Molecular Immunology

The B cell antigen receptor (BCR) comprises the membrane-bound immunoglobulin (mIg) molecule and the Ig-a/Ig-b heterodimer. By comparing the stability of the IgD-BCR and IgM-BCR in dierent detergent lysates, we ®nd that the IgD-BCR is more stable than the IgM-BCR. Analysis of chimeric mIgD molecules suggests that the dm transmembrane region is responsible for the more stable association of mIgD with the Ig-a/Ig-b heterodimer. Further, the dierential glycosylation of Iga molecules, in the two dierent BCR complexes, is determined solely by the ectodomains of the mIg molecules. The implications of these ®ndings for the intracellular transport and the signalling capacity of the BCRs are discussed.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.

Please cite this article in press as: Arockiaraj, J., et al., An anti-apoptotic B-cell lymphoma-2 (BCL-2) from Channa striatus: Sequence analysis and delayed and advanced gene expression in response to fungal, bacterial and poly I:C induction. Mol. Immunol. (2014), https://dx.a b s t r a c t B-cell lymphoma-2 (BCL-2) is a suppressor of apoptosis and inhibits the caspase dependent apoptosis pathway. In this study, we report molecular characterization of a cDNA sequence encoded of BCL-2 from striped murrel, Channa striatus. A partial cDNA sequence of CsBCL-2 was identified from the striped murrel cDNA library during annotation. Subsequently, the full length CsBCL-2 cDNA sequence was obtained by an internal sequencing method using a forward primer. The sequence contains 699 nucleotide base pairs which encode 232 amino acid residues. The domain and motif analysis revealed that the CsBCL-2 polypeptide consists of BCL-2 homologous domain BH4 at the N-terminal region between 4 and 21 and the BCL-2 homologous domains BH1, BH2 and BH3 between 87 and 187. The CsBCL-2 polypeptide sequence does not have a signal peptide region, but it consists of two novel transmembrane regions at 134-152 and 209-226. The sequence analysis showed that the CsBCL-2 has highest sequence identity (70%) with BCL-2 like protein 1 (BCL-2 L1) from pufferfish Takifugu rubripes. The phylogenetic analysis showed that the CsBCL-2 was situated in the BCL-2 L1 fish clade. The secondary analysis showed that the CsBCL-2 protein consists of 132 amino acid residues in the ␣-helical region and 100 amino acid residues in the random coil region. The validated 3D structure of CsBCL-2 showed the active residues Gly 135 and Arg 136 in the 7th ␣-helical position, whereas Trp 178 is in the 9th ␣-helical region. CsBCL-2 mRNA transcription is predominately present in spleen and is upregulated upon being induced with fungus Aphanomyces invadans, bacteria Aeromonas hydrophila, Escherichia coli LPS, Laminaria digitata beta-1,3-glucan and poly I:C. Overall, the CsBCL-2 mRNA transcription results indicate the potential involvement of CsBCL-2 in immune system of C. striatus. However, further research at proteomic level is necessary to examine these predictions.

The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C'): IgG3 and IgGl are very active. IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain (C,2) is critical for C' activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C"2. Mutants containing the N-terminus of the C,2 of IgG3 were as effective as wildtype IgG3 in Clq binding, Cl activation and terminal complex (MAC) formation, but had reduced ability to effect C/-mediated lysis. IgG2 and mutants containing the N-terminal portion of the CH2 of IgG2 were reduced compared to IgG3 in activating Cl, binding Clq and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C,,2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind Clq and activate the C' cascade although additional residues of C,2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C,2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C,2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders. pi;, 1997 Elsevier Science Ltd. All rights reserved.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement-and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Antigen recognition by ab T lymphocytes is mediated via the multisubunit TCR complex consisting of invariant CD3g,d,o and z chains associated with clonotypic TCRa and b molecules. Charged amino acids located centrally within the TCRa transmembrane region are necessary and sufficient for assembly with the CD3do heterodimer. Previously, we have shown that deletion of 6-12 amino acids from the carboxy terminus of the TCRa-chain dramatically abrogates surface TCR expression, suggesting that the distal portion of the TCRa transmembrane region contains information that regulates the assembly and/or intracellular transport of TCR complexes. We have examined in more detail the molecular basis for reduced TCR expression in T cells bearing truncated TCRa chains. We found that in contrast to wild-type (wt), variant TCRa proteins missing the last nine C-terminal amino acids did not associate with core CD3g,d,o chains and were not assembled into disulphide-linked ab heterodimers. The stability of newly synthesised wt and variant TCRa molecules was similar, showing that the abrogated surface TCR expression was not a consequence of impaired protein survival. Nevertheless, truncated TCRa chains still assembled with the chaperon protein calnexin in the endoplasmic reticulum, indicating that the distal portion of the TCRa transmembrane region is not essential for calnexin interaction. These data document a role for the distal portion of the TCRa transmembrane region in the assembly of TCR complexes and provide a molecular basis for reduced TCR expression in cells bearing truncated TCRa chains.

