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Background: Cow's milk allergy (CMA) is the most frequently diagnosed food allergy in infancy. In general, patients have a good prognosis because the majority acquire tolerance within the first years. Interventions have been proposed to accelerate tolerance and reduce morbidity. Probiotic supplementation could be effective through modulation of the immune system., Objective: We sought to determine whether supplementation with a combination of probiotics (Lactobacillus casei CRL431 and Bifidobacterium lactis Bb-12) accelerates tolerance to cow's milk (CM) in infants with CMA., Methods: We performed a double-blind, randomized, placebo-controlled trial in 119 infants with CMA. Infants received CRL431 and Bb-12 supplemented to their standard treatment of extensively hydrolyzed formula for 12 months. Primary outcome was clinical tolerance to CM at 6 and 12 months of treatment. Furthermore, we analyzed T- and B-lymphocyte subsets (CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), and CD20(+)) in peripheral blood at randomization and at 12 months with flow cytometry and examined the presence of viable probiotic strains in fecal samples., Results: The cumulative percentage of tolerance to CM at 6 and 12 months was similar in both groups: 56 (77%) in the probiotics group versus 54 (81%) in the placebo group. Infants in the placebo group had higher percentages of CD3(+) and CD3(+)CD4(+) lymphocytes compared with those seen in probiotic-treated infants. Probiotic intake was confirmed because probiotics were isolated from feces more often in treated infants than in the placebo group., Conclusion: Supplementation of CRL431 and Bb-12 to extensively hydrolyzed formula does not accelerate CM tolerance in infants with CMA.

We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.

Passive Heymann nephritis (PHN) in the rat is induced by the administration of heterologous antibodies to renal tubular epithelium (RTE). The nephritogenic capacity of anti-RTE resides primarily in the antibody fraction directed against a 330-kD glycoprotein (gp330). However, monospecific anti-gp330 antibodies are less nephritogenic than anti-RTE antibodies. This discrepancy led us to study a possible synergizing role for different antibody specificities present within anti-RTE. The enzyme dipeptidyl peptidase type IV (DPP IV) is, like gp330, present in RTE as well as in the glomerulus. Anti-DPP IV antibodies have been shown to induce an acute, transient proteinuria. In this study, we investigated the nephritogenic effect of separate or simultaneous administration of heterologous anti-DPP IV and anti-gp330 antibodies. Injection of anti-DPP IV antibodies resulted in a short-lived glomerular binding, and in a dose-dependent polyuria, albuminuria or proteinuria. Binding of anti-gp330 antibodies was slower, but much more stable. Albuminuria could only be observed in the heterologous phase. Combined injection did not alter antibody-binding kinetics of either antibody nor the albuminuria induced by anti-gp330. However, in the presence of anti-gp330 antibodies, a lower dose of anti-DPP IV was capable to induce transient albuminuria as compared to anti-DPP IV alone. In conclusion, anti-DPP IV and anti-gp330 antibodies bind to different sites in the glomerulus with different kinetics. The presence of anti-gp330 deposits facilitated anti-DPP-IV-induced renal injury.

Background: In murine chronic graft-versus-host disease, an experimental model for lupus nephritis, autoantibodies against renal tubular epithelium can be found. Part of these antibodies are directed against a constituent of renal tubular epithelium, the enzyme Dipeptidyl peptidase IV (DPP IV). DPP IV is present on the cell membrane of glomerular epithelial and endothelial cells, and plays an important role in cell-extracellular matrix interactions. In mice and rats, administration of heterologous anti-DPP IV antibodies can induce proteinuria and podocyte effacement., Experimental Design: In this study, the glomerular DPP IV enzyme activity was investigated in the course of graft-versus-host disease in (C57BL/10 x DBA/2) F1 hybrids both by light- and electron microscopy using a DPP IV-specific substratum., Results: Light microscopical examination revealed an overall reduction of DPP IV activity and an altered distribution pattern in glomeruli as early as 4 weeks after induction of graft-versus-host disease. Immunofluorescence studies using anti-DPP IV antibodies showed actual redistribution, excluding antibody-mediated enzyme inactivation. Enzyme electron microscopy revealed an irregular deposition of reaction product characterized by a patchy, "moth eaten" appearance of the endothelial and epithelial membranes. This process occurred simultaneously with the development of albuminuria and preceded the effacement of epithelial foot processes. In control mice, DPP IV showed a continuous distribution along endothelial and podocyte membranes., Conclusions: In view of these findings we postulate that impairment of the function of DPP IV as a non integrin adhesion molecule may be one of the causative factors underlying the structural and functional lesions observed in this model for lupus nephritis.

