Patent Application
20100113568
The present invention relates to the N-terminal lectin-like domain of thrombomodulin and its use for the prevention and/or the treatment of Lewis Y antigen related diseases or disorders.
Septic shock syndrome that results from excessive host immune responses induced by infectious organisms is a leading cause of death in hospitalized patients. Pathophysiological changes in sepsis involve the pathogen-induced uncontrolled release from immune cells, particularly monocytes and macrophages, of pro-inflammatory mediators including nitric oxide, tumor necrosis factor (TNF), and interleukins (ILs). Gram-negative bacterial infection is one of the major causes of systemic bacterial sepsis due to the lipopolysaccharide (LPS) constituent of the Gram-negative outer membrane. LPS induces a rapid increase of pro-inflammatory mediators, leading to lethal systemic tissue damage and multiple organ failure, which mimic the inflammatory responses of septic syndrome.
In mammals, membrane-bound CD14 and Toll-like receptor-4 (TLR4)-MD-2 participate in cellular recognition of LPS. Binding of LPS to TLR4 receptor triggers the activation of the members of the mitogen-activated protein kinase (MAPK) pathway including p38, p42/p44 extra-cellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). In resting, un-stimulated cells, nuclear factor (NF)-κB, a heterodimeric complex composed of 50- and 65-kDa (p50/p65) protein subunits, is retained as an inactive complex bound to inhibitory κBα (IκBα) in the cytoplasm. While the cells are under pro-inflammatory stimulation by LPS, phosphorylation and degradation of IκBα permit NF-κB nuclear translocation and promote the expression of inflammatory genes including inducible nitric oxide synthase (iNOS), TNF-α, and others.
Thrombomodulin (TM) is a 557 amino acid type I glycosylated transmembrane protein with an NH2-terminal lectin-like region (D1) followed by six epidermal growth factor (EGF)-like structures (D2), an O-glycosylation site-rich domain (D3), a transmembrane domain (D4), and a cytoplasmic tail domain (D5) (Weiler, H. et al. J Thromb Haemost. July 2003;1(7):1515-24). D2 EGF-like structures are responsible for the anticoagulant activity of TM via the alteration of the substrate specificity of thrombin. TM domain 2 (TMD2)-thrombin complex activates anticoagulant protein C (APC), which in turn inactivates procoagulant cofactors Va and VIIIa. On the other hand, TM expression occurs in cell types including keratinocytes, polymorphonuclear neutrophils (PMNs), monocytes, and endothelial cells, indicating that TM might have biological functions in addition to anticoagulation. Different biological functions of TM may also exist. TM domain functions as an angiogenic factor and anti-inflammatory agent through protein C dependent and independent mechanisms. Recently, the anti-inflammatory activity of TM domain 1 (TMD1) was implied, since mice with a deleted TM lectin-like domain (TMDEL/DEL) became more sensitive to the challenge with LPS, through the suppression of adhesion molecule expression via NFκB and MAPK signaling pathways (Conway, E. M. et al. J Exp Med. Sep. 2, 2002;196(5):565-77; U.S. Pat. No. 73,419,912). Moreover, it has been demonstrated that TMD1 sequesters high-mobility group-B1 (HMGB1) protein, a late cytokine mediator of lethal endotoxemia and sepsis, by interfering the binding of HMGB1 to receptor for advanced glycation end product (RAGE) (Abeyama, K. et al. J Clin Invest. May 2005;115(5):1267-74. Epub Apr. 14, 2005). TMD1 also interferes with complement activation and protects against arthritis.
TMD1 is structurally homologous to mammalian C-type lectins that generally consist of Ca2+-dependent carbohydrate receptor domains. Different C-type lectins contribute to various biological functions including cell adhesion, endocytosis, and pathogen neutralization and play an important role in the innate immune system.