Natural killer (NK) cells express receptors that are specific for MHC class I molecules. These receptors play a crucial role in regulating the lytic and cytokine expression capabilities of NK cells. In humans, three distinct families of genes have been defined that encode for receptors of HLA class I molecules. The first family identified consists of type I transmembrane molecules belonging to the immunoglobulin (Ig) superfamily and are called killer cell Ig-like receptors (KIR). A second group of receptors belonging to the Ig superfamily, named ILT (for immunoglobulin like transcripts), has more recently been described. ILTs are expressed mainly on B, T and myeloid cells, but some members of this group are also expressed on NK cells. They are also referred to as LIRs (for leukocyte Ig-like receptor) and MIRs (for macrophage Ig-like receptor). The ligands for the KIR and some of the ILT receptors include classical (class Ia) HLA class I molecules, as well as the nonclassical (class Ib) HLA-G molecule. The third family of HLA class I receptors are C-type lectin family members and are composed of heterodimers of CD94 covalently associated with a member of the NKG2 family of molecules. The ligand for most members is the nonclassical class I molecule HLA-E. NKG2D, a member of the NKG2 family, is expressed as a homodimer, along with the adaptor molecule DAP10. The ligands of NKG2D include the human class I like molecules MICA and MICB, and the recently described ULBPs. Each of these three families of receptors has individual members that can recognize identical or similar ligands yet signal for activation or inhibition of cellular functions. This dichotomy correlates with particular structural features present in the transmembrane and intracytoplasmic portions of these molecules.

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas a 2 -macroglobulin (a 2 M) inhibited neither. C1 inhibitor and a 2 M were found to be associated with the MBL complex. Upon incubation at 37°C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5Gl. 1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5Gl. 1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5Gl. 1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5Gl. 1 scFv also effectively blocked C5a generation. Finally. an intact humanized h5Gl.l antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5Gl. 1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G 1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases. (0 1997 Elsevier Science Ltd. All rights reserved.

An ELISA was developed to quantitate the level of antibodies to various cell surface antigens of the Gram positive bacterium, Streptococcus mutans. Whole cells and purified cell wall components of 5'. mutans, lipoteichoic acid (LTA) from Streptococcus pyogenes.

Previous studies have shown that IL-10 can induce the expression of the suppressor of cytokine signaling 3 (SOCS-3) mRNA in human monocytes and neutrophils, suggesting that the capacity of IL-10 to inhibit the expression of LPS-inducible proinflammatory genes may depend on SOCS-3 induction. However, no direct experimental evidence has been provided to support such hypothesis. Herein, we show that stable transfection of SOCS-3 into the mouse macrophage cell line J774 resulted in an inhibition of NO, TNF-␣, IL-6, and GM-CSF secretion in response to LPS at levels similar to those exerted by IL-10 in LPS-stimulated wild-type J774. Constitutive SOCS-3 expression also down-regulated the mRNA expression of inducible NO synthase and IL-6 and impaired the production of TNF-␣, mainly at a post-transcriptional level. In addition, SOCS-3-transfected cells displayed a constitutive expression of the IL-1R antagonist gene, consistent with the observation that IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells. Furthermore, in peritoneal macrophages harvested from mice carrying heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in repressing LPS-stimulated TNF-␣ and NO production. Taken together, our data show that SOCS-3 inhibits LPS-induced macrophage activation, strongly supporting the idea that it plays a role in the molecular mechanism by which IL-10 down-modulates the effector functions of LPS-activated macrophages. Finally, we show that forced expression of SOCS-3 significantly suppresses the ability of IL-10 to trigger tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as an LPS signal inhibitor and as a negative feedback regulator of IL-10/STAT3 signaling.