In active Heymann nephritis, an experimental autoimmune disease in the rat, gp330 is regarded as the main antigenic target. Immunization with detergent-solubilized renal tubular epithelium (RTE-DOC) has been shown to be less nephritogenic than immunization with crude RTE. In this study immunization with either crude RTE or affinity-purified gp330 did, but immunization with RTE-DOC did not induce proteinuria. Both a possible aberrant subclass distribution of anti-gp330 autoantibodies and the involvement of additional nephritogenic autoantigens such as DPP IV (gp90) or laminin could be excluded. Circulating anti-gp330 autoantibody titers were significantly higher in RTE-DOC-immunized rats than in RTE-immunized animals. In contrast, significantly more antibodies were shown to bind in the glomeruli in the latter group. The time of onset of abnormal proteinuria was shown to be related to the recognition of a particular V8 protease-induced 250 kD fragment of gp330 in Western blots. This study shows that a particular fragment-specific subset of autoantibodies against gp330 is involved in the glomerular damage in Heymann nephritis.

We investigated the pathogenesis of glomerulonephritis in mice that had been infected with Trypanosoma brucei. Polyclonal B cell activation was evaluated, with special attention to the presence of autoantibodies, which are known to be involved in other models for glomerulonephritis. The BALB/c mice studied developed severe albuminuria. Light-microscopy of the kidneys showed increase of mesangial matrix and thickening of the glomerular capillary walls. Immunofluorescence studies showed immunoglobulins in a mixed linear-granular pattern along the glomerular capillary wall and in the mesangium. Trypanosomal antigens were not found in these aggregates. Electron-microscopy revealed mesangial, subendothelial, and subepithelial electron-dense deposits. During the course of this infection, significant levels of antibodies directed against known nephritogenic renal autoantigens, i.e. GBM, laminin, RTE, and gp330, were measured in serum and glomerular eluates. These findings led us to conclude that mice infected with T. brucei and treated with diminazene aceturate develop an autoimmune-mediated glomerulonephritis, characterized by mesangial, subendothelial, and subepithelial immune aggregates. This murine model seems to offer a useful tool for the study of immunological aspects of polyclonal B cell activation in infection-related glomerular disease.

The present studies dealt with the pathogenesis of renal involvement in murine chronic graft-versus-host disease, which is a model for human systemic lupus erythematosus. The disease was induced in (C57BL10xDBA/2)F1 hybrids by injection of DBA/2 lymphocytes. The animals developed systemic disease accompanied by deposition of autoantibodies in the glomeruli and a lupus type of nephritis. Antibodies were eluted from glomeruli isolated during various stages of the disease by magnetic extraction from iron-perfused kidneys. For assessment of the specificity of the antibodies, we used indirect immunofluorescence, an enzyme-linked immunosorbent assay, and immunoblotting. In glomeruli from week 4, autoantibodies were found to be directed against several antigens, among which were the glomerular basement membrane component laminin and the glomerular enzyme dipeptidyl peptidase IV, whereas week 8 glomeruli also showed antibodies directed against nuclear antigens. Both laminin and dipeptidyl peptidase IV are known nephritogenic antigens occurring in renal tubular epithelial brush border preparations. Antibodies eluted from isolated glomeruli of diseased animals bound in a granular pattern along the glomerular capillary wall after in vivo transfer. Anti-renal tubular epithelial antibodies in the sera of diseased animals were affinity purified and injected into naive mice, which induced immune complex glomerulonephritis and proteinuria, thus confirming the nephritogenic role of these autoantibodies in this model.

This study analyses the expression by rat lymphocytes of six renal brush border (BB) antigens defined by a set of monoclonal antibodies and demonstrates that a 90 kD protein (gp 90), synthesized by BB and glomeruli is found on the surface of T and B cells. The other antigens, including the 330 kD protein involved in Heymann's nephritis--a model of epimembranous glomerulonephritis--are not detectable on the surface of lymphocytes. Immunochemical studies indicate that gp 90 and the related protein immunoprecipitated from thymocyte membranes co-migrate in SDS-PAGE. Quantitative binding analysis shows that the number of antigenic sites is in the same order of magnitude on thymocytes, spleen and bone-marrow lymphocytes as well as on two lines of pre-B and pre-T cells, but considerably lower on a highly differentiated helper T cell line.

Although membranous glomerulonephritis (MGN) has been long considered as a prototype of glomerulonephritis (GN) due to the deposition of circulating immune complexes (CIC), a growing body of evidence indicates that immune deposits can also be formed in situ and implicate antigens expressed by glomerular epithelial cells (GEC). Some of these antigens have recently been identified. The first one, gp330 - a 330-kd glycoprotein restricted to the coated pits of GEC and renal brush border (BB) - is responsible for Heymann nephritis, a rat model of MGN. However, it is absent in the human glomerulus and is therefore probably not involved in human cases of MGN, at least in those due to in situ formation of CIC. In addition, by raising monoclonal antibodies against rat and rabbit BB, we have isolated two other BB proteins also expressed on GEC. The latter, respectively identified as dipeptidyl peptidase IV (90 kd) and enkephalinase (85 kd), can serve as targets for the formation of short-lived immune deposits. Since they are also detected on human GEC, they might play a role in the pathogenesis of MGN in man.