Nectins form a family of integral molecules that belong to the immunoglobulin superfamily. Their ectodomain is made of three Ig-like domains (V, C, C). This family comprises at least five members, namely nectin1, -2, -3, -4, and poliovirus receptor (PVR), that are involved in different physiological and pathological processes. (i) Nectins are adhesion molecules localized at adherens junctions in epithelial cells. (ii) Some nectins act as poliovirus or alpha-herpesvirus receptors (nectin1). (iii) Nectin1 mutations are involved in orofacial developmental abnormalities in humans. Adhesion properties of nectins are mediated by Ca(2+)-independent homophilic and heterophilic processes through ectodomain trans-interactions. We have described a nectin trans-hetero-interaction network: nectin3 binds to nectin1, nectin2, and PVR; nectin1 also binds to nectin4. In the present study we compared the affinities of the different trans-interactions mediated by nectin1. We found that the K(D) of nectin1/nectin3 and nectin1/nectin4 interactions is 1 and 100 nm, respectively, whereas the K(D) of the nectin1-mediated homophilic interaction is 1 microm. We show that nectin1/nectin3 and nectin1/nectin4 trans-hetero-interactions were mediated through trans V to V domain interactions, whereas C domains contributed to increase the affinity of the interaction. Nectin3 and nectin4 share a common binding region in the nectin1 V domain: (i) nectin3 strongly competed with nectin4 binding, (ii) nectin3 and nectin4 binding to nectin1 was reduced by a number of monoclonal antibodies directed against the nectin1 V domain, and (iii) the glycoprotein D of herpes simplex virus-1 that binds to the V domain of nectin1 reduced nectin3 and nectin4 binding. Finally, using chimeric nectin1/PVR receptors where PVR V domain beta-strands were substituted with the corresponding regions of nectin1, the nectin3 and nectin4 minimal binding region on nectin1 V domain was mapped to the C-C'-C"-D beta-strands.

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19(+) cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I alpha/beta interferon (IFN-alpha/beta) receptor. Despite significant activation in the draining LN at day 3 after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80(+) or CD169(+) subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-alpha/beta receptor.

Human anti-pig antibodies were obtained by perfusing pig hearts (n = 4) and kidneys (n = 8) with human AB or O plasma followed by elution with 3 M NaSCN. The antibodies were screened against a panel of 132 synthetic carbohydrates conjugated to bovine serum albumin using an enzyme-linked immunoassay. An anti-immunoglobulin antibody was also used to detect immunoglobulin deposits on pig tissues. Four carbohydrate molecules with a terminal alpha-galactose residue bound all but one of the human anti-pig kidney antibodies and most of the anti-pig heart antibodies. These were: (i) alpha Gal(1-->3)beta Gal(1-->4)beta GlcNac (linear B type 2); (ii) alpha Gal(1-->3)beta Gal(1-->4)beta Glc (linear B type 6); (iii) alpha Gal(1-->3)beta Gal(B disaccharide); and (iv) alpha Gal(alpha-D-galactose). Immunoglobulin deposition was documented post-plasma perfusion in all pig hearts and particularly strongly in all pig kidneys. These results suggest that human anti-pig antibodies are mainly directed against alpha-galactosyl structures. Extracorporeal immunoadsorption of human plasma through columns of the specific synthetic carbohydrate(s) might lead to depletion of anti-pig antibodies and allow discordant xenografting in man. Alternatively, the infusion of the specific carbohydrate(s) for a period of several days might result in neutralization of the anti-pig antibodies and allow accommodation to take place.

