Double Staining Technique Research Articles

Evaluation of buffalo semen by Trypan blue/Giemsa staining and related fertility in vitro

The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls.

Comparison of Different Packing Materials for the Biofiltration of Air Toxics

Four different biofilter packing materials (two porous ceramics, perlite, and open pore polyurethane foam) were compared for the removal of toluene vapors. The focus was on evaluating performance at relatively short gas retention time (13.5 and 27 sec). The reactors were initially operated as biotrickling filters with continuous feeding and trickling of a nutrient solution. After significant plugging of the biotrickling filter beds with biomass was observed, the operation mode was switched to biofiltration with only periodic supply of mineral nutrients. This resulted in stable conditions, which allowed detailed investigations over >6 months. The reactor packed with cattle bone Porcelite (CBP), a ceramic material containing some macronutrients and micronutrients, exhibited the highest performance. The critical load (i.e., load at which 95% removal occurred) was 29 g m−3 hr−1 at a gas retention time of 13.5 sec and 66 g m−3 hr−1 at a gas retention time of 27 sec. After the long-term experiment, the packing materials were taken from the reactors and examined. The reactors were divided into three sections, top, middle, and bottom, to determine whether spatial differentiation of biomass occurred. The assays included a double-staining technique to count total and live microorganisms and determination of moisture, protein, and dry weight contents. Microbial community analysis was also conducted by denaturing gradient gel electrophoresis. The results showed that most reactors had a significant fraction of inactive biomass. Comparatively, the CBP biofilter held significantly higher densities of active biomass, which may be the reason for the higher toluene removal performance. The analyses suggest that favorable material properties and the nutrients slowly released by the CBP provided better environmental conditions for the process culture.

Comparative evaluation of the peri-implant bone tissue mineral density around unloaded titanium dental implants

The mechanical properties of bone are greatly influenced by the percentages of organic and mineral constituents. Nevertheless, the information about the mineral content on a microscopic scale in peri-implant bone is scarce. The aim of this work was to analyze the bone mineral density of peri-implant bone under different techniques. Five unloaded titanium dental implants with a micro-structured surface (three XiVE plus and two Frialit 2, DENTSPLY-Friadent, Mannheim, Germany) were retrieved from the mandible of five patients after a 6-month period. scanning electron microscopy with backscattered electron signal (BSE), light microscopy (LM) with a double staining technique, fluorescence microscopy and confocal laser microscopy were used for measuring microscopic mineral content variations in peri-implant bone. Histomorphometry and image intensity (grey level) were evaluated using a software package for image analysis. The low mineral density index (LMDI) for LM was of 29.2+/-3.1 (mean+/-S.D.), while the high mineral density index (HMDI) was of 88.2+/-3.6 (mean+/-S.D.). The one-way ANOVA analysis showed a significant difference (P<0.001) among the groups. The pairwise Holm-Sidak test identified the differences among HMDI indexes for both LM and SEM values and also for cross-evaluation of the LMDI and HMDI values. The comparison between LMDI indexes for both SEM and LM did not show any significance. The fluorescence microscopy analysis showed clearly the difference between old (high mineralized) and new (low mineralized) bone tissue near the implant surface. Under confocal laser microscopy the same sections showed the area of bone modelling closest to implant surface. In this study it was found that bone around unloaded implants showed a low mineral density index under all the investigation methods used. It was also found that the conventional LM technique with the double staining method was able to intensely stain the bone area with a low mineral content.

Contribution of Sensory Neurons to Sex Difference in the Development of Stress-Induced Gastric Mucosal Injury in Mice