Lewis Y antigen overexpressed on 70% of epithelial-derived tumors is a member of a family of blood group-related difucosylated oligosaccharides (Kuemmel A et al., Tumor Biol 2007;28:340-349). The epithelial-derived tumors include breast, pancreas, ovary, colon, gastric, and lung cancer. The high frequency of Lewis Y -expressing tumors, its high density and altered expression on the surface of tumor cells, and its relatively homogenous expression in primary and metastatic lesions have led to its selection as an antigenic target for a range of epithelial tumors, including breast cancer (Westwood J A et al. PNAS, 2005; 102(52): 19051-19056; Schlimok, G., et al. Eur. J. Cancer, 31A: 1799-1803, 1995; Tolcher, A. W., et al. J. Clin. Oncol., 17: 478-484, 1999). The antibodies recognizing Lewis Y antigen could eliminate carcinomas that express Ley (Kerrie Clarke et al. Clinical Cancer Research 6, 3621-3628, 2000; Erwin R. Boghaert et al. Clinical Cancer Research 10, 4538-4549, 2004).
The present invention provides a method for binding Lewis Y antigen of a subject, comprising administering to the subject an effective amount of nucleotide sequence encoding N-terminal lectin-like domain of thrombomodulin (TMD1), or its analogues.
The present invention also provides a method of detecting cancer cells, Gram-negative bacteria or LPS which over-express the Lewis Y antigen in a sample from a subject, said method comprising the steps of: administering a composition comprising a labeled nucleotide sequence encoding N-terminal lectin-like domain of thrombomodulin (TMD1), or labeled analogues.
The present invention provides a method for binding Lewis Y antigen of a subject, comprising administering to the subject an effective amount of nucleotide sequence encoding N-terminal lectin-like domain of thrombomodulin (TMD1), or its analogues. The Lewis Y antigen set forth is expressing in tumor cells, Gram-bacteria or LPS. In the embodiment, the tumor cells are epithelial-derived tumor, such as breast, pancreas, ovary, colon, gastric, and lung cancer. The method mentioned also prevents and treats tumor over-expressing Lewis Y antigen.
In this present invention, it is discovered that TMD1 binds to the LPS, inducing agglutination and enhancing bacteria phagocytosis by macrophages. It is shown that TMD1 functions as an anti-inflammatory factor in the early phase of systemic inflammation in sepsis induced by Gram-negative bacteria. Furthermore, it is demonstrated that TMD1 specifically interacts with bacteria carrying smooth-type LPS such as Klebsiella pneumoniae (K. pneumoniae), an important Gram-negative pathogen, and attenuates LPS- and K. pneumoniae-induced inflammatory responses and lethality by binding to LPS, blocking the downstream signal transduction. Thus, TMD1 may have an important protective function in neutralization of LPS, of value therapeutically in the treatment of septic shock syndrome and other inflammatory disease states. The biological function of TM in macrophages is also proposed.
The current invention demonstrates that TM protects against Gram-negative bacteria-mediated sepsis. The N-terminal domain of thrombomodulin (TMD1) binds to the Lewis Y antigen and LPS, inhibiting LPS-induced inflammatory responses. Moreover, TMD1 causes agglutination of Escherichia coli (E. coli and K. pneumoniae and enhances the macrophage phagocytosis of these Gram-negative bacteria. Administration of TMD1 protects the host by suppressing inflammatory responses in sepsis induced by LPS and Gram-negative bacteria. Therefore, recombinant TMD1 may be valuable in treatment of severe inflammation in sepsis, especially in infection caused by Gram-negative bacteria.
The present invention relates to a use of the recombinant TMD1 is valuable in treatment of severe inflammatory responses. This invention also relates to a method of TMD1 can be used to neutralize LPS, and hence TMD1 have an important protective function.
In a first embodiment the invention provides a method for treatment of LPS-induced or Gram-negative bacteria-induced inflammatory response comprising N-terminal lectin-like domain of thrombomodulin (TM) SEQ ID NO:1.
In the preferred embodiment, the inflammatory response is sepsis induced by LPS or Gram-negative bacteria. In a further preferred embodiment, the Gram-negative bacteria is E. coli or K. pneumoniae.