We previously showed that the in vitro transcription of a negatively supercoiled plasmid containing the murine IgA switch region caused the formation of fewer supercoiled conformers of the plasmid due to the presence of a stable RNA.DNA hybrid. Here, we demonstrate that the RNA.DNA hybrid is approximately 140 nucleotides, and it forms regardless of the initial topological state of the transcription template. Transcription of the switch region in a relaxed closed circular plasmid generates positively supercoiled plasmid conformers that revert to their original relaxed state when treated with RNase H. Conformers that have incorporated a stable RNA transcript are also observed when nicked circular and linear plasmids containing the IgA switch sequences are transcribed with 32P-labeled nucleotide triphosphates. Once formed, the RNA.DNA hybrid is stable to both thermal and superhelical stress, tolerating temperatures in excess of 95 degrees C and restraining approximately 12 positive supercoils in the plasmid.

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.

Gut-derived lymphocytes transiently migrate through the peripheral circulation before homing back to mucosal sites and can be detected using an ELISPOT-based antibody secreting cell (ASC) assay. Alternatively, transiently circulating lymphocytes may be cultured in vitro, and culture supernatants may be assayed for antigen-specific responses (antibody in lymphocyte supernatant [ALS] assay). The ALS assay has not been validated extensively in natural mucosal infection, nor has the ALS response been compared to the ASC assay and other cholera-specific immunological responses. Accordingly, we examined immune responses in 30 adult patients with acute cholera in Bangladesh, compared with 10 healthy controls, measuring ALS-immunoglobulin A (IgA), ASC-IgA, and serum and fecal IgA responses to two potent Vibrio cholerae immunogens, the nontoxic B subunit of cholera toxin (CtxB) and lipopolysaccharide (LPS) and a weaker V. cholerae immunogen, the mannose-sensitive hemagglutinin (MSHA). We found significant increases of anti-CtxB, anti-LPS, and anti-MSHA IgA in supernatants of lymphocytes cultured 7 days after onset of cholera using the ALS assay. We found that ALS and ASC responses correlated extremely well; both had comparable sensitivities as the vibriocidal responses, and both procedures were more sensitive than fecal IgA measurements. An advantage of the ALS assay for studying mucosal immune responses is the ability to freeze antibodies in supernatants for subsequent evaluation; like the ASC assay, the ALS assay can distinguish recent from remote mucosal infection, a distinction that may be difficult to make in endemic settings using other procedures.

Seventy individuals with ataxia telangiectasia were studied: 29 females and 41 males with an age range of 2 to 42 years. The majority (43/68) presented by 3 years of age with truncal ataxia. All had progressive, handicapping neurological symptoms exhibiting ataxia (70/70), ocular motor apraxia (70/70), an impassive face (70/70), dysarthria (70/70), chorea (68/70), dystonia (55/70) and peripheral neuropathy (50/70). Clinical immune deficiency was present in 43 of 70 patients. Ocular telangiectasia were seen in all but one case and excessive thinness in 54 of 70. The mean age of loss of walking was 10 years and of writing 8 years. All 60 tested showed increased sensitivity to ionizing irradiation, 43 of 48 had an elevated alpha-fetoprotein level and 14 of 21 had an immunoglobulin deficiency. Although there was a marked variation in disease findings sibs were always similar. The heterogeneity seen seems at odds with the unilocus linkage of ataxia telangiectasia to 11q23.

BACKGROUND

MicroRNAs (miRNAs) play important roles in the progression of renal fibrosis. We studied the urinary levels of miR-21, miR-29 family and miR-93, which are downstream mediators of the transforming growth factor-β(1) (TGF-β(1)), in patients with immunoglobulin A (IgA) nephropathy.

METHODS

We studied the urinary miRNA levels of 43 IgA nephropathy patients and 13 healthy controls.

RESULTS

The IgA nephropathy group had significantly lower urinary miR-29b and miR-29c, but higher miR-93 levels than controls. Proteinuria significantly correlated with urinary levels of miR-29b (r = -0.388, p = 0.003) and miR-29c (r = -0.409, p = 0.002). Glomerular filtration rate significantly correlated with urinary levels of miR-21 (r = 0.338, p = 0.028), miR-29b (r = 0.333, p = 0.031) and miR-29c (r = 0.304, p = 0.050). Urinary miR-93 level significantly correlated with glomerular scarring (r = -0.392, p = 0.010). Urinary miRNA level of SMAD3, but not TGF-β(1), correlated with urinary miR-21 (r = 0.624, p < 0.001), miR-29b (r = 0.566, p < 0.001), miR-29c (r = 0.619, p < 0.001) and miR-93 (r = 0.332, p = 0.032).

