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The effects of a high-pressure treatment on the IgE-specific binding activity and structural changes to bovine gamma globulin (BGG), a beef allergen, were investigated. The allergenicity of pressure-treated BGG was also evaluated. We found that the IgE-specific binding activity and allergenicity of BGG were decreased by the high-pressure treatment. Almost no significant change in secondary structure was apparent for pressurized BGG, but a change in the tertiary structure was detected. The decreased IgE-specific binding activity and allergenicity of pressurized BGG were probably due to changes in the tertiary structure caused by pressurization. The results of this study suggest that a high-pressure treatment would be an effective food-processing technique to reduce the allergenicity of BGG.

This paper describes general methods to eliminate or decrease the allergenicity of food protein, and also describes high pressure-induced elimination of the allergenicity of food protein, especially meat protein.The effect of high pressure treatment on the elimination of bovine serum albumin (BSA, the major beef allergen) allergenicity was evaluated on the basis of histamine release from human basophilic KU812F cells sensitized with sera from allergic patients, and the structural changes of BSA responsible for reducing allergenicity was estimated. BSA pressurized at pressures ranging from 300 to 600 MPa reduced histamine release from the cells sensitized with A5 serum with significance. The reducing effect of high-pressure treatment gradually increased with the increase of pressure applied to BSA. The pressure-induced structural changes of BSA were estimated by fluorescence spectra, circular dichroism (CD) spectra (the content of secondary structure), the amount of surface sulfhydryl (SH) group, and the surface aromatic hydrophobicity. The blue shift and decrease of the fluorescence of BSA gradually progressed with the increase of pressure applied. But no significant effect of pressure on CD spectra was observed. Pressure-treated BSA showed the maximum increase in the amount of SH group by pressure treatment at 100 MPa, and the aromatic hydrophobicity gradually decreased with the increase of high pressure applied. These results indicated that high pressure treatment induced the tertiary structural changes of BSA, but no effect on the secondary structure. We concluded that the pressure-induced elimination of BSA allergenicity seemed to be related to the tertiary structural change of BSA.

The sera of bovine gamma globulin (BGG) positive beef allergic patients were used in this study in order to investigate changes in IgE-specific binding activity with regard to beef extract altered by heat or high-pressure treatment. In inhibition-ELISA, the sample treated at 60 degrees C did not show any significant changes in the antigenicity of BGG, but the sample treated at 100 degrees C showed a decrease of the antigenicity. In the case of the treatment with heating at 100 degrees C, heat-coagulation occurred in the beef extract. The resulting supernatant and precipitate of the sample by centrifugation were analyzed by immunoblotting. Only the fraction of precipitate showed a specific binding activity with the sera. Based on this result, it was speculated that the persistent antigenicity found even after the treatment at 100 degrees C in inhibition-ELISA remained principally in the heat-coagulated fraction, which indicated the importance of the method of handling the heat-coagulation in heat treatment. High-pressure treatments (200 MPa-600 MPa) of beef extract did not show any significant changes in the binding with the sera.

SUMMARY: We have previously demonstrated the presence of detached podocytes in the urine in various glomerular diseases. In this study, we investigated the pathological significance of detachment of urinary podocytes (u-podocytes) in childhood IgA nephropathy. We investigated 28 children with IgA nephropathy. U-podocytes were detected in the urinary sediment by immunofluorescence. Renal pathological changes and urinalysis, including the u-podocyte test, were analysed. The expression of glomerular basement membrane (GBM) components, integrins and cytoskeleton components on urinary podocytes was investigated using dual immunofluorescence in 12 patients. The u-podocyte score paralleled the acute glomerular changes in renal biopsy samples. The infiltration of glomerular macrophages and polymorphonuclear leucocytes, and the number of urinary leucocytes, correlated highly with the u-podocyte score. Some of the u-podocytes expressed integrins and also components of the GBM. Detachment of podocytes reflects the presence of acute glomerular lesions. Infiltration of glomerular leucocytes is considered to be the cause of the detachment, partly because of the destruction of the GBM.

The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of approximately 200 kDa, approximately 67 kDa and approximately 60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the approximately 67 kDa component and the other had the approximately 200 kDa and approximately 60 kDa components. One of them (approximately 67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the approximately 200 kDa and approximately 60 kDa components were presumed to be glycoproteins. Bovine gamma (BGG:globulin M.W. approximately 160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the approximately 200 kDa and approximately 60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the approximately 200 kDa, approximately 67 kDa and approximately 60 kDa components in the beef extract had strong allergenicity: approximately 67 kDa was BSA, and approximately 200 kDa and approximately 60 kDa were presumably aggregated BGG and it's heavy chain, respectively.

Renal podocytes play an important role in glomerular filtration. We estimated podocyte injury by light microscopic examination of kidney specimens or by urinary excretion of substances related to podocytes. Fresh kidney sections from 69 patients and urinary sediments from 84 patients with various renal diseases were examined immunohistochemically using fluorescent labelling. Four kinds of monoclonal antibodies which recognize podocytes were used: (1) anti-podocalyxin (anti-PCX) antibody, (2) anti-C3b receptor (anti-C3bR) antibody, (3) anti-podocyte protein, 44 kilodalton, (anti-pp44) antibody, and (4) anti-alpha 3 integrin (anti-Int alpha3) antibody. Labelling of kidney sections by anti-C3bR (k-C3bR) was reduced in cases of severe glomerular injury, although there were no changes in k-PCX, k-pp44, or k-Int alpha3 labelling. PCX labelling of urinary sediments (u-PCX) was detected in nearly all cases of glomerulonephritis, u-C3bR was seen in some cases of glomerular injury, u-Int alpha3 was seen in only a small number of cases of severe glomerular injury, and u-pp44 was not detected in any urinary samples. u-PCX, u-C3bR, u-Int alpha3, and k-C3bR correlated with the degree of pathological change, the degree of proteinuria and hematuria. These results suggest that immunostaining kidney sections with anti-C3bR antibody and urinary sediments with anti-PCX, anti-C3bR, and anti-Int alpha3 antibodies might be useful in detecting podocyte injury.

The significance of the presence of podocytes in the urine was studied in various renal diseases in children. The podocytes were detected by immunofluorescence using monoclonal antibodies against the podocalyxin that is present on the surface of podocytes which serves as a glycocalyx. They were scored according to the numbers per partitioned area on cytospun urine sediments. Urine podocytes were absent in normal control, nonglomerular diseases such as urinary tract infection and nonglomerular hematuria, and glomerular, noninflammatory diseases such as minimal change nephrotic syndrome and membranous nephropathy. Conversely, the excretion of podocytes in the urine were detected in various glomerular, inflammatory diseases. A significantly higher level of the podocyte score was found in the acute state of glomerular diseases which was defined as within 6 months after disease onset. Positive correlations were obtained between the presence of urinary podocytes and the histological features of active extracapillary changes and mesangial proliferation. Urinary podocytes were examined monthly for 12 months in 7 cases with IgA nephropathy and 2 cases with Henoch-Schönlein purpura nephritis, and a consistently higher urinary podocyte score was observed in the patients with histological progression. The scoring of urinary podocytes was found to be useful clinically, as a diagnostic tool for glomerular or nonglomerular diseases, inflammatory or noninflammatory diseases, a marker for the estimation of the severity of active glomerular injury and also as a predictor of disease progression.