Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Morphine enhances the phosphorylation of a 58 kDa protein in mouse brain membranes.

Morphine and [D-Ala2,D-Leu5]enkephalinamide enhance the phosphorylation of a 58 kDa protein in mouse brain synaptosomal membranes. The enhancement of phosphorylation was inhibited by naloxone, an antagonist of morphine. The phosphorylated 58 kDa protein was retained on wheat-germ-agglutinin-agarose and morphinone-Affi-Gel 401 columns and biospecifically eluted out from the columns with N-acetyl-D-glucosamine and naloxone respectively. These results suggest a strong possibility that the opiate-binding protein undergoes phosphorylation by endogenous protein kinase. Since the molecular mass of a mu-type opioid receptor in mouse brain is suggested to be 58 kDa, coincident with those of rat brain and neuroblastoma x glioma hybrid cells, it is conceivable that the phosphorylated 58 kDa protein is a mu-type receptor.     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Change of incidence of antinuclear antibodies in serum of NOD mouse with aging

Extractable nuclear antigens (ENA) were prepared from liver of C57BL/6J mouse and analyzed by SDS PAGE Western-immunoblotting techniques. Some protein components of the ENA, with molecular weights of 94 K, 65 K, 32 K, and 26 K, reacted with antinuclear antibodies in the sera of NOD mice. Incidence of antinuclear antibodies in the sera of NOD mice with aging were measured by ELISA method using the ENA as antigen. The antinuclear antibodies were not detected in young NOD mice (10 weeks old). However, the incidence increased with aging and reached 100% in the female NOD mice of 40 weeks. In the male NOD mice, the incidence of antinuclear antibodies was delayed and low in comparison with that in female.     

Altered fractalkine cleavage potentially promotes local inflammation in NOD salivary gland

Introduction

In the nonobese diabetic (NOD) mouse model of Sjögren's syndrome, lymphocytic infiltration is preceded by an accumulation of dendritic cells in the submandibular glands (SMGs). NOD mice also exhibit an increased frequency of mature, fractalkine receptor (CX3C chemokine receptor [CX3CR]1) expressing monocytes, which are considered to be precursors for tissue dendritic cells. To unravel further the role played by fractalkine-CX3CR1 interactions in the salivary gland inflammation, we studied the expression of fractalkine in NOD SMGs.

Methods

We studied protein expression using Western blot analysis of whole tissue lysates. Protease activity was measured in salivary gland tissue lysates using fluorimetric substrates. Digestive capacity of enzymes was determined by in vitro incubation of recombinant enzyme and fractalkine, followed by protein staining and Western blot.

Results

Fractalkine was detected in salivary glands of both NOD and control mice at all ages. Western blot analysis showed fractalkine cleavage with increasing age, which was more pronounced in NOD mice. This cleavage resulted in a decrease in the 31 kDa form of the protein, and the generation of an approximately 19 kDa band. Furthermore, in NOD animals older than 15 weeks, we noted the presence of a unique approximately 17 kDa fragment. This cleavage was organ specific, because it did not occur in brain or pancreas. Increased gelatinase and α-secretase activity were detected in NOD SMG and contributed to cleavage of the 31 kDa protein. Because aberrant cleavage products may induce autoimmunity, we studied the presence of autoantibodies against fractalkine. Indeed, NOD mice exhibited significantly more antibodies against fractalkine than did control animals.

Conclusion

These data indicate that aberrant proteolytic activity in the NOD SMG results in increased fractalkine cleavage and generation of a unique fractalkine fragment. This specific cleavage may contribute to autoimmunity.     

Detection of nuclear protein antigens to antinuclear antibodies in serum of NOD mouse

Nuclear protein antigens to the antinuclear antibodies in serum of non-obese diabetic (NOD) mice were investigated. In the serum of diabetic NOD female mice (20 weeks old), the antinuclear antibodies were detected by indirect immunofluorescence assay using frozen sections of liver of C 57 BL/6 J or NOD mice as antigen. Nuclei were separated from the liver of C 57 BL/6 J mice and solubilized. Solubilized nuclear antigens were analyzed by SDS PAGE-Western immunoblotting techniques. Nuclear protein antigens with molecular weights of 26,000, 32,000 and 65,000 showed strongly positive reactions with the antinuclear antibodies in the serum of the NOD mouse.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.     