Serum levels of IgA, and IgAz were measured by solid phase radioimmunoassay in samples from 110 children between 3 months and 10 years of age. Both IgA, and IgAz were detectable in all samples, and both IgAi and IgAz increased with increasing age. The percent of total serum IgA that was IgA, did not change with age and was the same in samples from children (15.05 f 10.2%) as in samples from adults (15.86 + 7.98%). The proportion of serum IgA that was IgA, was much less variable within sibships than within the group at large (P < 0.005). In the 16 patients with IgA deficiency, the proportion of serum IgA that was IgA, or IgA, was highly variable. IgAt constituted more than 50% of the IgA in 5 patients and less than 7% of the IgA in an additional 5 patients. These findings suggest that regulation of serum concentrations of IgA, and IgA, is complex and influenced by genetic factors and probably other unidentified factors.

Complement receptor (CR) 1 on human erythrocytes (Es) and complement factor (CF) H on rodent platelets perform immune adherence, a function which allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1-Tg/CFH −/− mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing antigen, IgG and C3 bound to Es in CR1-Tg/CFH −/− mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30 kb of genomic DNA, and contains fourteen exons producing a 4.8 kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250 nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.

When an unknown amount of antigen is allowed to diffuse radially from a well in a uniformly thin layer of antibody-containing agar for a sufficient time to allow all antigen to combine, the final area reached by the precipitate is directly proportional to the amount of antigen employed, and inversely proportional to the concentration of antibody. It is also shown that the temperature at which the plates are incubated has no perceptible influence upon the results. By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens. In the experimental albumin-antialbumin system here described, the lower limit of the method was found to correspond to 0·0025 μg of antigen, and to an antigen concentrations of 1·25 μg per ml. The standard deviation of the antigen determinations was less than 2 per cent of the mean.

The majority of genes in the HLA region are directly or indirectly involved in immunological functions. They comprise HLA, HLA-related and non-HLA-related genes. Aberrant HLA expression patterns, including heterogeneous and negative HLA expression, are observed in specimens from head and neck squamous cell carcinoma (HNSCC). To explore the possible role of genes in the HLA region other than the classical HLA genes, susceptibility regions within the HLA region for HNSCC were defined in this study. Microsatellite analysis for 49 microsatellites dispersed throughout the HLA region, in combination with the DNA pooling approach of respectively one control DNA pool and three patient DNA pools, based upon the tumour location, offered an efficient method to define susceptibility regions. In the oral cavity three significant susceptibility regions were localized, one in the class I region (330 kb), and two in the class II region (170 and 210 kb). Eighteen genes from these regions were tested for their RNA expression in oral cavity tumour tissue and compared to expression in the surrounding healthy tissue. A significant increased MICA RNA expression in tumour tissues compared to healthy surrounding tissue and a significant decreased HSD17B8 RNA in tumour tissues compared to surrounding healthy tissue, particular in those tumours without lymph node metastasis, were observed. A trend for decreased RXR␤ and NOTCH4 RNA expression was observed in tumour tissue. In addition to the classical HLA genes, other genes within the HLA region define susceptibility for oral squamous cell carcinoma of the head and neck.

Complement analysis in the clinic is usually associated with the quantification of C3 and C4, measurement of C1-inhibitor and screening for complement activity. These analyses have been available in routine diagnostic laboratories for decades. In recent years, however, the field of complement analysis has expanded considerably, with the introduction of novel assays to detect complement activation products, and spreading still further towards genetic analysis to reveal the basis of complement deficiencies and identify mutations and polymorphisms associated with defined diseases such as atypical haemolytic uraemic syndrome and age related macular degeneration. Here we review the current status of complement analysis, including assays for the quantification of complement activity and complement activation products, together with genetic methods for the detection of deficiencies, mutations and polymorphisms. This is an area where significant developments have been made recently, paralleling the research advances into the role of complement in human disease. It is clear, however, that there is a need for consensus and standardisation of analytical methods. This will be a major challenge for the complement society in the future.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CDl9. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a K,, of 2.3 x 10 -'), compared with 4.2 x lOmy for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers. cl 1997 Elsevier Science Ltd. All rights reserved.