CONCLUSIONS

Urinary miR-29b and miR-29c levels correlated with proteinuria and renal function, while urinary miR-93 level correlated with glomerular scarring. More importantly, urinary levels of these miRNA targets significantly correlated with urinary SMAD3 level. Our results suggest that these miRNA targets are regulated by the TGF-β(1)/SMAD3 pathway and they may play important roles in the development of progressive renal fibrosis in IgA nephropathy.

Increased immunoglobulin A (IgA) antibodies to the Epstein-Barr virus (EBV) appear months to years before the clinical onset of nasopharyngeal carcinoma and define populations at high risk for this EBV-associated epithelial cancer common in south China. In the human HT-29 epithelial cell line, polymeric IgA (pIgA) specific for EBV promoted infection of the otherwise refractory epithelial cells. When bound to pIgA, EBV entered epithelial cells through secretory component-mediated IgA transport but no longer infected B lymphocytes. Such an immune-induced shift in EBV tissue tropism provides a paradigm for endogenous spread of EBV in the immune host that predicts infectious sequelae of epithelium.

The complete nucleotide sequence of the gene and mRNA coding for the constant (C) region of the secreted form of the BALB/c mouse IgA immunoglobulin alpha heavy (H) chain has been determined. As in other immunoglobulins, the three C region domains of the alpha protein, C alpha 1, C alpha 2, and C alpha 3 are coded in separate exons. However, the hinge region of C alpha is not coded on a separate exon as it is in other hinge-containing immunoglobulins. Instead, the alpha hinge is coded as a 5' extension of the C alpha 2 exon, and we suggest that it may have evolved by duplication leading to incorporation of an acceptor RNA splice site into the coding portion of the C alpha 2 exon. Extensions of this concept could provide an explanation for duplications in the human alpha 1 chain.

A computer program is described which, given a nucleotide or an amino acid sequence, outputs protein secondary structure prediction curves as well as hydrophobicity and charged-residue profiles. The program allows for cumulative averaging of properties (secondary structure propensities, hydrophobicity and charge profiles) from several homologous primary structures, a novel concept shown to improve the predictive accuracy. The use of the program is demonstrated on a set of nucleotide and amino acid sequences from human and murine histocompatibility antigens of class I and II. The last extracellular domains of both class I and II antigens (alpha 3 of class I, alpha 2 and beta 2 of class II) and the beta 2-microglobulin domain are predicted to consist of seven anti-parallel beta-strands, in accord with previous claims of homology between these domains and the constant domains of immunoglobulin chains. The remaining extracellular domains are all proposed to form an anti-parallel, four-stranded beta-sheet with one of its faces being covered by alpha-helices and/or structureless segments ("open face sandwiches").

We have isolated a myeloma genomic DNA clone containing the variable and constant regions of a mouse alpha chain. Restriction enzyme analyses and electron microscopic R loop mapping have demonstrated that the variable region is separated from the constant region by 6.8 kilobases of intervening DNA. In addition, two intervening DNA sequences of 100--200 bases separate the constant region into three approximately equal units. These intervening sequences may separate each of the segments coding for the three constant region domains of the alpha heavy chain. Southern blot analysis of embryo and myeloma DNA suggests that DNA rearrangement of heavy chain variable and constant regions occurs during the differentiation of antibody-producing cells.

Antigens and infectious agents that stimulate interferon alpha(IFN-alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN-alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells.

GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibalpha. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.