Evidence for Cd101 but not Fcgr1 as candidate for type 1 diabetes locus, Idd10

Among polygenes conferring susceptibility to type 1 diabetes in the NOD mouse, Idd10 on distal chromosome 3 has been shown to be important for disease susceptibility. In this study, we investigated the candidacy of Fcgr1 and Cd101 for Idd10, by congenic mapping and candidate gene sequencing. Among seven NOD-related strains studied, the IIS mouse was found to possess a recombinant Idd10 interval with the same sequence at Fcgr1 as the NOD mouse, but a different sequence at Cd101 from that in the NOD mouse with 10 amino acid substitutions. The frequency of type 1 diabetes in NOD mice congenic for IIS Idd10 (NOD.IISIdd10) was significantly reduced as compared to that in the NOD mouse, despite the presence of the identical Fcgr1 sequence. These data indicate that IIS mice possess a resistant allele at Idd10, and suggest that Cd101, but not Fcgr1, is responsible for the Idd10 effect.     

Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV)

The use of previously published primers to amplify the monodon baculovirus (MBV) polyhedrin gene sequence by polymerase chain reaction (PCR) from post larvae (PL) of Thai Penaeus monodon resulted in failure. As a result, the putative polyhedrin protein of MBV was isolated from infected PL by homogenization, differential centrifugation and density gradient centrifugation with verification by transmission electron microscopy (TEM). By SDS-PAGE, a single major protein band at 58 kDa was obtained from the putative polyhedrin fraction and this corresponded to a previous report of the molecular weight of polyhedrin from MBV. When used for N-terminal sequence analysis, the putative polyhedrin protein yielded a sequence of 25 amino acids (M F D D S M M M E N M D D L S G D Q K M V L T L A) that did not correspond to the deduced amino acid sequence derived from a previous report of a putative MBV polyhedrin gene amplicon. Despite this, a synthetic peptide of our 25 amino acid sequence (25Pmbv) was conjugated with bovine serum albumin and used as an antigen for antiserum production in mice. Using immunohistochemistry with tissue sections of PL infected with MBV or other viruses, the mouse anti-25Pmbv antiserum showed strong immunoreactivity to occlusion bodies of MBV only. It also showed strong reactivity to the 58 kDa putative polyhedrin protein in Western blots. Altogether, the results suggest that the 58 kDa protein is Thai MBV polyhedrin and that a previously reported MBV polyhedrin gene sequence may represent another protein or polyhedrin from a different variety of MBV.     

Human autoantibodies against a nucleolar protein

Autoimmune sera showing prominent immunofluorescence in nucleolus were selected and analysed by immunoblotting techniques. Immunoblots using a nucleolar extract as antigen source revealed sera recognizing a 38 kDa nucleolar protein. Low concentration of Actinomycin D, which inhibits the ribosomal RNA synthesis, caused a loss of fluorescence. This suggests that the nucleolar antigen may be associated with the assembly of packaging of the ribosomes. The present nucleolar antigen has properties similar to the previously described nucleolar phosphoprotein B23 of rat cells and the recently described nucleolar protein NO38 of mouse and Xenopus cells.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).     

Peptide dose, MHC affinity, and target self-antigen expression are critical for effective immunotherapy of nonobese diabetic mouse prediabetes

Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 microg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-A(g7) was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.     

Candida albicans fibrinogen binding mannoprotein: expression in clinical strains and immunogenicity in patients with candidiasis.

A 58 kDa cell wall-associated fibrinogen binding mannoprotein (mp58), previously characterized by our group in a Candida albicans laboratory strain (ATCC 26555), was found to be also present in the cell wall of clinical isolates of this fungus. Most strains examined appear to have functional mp58 species, as detected by their ability to bind fibrinogen. Western immunoblot analysis, with a monovalent polyclonal antibody generated against the mp58 species from strain ATCC 26555, revealed differences in recognition patterns depending on the strain tested and the culture conditions used. Serum samples from normal and Candida infected individuals were examined for the presence of antibodies against mp58 by Western immunoblotting. None of the sera from control individuals and patients suffering from superficial candidiasis contained antibodies against mp58. However, positive reactivity with this antigen and other cell wall constituents was detected for all sera from patients with confirmed systemic candidiasis. Together, these results suggest that mp58 could play an active role during infection and may be useful as a specific antigenic marker for candidiasis.     