Sensory neurons play a critical role in reducing stress-induced gastric mucosal injury by releasing calcitonin gene-related peptide (CGRP) through an increase in gastric mucosal levels of prostacyclin (PGI(2)). Because estrogen enhances nerve growth factor-mediated CGRP production in sensory neurons, we hypothesized that stress-induced gastric mucosal injury occurs less in females than in males. Gastric ulcer index, gastric myeloperoxidase activity, and gastric tissue levels of CGRP and 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were determined in male and female wild-type (CGRP(+/+)) mice and CGRP knockout (CGRP(-/-)) mice subjected to water-immersion restraint stress. In CGRP(+/+) mice, ulcer index and myeloperoxidase activities were lower and gastric tissue levels of CGRP and 6-keto-PGF(1alpha) were higher in female mice than in male mice, but there were no such sex differences in CGRP(-/-)mice. Sex differences in CGRP(+/+) mice were eliminated by pretreatment with SB366791 (500 microg/kg intraperitoneally), a vanilloid receptor antagonist, and by ovariectomy. Reversal of sex differences by ovariectomy was not observed in female CGRP(+/+) mice with estradiol replacement (1 mg . kg(-1). wk(-1) for 3 weeks). Levels of CGRP messenger RNA in dorsal root ganglion neurons isolated from female CGRP(+/+) mice were decreased by ovariectomy, and these decreases were reversed by estradiol replacement. Estrogen-mediated increases in CGRP levels in sensory neurons might contribute to reduce stress-induced gastric mucosal injury by attenuating inflammatory responses. This might at least partly explain the sex difference observed in the development of stress-induced gastric mucosal injury in mice.

The morphology of submicronsized core–shell latex particles: An electron microscopy study

The core–shell structure of a range of acrylic–acrylic latexes has been investigated by combining different specimen preparation methods with transmission electron microscopy (TEM), dark-field scanning transmission electron microscopy (DSTEM) and low-voltage scanning electron microscopy (LV-SEM), including the first reported use of LV-SEM to observe composite latex particles at ambient and subambient temperatures. Spin-coating of liquid latex dispersions directly onto TEM grids or SEM stubs is shown to be a relatively straightforward mean of avoiding film formation during specimen preparation. In conjunction with double staining techniques, it has been found to be particularly convenient for characterizing the fine structure of particles with diameters down to below 100 nm.

Distribution of growth hormone—like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus, branchiostoma belcheri *

Abstract Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exit in the nervous system and Hatschek's pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek's pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek's pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit. *Supported by the National Natural Science Foundation of China (Grant No. 30470224)

Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans

The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.

The Sialomucin CD34 Is a Marker of Lymphatic Endothelial Cells in Human Tumors

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.

Differential teratogenesis of all‐trans‐retinoic acid administered on gestational day 9.5 to SWV and C57BL/6N mice: Emphasis on limb dysmorphology

Mouse strain differences in teratologic response are well documented. However, because retinoids cause similar malformation syndromes across many species, the strain differences may be predicted to be minimal. The goals of this study were to characterize and explain the differences between the C57BL/6N and SWV mouse strains in terms of all-trans-retinoic acid (RA)-induced teratologic effects at the time of gestation that cause postaxial forelimb ectrodactyly. Visceral and skeletal malformations were determined by Wilson's sectioning and double-staining techniques, respectively; developmental staging was performed according to the somite count; and retinoid concentrations were assessed by HPLC. C57BL/6N mice were more susceptible than SWV mice to induction of embryolethality, cardiovascular defects, and forelimb ectrodactyly, whereas the opposite was true for the induction of ear, thymus, and tail agenesis, and cleft palate, gastroschisis, and anal atresia. As determined by somite counts, 1 strain intercross was developmentally advanced compared to the parental strains and the reciprocal cross. Retinoid susceptibility was equivalent between the reciprocal crosses for some malformations and determined by the maternal genotype for others. Toxicokinetic experiments showed that whole-embryo peak retinoid concentrations did not differ between the strains, but the area under the curve (AUC) for all-trans-RA was 1.3 times higher in C57BL/6N than in SWV embryos. The malformation spectrum induced by RA was strain-specific, and the strain sensitivity for forelimb ectrodactyly was consistent with all previously tested teratogenic agents (i.e., C57BL/6N was more sensitive than SWV). The strain differences in teratologic effects were not explained by developmental timing differences or toxicokinetic differences at the whole-embryo level.

