Immunohistochemical detection of immunoglobulins and complements in formaldehyde-fixed and paraffin-embedded renal biopsy tissues; an adjunct for diagnosis of glomerulonephritis

BACKGROUND: The present study was undertaken to demonstrate the deposition of immunoglobulins or complements in formaldehyde-fixed and paraffin-embedded renal biopsy tissues through the unmasking of antigens with microwave treatment plus protease digestion or trypsin digestion. METHODS: Biopsy samples from patients with IgA nephritis (n = 7), lupus nephritis (7), membranous nephropathy (7) and mesangiocapillary glomerulonephritis (3) were used. Antigen unmasking was performed with (i) microwave treatment plus protease digestion for 10, 30 or 60 min, or (ii) digestion with 0.25% trypsin for 60 or 120 min. RESULTS: Microwave treatment plus protease digestion for 30 or 60 min and trypsin digestion for 120 min provided good results for the unmasking of immunoglobulins in glomeruli with structural preservation. The IgA deposits in IgA nephritis and IgG deposits in lupus nephritis and membranous nephropathy were clearly revealed in more than 80% of cases by both pretreatments. Microwave treatment plus protease digestion for 30 min revealed the deposition of C3 in all cases of mesangiocapillary glomerulonephritis and lupus nephritis and was superior to trypsin digestion. Characteristic patterns of C3 deposition were observed for these forms of glomerulonephritis, although C3 deposits in membranous nephropathy were detected in only 50% of cases. It was not possible to unmask all of the antigens in the glomeruli, especially those with weak immunofluorescence. CONCLUSION: Microwave treatment plus protease digestion is effective for the unmasking of antigens in paraffin sections and as useful for the diagnosis of immune-mediated glomerulonephritis as trypsin digestion.     

Unilateral optic nerve crush induces bilateral retinal glial cell proliferation

The post-injury responses of retinal ganglion cells elicit a number of glial reactions which have not been completely understood. The bilateral pattern of non-neuronal retinal cell proliferation was examined in association with the differential fates of unilaterally injured adult retinal ganglion cells by means of bromodeoxyuridine (BrdU) immunocytochemistry. Lateralization of the glioproliferative events was studied by analysing both the experimental and the uninjured contralateral as well as matched retinas of sham-operated animals. Control adult rat retina included very few BrdU-positive cells within the nerve fibre and ganglion cell layers; however, experimental retinas of degenerating groups exhibited statistically significantly higher densities of newborn cells in most layers. Clusters of labelled cells were found in the inner plexiform layer related to OX-42 staining, indicating their microglial nature. Indeed, double-labelling experiments, after short-term unilateral optic nerve crushing, identified proliferating retinal glial cells in vivo. Both types of glia, astroglial and microglial cells, exhibited BrdU-positive labelling in injured as well as uninjured experimental rat retinas. Moreover, microglial proliferating cells were also identified in explanted retinal pieces after 2 days in culture. Affected and contralateral retinas responded similarly to the unilateral experimental manipulations applied with respect to BrdU labelling. The acute glial responses observed suggest that bilateral glial proliferation might represent a common response related to degeneration events in both retinas, i.e. ipsi- and contralateral to the experimental injury.     

Studies on protocadherin-2 expression in the human fetal central nervous system

Protocadherin (Pcad) is a group of molecules obtained by polymerase chain reaction (PCR) utilizing the sequence that is well preserved in the extracellular domain of cadherin. Sano et al. analyzed Pcad (PC42,43) that had been cloned from rats, and found that it basically had homology to cadherin, but contained more than six cadherin repeats with a completely different intracellular domains (Sano et al. 1993). In the present study, of the Pcad (Pcad-1,2) cloned from a human cDNA library, as-yet-unspecified Pcad-2 was analyzed for expression in the human fetal central nervous system (CNS). Northern blot analysis of adult human tissue showed that Pcad-2 was expressed in the brain and the placenta, and that Pcad-2 mRNA was expressed in actively dividing neural tumor cell lines. Monoclonal antibodies against Pcad-2 were then made, and the CNS of fetuses were immunohistochemically stained. The expression was hardly visible at the 6th week of pregnancy, and began to become visible along the nerve fiber in the brain stem at the 8th week, and spread over the entire brain at the 11th week. At the 18th week, however, expression in the nerve fascicles, which had been visible by that time, was no longer visible or had decreased. These results suggest that Pcad-2 appears relatively early in the critical stage of development of the fetal CNS, and is involved in the induction, fasciculation, and extension of axons.     

Can p53 staining be used to identify patients with aggressive superficial bladder cancer?

