By means of double fluorescence staining experiments, intracellular alpha-actinin was found to accumulate under caps and patches induced in several cells by a variety of ligands. This phenomenon was demonstrated in lymphocytes and lymphoma cells treated with anti-H-2 sera; spleen lymphocytes treated with concanavalin A or anti-immunoglobulin antibodies, and VSV-infected mouse fibroblast line MC57 treated with antiserum against viral antigens. It occurred during both rapid and slow capping processes, and could be obtained by either direct or indirect ligand-induced redistribution. These observations were carried out on whole cells. For other cytoskeletal proteins such as filamin, tropomyosin and myosin, a similar accumulation under caps was not readily apparent using whole cell mounts, although earlier experiments with frozen-sectioned cells had shown such an enrichment of myosin (as well as actin). The enrichment of alpha-actinin under the clustered surface molecules was already apparent in early stages (patching) of the capping process, with or without 10 mM sodium azide present. Prolonged incubation of the cells with the different ligands resulted in endocytosis of the ligand-receptor complex. alpha-Actinin was not associated with the inernalized complex, however, suggesting that it may dissociate from the patched or capped surface structures at some stage during endocytosis.

Asthma is a chronic life-threatening disease of worldwide importance. Although allergic asthma and related atopic conditions correlate strongly with immune sensitization to house dust mites, it is unclear why antigens from mites provoke such powerful allergic immune responses. We have characterized the protease activity of Der p I, the group I protease allergen of the house dust mite Dermatophagoides pteronyssinus, and here report that it cleaves the low-affinity immunoglobulin (Ig) E Fc receptor (CD23) from the surface of human B lymphocytes. Der p I selectively cleaves CD23 and has no effect on the expression of any other B cell surface molecules tested. We speculate that this loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating an important feedback inhibitory mechanism that normally limits IgE synthesis. Furthermore, since soluble CD23 is reported to promote IgE production, fragments of CD23 released by Der p I may directly enhance the synthesis of IgE. alpha 1-Antiprotease, a pulmonary antiprotease, is also shown to inhibit the cleavage of CD23 by Der p I. This may be significant in the etiopathogenesis of asthma, because other indoor pollutants associated with asthma are known to potently inhibit this antiprotease. These data suggest that the proteolytic activity of Der p I, the group I allergen of the house dust mite D. pteronyssinus, is mechanistically linked to the potent allergenicity of house dust mites. Furthermore, inhibition of Der p I by alpha 1-antiprotease suggests a mechanism by which confounding factors, such as tobacco smoke, may act as a risk factor for allergic asthma.

C57BL/6 mice bearing either a transplantable methylcholanthrene-induced sarcoma or Lewis lung adenocarcinoma were passively immunized every other day with a rabbit immunoglobulin fraction raised against murine cachectin/tumor necrosis factor-alpha. Mice bearing methylcholanthrene-induced sarcoma developed tumor-associated hypophagia that was attenuated by anticachectin immunoglobulin treatment. In the same tumor-bearing animals, anticachectin treatment also significantly reduced the extent of carcass protein and fat loss, and reduced tumor weight. Mice bearing Lewis lung adenocarcinoma did not develop significant anorexia or carcass lean tissue depletion as tumor growth progressed, but they lost carcass lipid. Treatment of Lewis lung adenocarcinoma bearing mice with anticachectin antibodies diminished the degree of carcass lipid depletion and prevented plasma hypertriglyceridemia. However, in both tumor models, anticachectin treatment did not affect either the development of anemia, hypoalbuminemia or the increase in serum amyloid P concentrations seen with increasing tumor burden. We conclude that an endogenous cachectin response, inhibitable by exogenously administered antibody, contributes to anorexia and to changes in body fat and protein metabolism in these tumor-bearing animals. Neutralizing endogenous cachectin production with antibodies offers the potential to reduce tissue wasting that is frequently associated with neoplastic disease, but it does not appear to affect all of the hematologic and acute phase responses in these murine tumor models.

