/007,806 filed Nov. 9, 2001 which is a CIP of U.S. Ser. No. 09/495,724 filed Feb. 1, 2000, both incorporated in their entirety by reference.
The invention described herein may be manufactured, used and licensed by or for the U.S. Government.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10007806 | Nov 2001 | US |
| Child | 11000615 | Dec 2004 | US |
| Parent | 09495724 | Feb 2000 | US |
| Child | 10007806 | Nov 2001 | US |