Morphologic Research of Microcirculation Patterns in Human and Animal Melanoma

The pattern and distribution of microcirculation of malignant melanoma were studied in human malignant melanoma tumor samples and in an animal model by staining of paraffin-embedded sections and transelectron microscopy. Blood supply models for melanoma were studied with immunohistochemical and periodic acid-Schiff (PAS) double-staining technique. New sections were made from 190 paraffin-embedded melanoma samples, and immunohistochemical staining of the platelet-endothelial cell adhesive molecule (CD31 antigen) and PAS staining were conducted to confirm different microcirculation patterns of melanoma. Furthermore, malignant melanoma cells B16 and LiBr were injected into the groin of C57 mice and into the abdominal cavity of SCID mice, respectively. Tumors with vasculogenic mimicry (VM) were stained with PAS and CD31 to study the morphology and distribution of VM in mice melanoma. A diluted suspension of activated carbon was injected into the circulation of mice previously inoculated in the groin with B16 melanoma cells. Tumor tissue with VM was observed under electron microscopy. There were three kinds of microcirculation pattern in human and animal melanoma. The walls of VM were positive for PAS staining and negative for CD31 staining in the tumor tissues. The distribution of VM and mosaic vessels was not uniform and appeared in patches. VM along with endothelium-dependent vessels and mosaic vessels sustained the blood supply for the tumors. The results from electron microscopy validated the presence of three patterns. The results obtained using activated carbon as a tracer showed that VM and mosaic vessels connect with the host blood circulation. VM and mosaic vessels exist in malignant melanoma. Tumor cells can obtain oxygen and nutriment through VM and mosaic vessels besides endothelium-dependent vessels.

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.

Regulation of Fibroblast Growth Factor-2 Activity by Human Ovarian Cancer Tumor Endothelium

Fibroblast growth factor-2 (FGF-2) is a potent angiogenic cytokine that is dependent on heparan sulfate for its biological activity. We have investigated the relationship among heparan sulfate, FGF-2, and the signal-transducing receptors in human, advanced-stage, serous ovarian adenocarcinoma. Using a unique molecular probe, FR1c-Ap, which consisted of a soluble FGF receptor 1 isoform IIIc covalently linked to an alkaline phosphatase moiety, the distribution of heparan sulfate that had the ability to support the formation of a heparan sulfate/FGF-2/FGFR1 isoform IIIc alkaline phosphatase heparan sulfate construct complex was determined. This may be taken as a surrogate marker for the distribution of biologically active heparan sulfate and was distributed predominantly in endothelial cells and stroma but was absent from adenocarcinoma cells. In situ hybridization revealed the expression of FGFR1 mRNA in the endothelium and reverse transcription-PCR confirmed the presence of FGFR1 isoform IIIc but not isoform IIIb. The presence of FGF-2 around tumor endothelium was detected through immunohistochemistry. Double-staining techniques showed that heparan sulfate was found predominantly at the basal aspect of the endothelium and suggested that syndecan-3 might function as one of the proteoglycans involved in FGF-2 signaling in the endothelium. The data suggest that the entire extracellular signaling apparatus, consisting of FGF-2, biologically active heparan sulfate, and FGFRs capable of responding to FGF-2, is present in ovarian cancer endothelium, thereby highlighting the cytokine and its cognate receptor as potential targets for the antiangiogenic treatment of this disease.

GABAergic neurons express μ-opioid receptors in the ventrolateral orbital cortex of the rat

Behavioral studies have indicated that GABAergic modulation is involved in the opioid-induced antinociception in the ventrolateral orbital cortex (VLO). The aim of the current study was to examine whether the GABAergic neurons in the rat VLO expressed μ-opioid receptor subtype 1 (MOR1). This study employed immunofluorescence histochemical double-staining technique and showed that a considerable amount of GABA- and MOR1-like immunoreactive neurons existed in layers II–VI in the VLO. Of these GABA-like immunoreactive neurons, 92.0% of them showed MOR1-like immunoreactivities. Similarly, 80.2% of MOR1-like immuoreactive neurons also exhibited GABA-like immunoreactivities. These results provide morphological evidence that opioid-induced antinociception in the VLO might be due to an inhibitory effect by opioid via MOR1 on GABAergic neurons, resulting in disinhibition of VLO projection neurons and leading to activation of the VLO-PAG brainstem descending pain control system to depress the nociceptive inputs at the spinal cord level.