The contribution of secretory component to the stability of secretory IgA against proteolysis has been studied by a new approach, i.e., by comparing the proteolytic degradation of the complexes formed in vitro between these proteins and secretory component. The results show that attachment of secretory components to the dimeric backbone of the secretory IgA molecule is accompanied by a significantly increased resistance of this backbone against digestion by both trypsin and pepsin. This protective effect may be a physiologic function of secretory component or may be due merely to unspecific blocking by secretory component of one or more sensitive peptide bonds in the IgA backbone.

Comparisons of nucleotide sequences of several pseudogenes described to date, including alpha- and beta-globin and immunoglobulin kappa-type variable domain pseudogenes, with those of functional counterparts revealed that pseudogenes accumulate mutations at an extremely high rate uniformly over their entirety. It is remarkable that the evolutionary rate exceeds the rate of changes between synonymous codons, the highest known rate, in functional genes. Because no pseudogenes appear to function, this result strongly supports the neutral theory. In addition this result apparently indicates the presence of selective pressure against changes between synonymous codons in functional genes. Close examinations of codon utilization patterns in pseudogenes and functional genes revealed a significant correlation between the rate of changes at synonymous codon sites and the strength of bias in code word usage. This implies that even synonymous codon changes are not completely free from selective pressure but are constrained in part, although presumably weakly, depending on the degree of bias in code word usage. We also reexamined alignment between mouse beta h3 (pseudogene) and beta maj sequences and found a unique structure of the beta h3 that is homologous in sequence to the beta maj gene overall but contains a long deletion (about 150 base pairs) in the middle of the gene.

Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.

The characteristics and functions of microbial IgA proteases are reviewed. These enzymes represent a structurally heterogeneous group of proteins that are secreted into the extracellular environment by bacteria capable of causing human disease. The IgA proteases, which vary in their requirements for metal ions, are neutral endopeptidases whose role in the infectious process is not known but whose pronounced substrate specificity for human proteins of the IgA1 subclass has repeatedly been demonstrated. As reagents, the IgA proteases are useful in cleaving IgA molecules to yield intact Fc alpha and Fab alpha fragments that will allow the study of the structure and function of the two large regions of IgA immunoglobulin proteins. The role, if any, of these enzymes in promoting infection by pathogenic members of the genera Neisseria, Hemophilus, and Streptococcus is not known, although the secretory immune system is primarily mediated by antibodies of the IgA isotype, among which are IgA1 subclass proteins, and these proteins are susceptible to cleavage by IgA protease. The determination of the role of these enzymes in the pathogenesis of human infection must await clearer understanding of antigenicity and antibody function at secretory sites and of the relative roles of the two subclasses of human IgA in immune defense.

CD31 (PECAM-1) is a member of the immunoglobulin gene superfamily (IgSF) and has an important role in a number of endothelial cell functions including angiogenesis, inflammation, integrin activation and cell-cell adhesion. CD31 has both homotypic and heterotypic adhesive properties and in common with other IgSF members contains multiple functional domains. Using chimaeric fusion proteins of CD31 and a panel of haematopoietic cell lines we show that CD31 can bind cells in a predominantly homotypic or heterotypic manner depending on the cell line used. Heterotypic binding was found to be cation and temperature dependent and enhanced by Mn2+: all features of integrin mediated binding. Using a panel of anti-CD31 and anti-integrin antibodies we show that alpha v beta 3 is a ligand for CD31 on the monocytic cell line U937. The specificity of the interaction between alpha v beta 3 and CD31 was further confirmed by solid phase binding assays and the use of alpha v beta 3 transfected cells which bound CD31 specifically. Furthermore, we have mapped the binding site for alpha v beta 3 to domains 1 and 2 of CD31. The interaction of CD31 with alpha v beta 3 may be important in many aspects of endothelial function including leukocyte-endothelial transmigration and angiogenesis.