‘Inflammation’ as used herein means the local reaction to injury of living tissues, especially the local reaction of the small blood vessels, their contents, and their associated structures. The passage of blood constituents through the vessel walls (extravasation) into the tissues is the hallmark of inflammation. Generally, inflammation starts with an enhanced leukocyte adhesion to the endothelial wall and results in leukocyte extravasation into tissues or organs.
In fact, any noxious process that damages living tissue infection with bacteria, excessive heat, cold, mechanical injury such as crushing, acids, alkalis, irradiation, or infection with viruses can cause inflammation irrespective of the organ or tissue involved. It should be clear that diseases of animals and man classed as ‘inflammatory diseases’ comprising arthritis, skin inflammation, peritonitis, injury associated with ischemia/reperfusion (eg. heart, liver, kidney, brain), inflammatory pulmonary disorders (including for example, asthma, bronchitis, adult respiratory distress syndrome (ARDS), vasculitis, atherosclerosis, nephritis, skin wound healing, sepsis, and local and systemic infections.
The term ‘recombinant DNA vector’ as used herein refers to DNA sequences comprising a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. a mammal). DNA sequences necessary for the expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome-binding site and possibly other sequences; Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
The recombinant protein may be purified by any conventional protein purification procedure close to homogeneity and/or be mixed with additives. The recombinant protein may be manufactured using recombinant expression systems comprising bacterial cells, yeast cells, animal cells, insect cells, plant cells or transgenic animals or plants.
The term ‘Gram-negative bacteria’ as used herein are those that do not retain crystal violet dye in the Gram staining protocol. Gram-positive bacteria will retain the dark blue dye after an alcohol wash. In a Gram stain test, a counter stain is added after the crystal violet, which colors all Gram-negative bacteria a red or pink color. The test itself is useful in classifying two distinctly different types of bacteria based on structural differences in their cell walls. Many species of Gram-negative bacteria are pathogenic, meaning they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer.
The term ‘Agglutination’ as used herein is the clumping of particles. This occurs in biology in three main examples: 1. The clumping of cells such as bacteria or red blood cells, in the presence of an antibody. The antibody or other molecule binds with multiple particles, and joining them. 2. The coalescing of small particles that are suspended in solution; these larger masses are then (usually) precipitated. 3. An allergic reaction type occurrence where cells become more compacted together to prevent foreign materials entering them. This is usually the result of an antigen in the vicinity of the cells. The agglutination of bacteria of the present invention facilitates phagocytosis of the bacteria by macrophages.
In another embodiment, the present invention provides a method of preventing or treating an inflammatory disease or disorder induced by LPS or Gram-negative bacteria comprising administering a subject in need of such prevention or treatment with nucleotide sequence encoding N-terminal lectin-like domain of thrombomodulin (TMD1), TMD1 or analogues thereof.
In the preferred embodiment, the disease is sepsis induced by LPS or Gram-negative bacteria. The disease or disorder is prevented or treated by binding N-terminal lectin-like domain of thrombomodulin (TMD1) to LPS or Gram-negative bacteria to induce agglutination or opsonization of bacteria. Thus, the disease or disorder is prevented or treated by reducing LPS-induced production of the inflammatory mediators TNF-α or NO in macrophages and by suppressing signal pathways involved in LPS-induced inflammatory responses.
In the further preferred embodiment, the disease or disorder is prevented or treated by interaction TMD1. TMD1 has a function as a natural opsonic moiety for innate immunity against Gram-negative bacteria. The induction of opsonization is made by binding TMD1 to Lewis Y antigen in LPS or cell membrane, and the induction of agglutination of bacteria facilitates phagocytosis of the bacteria by macrophages. Those responses alleviate PMN infiltration in lungs or preserves kidney function. The signal pathways involved in LPS-induced inflammatory responses includes phosphorylation of ERK and P38, degradation of IκB and nuclear translocation of NF-κB and increasing expression of inducible nitric oxide synthase (iNOS) and TNF-α. Besides, the disease or disorder is prevented by interaction TMD1 with high mobility group B1 (HMGB1).