In addition to conventional antibodies (Abs), camelids possess Abs consisting of only heavy chains. The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding. Earlier analysis of 47 VHHs showed sequence features unique to VHH domains. These include the presence of characteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently constrained by an interloop disulphide bond. Here, we describe a large (152) set of Lama glama VHH cDNAs. Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies. Three subfamilies are absent in dromedaries, which have been the primary source of VHHs thus far. Comparison of these subfamilies to conventional VH regions reveals new features characteristic of VHHs and shows that many features earlier regarded as characteristic of VHHs in general are actually subfamily specific. A long CDR3 with a concomitant putative additional disulphide bond is only observed in two VHH subfamilies. Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in conventional VH domains. The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability. Since different VHH-characteristic residues are observed in all four subfamilies, these subfamilies must have evolved independently from classical VH domains.

The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, https://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver Motif-Mate (https://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins.

Retinoic acid-inducible protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytosolic viral RNA sensors that induce type I interferon production (IFN). In this study, we found that MDA5 undergoes inducible SUMOylation by small ubiquitin-like modifier-1 (SUMO-1) in response to polyI:C stimulation. Enhanced SUMOylation of MDA5 by exogenously expressed SUMO-conjugating enzyme Ubc9 correlated with upregulation of IFN expression and repressed virus replication. Conversely, overexpression of a SUMOylation-deficient mutant of Ubc9 or knockdown of endogenous Ubc9 reduced IFN production. Furthermore, we showed that PIAS2␤, a SUMOylation E3 ligase, could specifically interact with and enhance the SUMOylation of MDA5. Consequently, PIAS2␤ knockdown reduced the SUMOylation of MDA5 and the IFN production. Collectively, these findings suggest that SUMO-1 modification of MDA5 possibly via PIAS2␤ may play a role in the MDA5-mediated IFN response to viral infections.

The extent of tropical wetlands, the magnitude of loss, and the related socioeconomic ramifications of the destruction of Indonesian wetlands are of global significance. The carbon density and rates of land-cover change in these ecosystems are amongst the highest of any forest type on Earth. Therefore, addressing interrelated issues of climate change and land use could be valuable in generating new options on how mangroves and peatlands should be best managed. This paper is produced by bringing together Indonesian and international scientists from diverse backgrounds and with diverse experiences in both freshwater and coastal tropical wetlands. It describes the state of the science, significant research needs, and potential transdisciplinary approaches necessary to implement climate change adaptation and mitigation strategies.

Sjögren&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s syndrome (SjS) is a human autoimmune disease characterized by the loss of exocrine function as a result of a chronic immune attack directed primarily against the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). NOD.B10-H2b mice manifest many features of SjS, exhibiting exocrine gland dysfunction concomitant with leukocyte infiltration of the salivary and lacrimal glands. Recent studies have shown that both SjS patients and NOD.B10-H2b mice exhibit increased B lymphocyte survival, B cell hyper-reactivity and hyper-gammaglobulinemia with high production of autoantibodies. To study the possible influence of complement on the development and expression of SjS-like disease of the NOD.B10-H2b, we have utilized a prophylactic treatment with CVF known to interfere with the action of complement C3 factor. NOD.B10-H2b mice, injected with CVF starting at 10 weeks of age, a time when leukocyte infiltration is expected to begin, failed to develop salivary dysfunction out to 24 weeks of age, a time when reduced salivary flow rates are known to occur in non-treated animals. Concomitant with retention of salivary exocrine function, CVF-treated mice showed reduced levels of leukocytic infiltration, reduction of anti-nuclear autoantibodies and major alterations in the B lymphocyte profiles while maintaining general pathophysiological measures of disease. These data suggest that C3 plays a significant role in development and onset of SjS-like disease, yet additional studies need to be carried out to identify the precise mode of action.

Despite much investigation, the nature of the primary disturbances that culminate in the production of pathogenic autoantibodies remains imprecise. However, major advances in the understanding of the genetics, the cellular and the molecular basis of pathogenic autoreactivity have been achieved in recent years. Not only B cells play a paramount role in systemic autoimmunity, but their role is not limited to secretion of autoantibodies. Under certain experimental conditions, B cells can activate memory T cells, and can process and present self-antigens to naive T cells, implying the existence of an antibody-independent mechanism for tissue injury in systemic autoimmune diseases, such as lupus. In both the mouse and the human disease, B cells secreting autoantibodies exhibit features which suggest that they are selected by specific autoantigens. Factors, such as BAFF, that support differentiation of selected B cells into mature long-lived B cells may be critical in generating deleterio...