A DNA region not associated with conventional immunoglobulin gene rearrangement is rearranged in many lymphoid tumors. This region, designated here as lymphoid rearranging (LyR) DNA, was cloned from plasmacytoma J558 in which it had recombined 5' to a constant (C) region of the alpha heavy (H) chain gene, C alpha, within a switch (S) region, S alpha, involved in the switching of CH genes. Sequence determination established that LyR DNA had recombined within a S alpha recombination unit. LyR DNA does not originate from the H chain locus, and discordance between LyR DNA and CH copy number in certain lines suggests that LyR DNA probably derives from another chromosome. LyR DNA rearrangement is a characteristic of tumors of mature B cells; it was detected in 24 of 28 plasmacytomas and B-cell lymphomas, usually as LyR-S alpha, but not in 11 Abelson retrovirus-induced lymphomas of B-cell precursors nor detectably in normal B cells. In contrast, rearrangement was observed in only 3 of 18 T-cell lymphomas, and none of seven nonlymphoid lines. Most tumor lines (49 of 52), whether lymphoid or not, contained a low level of polyadenylylated LyR transcript(s), but several new RNA species with differences in their 5' regions appeared in B-cell lines in which LyR DNA was rearranged, suggesting that rearrangement may activate a new promoter or mode of splicing. The results suggest that the LyR-S alpha rearrangement represents a translocation to chromosome 12 that alters expression of LyR-encoded genes; hence, it may have participated in lymphoid tumor oncogenesis.

Voltage-gated Na(+) channels (VGSCs), predominantly the 'neonatal' splice form of Na(v)1.5 (nNa(v)1.5), are upregulated in metastatic breast cancer (BCa) and potentiate metastatic cell behaviours. VGSCs comprise one pore-forming alpha subunit and one or more beta subunits. The latter modulate VGSC expression and gating, and can function as cell adhesion molecules of the immunoglobulin superfamily. The aims of this study were (1) to determine which beta subunits were expressed in weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 human BCa cells, and (2) to investigate the possible role of beta subunits in adhesion and migration. In both cell lines, the beta subunit mRNA expression profile was SCN1B (encoding beta1>>)SCN4B (encoding beta4>>SCN2B (encoding beta2); SCN3B (encoding beta3) was not detected. MCF-7 cells had much higher levels of all beta subunit mRNAs than MDA-MB-231 cells, and beta1 mRNA was the most abundant. Similarly, beta1 protein was strongly expressed in MCF-7 and barely detectable in MDA-MB-231 cells. In MCF-7 cells transfected with siRNA targeting beta1, adhesion was reduced by 35%, while migration was increased by 121%. The increase in migration was reversed by tetrodotoxin (TTX). In addition, levels of nNa(v)1.5 mRNA and protein were increased following beta1 down-regulation. Stable expression of beta1 in MDA-MB-231 cells increased functional VGSC activity, process length and adhesion, and reduced lateral motility and proliferation. We conclude that beta1 is a novel cell adhesion molecule in BCa cells and can control VGSC (nNa(v)1.5) expression and, concomitantly, cellular migration.

Membrane immunoglobulin M (mIgM) and mIgD are major B-lymphocyte antigen receptors, which function by internalizing antigens for processing and presentation to T cells and by transducing essential signals for proliferation and differentiation. Although ligation of mIgM or mIgD results in rapid activation of a phospholipase C and a tyrosine kinase(s), these receptors have cytoplasmic tails of only three amino acid residues (Lys-Val-Lys), which seem ill suited for direct physical coupling with cytoplasmic signal transduction structures. In this report, we identify the alpha, beta, and gamma components of the mIgM-associated phosphoprotein complex, which may play a role in signal transduction. Proteolytic peptide mapping demonstrated that the IgM-alpha chain differs from Ig-beta and Ig-gamma. The chains were purified, and amino-terminal sequencing revealed identity with two previously cloned B-cell-specific genes. One component, IgM-alpha, is a product of the mb-1 gene, and the two additional components, Ig-beta and Ig-gamma, are products of the B29 gene. Immunoblotting analysis using rabbit antibodies prepared against predicted peptide sequences of each gene product confirmed the identification of these mIgM-associated proteins. The deduced sequence indicates that these receptor subunits lack inherent protein kinase domains but include common tyrosine-containing sequence motifs, which are likely sites of induced tyrosine phosphorylation.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.