OBJECTIVE

To investigate the prevalence of celiac disease in a large cohort of children and adolescents at the onset of type 1 diabetes and the occurrence of new cases during a 6-year follow-up.

METHODS

We prospectively studied, by repeated serologic screening, 274 consecutive patients at the onset of type 1 diabetes (age [mean +/- standard deviation]: 8.28 +/- 4.65 years) for 6 subsequent years. One patient had a diagnosis of celiac disease before the onset of diabetes. The immunoglobulin A-antiendomysium antibody test was selected as the screening test; patients with positive results (++ or +++) or with 2 consecutive weak positive tests (+) were considered appropriate for the jejunal biopsy.

RESULTS

At diabetes onset, 15 (5.5%) of 273 patients tested positive with the antiendomysium test; jejunal biopsy was performed in 10, and celiac disease was diagnosed in 9. The prevalence of biopsy-confirmed celiac disease at the manifestation of diabetes was 3.6% (10 of 274 patients). Twelve more patients with a negative antiendomysium antibody test at diabetes onset tested positive during the follow-up within 4 years; 10 of them had biopsies performed, and 7 had celiac disease. Therefore, the overall prevalence of biopsy-confirmed celiac disease in the entire cohort of patients was 6.2%. The age at diabetes onset in patients with and without celiac disease was not different (7.88 +/- 5.69 vs 8.3 +/- 4.58 years). The majority of cases of celiac disease were asymptomatic in their presentation, and no signs of overt malnutrition were documented.

CONCLUSIONS

The prevalence of celiac disease in patients with type 1 diabetes is approximately 20 times higher than in the general population. Sixty percent of cases are already present at diabetes onset, mostly undetected, but an additional 40% of patients develop celiac disease a few years after diabetes onset. Extending screening programs for celiac disease after the onset of type 1 diabetes is recommended, even in the absence of clinical symptoms.

Cells that produce interleukin-6 (IL-6) require the presence of signaling molecules since this cytokine is not normally constitutively expressed. It is now established that astrocytes produce IL-6; however, the precise inducing molecules and the kinetics of their action have not yet been clearly identified. In the current study, we show that either interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) exert a strong inducing signal for IL-6 in primary rat astrocytes. When the two cytokines are added together the response is synergistic, suggesting that each cytokine may induce IL-6 gene expression by different pathways. Interferon-gamma (IFN-gamma) does not affect IL-6 expression although if it is added in conjunction with IL-1 beta, an augmented induction of IL-6 occurs. In addition to the cytokines, bacterial lipopolysaccharide (LPS) and the calcium ionophore, A23187, induce IL-6 expression. IL-6 expression can be blocked by the glucocorticoid analogue, dexamethasone. IL-6 induction by LPS/Ca2+ ionophore is more sensitive to the suppressive effects of dexamethasone than is IL-6 induction by TNF-alpha/IL-1 beta. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased levels of IL-6 mRNA in both unstimulated and stimulated astrocytes, indicating that ongoing protein synthesis is not required for astrocyte IL-6 gene expression. We propose that astrocyte-produced IL-6 may have a role in augmenting intracerebral immune responses in neurological diseases such as multiple sclerosis (MS), AIDS dementia complex (ADC), and viral infections. These diseases are characterized by infiltration of lymphoid and mononuclear cells into the central nervous system (CNS), and intrathecal production of immunoglobulins. IL-6 may act to promote terminal differentiation of B cells in the CNS, leading to immunoglobulin synthesis.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

BACKGROUND

The Oxford classification of immunoglobulin A (IgA) nephropathy (IgAN) provides a histopathologic grading system that is associated with kidney disease outcomes independent of clinical features. We evaluated the Oxford IgAN classification in a large cohort of patients from China.

METHODS

Retrospective study.

METHODS

1,026 adults with IgAN from 18 referral centers in China. Inclusion criteria and statistical analysis were similar to the Oxford study.