Early Th1 response in unprimed nonobese diabetic mice to the tyrosine phosphatase-like insulinoma-associated protein 2, an autoantigen in type 1 diabetes

The insulinoma-associated protein 2 (IA-2) is a phosphatase-like autoantigen inducing T and B cell responses associated with human insulin-dependent diabetes mellitus (IDDM). We now report that T cell responses to IA-2 can also be detected in the nonobese diabetic (NOD) mouse, a model of human IDDM. Cytokine secretion in response to purified mouse rIA-2, characterized by high IFN-gamma and relatively low IL-10 and IL-6 secretion, was elicited in spleen cells from unprimed NOD mice. Conversely, no response to IA-2 was induced in spleen cells from BALB/c, C57BL/6, or Biozzi AB/H mice that express, like NOD, the I-A(g7) class II molecule, but are not susceptible to spontaneous IDDM. The IA-2-induced IFN-gamma response in NOD spleen cells could already be detected at 3 wk and peaked at 8 wk of age, whereas the IL-10 secretion was maximal at 4 wk of age and then waned. IA-2-dependent IFN-gamma secretion was induced in CD4(+) cells from spleen as well as pancreatic and mesenteric lymph nodes. It required Ag presentation by I-A(g7) molecules and engagement of the CD4 coreceptor. Interestingly, cytokines were produced in the absence of cell proliferation and IL-2 secretion. The biological relevance of the response to IA-2 is indicated by the enhanced IDDM following a single injection of the recombinant protein emulsified in IFA into 18-day-old NOD mice. In addition, IFN-gamma production in response to IA-2 and IDDM acceleration could be induced by IL-12 administration to 12-day-old NOD mice. These results identify IA-2 as an early T cell-inducing autoantigen in the NOD mouse and indicate a role for the IA-2-induced Th1 cell response in IDDM pathogenesis.     

Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.     

Primary structure of the human 4F2 antigen heavy chain predicts a transmembrane protein with a cytoplasmic NH2 terminus

The human cell surface antigen 4F2 is a disulfide-linked heterodimer consisting of a glycosylated heavy chain and a nonglycosylated light chain. The antigen is ubiquitously expressed on proliferating cells but only in resting cells from certain tissues. Its function has been proposed to relate to cellular Ca2+/Na+ exchange. We describe the molecular cloning of the 4F2 heavy chain gene and cDNA by a gene transfer approach. Part of the gene was isolated from a genomic lambda library constructed with DNA of a secondary transfectant L cell line that expresses 4F2 antigen. A gene-specific probe derived from the phage inserts was used to isolate two full length cDNA clones. Both cDNA clones directed the expression of 4F2 antigen in transfected mouse L cells. The 4F2 antigen heavy chain gene specifies a 2.1-kilobase mRNA with an open reading frame coding for a 529-residue protein of 58 kDa. The protein lacks an NH2-terminal signal peptide but contains an internal transmembrane-spanning region and four potential glycosylation sites in its COOH-terminal domain. We predict that the 4F2 antigen heavy chain is a transmembrane protein with a cytoplasmic NH2 terminus of 81 amino acids. The antigen shows no homology to known protein sequences.     

Incomplete Freund's adjuvant reduces diabetes in the non-obese diabetic mouse.

As the study of type 1 diabetes moves towards preventive therapy, the role of adjuvants needs to be addressed. Incomplete Freund's adjuvant (IFA) is thought of as "immunologically inert" as, unlike complete FA (CFA), it has no components designed to provoke an immune response. We investigated the effect of IFA as an immunomodulator on the disease process leading to type 1 diabetes in the non-obese diabetic (NOD) mouse. 24 NOD mice were injected intradermally (i.d.) at 8 and 12 weeks of age with a 1:1 mixture of IFA and saline; 24 controls received saline alone. Splenocytes were tested against antigens thought to be involved in the disease process, namely insulin, a GAD peptide, a beta-casein peptide, a Glut-2 peptide and concanavalin A (ConA) as a non-specific antigen. In the IFA experiment diabetes incidence was 13% compared to 38% in the controls (p < 0.05). In vitro, splenocytes from IFA treated animals showed non-specific immunosuppression with ConA (p < 0.01), whereas the response to 1-casein and Glut-2 was raised in IFA treated animals with respect to controls. ELISA using supernatants from IFA treated animals, showed a typical Th2 cytokine pattern, whereas controls showed a Th1 pattern. In conclusion, IFA alone can reduce diabetes incidence in the NOD mouse apparently by modulating the immune response towards beta-cell related specific antigens. As IFA has been adopted as an adjuvant in preventive trials in the NOD mouse, this might have implications for the interpretation of previous and future results.