In addition to the assembled coding regions of immunoglobulin and T-cell receptor (TCR) genes, the V(D)J recombination reaction can in principle generate three types of by-products in normal developing lymphocytes: broken DNA molecules that terminate in a recombination signal sequence or a coding region (termed signal or coding end molecules, respectively) and DNA molecules containing fused recombination signal sequences (termed reciprocal products). Using a quantitative Southern blot analysis of the murine TCR alpha locus, we demonstrate that substantial amounts of signal end molecules and reciprocal products, but not coding end molecules, exist in thymocytes, while peripheral T cells contain substantial amounts of reciprocal products. At the 5' end of the J alpha locus, 20% of thymus DNA exists as signal end molecules. An additional 30 to 40% of the TCR alpha/delta locus exists as remarkably stable reciprocal products throughout T-cell development, with the consequence that the TCR C delta region is substantially retained in alpha beta committed T cells. The disappearance of the broken DNA molecules occurs in the same developmental transition as termination of expression of the recombination activating genes, RAG-1 and RAG-2. These findings raise important questions concerning the mechanism of V(D)J recombination and the maintenance of genome integrity during lymphoid development.

Adaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virus-infected secretory epithelial cells without causing tissue damage. Resistance against toxin-producing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to long-standing serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents.

The protozoan pathogen Giardia is an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controlling Giardia infection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls with Giardia muris or Giardia lamblia GS/M-83-H7. We found that IgA-deficient mice could not eradicate either G. muris or G. lamblia infection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clear G. muris infections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies cleared G. muris infection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to control G. muris infection, show that IgA-dependent host defenses are central for eradicating Giardia spp. Moreover, B-cell-dependent but IgA-independent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.

Secretory immunoglobulin A (IgA) antibodies (sIgA) directed against cholera toxin (CT) and surface components of Vibrio cholerae are associated with protection against cholera, but the relative importance of specific sIgAs in protection is unknown. A monoclonal IgA directed against the V. cholerae lipopolysaccharide (LPS), secreted into the intestines of neonatal mice bearing hybridoma tumors, was previously shown to provide protection against a lethal oral dose of 10(7) V. cholerae cells. We show here that a single oral dose of 5 to 50 micrograms of the monoclonal anti-LPS IgA, given within 2 h before V. cholerae challenge, protected neonatal mice against challenge. In contrast, an oral dose of 80 micrograms of monoclonal IgA directed against CT B subunit (CTB) failed to protect against V. cholerae challenge. A total of 80 micrograms of monoclonal anti-CTB IgA given orally protected neonatal mice from a lethal (5-micrograms) oral dose of CT. Secretion of the same anti-CTB IgA antibodies into the intestines of mice bearing IgA hybridoma backpack tumors, however, failed to protect against lethal oral doses of either CT (5 micrograms) or V. cholerae (10(7) cells). Furthermore, monoclonal anti-CTB IgA, either delivered orally or secreted onto mucosal surfaces in mice bearing hybridoma tumors, did not significantly enhance protection over that provided by oral anti-LPS IgA alone. These results demonstrate that anti-LPS sIgA is much more effective than anti-CT IgA in prevention of V. cholerae-induced diarrheal disease.

Anti-neutrophil cytoplasmic antibodies (ANCA) activate primed human polymorphonuclear neutrophils (PMN) in vitro, resulting in a respiratory burst and degranulation. In this study, the hypotheses that the initiation of this process requires engagement of the F(ab')2 portion of ANCA, and that crosslinking of ANCA target antigens is necessary to trigger superoxide (O2-) release, were explored. It is speculated that Fc gamma receptor engagement is a modulator of ANCA-mediated activation. Flow cytometry demonstrated that intact human ANCA immunoglobulin (Ig), their corresponding F(ab')2 and Fab fragments, as well as a murine monoclonal to human PR3 and its F(ab')2 fragment, bind to ANCA antigens on the surface of PMN primed with tumor necrosis factor (TNF) alpha. Intact Ig of patients with PR3-ANCA or with MPO-ANCA stimulate O2- release from TNF alpha-primed normal PMN (2.6 +/- 3.57 to 15.3 +/- 7.39 nmol O2-/2.5 x 10(6) PMN/30 min). Corresponding F(ab')2 fragments result in similar O2- production (10.2 +/- 4.34 to 36.9 nmol) in a dose-dependent manner. ANCA Fab fragments do not stimulate O2- generation until these fragments are crosslinked with F(ab')2 of goat anti-human Ig F(ab')2, or when fragments are biotinylated and crosslinked with avidin. In contrast with these human autoantibody data, a mouse monoclonal anti-human PR3 antibody (25.7 +/- 8.55 nmol O2-), but not its corresponding F(ab')2 fragment, activates TNF alpha-treated human PMN. When the Fc gamma IIa receptors were blocked, superoxide production was reduced by 33% using human PR3-ANCA Ig (P < 0.05). In conclusion, PMN activation by ANCA occurs when intact ANCA or ANCA F(ab')2 fragments crosslink target antigens on the neutrophil cell surface. ANCA F(ab') fragments result in PMN activation when crosslinked by secondary reagents.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.

