Expression of nestin in the podocytes of normal and diseased human kidneys

The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.     

Expression of human pendrin in diseased thyroids.

We examined pendrin expression in various diseased thyroid tissues by immunohistochemistry (IHC) using antiserum raised against human pendrin and by real-time quantitative RT-PCR. In normal thyroids the antiserum reacted with the apical membrane of follicular cells and its immunoreactivity was faint. In Graves' thyroids, the IHC expression of pendrin appeared in a pattern similar to that of normal thyroids but it was more extensive and stronger, especially in areas showing marked proliferation of follicular cells. The immunoreactivities of pendrin in nodular goiters varied from case to case. In follicular adenomas, pendrin was localized in the follicle-forming parts of the tumor but was negative in trabecular parts. Pendrin was negative in all follicular carcinomas, papillary carcinomas, and in one case of medullary carcinoma. In quantitive mRNA analysis, the relative values of pendrin mRNA were significantly low in papillary carcinoma (p<0.01), whereas the values in other diseased thyroids were not significantly different from those in normal thyroids. These results suggest that pendrin may play a role in thyroid hormone production as the apical porter of chloride/iodide and investigation of pendrin leads to a better understanding of functional aspects of the iodine transportation system in thyroid diseases.     

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells

We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.     

Tamm-Horsfall protein antibody in patients with end-stage kidney disease.

Circulating antibody to Tamm-Horstall protein (THP) was measured using a radioimmunoassay in forty-five patients on maintenance hemodialysis and compared to levels of antibody titers measured in sera from ten healthy controls. The etiology of the end-stage kidney disease in the patient population was polycystic kidney disease in thirteen, glomerulonephritis in fourteen, diabetic nephropathy in nine, interstial nephritis and chronic pyelonephritis in three each, multiple myeloma in two, and urinary tract obstruction in one. Four patients had significantly elevated titers of antibody to THP but shared no other unifying characteristics. The results also indicate that none of the groups studied had mean antibody titers significantly different from controls. Furthermore, no general trend was apparent between levels of antibody to THP and number of months on dialysis. Observations made during the study revealed that heparinized samples of blood had lower titers of antibody to THP than did non-heparinized samples from the same patient. This finding was repeated when other anti-coagulants, i.e., ethylenediaminetetraacetate (EDTA) and sodium citrate, were used. Titers returned toward normal when CaCl2 was added back to samples anticoagulated with EDTA and sodium citrate. This suggests that clotting factors, probably fibrinogen, interfered with the measurement of antibody titers. Therefore, only serum should be used in further investigations of THP antibody using this assay.     

Histochemistry of normal and diseased human lymph nodes.

Gomori's metal precipitate technique was used to demonstrate the phosphatase activity of the human cervical lymph node in health and disease, using four different phosphate esters (sodium beta-glycerophosphate and adenosine triphosphate at pH 9, riboflavin 5'-phosphate at pH 9.2 and 5'-monophosphoric acid at pH 8.3). In fetal lymph nodes, using 5'-monophosphoric acid, an outstanding positive activity was noticed in the lymphatic follicles. With the other three substrates there was either no nodular reaction or just a narrow rim of positive activity around the follicles, the internodular tissue being negative with all four substrates used. With chronic non-specific lymphadenitis the enzyme hydrolysing the three substrates (beta-glycerophosphate, riboflavin 5'-phosphate and adenosine triphosphate) began to make their appearance. It seems that with lymphadenitis, a qualitative change of the phosphatase activity takes place. A special characteristic pattern of phosphatase activity has been described in both 'early' and 'caseating' tuberculous lymphadenitis. In malignant lymphomas it was noticed that no activity was encountered with any of the four substrates in reticulum cell sarcoma. However, in lymphosarcoma a positive activity was obtained when either beta-glycerophosphate or adenosine triphosphate substrates was used, to the extent that one can depend upon this characteristic phosphatase activity in differentiating between reticulum cell sarcoma and lymphosarcoma. However, no enzymatic activity was obtained when the other two phosphate esters were used.     

Histochemical demonstration of alkaline phosphatase in human large intestine, normal and diseased.