Cell Cycle Analysis of Waldenström's Macroglobulinemia

Little is known about the DNA cell content and cell cycle characteristics of immunoglobulin (Ig) M monoclonal gammopathies. The autonomous clone appears to be rather heterogeneous, from mature B lymphocytes to plasma cells (PCs). We have evaluated the DNA cell content of 27 patients with IgM monoclonal gammopathies: 18 of them had Waldenström's macroglobulinemia (WM), and 9 were diagnosed with IgM-monoclonal gammopathy of undetermined significance (MGUS). To specifically analyze the cell cycle of the B lymphocyte and PC populations, we used a flow-cytometric double-staining technique with CD19/CD20/CD22 propidium iodide for B lymphocytes and CD38/CD138 propidium iodide for PCs. In 26 of 27 patients, both subsets of tumor cells (B lymphocyte and PC) showed a diploid DNA cell content (DNA index, 1). The median percentage of proliferating B lymphocytes, S-phase + G2/M-phase, was 1.8% (range, 0.4%-4.1%). This proliferative activity was significantly lower than that observed in nonmalignant cells (5.7%; range, 0.1%- 14.2%; P = 0.004) in the same sample. No differences were observed when comparing the proliferative activity of WM with that of IgM MGUS (median, 1.7% vs. 2.2%, respectively). Cell cycle characteristics of PCs were simultaneously evaluated in 9 patients, with 1.8% cells in S phase or G2/M phase. In summary, the cell cycle analysis showed that IgM monoclonal gammopathies are low-proliferative disorders, with a DNA ploidy pattern (diploid) clearly different from that of multiple myeloma.

1223 多方向重力場に置かれた三次元培養骨細胞の機能調整(G02-3 骨梁構造とリモデリング,G02 バイオエンジニアリング)

Microgravity induces osteoporosis. Osteocytes are considered to act as a mechanosensor in bone tissue. They are the most abundant cells in bone and distribute throughout the bone matrix. However, very little is known about detail mechanism of osteoporosis occurred under microgravity. We have already reported that microgravity influenced cell proliferation, cell-to-cell communication and differentiation of bone marrow cells when osteocyte-like cell line MLO-Y4 cells were cultured in monolayer and subjected to multi-dimensional gravity. However, osteocytes in vivo were deep embedded in bone matrix. Therefore, the aim of this study was to examine the effects of multi-dimensional gravity on osteocytes in three-dimensional culture. MLO-Y4 cells were cultured in type I collagen gel, and then exposed to multi-dimensional gravity for 24h. Double fluorescence staining technique revealed that cell-to-cell communications via gap junction were significantly decreased in multi-dimensional gravity. Bone marrow cells were cultured for a week with using conditioned medium (CM) obtained from osteocyte culture under multidimensional gravity. TRAP (tartrate-resistant acid phosphatase) activity was significantly increased by adding CM from osteocyte culture exposed to multi-dimensional gravity.

Overexpression of HSP-72 confers cytoprotection in experimental peritoneal dialysis

Peritoneal dialysis is complicated by mesothelial cell injury due to low biocompatibility of peritoneal dialysis fluid (PDF). We have previously demonstrated that heat shock protein (HSP)-72 is potently up-regulated in response to PDF exposure of mesothelial cells in in vitro and in vivo models of peritoneal dialysis. The aim of this study was to evaluate potential cytoprotective effects of overexpression of HSP-72. Cytoprotection was assessed by comparing cellular viability between pretreated versus nonpretreated human mesothelial cells (Met 5a; ATCC, Manassas, VA, USA, and primary cell cultures) subjected to extended, usually lethal PDF exposure times (120 min, CAPD2; Fresenius, Bad Homburg, Germany). Pretreatment was performed with exposure to PDF (60 min, CAPD2; Fresenius) or heat (15 min, 41.5 degrees C), and by transient transfection with HSP-72. When mesothelial cells were pretreated by nonlethal exposure to PDF or heat, HSP-72 was markedly up-regulated (>5-fold, P < 0.01). Pretreated human mesothelial cells were significantly protected against subsequent "lethal" exposures to PDF, as assessed by dye exclusion (>50% reduction, P < 0.05) and lactate dehydrogenase (LDH) release (>30% reduction, P < 0.05). Comparable cytoprotection (50% reduction by dye exclusion) was indicated by overexpression of HSP-72 in cultered human mesothelial cells (>5-fold) after transient transfection with HSP-72. This cytoprotection was confirmed at a cellular basis by double staining techniques with HSP-72 and ApopTag (apoptosis detection kit). Our study therefore shows that the mesothelial stress response confers cytoprotection in experimental peritoneal dialysis, mediated by the induction of HSP-72, and that the stimulus of the pretreatment does not have to be identical to the subsequent injury. These data offer the basis for an attractive novel therapeutic approach against PDF toxicity.

Effect of hTERT Antisense on Cisplatin Sensitivity of Acute Lymphoblastic Leukemic Cells.