Dendritic cells (DCs) play critical roles in immune responses and can be distinguished in two major subsets, myeloid and plasmacytoid DCs. Although the presence of DC in all peripheral organs, including the kidney, has been well documented, no accurate estimates of DC subsets in human kidneys have been reported. This study shows a detailed analysis of DC subsets in cryosections of human renal tissue. The cortex of normal kidneys contains at least two different HLA-DR(+) myeloid DC subtypes characterized by BDCA-1(+)DC-SIGN(+) and BDCA-1(+)DC-SIGN(-). The staining for DC-SIGN completely overlapped with CD68 in the renal interstitium. Unexpectedly, BDCA-2(+)DC-SIGN(-) plasmacytoid DCs are also abundantly present. Both subsets are located in the tubulo-interstitium often with a high frequency around, but rarely observed within glomeruli. Quantification of BDCA-1(+), DC-SIGN(+), and BDCA-2(+) cells in normal human renal tissue (pretransplant biopsy living donors; n=21) revealed that BDCA-1 is about four times as frequently present as BDCA-2. A preliminary cross-sectional analysis of DC in diseased kidneys, including rejection and immunoglobulin A nephropathy, revealed that the number of DC as well as their anatomical distribution might change under pathophysiological conditions. In conclusion, we show that human kidneys contain a dense network of myeloid and plasmacytoid DCs and provide the tools for phenotyping and enumeration of these cells to better understand interindividual differences in immune responses.

BACKGROUND

A potential contribution of local activation of the renin-angiotensin system (RAS) to the pathogenesis of renal injury has been indicated by evidence for blood pressure-independent renoprotective effects of angiotensin II (AngII) receptor blockers (ARBs). The present study was performed to test the hypothesis that urinary angiotensinogen provides a specific index of intrarenal RAS status in patients with immunoglobulin A (IgA) nephropathy.

METHODS

This paper is a survey of urine specimens from three groups: healthy volunteers, patients with IgA nephropathy and patients with minor glomerular abnormality (MGA). Patients with hypertension, diabetes, reduced glomerular filtration rate and/or who were under any medication were excluded from this study. Urinary angiotensinogen levels were measured by a sandwich enzyme-linked immunosorbent assay system.

RESULTS

Urinary angiotensinogen levels were not different between healthy volunteers and patients with MGA. However, urinary angiotensinogen levels, renal tissue angiotensinogen expression and AngII immunoreactivity were significantly higher in patients with IgA nephropathy than in patients with MGA. Baseline urinary angiotensinogen levels were positively correlated with renal angiotensinogen gene expression and AngII immunoreactivity but not with plasma renin activity or the urinary protein excretion rate. In patients with IgA nephropathy, treatment with an ARB, valsartan (40 mg/day), significantly increased renal plasma flow and decreased filtration fraction, which were associated with reductions in urinary angiotensinogen levels.

CONCLUSIONS

These data indicate that urinary angiotensinogen is a powerful tool for determining intrarenal RAS status and associated renal derangement in patients with IgA nephropathy.

OBJECTIVE

We studied the coadjuvant capability of oral consumption of the breast-milk-isolated strain Lactobacillus fermentum (CECT5716) for an anti-influenza vaccine.

METHODS

A randomized, double-blinded, placebo-controlled human clinical trial including 50 volunteers (31 male and 19 female) was performed to address the immunologic effects of an intramuscular anti-influenza vaccine in adults (33.0 +/- 7.7 y old). Fifty percent of volunteers received an oral daily dose of methylcellulose (placebo) or probiotic bacteria (1 x 10(10) colony-forming units/d) 2 wk before vaccination and 2 wk after vaccination.