The present invention also provides a method of detecting cancer cells, Gram-negative bacteria or LPS which over-express the Lewis Y antigen in a sample from a subject, said method comprising the steps of: administering a composition comprising a labeled nucleotide sequence encoding N-terminal lectin-like domain of thrombomodulin (TMD1), or labeled analogues. In the preferred embodiment, the cancer cells which express the Lewis Y antigen is breast, pancreas, ovary, colon, gastric, or lung cancer. The sample set forth is derived from blood, tissue, body fluid, cell, or excrement from the subject. In the embodiment of this invention, the subject is mammal. In the preferred embodiment of this invention, the subject is human. The labeled marker in the TMD1 or its analogues is detectable marker, such as fluorescent molecules, radioactive molecules, chromogenic molecules, biotin, acridinium ester and acridinium-9-carboxamide.
A “nucleotide sequence” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary or quaternary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
The term ‘subject’ as used herein is any animal who receives medical attention, care, or treatment. In particularly, the animal is a human. The human is the most often ill or injured and in need of treatment by a physician or other medical professional.
The term ‘analogues’ as used herein are compounds in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Another use of the term in biochemistry refers to a substance which is similar in structure to another substance. The analogues of TMD1 used herein includes lectin domain of TM from different animals including murine but not limited to and the modification known to the art of people with knowledge of the field such as point mutation and chemical modification such as PEGylation and glycosylation
The term ‘innate immunity’ as used herein comprises the cells and mechanisms that defend the host from infection by other organisms, in a non-specific manner. This means that the cells of the innate system recognize, and respond to, pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.
The term ‘opsonization’ as used herein is any molecule that acts as a binding enhancer for the process of phagocytosis, for example, by coating the negatively-charged molecules on the membrane. Both the membrane of a phagocytising cell, as well as its target, have a negative charge (Zeta-potential), making it difficult for the two cells to come close together. During the process of opsonization, antigens are bound by antibody and/or complement molecules. Phagocytic cells express receptors that bind opsonin molecules. With the antigen coated in these molecules, binding of the antigen to the phagocyte is greatly enhanced. Most phagocytic binding cannot occur without opsonization of the antigen. Furthermore, opsonization of the antigen and subsequent binding to an activated phagocyte will cause increased expression of complement receptors on neighboring phagocytes. Examples of opsonin molecules include the IgG and IgA antibodies and the C3b, C4b, and iC3b components of the complement system.
Material and Methods
1. Preparation of Recombinant TMD (rTMD) Proteins Using Both Pichia and Mammalian Protein Expression Systems.
The pPICZαA and pCR3-EK vectors were used for expression and secretion of human rTMD proteins containing 6×His tag and c-Myc epitope for purification and for protein detection in the Pichia pastoris and human embryonic kidney 293 (HEK293) mammalian protein expression systems. Briefly, DNA fragments coding for TMD1 (residues Ala1-Ala155), TMD2/EGF123 (residues Ala224-Glu346), and TMD23 (residues Ala224-Ser497) were obtained by a PCR of human umbilical vein endothelial cell cDNA using specific primers. The primer sequences used for construction were as follows: TMD1 (TMD1-sense: SEQ ID NO: 2 and TMD1-antisense: SEQ ID NO: 3), TMD23 (TMD2-sense: SEQ ID NO: 4 and TMD3-antisense: SEQ ID NO: 5), and TMD2/EGF123 (TMD2-sense and TMD2/EGF3-antisense: SEQ ID NO: 6). An enterokinase cutting site between the rTMD sequence and His/c-Myc tag allowed subsequent removal of the tag sequence. Yeast fermentation medium or HEK293 cell conditioned medium containing expressed rTMD proteins was applied to a nickel-chelating Sepharose column (Amersham Pharmacia Biotech., Piscataway, N.J.), and rTMD-containing fractions were eluted in an imidazole gradient. The purified rTMD proteins were examined by SDS-PAGE and Western blotting, and were verified by protein identification with mass spectrometry analysis. The N-terminal amino acid sequence of rTMD proteins was also determined by Edman degradation with a model 477A sequencer (Applied Biosystems, Foster City, Calif.). The molecular masses of rTMD proteins containing fusion peptides were predicted using the ExPAsy website for calculating protein concentration (rTMD1: 22,902 Da, rTMD2/EGF123: 16,473 Da, and rTMD23: 32,823 Da). A non-tagged rTMD1 was prepared by incubation of 1 mg rTMD1 with 5 ng enterokinase (New England Biolab, Beverly, Mass.) at 4° C. for 16 hours. After incubation, the sample was applied to a nickel-chelating Sepharose column and the non-bound fraction containing the non-tagged rTMD1 was collected. The sample was further treated with immobilized soy bean trypsin inhibitor-Sepharose gel to adsorb the residual enterokinase. Each sample was analyzed before and after enterokinase treatment by SDS-PAGE and Western blot, and non-tagged rTMD1 having a molecular mass 2000 Da less than the original tagged rTMD1 was obtained.