Surfactant proteins, SP-A and SP-D, are collagen-containing C-type (calcium dependent) lectins called collectins, which contribute significantly to surfactant homeostasis and pulmonary immunity. These highly versatile innate immune molecules are involved in a range of immune functions including viral neutralization, clearance of bacteria, fungi and apoptotic and necrotic cells, down regulation of allergic reaction and resolution of inflammation. Their basic structures include a triple-helical collagen region and a C-terminal homotrimeric lectin or carbohydrate recognition domain (CRD). The trimeric CRDs can recognize carbohydrate or charge patterns on microbes, allergens and dying cells, while the collagen region can interact with receptor molecules present on a variety of immune cells in order to initiate clearance mechanisms. Studies involving gene knock-out mice, murine models of lung hypersensitivity and infection, and functional characterization of cell surface receptors have revealed the diverse roles of SP-A and SP-D in the control of lung inflammation. A recently proposed model based on studies with the calreticulin-CD91 complex as a receptor for SP-A and SP-D has suggested an anti-inflammatory role for SP-A and SP-D in naïve lungs which would help minimise the potential damage that continual low level exposure to pathogens, allergens and apoptosis can cause. However, when the lungs are overwhelmed with exogenous insults, SP-A and SP-D can assume pro-inflammatory roles in order to complement pulmonary innate and adaptive immunity. This review is an update on the structural and functional aspects of SP-A and SP-D, with emphasis on their roles in controlling pulmonary infection, allergy and inflammation. We also try to put in perspective the controversial subject of the candidate receptor molecules for SP-A and SP-D.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5Gl. 1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5Gl. 1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5Gl. 1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5Gl. 1 scFv also effectively blocked C5a generation. Finally. an intact humanized h5Gl.l antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5Gl. 1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G 1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases. (0 1997 Elsevier Science Ltd. All rights reserved.

Consanguineous marriages are common in many countries of the Middle East including Lebanon. Their impact on the repartition of kidney diseases and on the risk for familial nephritis is not known. We surveyed all of the dialysis centers in Lebanon. Nine hundred and twenty-five (925) patients and their private physicians were asked to answer a questionnaire. More than half of the hemodialysis (HD) patients had an unknown etiology of their kidney disease. Diabetes, polycystic kidney disease (PKD), chronic pyelonephritis and nephrosclerosis (NS) were the most commonly documented diagnoses. Consanguinity was present in 26% of the total HD population. More consanguineous patients with unknown renal etiology were diagnosed with their kidney diseases and initiated on dialysis before the age of 30 when compared with their non-consanguineous counter-parts (45% versus 33%, P < 0.02 and 42% versus 27%, P < 0.01), respectively. Similarly, consanguineous polycystic patients were diagnosed and started earlier on dialysis when compared with the non-consanguineous population (34% versus 12%, P < 0.05 and 28% versus 8%, P < 0.05), respectively suggesting a different disease pattern. Furthermore, the risk for family history of kidney disease was noticeable in the non-consanguineous population and significantly higher among the consanguineous patients (12% versus 18%, P < 0.04). Consanguinity-associated kidney diseases affected all religious communities, in particular the Muslim and the Druze (36 and 39%), respectively versus 17% of the Christian community. Certain geographical areas were more involved than others such as the North, South and the Bekaa with the highest percentage (40%) in the latter. Socio-economical level was not a contributing factor. We conclude that the documentation of the underlying etiology in end-stage renal diseases (ESRD) seems to be deficient. Furthermore, consanguinity is prevalent in the Lebanese dialysis patients population, in particular the Muslim and the Druze communities. Consanguinity-associated kidney diseases pattern seems to differ from that of the general HD population by disease diagnosis and initiation at a younger age and a significantly higher risk for familial renal disease. It is a cultural phenomenon prevalent predominantly in the rural areas. We recommend a multi-approach including educational, informative and probably legislative strategy in order to limit and hopefully discourage consanguineous marriages.

