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Vinevaxtrade; (containing a mixture of strains of the fungus Trichoderma harzianum) is normally applied as soon as possible after grapevine pruning However using a range of six timings after pruning from immediate to 4 days later it was shown that the best penetration of the fungi into the wood was with treatment at approximately 5 h after pruning A novel method of measuring penetration and vigour of the growth of Trichoderma spp into grape wood is described A second trial using a range of concentrations of Vinevaxtrade; (at and below label rates) was applied as a general cover spray over dormant pruned vines The percentage incidence and vigour of penetration of the T harzianum into canes was concentration dependent and lower than the targeted handapplied treatment earlier in the season Therefore targeted timely applications of the product appear to be best for good wood penetration of T harzianum

The reactive nitrogen intermediate, nitric oxide (NO) is important in host defense against both NO-sensitive microorganisms and tumor cells. Macrophages are one of the chief inflammatory sources, especially when stimulated with the combination of LPS and interferonγ (IFNγ). It is not known, however, whether IFNγ-mediated augmentation of LPS-induced production of NO is the result of greater production by all cells or to the recruitment of more producer macrophages within a given population. This question was addressed, first, by stimulating mouse macrophages (either bone marrow culture-derived, inflammatory peritoneal or those of the cell line, RAW 264.7) with up to 10 U/ml IFNγ for as long as 24 h. Under these conditions, there was little or no production of NO and rare or no cells were immunocytochemically positive for the inducible form of nitric oxide synthase (iNOS), which catalyzes the production of NO. Populations similarly exposed to 1 ng/ml LPS were low producers of NO and contained somewhat more, but still only a few (< 15%), iNOS-positive cells. In contrast, as the concentration of IFNγ was increased (≥ 1 U/ml) in the presence of a constant amount of LPS (1 ng/ml), the principal effect was to increase both the production of NO and the number of iNOS-positive macrophages. The amount of iNOS expressed by some cells also appeared to be increased. Two important conclusions can be drawn from these findings: (1) there is heterogeneity in mouse macrophage populations with respect to the production of iNOS; and (2) increasing concentrations of IFNγ appear to augment LPS-induced secretion of NO by recruiting increasingly greater numbers of macrophages into the production of iNOS. Such results potentially provide important clues as to how IFNγ may be acting at the subcellular level to enhance iNOS synthesis.

Three antigenic fractions were isolated from collagenase-solubilised human glomerular basement membrane utilising affinity columns containing purified human anti-glomerular basement membrane autoantibodies. The three fractions were able to inhibit the binding of sera containing anti-glomerular basement membrane autoantibodies when measured by solid phase radio-immunoassay. Amino acid and carbohydrate analyses revealed differences in the composition of the three fractions and were consistent with them being derived from the non-collagenous components of glomerular basement membrane.

A solid phase radioimmunoassay for the identification of human urine has been developed, utilizing affinity-purified rabbit anti-human Tamm-Horsfall protein. This antiserum exhibited no cross-reactivity with human body fluids and showed some specificity for human urine over urine from several animal species. Conditions for the reliable identification of human urine in stains on cotton cloth have been determined. The effects of age and storage conditions on this and other materials are also described.

Administration of 6 mM silver nitrate in the drinking water of mice resulted in deposition within the glomerular basement membrane of silver granules which were detected on electron microscopy after 12 days. Larger aggregates were detected in the basement membrane and mesangium when silver ingestion was extended to 14 weeks. The silver deposits did not alter significantly over a period of 21 weeks after silver nitrate ingestion was stopped.

Systematic study of groups of NZB/W mice aged from 1-8 mth showed progressive development of antibodies to ssDNA and dsDNA, accompanied at 5 mth by the appearance of serum immune complexes as detected by Clq liquid phase binding and immune deposits in glomeruli. Cellular outgrowths in cultures of isolated glomeruli showed increased numbers of phagocytic cells as well as epithelial and mesangial cells, reaching a maximum in mice aged 7 mth. These numbers exceeded those found in control CBA mice and, while consistent with the hypothesis that phagocytic cells are involved in the processing of immune complexes lodged in the glomeruli, also reflected the hypercellularity of the glomerular lesions.

