Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Coronavirus Receptors as Immune Modulators.

The Coronaviridae family includes the seven known human coronaviruses (CoV) that cause mild to moderate respiratory infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) as well as severe illness and death (MERS-CoV, SARS-CoV, SARS-CoV-2). Severe infections induce hyperinflammatory responses that are often intensified by host adaptive immune pathways to profoundly advance disease severity. Proinflammatory responses are triggered by CoV entry mediated by host cell surface receptors. Interestingly, five of the seven strains use three cell surface metallopeptidases (CD13, CD26, and ACE2) as receptors, whereas the others employ O-acetylated-sialic acid (a key feature of metallopeptidases) for entry. Why CoV evolved to use peptidases as their receptors is unknown, but the peptidase activities of the receptors are dispensable, suggesting the virus uses/benefits from other functions of these molecules. Indeed, these receptors participate in the immune modulatory pathways that contribute to the pathological hyperinflammatory response. This review will focus on the role of CoV receptors in modulating immune responses.

SslE (YghJ), a Cell-Associated and Secreted Lipoprotein of Neonatal Septicemic Escherichia coli, Induces Toll-Like Receptor 2-Dependent Macrophage Activation and Proinflammation through NF-κB and MAP Kinase Signaling.

SslE (YghJ), a cell surface-associated and secreted lipoprotein, was identified as a potential vaccine candidate for extraintestinal pathogenic Escherichia coli, providing nearly complete protection from sepsis in a mouse model. We earlier found that SslE from neonatal septicemic E. coli could trigger the secretion of various proinflammatory cytokines in murine macrophages, the signaling pathway of which is still obscure. In this study, we showed that SslE specifically binds to Toll-like receptor 2 (TLR2)/TLR1 heterodimers and recruits downstream adaptors MyD88, TIRAP, and TRAF6. In addition, SslE stimulates nuclear translocation of NF-κB and activates different mitogen-activated protein (MAP) kinase signaling cascades specific to the secretion of each cytokine in murine macrophages, which becomes impaired in TLR2 small interfering RNA (siRNA)-transfected cells and in cells blocked with a monoclonal antibody (MAb) against TLR2, suggesting the involvement of TLR2 in NF-κB and MAP kinase activation and subsequent cytokine secretion. Furthermore, our study is the first to show that SslE can stimulate TLR2-dependent production of other proinflammatory hallmarks, such as reactive nitrogen and oxygen species as well as type 1 chemokines, which contribute to the anti-infection immune response of the host. Also, the overexpression of major histocompatibility complex class II (MHC II) and other costimulatory molecules (CD80 and CD86) in macrophages essentially indicates that SslE promotes macrophage activation and M1 polarization, which are crucial in framing the host's innate immune response to this protein, and hence, SslE could be a potent immunotherapeutic target against E. coli sepsis.

Tumor Necrosis Factor: Function, Release and Clearance.

Tumor Necrosis Factor (TNF) is a multifunctional cytokine. It plays an important role in the pathophysiology of several diseases. Recently, it has been discovered that TNF is circulating in two different forms, a bioactive form and an immunologically detectable form. These two forms of TNF show different clearance kinetics. The immunological form is supposed to be an inactivated TNF protein. For this inactivation, proteolytic degradation or TNF binding by inactivating proteins is necessary. In this review we have focused on TNF inactivation by TNF binding proteins. Recent data show that there are soluble TNF receptors circulating which can bind and inactivate TNF. These receptors are membrane-bound TNF receptors which have been proteolytically cleaved from the cell membrane. Two TNF receptors are circulating, the soluble TNF receptor of 55 kDa (P55) and the receptor of 75 kDa (P75). The receptors are held responsible not only for inactivation of the TNF, but also for the clearance of TNF. Recent data show that the kidney is the most important organ for TNF clearance, followed by the liver. All other organs are of less importance. In this review, function, release, and clearance of TNF are discussed.

Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection.

Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection.

Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors.

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System.

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹â»³8 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹â»³8 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen.

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

Role of metalloproteases in vaccinia virus epitope processing for transporter associated with antigen processing (TAP)-independent human leukocyte antigen (HLA)-B7 class I antigen presentation.

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8(+) lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8(+) T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections.

Activation of cytokine expression occurs through the TNFα/NF-κB-mediated pathway in birnavirus-infected cells.

The infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family of viruses and causes acute contagious diseases in a number of economically important freshwater and marine fish. In this study, we infected zebrafish embryonic cells (ZF4) with IPNV and analyzed the gene expression patterns of normal and infected cells using quantitative real-time PCR. We identified a number of immune response genes, including ifna, ifng, mx, irf1, irf2, irf4, tnfa, tnfb, il-1b, il-15, il-26, ccl4 and mmp family genes, that are induced after viral infection. Transcriptional regulators, including cebpb, junb, nfkb and stat1, stat4 and stat5, were also upregulated in IPNV-infected cells. In addition, we used Pathway Studio software to identify TNFα as having the greatest downstream influence among these altered genes. Treating virus-infected cells with an siRNA targeting TNFα inhibited NF-κB expression. To further interrupt the TNFα/NF-κB-mediated pathway, the expression levels of cytokines and metalloproteinases were inhibited in IPNV-infected cells. These data suggest that, during IPNV infection, the expression of cytokines and metalloproteinases might be initiated through the TNFα/NF-κB-mediated pathway. The modulation of TNFα/NF-κB-related mechanisms may provide a therapeutic strategy for inhibiting viral infection in teleosts.

Soluble MHC I and soluble MIC molecules: potential therapeutic targets for cancer.

