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Biomedical subjects

J S Hunt

Publications and source records attributed to J S Hunt.

At least 127 records · Page 7Linked to original sources

Ia-positive macrophages bind and internalize viable lymphocytes in murine thymus.

Macrophages have been shown to be present in thymus throughout its development. In the present study monoclonal and polyclonal antimacrophage reagents were used to identify, quantitate, and determine the distribution of thymic macrophages. Those studies demonstrated that significant numbers of macrophages were evenly distributed throughout the cortex and medulla, and that macrophages account for most, if not all, Ia positivity in murine thymus. Suspensions of thymic cells prepared by enzyme digestion contained 2-4% macrophage antigen-positive cells, over 95% of which were I-Ak positive in double-labeling studies. Removal of lymphocytes and macrophages left only epithelial cells and those failed to label for Ia. Subsequent to mild enzymatic digestion, up to 80% of the thymic macrophages were in the form of lymphocyte/macrophage rosettes. Morphologic evaluation of the thymocyte rosettes revealed that some of the macrophages contained internalized lymphocytes. The proportion of macrophages with internalized lymphocytes generally was less than 10%, but during the first 4 weeks of life values often approached 30%. Nurse cells, which were shown through double labeling to express both Ia and macrophage-associated antigen, were included in the population of rosetted cells which had internalized lymphocytes. The results demonstrated that there is a high level of interaction between lymphocytes and Ia-positive macrophages in the thymus which is greatest during the immediate postnatal period.

Age Factors↗

Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

Adsorption↗

Localization and characterization of macrophages in murine uterus.

Macrophages are known to be present in the murine uterus and are known to be among those cells comprising the uterine decidual response to pregnancy. The extent of macrophage involvement in the decidual response has not been documented, and there are unresolved questions regarding expression of markers normally associated with macrophages on cells within the decidua. Using tissue immunohistology, macrophages were identified in virgin and pregnant murine uteri. A significant increase in macrophage density was noted during all stages of pregnancy. When uteri from virgin and pregnant mice were enzymatically digested, 10% of uterine cells from virgin and 22% from pregnant mice expressed macrophage markers (binding of rabbit antimouse macrophage serum, Fc gamma receptor expression). Double labeling immunofluorescence demonstrated that the two markers were associated with the same cells. Those results were confirmed in "panning" experiments using a monoclonal antimouse macrophage reagent. In cell suspensions from pregnant murine (C3H/HeN) uteri, 50% of cells exhibiting macrophage markers were I-Ak positive, and macrophages accounted for nearly all I-Ak positive cells in uterine cell suspensions. The results of this study demonstrate that the murine decidual response to pregnancy includes an increase in Fc gamma receptor-bearing macrophages and that a relatively high percentage of those macrophages are Ia positive.

Animals↗

A system for accurate immunolocalization of Tamm-Horsfall protein in renal biopsies.

The distribution of Tamm-Horsfall protein (THP) was studied in the human kidney using formalin-fixed, paraffin-embedded sections with a monoclonal antibody specific for human THP applied in conjunction with a modified dinitrophenyl hapten sandwich staining (DHSS) procedure. The method was found to be highly sensitive producing very strong specific staining at antibody dilutions up to 1 in 64 000. Counterstaining with Haematoxylin and Eosin was possible without significant masking of the specific staining. This provided excellent structural definition of the background tissue which proved especially important in the study of THP localization in randomly oriented biopsy material. THP was found in all segments of the thick ascending limbs of loops of Henle, most segments of distal convoluted tubules and occasionally in distended collecting ducts and in the glomerular capsular space. Maculae densae did not contain THP. The combination of the modified DHSS procedure and the human THP specific antibody represents a highly sensitive and reliable method for specific staining of the THP in kidney sections.

Antibodies, Monoclonal↗

Relationship of glomerular phagocytic cells to immune complexes and DNA autoantibodies in NZB/W mice.

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.

Animals↗

Degradation of glomerular basement membrane by human neutrophils in vitro.

The glomerular basement membrane is susceptible to immunologic injury when immune complexes or anti-basement-membrane antibodies become lodged in its network. We have studied the digestion of glomerular basement membrane prepared from normal human kidney by isolated neutrophils. In the absence of immunoglobulin aggregates or immune complexes, there was little evidence of neutrophil adherence to the membrane, of release of lysosomal enzymes, or of digestion. However, when the basement membrane contained immunoglobin G (IgG) aggregates generated in situ by heating the membrane impregnated with IgG to 63 degrees C, electron micrographs showed neutrophils adherent to the basement-membrane surface and phagocytosis of smaller fragments. Lysosomal enzymes were detectable in the extracellular medium, and measurements of either total protein or hydroxyproline solubilized showed digestion of 80 micrograms basement membrane/h per 10(7) cells. Hydroxyproline solubilization was almost totally inhibited by phenylmethylsulphonyl fluoride, indicating that the neutrophil serine proteinases, elastase and cathepsin G are responsible for degradation. These findings provide direct evidence for the digestion of extracellular matrix by neutrophils stimulated in situ by deposited immune complexes as a contributor to inflammatory tissue damage.

Antigen-Antibody Complex↗

Macrophages in murine uterus are immunosuppressive.