A method has been developed for the detection of C1q binding immune complexes in serum in which microculture plates are used as the solid-phase matrix for adsorption of C1q. This micromethod used only one-tenth of the amount of both C1q and [ 125 I]anti-human immunoglobulin per test and enabled 7 times as many samples to be tested in triplicate in comparison with the number performed in duplicate by the standard tube assay. 125 I-labelled C1q studies showed that adsorption varied with the brand of microplate used, some types of plate binding up to 66% of the labelled material. This is considerably more than that bound by the polystyrene tubes generally used for this assay. The increased capacity of the method allowed the binding to plates coated with the same batch of C1q to be assessed at various times after storage for 8–10 weeks at both 4°C and −70°C. A marked decrease in binding to C1q of aggregates of IgG was observed on storage for longer periods. Results with different batches of aggregated IgG which had been ultracentrifuged suggested that this may be used as an effective standard, stable on storage at −70°C. A comparison with the standard tube method of aggregated IgG, normal control sera and sera from patients with SLE showed that the micromethod adaptation of the C1q solid-phase binding radioimmunoassay is more economical and easier to perform and does not impair accuracy or standardization.

A mesangiopathic glomerulonephritis with prominent mesangial immunoglobulin deposits was induced in mice by single or multiple intraperitoneal injections of bovine serum albumin. The silver-labelled basement membrane in mice which had ingested 6mM silver nitrate in drinking water proved a useful marker indetermining that the immune deposits were on the intracapillary aspect of the basement membrane.

Glomerular cultures from mice have been established and the various cells associated with them observed microscopically by phase contrast time-lapse cinematography, after histological staining and ultrastructurally with the transmission and scanning electron microscope. The most common cell types were large 80–200 µm diameter irregular polyhedral cells appearing as flattened plate-like structures morphologically, possibly of epithelial origin. Smaller 70–100 µm diameter fusiform cells possibly derived from either endothelium or mesangium were also often seen. A third cell type, small 10–20 µm diameter circular, motile and actively phagocytic was especially noticeable in cultures of hybrid New Zealand Black/New Zealand White mice but was not present in appreciable numbers in control cultures from strains without renal disease. This third cell type appeared to have Fc receptors, stained positively with non-specific esterase and by indirect immunofluorescence with specific rabbit anti-mouse macrophage serum and had the morphological appearance of a macrophage by scanning and transmission electron microscopy. It is concluded that cells of macrophage origin are associated with the immune complex glomerulonephritis in these hybrid mice.

A simple, economical, macrophage migration inhibition assay enabling multiple tests to be performed using microculture plates has been developed. The system confers many advantages over the standard in vitro assay of macrophage migration inhibition factor. It requires only 5 X 10(5) macrophages for each assay, small volumes (100 microliter) of test factors are consumed, 96 tests in each culture plate are easily assessed microscopically and the technique is easily and reproducibly executed. An adherent monolayer of activated fresh mouse peritoneal exudate cells (PEC) is established in each microculture well. A 'starting line' is scored across the monolayer by the application of a trimmed razor blade and the cells are dislodged and aspirated from one side of the line. Test factors from cultures of stimulated lymphocytes were added to the 'wounded' monolayer and the migration of macrophages from test and control cultures was measured after staining. The cells across the line were counted after incubation. Miration was shown to be time and temperature dependent, markedly stimulated by colchicine and appeared to associate with tuberculin antigen (PPD) skin testing and lymphocyte transformation in human subjects.

1. Binding between the radiolabeled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm–Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis. 2. The diuretic action and binding to urinary Tamm–Horsfall glycoprotein of the radiolabeled diuretics in vivo, after intravenous administration, were examined in rabbits. 3. In vitro, all three radiolabeled diuretics bound strongly to human serum albumin, but not to Tamm–Horsfall glycoprotein. 4. Radiolabeled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm–Horsfall glycoprotein was seen in vivo. 5. Binding to Tamm–Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.