It has become clear that soluble MHC I (sMHC I) and soluble MIC (sMIC), which are highly elevated in sera of cancer patients, can be viewed to be tolerogenic, and that metalloproteinases are involved in their generation process. In this review, an overview is provided of the recent progress made in the sMHC I and sMIC fields, with emphasis on their structure, formation, and function, and the key-questions that still await answers are addressed. Understanding better their formation mechanism, it will become more feasible to modulate the immune responses in cancer patients by targeting molecules involved in their generation process.

Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients.

BACKGROUND: To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays. METHODS: Tears were collected from 12 normal subjects (CT) and 24 active VKC patients. Tears were centrifuged and successively probed using three microwell plate arrays specific for: (i) cytokines: interleukin (IL)-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, interferon-gamma and tumour necrosis factor-alpha; (ii) growth factors: basic fibroblast growth factor (bFGF), platelet-derived growth factor, thrombopoietin, angiopoietin-2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratocyte growth factor, tissue inhibitor of metalloprotease (TIMP)-1 and heparin-binding epithelial growth factor (HB-EGF) and (iii) matrix metalloprotease (MMP)-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2. RESULTS: Interleukin-8 signals were detected in all CT and highly detected in all VKC samples. The Th2-type cytokines, IL-4, IL-5 and IL-10 were detected only in tears of VKC patients. Signals for bFGF, HB-EGF, VEGF and HGF were detected in 41-87% of VKC samples and in few CT samples. Only TIMP-1 and TIMP-2 were found in all normal and patient tear samples, whereas MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 were highly present in all VKC samples. CONCLUSIONS: Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation.

Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis.

We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii.

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.

Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus.

Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.

Towards a metalloprotease-DNA vaccine against piscine cryptobiosis caused by Cryptobia salmositica.

Cysteine protease is a metabolic enzyme, whereas metalloprotease is the virulent factor in cryptobiosis caused by Cryptobia salmositica. Recombinant DNA vaccines were produced with the insertion of either the metalloprotease or cysteine protease gene of C. salmositica into plasmid vectors (pEGFP-N). As expected, fishes (Oncorhynchus mykiss and Salmo salar) injected intramuscularly with the metalloprotease-DNA (MP-DNA) vaccine (50 microg/fish) were consistently more anemic (lower packed cell volume, PCV) than controls (injected only with the plasmid) at 3-5 weeks post-inoculation. Also, there were no difference in PCV between fish injected with the cysteine-DNA plasmids and the controls. In addition, agglutinating antibodies against Cryptobia were detected only in the blood of MP-DNA-vaccinated fish at 5-7 weeks post-vaccination and not in cysteine-DNA plasmids and the control groups. MP-DNA-vaccinated fish when challenged with the pathogen had consistently lower parasitemia, delayed peak parasitemia, and faster recovery compared with the controls. All fish vaccinated with attenuated strain were protected when challenged with the pathogen; this positive control group confirmed that the two vaccines operate through different mechanisms.

Neutralization of hemorrhagic snake venom metalloproteinase HR1a from Protobothrops flavoviridis by human monoclonal antibody.

Human monoclonal antibodies (HuMAbs) against HR1a from Protobothrops (formerly Trimeresurus) flavoviridis venom were obtained by the fusion of SP2/0-Ag14 myeloma cells and spleen cells from KM mice immunized with purified HR1a. The ability of HuMAbs to neutralize the HR1a was determined by in vitro neutralization assay and by neutralization of the hemorrhagic activity. The initial screening of over 300 hybridoma fusion wells resulted in the establishment of 80 HR1a-reactive hybridomas. Of the reactive clones, HuMAb HR1a-7 and HR1a-18 neutralized both proteolytic and hemorrhagic activity of HR1a. Mapping of epitope recognized by the reactive clones was performed by using an ELISA that measured antibody binding to overlapping peptides (15 amino acid peptide offset frameshifted by three residues) covering the metalloproteinase domain sequence of HR1a. HuMAbs HR1a-7 and HR1a-18 neutralized HR1a by reacting with peptides of EQQRYLNNFRFIELV and IVNTLNETYRYL. The three-dimensional structure of HR1a based on a homology modeling predicted that these two epitopes are surface exposed.

Recent advances in the molecular pathogenesis of hereditary recurrent fevers.

PURPOSE OF REVIEW: To discuss recent developments in the molecular basis of several hereditary recurrent fever syndromes, specifically the cryopyrin-associated periodic syndromes, familial Mediterranean fever and the tumor necrosis factor receptor associated periodic syndrome. RECENT FINDINGS: Mutations of CIAS1, the gene encoding cryopyrin/NALP3, lead to a spectrum of disease states termed the cryopyrinopathies. Recently, cryopyrin-deficient mice have been used to show that the protein is a key regulator of interleukin-1beta production that functions by recognizing stimuli such as bacterial RNA and infectious agents. Tumor necrosis factor receptor-associated periodic syndrome was initially thought to be caused by deficient metalloprotease-induced tumor necrosis factor receptor shedding, however new findings suggest that mutations in this receptor may result in inappropriate protein folding, leading to a host of other functional abnormalities that may cause inflammatory disease. Finally, data are emerging that address the possible function of the C-terminal B30.2 domain of pyrin, the familial Mediterranean fever protein. This motif has recently been shown to interact with and inhibit caspase-1, and the modeled structure of this complex highlights how mutations may affect the binding interface. SUMMARY: Recent reports have advanced our understanding of the structural and functional biology underlying the hereditary recurrent fevers, and are beginning to suggest possible mechanisms by which specific mutations cause disease.

TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells.

NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.