The mechanisms by which the fetal allograft is protected from a maternal anti-fetal immune response are not understood. This study was designed to examine the possibility that tissues near the developing fetus contain immunoregulatory cells and to begin the process of identification of those cells. Dispersed uterine cell suspensions from pregnant Swiss/Webster mice consistently inhibited the responses of normal murine spleen cells to the polyclonal mitogen phytohemagglutinin (PHA). These suspensions contained few lymphocytes (mean 1%), but abundant macrophages (mean 28%), identified by morphology and Fc gamma-receptor expression. Depletion of Fc gamma-receptor-positive cells restored spleen cell (SC) responses to PHA to near normal levels and partial depletion of adherent cells provided varying degrees of relief of the observed suppression. Adherent cells (greater than 95% macrophages) recovered from plastic surfaces were highly immunosuppressive. Suppressor cells appeared to interfere with both early and late stages of spleen cell proliferative responses. The results suggest that cells with some characteristics of macrophages within tissues near the maternal-fetal interface may create a local environment prohibitive to maternal lymphocyte proliferation.

Animals↗

Evaluation of human chorionic trophoblast cells and placental macrophages as stimulators of maternal lymphocyte proliferation in vitro.

Dispersed cell suspensions of human chorion membrane and placentae were obtained by enzyme digestion and the cells examined for HLA expression and for the ability to stimulate immune cell proliferation in vitro. Chorion cells with the characteristics of trophoblast were HLA-A, B, C and Ia negative following tissue digestion whereas placental cells, primarily Fc gamma R positive macrophages, were HLA-A, B, C positive and were frequently Ia positive. When chorion and placental cell suspensions were used as stimulator cells in one-way mixed cell cultures (MCC) with maternal mononuclear leukocytes (MNL) as responder cells, chorion cells were not normally stimulatory (mean stimulation index (SI), 2.7) whereas placental cells usually were (mean SI, 11.5). No evidence for active suppression by chorion cells was obtained in a group of experiments designed to detect suppressive activity. The results support the concept of the trophoblast layer as an immunologically inert barrier between the mother and the fetus.

Chorion↗

Dose-response effects of tropicamide HCl.

Using double-masking procedures, the mydriatic and cycloplegic effects of 0.25, 0.5, 0.75, and 1.0% tropicamide were studied. All doses produced clinically useful mydriasis of at least 6-mm pupils. Using testing conditions of both normal (35 ft-c) and bright (150 ft-c) illumination, no mydriatic dose-response differences for the four concentrations were found. Cycloplegic effects were dose related. A single drop of either 0.75 or 1.0% tropicamide reduced the amplitude of accommodation to about 1.5 D, which is adequate for a cycloplegic refraction.

Adult↗

Immune complexes facilitate uptake of antigen from the gut.

Radiolabeled DNP-ovalbumin was injected with and without DNP-specific antibody into ligated gut segments of neonatal rats. The transfer of this labeled protein to mesenteric lymph nodes and binding to gut tissue was examined. When the antigen, DNP-ovalbumin, was present in the gut in an immune complex from a selective localization of antigen in the mesenteric lymph node was observed. These data suggest that immune complexes in milk could potentially modulate neonatal immunological development.

Animals↗

Mouse glomerular culture.

A method is described for the culture of isolated intact mouse glomeruli. Cultures showed adherent glomeruli after 2 days and from 2-4 days a monolayer of cells began to develop around the points of adherence. The predominant cell (Type I) was 80-200 micrometer in diameter with cytoplasmic extensions, later becoming polyhedral and often multinucleated. Type II spindle cells, 70-100 micrometer in length, were less prominent in the earlier stages and later formed sheaves between Type I cells. Small phagocytic Type III cells, 10-20 micrometer in diameter, were not prominent in cultures from normal kidneys and tended to form small clusters on top of the Type I cell monolayer.

Animals↗

Phagocytic cells in glomerular cultures from NZB/W mice.

Comparative studies of glomerular cultures from NZB/W and normal control mice aged 2, 4, 6, 8, 10 and 12 months revealed a significant increase in the number of phagocytic cells in cultures from NZB/W mice at all ages. These cells on electron microscopy had the appearance of macrophages and on immunofluorescence showed Fc receptors and reacted with rabbit anti-mouse macrophage serum. The cells appear to be of blood monocyte origin and it is likely that they play a role in the pathogenesis in NZB/W mice of the glomerulopathy associated with clearance of immune complexes.

Animals↗

Characterization of human erythrocyte alloantibodies by IgG subclass and monocyte interaction.

Human blood group antibodies of 11 specificities were examined for ability to mediate interaction with normal blood monocytes, IgG subclass composition, and titer score. Most antibodies were normally non-complement-binding (except anti-JKa) and clinically significant (except anti-Lub). A simple and relatively rapid in-vitro assay for the visualization of sensitized erythrocyte-monocyte interaction is described. Capillary procedures were adapted for IgG subclass composition determinations. Standard serologic tests were used to assess antibody titer score. A relationship appeared to exist between increased antibody titer score and increased interaction of sensitized erythrocytes (RBC) with normal blood monocytes (MNL), insofar as the mean titer score for antibodies that did mediate significant interaction was 52, whereas titer scores for antibodies that did not averaged 25. One or more examples within all antibody specificity, mediated significant RBC-MNL interaction. No relationship was observed between IgG subclass composition and mediation of RBC-MNL interaction except that all antibodies that did mediate interaction contained IgG1 or IgG3. Characteristic patterns of IgG subclass composition were observed for many antibody specificity groups, although too few examples were tested for valid statistical analysis. This study identifies two features of common blood group antibody populations that are instrumental in mediating erythrocyte destruction by monocytes in vitro. Similar information may be of value in predicting the result of erythrocytes transfused to patients with antibodies of unknown clinical significance.

Cell Adhesion↗