We have developed a novel single-chain Ep-CAM-/CD3-bispecific single-chain antibody construct designated MT110. MT110 redirected unstimulated human peripheral T cells to induce the specific lysis of every Ep-CAM-expressing tumor cell line tested. MT110 induced a costimulation independent polyclonal activation of CD4-and CD8-positive T cells as seen by de novo expression of CD69 and CD25, and secretion of interferon gamma, tumor necrosis factor alpha, and interleukins 2, 4 and 10. CD8-positive T cells made the major contribution to redirected tumor cell lysis by MT110. With a delay, CD4-positive cells could also contribute presumably as consequence of a dramatic upregulation of granzyme B expression. MT110 was highly efficacious in a NOD/SCID mouse model with subcutaneously growing SW480 human colon cancer cells. Five daily doses of 1 g MT110 on days 0-4 completely prevented tumor outgrowth in all mice treated. The bispecific antibody construct also led to a durable eradication of established tumors in all mice treated with 1 g doses of MT110 on days 8-12 after tumor inoculation. Finally, MT110 could eradicate patient-derived metastatic ovarian cancer tissue growing under the skin of NOD/SCID mice. MT110 appears as an attractive bispecific antibody candidate for treatment of human Ep-CAM-overexpressing carcinomas.

The use of nanoscale materials is growing exponentially as concerns rise about the human hazards to it. It is assumed that living beings are coevolved with nanoparticles ever since the origin of life on earth and therefore, they must have developed the defense and toxicity mitigating mechanisms for nanoparticles. Although having peculiar properties these new materials also present new health risks upon interacting with biological systems. Zinc oxide is the most widely used nanoparticles among various nanomaterials. Recently, these nanoparticles have been shown to specifically kill cancerous cells; therefore, it is believed that these nanoparticles may be used as an alternative anti-tumor agent. However, it is also known that these nanoparticles pose several deleterious effects to living beings. It is therefore critical to understand the nature and origin of the toxicity imposed by these nanomaterials. Keeping these points in mind the present review provides updated information on various aspects of toxicities induced by these engineered nanoparticles.

Teleost fish represent a transition point on the phylogenetic spectrum between invertebrates that depend only on innate immunity and mammals that heavily depend on adaptive immunity. The major mechanisms of the teleost fish innate immune response are suggested to be similar to mammals, although fine details of the process require further studies. Within the innate immune response the type I interferon (IFN) system is an essential innate antiviral component that protects fish from some virus infections. The current progress of cloning and functional characterization of fish antiviral genes is promising in further elucidation of the fish antiviral response. The adaptive immune system of fish utilizes cellular components more or less similar to mammals. Teleost fish produce IgM as a primary antibody response and lack isotype switching to mount virus-specific antibodies during the infection process. Despite this, the development of successful fish rhabdoviral vaccines suggest that vaccination may prove to be an effective way of promoting fish adaptive immune responses to viruses. This paper reviews the bony fish antiviral response with specific discussion on the evolutionary mechanisms that allow aquatic viruses to co-exist with their host. Detailed aspects of the teleost type I IFN system are also addressed.

Scabies is a disease of considerable importance in the developing world and among disadvantaged people such as the Aborigines in Australia, as it underlies streptococcal skin infections. It has been neglected at the molecular level until very recently. However, 43,776 expressed sequence tags have now been generated at the Australian Genome Research Facility from a cDNA library we constructed. Unexpectedly,

Although human and murine antibodies are similar when considering their diversification strategies, they differ in the proportion by which K and ,i type chains are present in their receptive V, repertoires. It has been shown that this difference implies a divergence in the structural repertoire of the ti and i genes of these species. Nonetheless, the differences in V, have not been systematically studied. In this paper a systematic characterization of the V, structural repertoire of mice is made. so that a comparison with the V, structural repertoire of humans. described in detail elsewhere. could be properly accomplished. Our study shows the structural repertoire of mice to be dominated by canonical structure class I-2 (-60%) while in humans the dominant one is class l--3 ( -40%). Analysis of the evolutionary relationships between human and mice suggest that this divergence may have a functional meaning. The implications of such findings are discussed. 1~' 1997 Elsevier Science Ltd. All rights reserved.

It has been proven that both resting and activated mast cells (MCs) and basophils are able to induce a significant increase in proliferation and survival of naïve and activated B cells, and their differentiation into antibody-producing cells. The immunological context in which this regulation occurs is of particular interest and the idea that these innate cells induce antibody class switching and production is increasingly gaining ground. This direct role of MCs and basophils in acquired immunity requires cell to cell contact as well as soluble factors and exosomes. Here, we review our current understanding of the interaction between B cells and MCs or basophils as well as the evidence supporting B lymphocyte-MC/basophil crosstalk in pathological settings. Furthermore, we underline the obscure aspects of this interaction that could serve as important starting points for future research in the field of MC and basophil biology in the peculiar context of the connection between innate and adaptive immunity.