METHODS

Histologic findings of mesangial hypercellularity score, endocapillary proliferation, segmental sclerosis or adhesion, crescents, necrosis, and tubular atrophy/interstitial fibrosis. Clinical features, blood pressure, estimated glomerular filtration rate (eGFR), proteinuria, and treatment modalities.

RESULTS

Time to a 50% reduction in eGFR or end-stage renal disease (the combined event); the rate of eGFR decline (slope of eGFR); proteinuria during follow-up.

RESULTS

Compared with the Oxford cohort, the Chinese cohort had a lower proportion of patients with mesangial hypercellularity (43%) and endocapillary proliferation (11%), higher proportion with segmental sclerosis or adhesion (83%) and necrosis (15%), and similar proportion with crescents (48%) and tubular atrophy/interstitial fibrosis (moderate, 24%; severe, 3.3%). During a median follow-up of 53 (25th-75th percentile, 36-67) months, 159 (15.5%) patients reached the combined event. Our study showed that patients with a mesangial hypercellularity score higher than 0.5 were associated with a 2.0-fold (95% CI, 1.5-2.8; P<0.001) higher risk of the combined event than patients with a score of 0.5 or lower. Patients with tubular atrophy/interstitial fibrosis of 25%-50% and >50% versus <25% were associated with a 3.7-fold (95% CI, 2.6-5.1; P<0.001) and 15.1-fold (95% CI, 9.5-24.2; P<0.001) higher risk of the combined event, respectively. Endocapillary proliferation, glomerular crescents, and necrosis were not significant.

CONCLUSIONS

Retrospective study; the therapeutic interventions were miscellaneous.

CONCLUSIONS

We confirmed the associations of mesangial hypercellularity and tubular atrophy/interstitial fibrosis with kidney disease outcomes.

Events of cell proliferation have been assessed by thymidine labeling index (TLI) in morphologically normal breast lobules from women of reproductive age. TLI is higher during the second half of the menstrual cycle both in women with natural menstrual cycles and in those with artificial cycles due to oral contraceptive (OC) use. TLI varies between 0.04% and 5.7% and declines with increasing age. There is no difference in mean TLI between OC users and nonusers if correction is made for the difference in age distribution between these two groups. Immunohistochemical detection of immunoglobulin A (IgA), secretory component (SC), and alpha-lactalbumin shows more frequent positive staining during artificial than natural menstrual cycles, and positive staining tends to be associated with higher TLI values. No significant variation in staining with cycle phase is detected in natural or artificial cycles. These results show that breast tissue does not resemble endometrium in the relationships that exist between proliferation, secretion, and menstrual cycle phase.