2. Cells and Cell Culture.
Human leukemia monocytic THP-1 cells and murine macrophage cell line RAW 264.7 were obtained from the American Type Culture Collection. THP-1 cells were grown in suspension in RPMI-1640 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 0.1% β-mercaptoethanol, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (FBS). RAW 264.7 cells were grown as an adhered layer in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FBS under a humidified 5% CO2 atmosphere at 37° C. For differentiation, THP-1 cells were plated in medium containing 10 nM PMA and allowed to adhere for 18 h.
3. Inflammatory Mediator Assays.
TNF-α in conditioned medium or mouse serum was measured by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn.). Accumulation of nitrite in the medium or mouse serum was determined by a colorimetric assay using Griess reagent (Sigma-Aldrich). Blood urea nitrogen (BUN) and creatinine were measured with a Roche D&P modular (Roche Diagnostic Systems, Branchburg, N.J.). Assays were performed according to the manufacturer's protocol.
4. Western Blotting.
rTMD1 (0-50 μg/mL, equals 0-2.18 nmole/mL) pre-incubated without or with Escherichia coli (E. coli) LPS (LPS O111:B4, Sigma-Aldrich) in the absence or presence of Lewis Y (Ley; Dextra Laboratories, Reading, UK) for 30 minutes was used to stimulate THP-1 or RAW 264.7 cells. Incubation period included 15 minutes for pIκB and IκB (B-9 and C-21, Santa Cruz Biotechnology, Santa Cruz, Calif., unless otherwise noted), 20 minutes for p38 (C-20) and pp38 (D-8), 30 minutes for ERK1/2 (K-23), pERK1/2 (E-4), lamin B2 (E-3, Zymed Laboratories, San Francisco, Calif.), NF-κB p50 (C-19) and NF-κB p65 nuclear translocation (C-20), and 24 hours for iNOS expression using a specific antibody (N-20).