Approximately 10% of patients with superficial bladder cancer (pTa/pT1) recur with life-threatening muscle-invasive disease. Identification of these patients has been a major goal of bladder cancer research. In 1994, it was suggested that p53 immunostaining could identify the cancers that would progress and it was proposed that tumours that stain for p53 should be treated aggressively with radiotherapy or cystectomy. Despite the hundreds of studies published since on the relationship between p53 and progression in superficial bladder cancer, the clinical utility of p53 immunostaining has not been resolved because of limitations concerning the numbers of patients and the length of follow-up. This study set out to overcome these limitations by using tissue from a large multicentre trial that recruited 502 patients with a median follow-up of 10 years. Each of 34 patients that had progressed with >/= pT2 disease or had distant metastases or had died from bladder cancer was compared with one or two matched controls. Sections were stained with a mouse monoclonal antibody to p53, pAb1801. In agreement with many of the earlier studies, p53 immunostaining had prognostic significance. The adjusted hazard ratio for time to progression for the pAb1801-positive versus negative group was 2.5, with 95% confidence intervals of 1.05-5.98 (p = 0.039). The other major risk factor that is associated with progression of superficial bladder cancer is pT1G3 disease. Of the 42 pT1G3 cancers, 14 (33%) progressed. The proportion of cancers with p53 staining that progressed was similar to the proportion of pT1G3 cancers that progressed, but neither the sensitivity nor the specificity of association of p53 staining with progression is sufficient to recommend cystectomy in individual patients.     

Changes in Cytochemistry of Sensory and Nonsensory Cells in Gentamicin-Treated Cochleas

Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.     

Characterization of green fluorescent protein–expressing retinal cone bipolar cells in a 5-hydroxytryptamine receptor 2a transgenic mouse line

Retinal bipolar cells relay visual information from photoreceptors to third-order retinal neurons. Bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The identification of molecular markers that can label specific retinal bipolar cells could facilitate the investigation of bipolar cell functions in the retina. Transgenic mice with specific cell type(s) labeled with green fluorescent protein (GFP) have become a powerful tool for morphological and functional studies of neurons in the CNS, including the retina. In this study, we report a 5-hydroxytryptamine receptor 2a (5-HTR2a) transgenic mouse line in which expression of GFP was observed in two populations of bipolar cells in the retina. Based on the terminal stratification and immunostaining, all the strongly GFP-labeled bipolar cells were found to be type 4 cone bipolar cells. A small population of weakly labeled bipolar cells was also observed, which may represent type 8 or 9 cone bipolar cells. GFP expression in retinal cone bipolar cells was seen as early as postnatal day 5. In addition, despite severe retinal degeneration due to the presence of the rd1 mutation in this transgenic line, the density of GFP-labeled cone bipolar cells remained stable up to at least 6 months of age. This transgenic mouse line will be a useful tool for the study of type 4 cone bipolar cells in the retina under both normal and disease conditions.     

活体免疫染色评估眼底新生血管实验研究

目的 探寻更好地对视网膜新生血管(RNV)和脉络膜新生血管(CNV)及其渗漏作定性和定量分析的方法 .方法 13只小鼠随机分为RNV评估组(n=7)和CNV评估组(n=6).①RNV评估:取3只小鼠作为正常对照组,其余4只制作成未成熟视网膜病变(ROP)模型,分别于出生后第16天双眼内注射FITC标记的抗体或未经FITC标记的抗体以及空白组,12 h后直接视网膜铺片或用带荧光的二抗孵育后再铺片镜检.②CNV渗漏评估:激光诱导6只小鼠(12眼)制作CNV模型,随机平分为两组,分别于光凝后第7和14天眼内注射非荧光标记的抗体,12 h后腹腔内注射荧光素钠,并定量分析CNV面积及其渗漏面积.结果 FITC标记抗体组的视网膜表现出高荧光、高清晰度的视网膜结构血管和RNV,而非FITC标记抗体组的视网膜只选择性染色RNV,几乎无干扰荧光背景,可观察到细微变化,利于应用图像软件对RNV图片进行定性和定量分析.应用SPOT图像软件把CNV染色的图片与CNV渗漏荧光的图片进行叠加,能够明确区分CNV及其渗漏面积;第14天与第7天CNV面积及其渗漏面积比较差异有统计学意义(P<0.05).结论 使用活体免疫染色技术可以方便地观察视网膜血管细微结构,并精准地定性和定量分析RNV和CNV渗漏.     

Heidenhain variant of Creutzfeldt-Jakob disease: An autopsy study from India

Prion diseases are rare, progressive and fatal neurodegenerative diseases characterized by long incubation period and short clinical course. We present a rare case of Heidenhain variant of Creutzfeldt-Jakob disease, occurring in a 55-year-old lady presenting with dementia, cortical blindness, and myoclonic jerks. She succumbed to the disease within 8 weeks of onset of symptoms. MRI revealed hyperintense signals on T2WI and fluid attenuated inversion recovery (FLAIR) images in basal ganglia and fronto-temporal and parietal cortex, sparing thalamus, striate cortex and globus pallidum. Abundant abnormal prion protein deposits (PrP^sc) were detected in caudate, putamen, thalamus, cingulate and striate cortex, in comparison to frontal and parietal cortex while no deposits were found in globus pallidum. MRI changes did not correlate with degree of spongy change, gliosis or prion protein deposition. The cause for abnormal signal changes in MRI and FLAIR images remains unclear.