RESULTS

Two weeks after vaccination there was an increase in the proportion of natural killer cells in the probiotic group but not in the placebo group. The vaccination induced an increase in T-helper type 1 cytokine concentrations and in T-helper and T-cytotoxic proportions in both groups; however, the probiotic group showed a significant higher induction in some of these parameters. Regarding the humoral effects, induction of antibody response in the placebo group could not be detected. In the case of the probiotic group, a significant increase in antigen specific immunoglobulin A was detected. Although an increase in total immunoglobulin M was observed, changes in anti-influenza antigen specific immunoglobulin M were not observed. The incidence of an influenza-like illness during 5 mo after vaccination (October to February) was lower in the group consuming the probiotic bacteria.

CONCLUSIONS

Oral administration of the strain L. fermentum CECT5716 potentates the immunologic response of an anti-influenza vaccine and may provide enhanced systemic protection from infection by increasing the T-helper type 1 response and virus-neutralizing antibodies.

Efficiency of nutrient utilization is high in neonates with normal birth weights but is reduced in those with intrauterine growth restriction (IUGR). However, the underlying mechanisms are largely unknown. This study was conducted with the piglet model and proteomics technology to test the hypothesis that IUGR affects expression of key proteins that regulate growth and development of the small intestine, liver, and muscle, the major organs involved in the digestion, absorption, and metabolism of dietary nutrients. Jejunum, liver, and gastrocnemius muscle were obtained from IUGR and normal birth-weight piglets at birth for analysis of proteomes using the 2-dimensional-PAGE MS technology. The results indicate that IUGR decreased the levels of proteins that regulate immune function (immunoglobulins and annexin A1), oxidative defense (peroxiredoxin 1, transferrin, and zeta-crystallin), intermediary metabolism (creatine kinase, alcohol dehydrogenase, L-lactate dehydrogenase, prostaglandin F synthase, apolipoprotein AI, catecho O-methyltransferase, and phosphoglycerate kinase 1), protein synthesis (eukaryotic translation initiation factor-3), and tissue growth (beta-actin, desmin, and keratin 10) in a tissue-specific manner. In addition, IUGR increased the levels of proteins that are involved in proteolysis (proteasome alpha-5 and alpha-1 subunits), response to oxidative stress (scavenger-receptor protein and alpha-1 acid glycoprotein), and ATP hydrolysis (F1-ATPase). These novel findings suggest that cellular signaling defects, redox imbalance, reduced protein synthesis, and enhanced proteolysis may be the major mechanisms responsible for abnormal absorption and metabolism of nutrients, as well as reduced growth and impaired development of the small intestine, liver, and muscle in IUGR neonates.

We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.

BACKGROUND

The study aim was, for the first time, to conduct a multicenter randomized controlled trial to evaluate the effect of tonsillectomy in patients with IgA nephropathy (IgAN).

METHODS

Patients with biopsy-proven IgAN, proteinuria and low serum creatinine were randomly allocated to receive tonsillectomy combined with steroid pulses (Group A; n = 33) or steroid pulses alone (Group B; n = 39). The primary end points were urinary protein excretion and the disappearance of proteinuria and/or hematuria.

RESULTS

During 12 months from baseline, the percentage decrease in urinary protein excretion was significantly larger in Group A than that in Group B (P < 0.05). However, the frequency of the disappearance of proteinuria, hematuria, or both (clinical remission) at 12 months was not statistically different between the groups. Logistic regression analyses revealed the assigned treatment was a significant, independent factor contributing to the disappearance of proteinuria (odds ratio 2.98, 95% CI 1.01-8.83, P = 0.049), but did not identify an independent factor in achieving the disappearance of hematuria or clinical remission.

CONCLUSIONS

The results indicate tonsillectomy combined with steroid pulse therapy has no beneficial effect over steroid pulses alone to attenuate hematuria and to increase the incidence of clinical remission. Although the antiproteinuric effect was significantly greater in combined therapy, the difference was marginal, and its impact on the renal functional outcome remains to be clarified.