Recently, an acute phase (AP) protein response has been reported in major depression. In order to examine whether an AP response occurs in other psychiatric disorders, such as schizophrenia and mania, the authors measured plasma AP reactants, such as haptoglobin (Hp), immunoglobulin G (IgG), IgM, fibrinogen (Fb), complement component 3 (C3C), C4, alpha 1-antitrypsin (alpha 1 AT), alpha 1-acid-glycoprotein (alpha 1S) and hemopexin (Hpx), in 27 schizophrenic, 23 manic, 29 major depressed and 21 normal subjects. Schizophrenic patients had significantly higher plasma Hp, Fb, C3C, C4, alpha 1S and Hpx than normal controls. Manic subjects showed significantly higher plasma Hp, Fb, alpha 1S and Hpx than normal volunteers. Depressed subjects had significantly higher plasma Hp, Fb, C3C, C4 and alpha 1S than normal controls. Overall, the above disorders in AP reactants were more pronounced in schizophrenic than in depressed subjects. No significant differences in the above AP reactants could be found between normal volunteers, and schizophrenic, manic or depressed patients who underwent chronic treatment with psychotropic drugs. Plasma Hp, Fb, C3C, C4, alpha 1S, and Hpx were significantly higher in schizophrenic, manic and depressed patients who were non-medicated than in those who were treated with antidepressants, antipsychotics or lithium. The results suggest that not only major depression but also schizophrenia and mania are accompanied by an AP response, and that the latter may be suppressed by (sub)chronic treatment with psychotropic drugs.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Here, we identify a cDNA encoding a novel mucin protein, shown previously to be up-regulated in IgAN patients, from a human kidney cDNA library. This protein contains a mucin tandem repeat of 19 amino acids consisting of many threonine, serine, and proline residues and likely to be extensively O-glycosylated; thus, this gene was classified in the mucin family and named MUC20. The human MUC20 gene contains at least four exons and is localized close to MUC4 on chromosome 3q29. We found variations in repeat numbers in the mucin tandem domain, suggesting polymorphism of this region. Northern blot and reverse transcription-PCR analyses revealed that human MUC20 mRNA was expressed most highly in kidney and moderately in placenta, colon, lung, prostate, and liver. Immunohistochemical analysis of human kidney revealed that MUC20 protein was localized in the proximal tubules. Immunoblotting analysis of MUC20 proteins produced in Madin-Darby canine kidney and HEK293 cells indicated the localization of MUC20 protein in a membrane fraction and extensive posttranslational modification. Immunoelectron microscopy of MUC20-producing Madin-Darby canine kidney cells demonstrated that MUC20 protein was localized on the plasma membrane. Expression of MUC20 mRNA in a human kidney cell line was up-regulated by tumor necrosis factor-alpha, phorbol 12-myristate 13-acetate, or lipopolysaccharide. Two species of MUC20 mRNA (hMUC20-L and hMUC20-S), resulting from alternative transcription, were identified in human tissue, whereas only one variant was observed in mouse tissues. Mouse MUC20 mRNA was expressed in the epithelial cells of proximal tubules, and the expression increased dramatically with the progression of lupus nephritis in the kidney of MRL/MpJ-lpr/lpr mice. Moreover, the expression of mouse MUC20 was augmented in renal tissues acutely injured by cisplatin or unilateral ureteral obstruction. These characteristics suggest that the production of MUC20 is correlated with development and progression of IgAN and other renal injuries.

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.

Currently, little is known about the function of arginine in the homeostasis of the intestinal immune system. This study was conducted to test the hypothesis that dietary arginine supplementation may alter intestinal microbiota and innate immunity in mice. Mice were fed a basal diet (containing 0.93% l-arginine; grams per gram) or the basal diet supplemented with 0.5% l-arginine for 14 d. We studied the composition of intestinal microbiota, the activation of innate immunity, and the expression of toll-like receptors (Tlrs), proinflammatory cytokines, and antimicrobials in the jejunum, ileum, or colon of mice. Signal transduction pathway activation in the jejunum and ileum, including TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide-3 kinase (PI3K)/PI3K-protein kinase B (Akt), was analyzed by Western blotting. Quantitative polymerase chain reaction analysis revealed that arginine supplementation induced (P < 0.05) a shift in the Firmicutes-to-Bacteroidetes ratio to favor Bacteroidetes in the jejunum (0.33 ± 0.04 vs. 1.0 ± 0.22) and ileum (0.20 ± 0.08 vs. 1.0 ± 0.27) compared with the control group. This finding coincided with greater (P < 0.05) activation of the innate immune system, including TLR signaling, as well as expression of proinflammatory cytokines, ​secretory immunoglobulin A, mucins, and Paneth antimicrobials in the jejunum and ileum. Finally, arginine supplementation reduced (P < 0.05) expression of the proteins for NF-κB, MAPK, and PI3K-Akt signaling pathways but activated (P < 0.05) p38 and c-Jun N-terminal protein kinase in the jejunum and the ileum, respectively. Collectively, dietary arginine supplementation of mice changes the intestinal microbiota, contributing to the activation of intestinal innate immunity through NF-κB, MAPK, and PI3K-phosphorylated Akt signaling pathways.

Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.