5. Animals and Systemic Sepsis Models.
Male FVB mice (8-10 weeks old) were used to model sepsis in vivo. The Institutional Animal Care and Use Committee of the National Cheng Kung University approved procedures. In the chronic endotoxemia model, various concentrations of rTMD1 or rTMD proteins in equi-molar amounts were administrated by tail intravenous (i.v.) injection and LPS (20 mg/kg) or K. pneumoniae (5×102 CFU/mouse, BCRC) was administrated by intraperitoneal (i.p.) injection. After 6 or 12 hours, mice were sacrificed by pentobarbital anesthesia (50 mg/kg; i.p.). Sera were collected and assayed for TNF-α, NO, BUN, and creatinine, and lung and kidney tissues were removed, fixed with formalin, and embedded in paraffin for histochemical examination. In the lethal sepsis model, rTMD1 (2 mg/kg, equals 87.3 nmole/kg) was i.v.-administrated at 0, 6, 12, and 24 hours after i.p. administration of LPS (40 mg/kg). The half-life of rTMD1 in the circulation was determined by i.v. injection of rTMD1 (10 mg/kg, equals 436.64 nmole/kg) and serum samples were collected at various time intervals. The levels of rTMD1 in the collected sera were determined by a sandwich ELISA which employed anti-c-Myc monoclonal antibody and TM-H300 as capture and detection antibodies (both from Santa Cruz Biotechnology), respectively. For the lethal bacteremia model, rTMD1 (10 mg/kg) was administrated prior to i.p. injection of K. pneumoniae (5×103 CFU/mouse). Mortality was monitored every 6-12 hours until all mice in either experimental group died. Experimental procedure of determining LPS half-life is as following. LPS (20 mg/kg) was i.p.-administrated to male FVB mice without or with rTMD1 (10 mg/kg; i.v.). Serum samples were collected at various time intervals and the amount of LPS was determined by Limulus amebocyte lysate test (Associates of Cape Cod, E. Falmouth, Mass.). A further experiment was performed to determine whether rTMD1 affects bacterial clearance. K. pneumoniae (5×102 CFU/mouse) was i.p.-administrated to male FVB mice without or with rTMD1 (10 mg/kg; i.v.), and blood samples were collected at various time intervals. Outgrowth of K. pneumoniae was quantified by plating serial dilutions of blood samples on blood agar plates and enumerating colonies after overnight incubation at 37° C.
6. K. pneumoniae and LPS Binding Assays.
K. pneumoniae (3×105 CFU/well), E. coli O111:B4 smooth-type LPS (5 μg/well), or bovine serum albumin (BSA, 5 μg/well) in 100 μL of bicarbonate buffer (pH 9.6) were coated onto wells of high-binding microtiter plate (Corning Costar, Cambridge, Mass.). Nonspecific binding was blocked with binding buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM CaCl2) containing 50 mg/mL BSA. Various concentrations of rTMD proteins without or with CD14 (10 μg/mL; R&D Systems) in binding buffer containing 1 mg/mL BSA were added to wells and incubated for 2 hours. In some experiments, 0.2 M mannose, 5 mM EDTA, or Ley were included in the binding buffer to compete with LPS and rTMD1 binding. Antibody against the c-Myc epitope of the rTMD proteins was added to wells and incubated for 2 hours, and peroxidase-labeled secondary antibody followed. Binding of rTMD proteins was detected by measuring absorbance at 450 nm.
7. Effect of rTMD1 on Bacterial Agglutination.
K. pneumoniae and E. coli DH5α (BCRC) were washed twice in 0.1 M sodium bicarbonate buffer (pH 9.6) and incubated with 0.1 mg/mL fluorescein isothiocyanate (FITC) at 37° C. in the dark. Each of 20 μg/mL of rTMD1, non-tagged rTMD1, mammalian rTMD1, and recombinant TM domains 2 and 3 (rTMD23) was co-incubated with FITC-labeled bacteria (1×105 CFU/mL) in buffer (20 mM Tris, pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 1 mg/mL BSA) with or without 0.2 M mannose or 5 mM EDTA. Samples were individually placed on a glass slide and fluorescently-labeled bacteria were observed microscopically to evaluate agglutination.
8. THP-1 Phagocytosis Assay.
FITC-labeled K. pneumoniae (1×105 CFU/well) were pre-incubated with rTMD1 at 37° C. for 30 minutes before adding to differentiated THP-1 cells. After incubation for 2 hours the THP-1 cells were washed twice with ice-cold PBS prior to fluorescence microscopy, or were further trypsinized, resuspended in PBS, and analyzed in a fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, Calif.) to measure THP-1 cell intracellular fluorescence. Green fluorescence data from 10,000 events (cells) per condition collected at 530 nm on a log scale were analyzed with CellQuest™ software (Becton Dickinson).
9. rTMD1 Ligand Analysis.