The soluble material produced from a 96-hour digest of sonicated human glomerular basement membranes with purified collagenase was shown to contain at least 12 components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with molecular weights ranging from 20×103 to greater than 200 ×103 daltons. Five of these components produced antigenic peaks after two-dimensional immunoelectrophoresis into agar plates containing rabbit antiserum to the solubilised glomerular basement membrane. Two groups of antigenic components were demonstrated in these gels by two-dimensional immunoelectrophoresis autoradiographic techniques utilising 125I-labelled collagenase-digested human glomerular basement membranes reacted with antibodies eluted at acid pH from the kidney of the Goodpasture’s patient. This acid eluate was shown to contain contaminants of α1-antitrypsin and albumin co-eluting with the antibodies bound to the glomerular basement membrane. After removal of these contaminants, the antibodies were linked to cyanogen bromide-activated Sepharose and used to affinity purify four antigenic fractions from the collagenase-digested glomerular basement membrane. These fractions were eluted by 0.2 Mglycine pH 2.8 and with 0.5 Mammonium thiocyanate and had molecular weights of 22–25,43–45,65–70 and 205 ×103 daltons. The smaller molecular weight components were shown to be common to both included and excluded fractions obtained by Ultragel AcA 44 chromatography of the digested material in 10 mM phosphate pH 8. This suggests that the larger molecular weight component would be an aggregate of a smaller component. Preliminary carbohydrate and amino acid analyses indicated that the different elution procedures elicited antigens with different chemical characteristics.

Thirty separate preparations of human Tamm-Horsfall urinary glycoprotein (THP) prepared from pooled urine of normal individuals were assessed for the capacity to stimulate in vitro blast transformation with normal human peripheral blood lymphocytes. Detailed studies were performed on some of these preparations by various biochemical, ultrastructural and in vitro techniques. Heterogeneity amongst the sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profiles of several electrophoresis (2D-IEP). Gas liquid chromatographic analysis (GLC) revealed significant amounts of glucose in two of these samples. Ultrastructural studies using direct staining or platinum carbon shadowed specimens confirmed the fibrillar polymeric nature of soluble THP in vitro studies with human peripheral blood lymphocyte populations showed THP to have mitogenic properties towards B cells associated with the production of intracytoplasmic immunoglobulin. THP also stimulated the migration of mouse peritoneal exudate macrophages in a micro macrophages migration assay.

Erythrocytes, serum, and blood monocytes from patients with erythrocyte autoantibodies were examined by in vitro serologic and cell culture procedures in order to evaluate factors modulating patient mononuclear phagocyte activity. Monocyte recognition of autoantibody-coated erythrocytes was influenced both by antibody titer score and by the monocyte source. The results suggest that patients with low serum autoantibody titer scores and normal monocyte behavior are less likely to destroy autologous or normal donor erythrocytes than are patients with high-serum antibody titer scores or enhanced monocyte activity. We conclude that patient, rather than normal donor monocytes, must be used in cellular assays designed to predict the fate of erythrocytes transfused into patients with erythrocyte autoantibodies.

Tamm-Horsfall protein was detected within the capsular space by immunofluorescence in 7 of 72 consecutive patients on whom renal immunopathological studies were performed. Three patients showed prominent aggregates or crescentic collections affecting 30-50% glomeruli; the remaining four patients showed smaller aggregates between lobules. All patients showed pathological evidence of tubulointerstitial disease. It is suggested that Tamm-Horsfall protein in the capsular space is a sign of intratubular urinary backflow and that Tamm-Horsfall antiserum is a useful addition to the reagents used in the immunofluorescence study of renal biopsies.

Three assay systems-EAIgG rosette formation, 51Cr release, and erythrophagocytosis-were used to quantitate interaction between antibody-coated human erythrocytes and normal blood monocytes. The three methods were compared in terms of time requirements and sensitivity. Erythrophagocytosis required more time to perform (2 hours) than did rosette tests (30 minutes) but less than minimum 51Cr release assays (5.5 hours). Erythrophagocytosis was 20-fold more sensitive than either of the other two procedures. Results obtained with purified IgG anti-D and with antibodies induced by transfusion or pregnancy were similar.

Tamm-Horsfall protein (THP), which is a prominent component of urinary casts, is a glycoprotein with a molecular weight of 7×10 6 composed of subunits of 10 5 daltons and contains approximately 23% carbohydrate. Electron microscopic studies after adsorption with horseradish peroxidase reveal a zig-zag pattern of periodicity 21 nm, and a flattened helical structure is proposed. Biologically some THP preparations stimulate cell division in normal human lymphocytes. Recent work suggests this may reflect the presence of glucose in stimulatory preparations. This mitogenic property must be taken into account in evaluating cellular autoimmunity against THP in patients with renal disease. As THP is secreted by cells in the ascending limb of the loop of Henle and distal convoluted tubule, its detection in Bowman's space may prove to be a useful sign of intrarenal urinary reflux.