IgA is found in both mucosal secretions and serum and is the dominant immunoglobulin isotype produced in humans. It exists in different molecular forms, namely monomeric IgA, dimeric IgA, polymeric IgA and secretory IgA, all exhibiting interactions with FcalphaRI/CD89 to some extent. CD89 is an activating, gamma-chain associated, Fc receptor for IgA expressed on myeloid cells. Here, we investigated the interaction of monomeric and polymeric IgA purified from human serum with CD89 using surface plasmon resonance. The results demonstrate a similar association for monomeric and polymeric IgA with CD89. In contrast, monomeric IgA dissociated more rapidly from CD89 than polymeric IgA. Removal of N-glycans from mIgA resulted is an increased association with CD89, whereas the dissociation was more rapid, resulting in binding comparable to that of untreated monomeric IgA. We conclude that the initial interaction of monomeric and polymeric IgA with CD89 is similar, whereas monomeric IgA diss...

Plasmodium vivax represents the most widespread malaria parasite worldwide. Although it does not result in as high a mortality rate as P. falciparum, it inflicts debilitating morbidity and consequent economic impact in endemic communities. In addition, the relapsing behavior of this malaria parasite and the recent resistance to anti-malarials contribute to making its control more difficult. Although the biology of P. vivax is different from that of P. falciparum and the human immune response to this parasite species has been rather poorly studied, significant progress is being made to develop a P. vivax-specific vaccine based on the information and experience gained in the search for a P. falciparum vaccine. We have devoted great effort to antigenically characterize the P. vivax CS protein and to test its immunogenicity using the Aotus monkey model. Together with other groups we are also assessing the immunogenicity and protective efficacy of the asexual blood stage vaccine candidates MSP-1 and DBP in the monkey model, as well as the immunogenicity of Pvs25 and Pvs28 ookinete surface proteins. The transmission-blocking efficacy of the responses induced by these latter antigens is being assessed using Anopheles albimanus mosquitoes. The current status of these vaccine candidates and other antigens currently being studied is described.

We determined the interferon-gamma (IFN-gamma) cDNA sequence from three porcine breeds, Duroc, Landrance/Duroc hybrid, and Landrance breeds. Five single nucleotide polymorphisms (SNPs) of porcine IFN-gamma (PoIFN-gamma) were identified, respectively, at positions 269 (A/G), 376 (C/T), 426 (T/C), and 465 (T/C) of the coding sequence in Landrance/Duroc hybrid, and at position 251 (A/G) in Landrance breed. Among them, A269G and A251G polymorphisms resulted in Q67R and K61R replacements in the mature protein. PoIFN-gamma cDNAs of Duroc breed (PoIFN-gamma-W) and Landrance/Duroc hybrid (PoIFN-gamma-M), which, respectively, encoded Q67 and R67, were introduced into a prokaryotic expression vector pET32 to express recombinant PoIFN-gamma-W (rPoIFN-gamma-W) and rPoIFN-gamma-M protein variants in Escherichia coli. The identity of both protein variants was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We then compared bioactivities of these two recombinant proteins. Although both recombinant protein variants exhibited comparable activities in antiproliferation of PK-15 cells and in nitric oxide (NO) induction of porcine peripheral monocytes, antiviral activity of rPoIFN-gamma-W protein was significantly higher (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001) than that of rPoIFN-gamma-M protein in a plaque inhibition assay using pseudorabies virus (PRV). IC50 values of rPoIFN-gamma-W and rPoIFN-gamma-M protein in anti-PRV assay were determined as 5.3+/-1.3 and 9.3+/-4.3nM, respectively. In conclusion, we have identified five novel SNPs in PoIFN-gamma cDNA, including two missense polymorphisms that result in Q67R and K61R replacements. Our results further demonstrate that Q67R can markedly reduce antiviral activity of the PoIFN-gamma protein. This is the first report that shows the functional SNP in the coding region of IFN-gamma. In the future, it is imperative to determine whether Q67R replacement in IFN-gamma may have disease association.