AIM: To investigate the effect of human telomerase reverse transcriptase(hTERT) antisense phosphorothioate oligodeoxynucleotide(AS PS-ODN) on enhancing cisplatin sensitivity in acute lymphoblastic leukemic cells.METHODS: The expression levels of hTERT protein were detected by immunofluorescence using fluoresce isothiocyanate(FITC) lable. Cell surviving fraction was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis.RESULTS: The expression of hTERT protein was inhibited after treated by hTERT AS PS-ODN. Treatment with cisplatin after 24h of exposure to AS PS-ODN had significantly reduced the number of viable ALL cells. However, there was no difference on ALL cells survival between sense oligodeoxynucleotide(S PS-ODN)/CDDP combination and CDDP treated cells alone. In morphological observation of apoptotic cells using Hoechst 33258 and PI double staining techniques, cells displayed classic apoptotic changes treated with CDDP or CDDP combined with hTERT AS PS-ODN or S PS-ODN at 48h. Apoptotic rates of cells added CDDP and AS PS-ODN were higher than that of cells added CDDP only(P<0.05). Apoptotic rates of cells added CDDP and S PS-ODN were similar with that of cells added CDDP only(P>0.05).CONCLUSION: hTERT AS-ODN inhibited the expression of hTERT protein and increased the CDDP induced apoptosis in primer acute lymphoblastic leukemic cells.

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats.

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.

The immunohistochemical assessment of hypoxia, vascularity and proliferation in bladder carcinoma

Background and purpose Hypoxia and proliferation are important determinants of radiation responsiveness; prospective measures of these before radiotherapy may enable individualisation of treatment schedules. Immunohistochemical techniques offer a potential means of achieving this in routine biopsy material. Material and methods Cellular hypoxia as measured by pimonidazole fixation and immunohistochemistry has been evaluated in a series of human bladder cancers with dual staining of sections for pimonidazole and either the vascular markers, CD31/34, or proliferation markers, Ki-67 or cyclin A. Twenty one tumour specimens were examined suitable for the double staining technique. Results The median hypoxic fraction was 9% (range 0–38). Seven tumours did not stain for pimonidazole and 11 exhibited necrosis. The mean vascular density ranged from 16.7 to 160.6 vessels per mm 2. The median hot spot count was 30 (range 16–43). There was a statistically significant increase in vessel density in hypoxic compared to oxic regions measured by both vessel density ( P=0.02) and hot spot count ( P=0.004). Proliferation indices decreased from oxic to hypoxic areas close to blood vessels. Conclusions We have demonstrated that bladder cancer exhibits a range of hypoxia, proliferation and vascular density which may be used to form the basis for patient selection for hypoxia modification, accelerated radiotherapy and vascular targeting agents.

The in situ expression of interleukin-8 in the normal human kidney and in different morphological forms of glomerulonephritis

Background: Interleukin-8 (IL-8) is considered a deleterious chemokine involved in renal injury in glomerulonephritis (GN). IL-8 may be released as a 77-amino acid (AA) peptide or 72-AA protein. Methods: We evaluated gene and protein expression of IL-8 in 53 renal biopsy specimens from patients with GN and 9 control kidneys. Nonradioactive in situ hybridization and reverse-transcriptase polymerase chain reaction (RT-PCR) were applied to detect IL-8 messenger RNA (mRNA). In immunohistochemistry, a double-staining technique with the use of antibodies against the 77-AA and 72-AA forms of IL-8, as well as glomerular cell antigens, was used. Results: By in situ hybridization, IL-8 mRNA was detected in normal glomerular, tubular, and some interstitial cells. The RT-PCR study showed that IL-8 mRNA expression in control kidneys significantly exceeds that in specimens with GN (0.89 ± 0.82 versus 0.21 ± 0.20; P < 0.003). In control kidneys, major sources of 77-AA IL-8 were podocytes and endothelial cells of interstitial vessels, whereas tubular epithelial cells expressed minute amounts of 72-AA IL-8. In GN specimens, podocyte expression of 72-AA IL-8 varied notably, with the greatest level found in minimal change disease and the lowest level found in acute endocapillary GN. Conversely, increased glomerular expression of the 72-AA form of IL-8 was a general feature of GN, with its level significantly exceeding that of the 77-AA form in acute endocapillary GN (P < 0.01). Conclusion: Our results suggest that intrinsic glomerular cell production of IL-8, in particular the 77-AA form, may be relevant for preservation of the glomerular architecture.