Alkaline phosphatase activity has been investigated by histochemical methods in normal and diseased human large intestine. The tissues were constantly maintained at 4 degrees C or below. Specimens were either frozen in liquid nitrogen, freeze-dried and embedded in glycol methacrylate for sectioning at 2 mu, or, fixed in ice-cold formol-calcium for frozen sectioning at 10 mu. The simultaneous coupling azo dye method using the substrates sodium alpha-naphthyl phosphate and Naphthol AS-BI phosphate, resulted in the demonstration of alkaline phosphatase activity in the surface epithelial cells, and the middle and upper crypts, of normal and transitional mucosa.     

TRAIL,DR5 and caspase 3-dependent apoptosis in vessels of diseased human temporomandibular joint disc. An immunohistochemical study

To evaluate the apoptosis involvement in the angiogenesis as a self-limiting process in patients with temporomandibular joint (TMJ) degenerated disc vessels, we assessed, by immunohistochemistry, the detection of TRAIL, its death receptor DR5 and caspase 3. TRAIL, its death receptor DR5 and caspase 3 expression were studied by immunohistochemistry in 15 TMJ discs displaced without reduction and in 4 unaffected discs. These apoptosis molecules were detected in the intima and media layers of newly formed vessels affected discs. In conclusion, vessels apoptosis activation in TMJ disc with ID could be regarded as a self-limiting process that try to leads to vessel regression; in this way an inhibition of angiogenic vessels may prove a key strategy in limiting pathological angiogenesis, by cutting off blood supply to tumors, or by reducing harmful inflammation.Key words: temporomandibular joint disc, vessels, apoptosis.     

Localization and characterization of antithrombin in human kidneys.

Antithrombin is a serine protease inhibitor that is critical in maintaining a thromboresistant vasculature. The association between low serum antithrombin concentration and renal disease suggests that the kidney plays a role in the conservation of plasma antithrombin. We used immunohistochemical techniques to determine the spatial distribution, heparin binding characteristics, and intracellular and intercellular localization of antithrombin in biopsy specimens (n = 53) of human donor kidneys obtained at the time of transplantation. In the renal cortex, double antibody techniques demonstrated the presence of intracellular antithrombin in proximal tubule epithelial cells. The reactivity was granular and was co-localized with vesicle-like structures. Distal and collecting tubules did not demonstrate intraepithelial antithrombin reactivity. No tubule structures in the medullary region demonstrated intracellular antithrombin, but all these structures showed intense basement membrane antithrombin reactivity. Double antibody techniques also demonstrated that the heparin binding domain of intraepithelial antithrombin was occupied. Semiquantitative scores for intraepithelial antithrombin were significantly decreased in renal biopsy specimens obtained 30 min after anastomosis compared with biopsies from the same organ obtained before anastomosis. These findings suggest that antithrombin, probably in association with heparin or heparan sulfate, is internalized by renal proximal epithelial cells. Although the ultimate fate of intraepithelial antithrombin is not known, this may represent a mechanism by which the kidney helps to maintain plasma antithrombin concentrations.     

Prolyl hydroxylase activity in normal and diseased human liver.

Prolyl hydroxylase activity was determined in liver biopsy samples obtained from 10 patients. The liver prolyl hydroxylase values in patients with active hepatitis distribute into two numerical populations based on the extent of elevation over control. The first of these groups includes those with enzyme levels elevated approximately 2.5-fold over normal. Included in this group are patients with active (but nonagrressive) hepatitis and patients where advanced portal fibrosis is already established. The second group where prolyl hydroxylase is elevated approximately nine-fold is comprised of two patients with advanced clinical symptoms of active alcoholic hepatitis with evidence of aggressive cirrhosis but with only early minimal evidence of existing fibrosis.     

Collapse and viscoelasticity of diseased human arteries

A laboratory study of the hydrostatic collapse of diseased tibial arteries demonstrated hysteresis in the pressure-flow behaviour which resembled that seen in the stress-strain relations of the arterial tissue. The pressures at which the vessels collapsed were found to be considerably lower than expected on the basis of theoretical elastic models. Also, the pressures at which the vessels reopened were consistently lower than the pressures at which they collapsed. These findings were explained on the basis of viscoelasticity. The difference between collapse and opening pressure may provide insight into the mechanical properties of vessels, and a clue to errors in non-invasive measurements of blood pressure which depend upon collapse of arteries.     

Fluorescent labeling of antibody fragments using split GFP

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.