Biotin-polyacrylamide (Biotin-PAA)-sugars (GlycoTech, Rockville, Mass.), rTMD1, rabbit anti-mouse IgG antibody (Zymed), mouse anti-6×His tag antibody (Abcam, Cambridge, UK), and streptavidin-coated donor and protein A conjugated acceptor beads (PerkinElmer, Boston, Mass.) were diluted with assay buffer containing 25 mM Tris (pH 7.0), 25 mM CaCl2, and 1 mg/mL BSA to appropriate concentrations. The anti-6×His tag antibody, rabbit anti-mouse IgG antibody, and acceptor beads were incubated with assay buffer for 1 hour at 25° C. before use (as the acceptor mixture). Biotin-PAA-sugars, rTMD1, and donor beads were separately added to wells of ProxiPlate-384 assay plates (PerkinElmer) and incubated at 25° C. for 1 hour. An aliquot of the acceptor mixture was then added to the wells and incubated at 25° C. for another 2 hours. The results were read on a PerkinElmer Envision instrument using the AlphaScreen program. All the procedures and incubations were carried out in the dark.
10. Analysis of Lewis Antigens in E. coli LPS O111:B4.
E. coli O111:B4 LPS (5 μg/well) was coated onto wells of a high-binding microtiter plate. After the nonspecific binding blocked with binding buffer containing 50 mg/mL BSA, 5 μg/mL of various kinds of antibodies (Abcam) against Lewis a (Lea), Lewis b (Leb), Lewis X (Lex), and Ley were added to wells with binding buffer containing 1 mg/mL BSA and incubated at 37° C. for 2 hours prior to incubation with goat anti-mouse horseradish peroxidase-conjugated IgG or IgM at 37° C. for 2 hours. The peroxidase reaction was performed using 3,3′,5,5′-tetramethylbenzidine as a substrate and was stopped by 2N H2SO4. The products were detected by measuring absorbance at 450 nm.
11. Statistical Analyses.
Survival data were analyzed by log-rank test. Data expressed as the mean±standard deviation (SD). Statistical significance was analyzed by unpaired Student's t test. Differences between more than two groups were compared by one-way analysis of variance or two-way analysis of variance and following Bonferroni's post hoc test, with P<0.05 considered statistically significant.
To test whether TMD1 has anti-inflammatory property, rTMD proteins were prepared by using both Pichia pastoris and mammalian protein expression systems. The rTMD1 proteins obtained from both systems had similar molecular mass with glycosylation modification, which was about 35 kDa and as assayed by silver staining and Western blotting. The anti-inflammatory effect of rTMD proteins in RAW 264.7 and THP-1 cells stimulated with LPS was tested first. Pichia-expressed rTMD1 but not recombinant TMD2 containing EGF like domain 1, 2, 3 (rTMD2/EGF123) dose-dependently inhibited TNF-α production in RAW 264.7 cells stimulated with LPS (
Phosphorylation and degradation of IκBα occurred in RAW 264.7 cells stimulated with LPS (100 ng/mL). The effect was totally reversed by rTMD1 (50 μg/mL, equals 2.18 nmole/mL) (
Since rTMD1 could inhibit LPS-induced inflammatory mediator productions and signaling pathways, rTMD1 might function as a therapeutic agent to reduce the inflammatory response and lethality induced by LPS in vivo. TNF-α and NO levels were increased in mice 6 hours after i.p. administration of 20 mg/kg LPS, relative to control mice. Mice receiving an i.v. injection of rTMD1 (1-5 mg/kg, equals 43.66-218.3 nmole/kg) had significantly decreasing levels of TNF-α and NO (
To test whether rTMD1 protects against lethality induced by Gram-negative bacteria, systemic sepsis was induced in mice by injecting i.p. with K. pneumoniae and treating with rTMD1 (
To test whether TMD1 is capable of specific binding to Gram-negative bacteria and LPS, rTMD1 and rTMD23 were used for binding with K. pneumoniae, LPS, or BSA. rTMD1 but not rTMD23 could specifically bind to K. pneumoniae (
Since TMD1 mediates cell-cell adhesion by binding to carbohydrate ligands17 and rTMD1 functions in clearance of LPS and K. pneumoniae (
To identify the ligand specificity of TMD1, a panel of carbohydrate ligands (
