HRP-F1McAb酶免疫染色技术检测小鼠脏器鼠疫F1抗原应用效果评价

目的：评价辣根过氧化酶标记鼠疫F1单克隆抗体（HRP‐F1McAb）酶免疫染色技术检测小鼠脏器鼠疫F1抗原的应用效果。方法通过HRP‐F1McAb酶免疫染色法检测鼠疫菌EV株感染小鼠脏器标本和对照小鼠脏器标本,同时用细菌学、RIHA和ELISA做平行检测。结果 HRP‐F1McAb酶免疫染色法与细菌学一致率为96．36％,阳性检出率差异无统计学意义（χ2＝0,P＞0．05）;HRP‐F1McAb酶免疫染色法和RIHA一致率为98．18％,阳性检出率差异无统计学意义（χ2＝0,P＞0．05）;HRP‐F1McAb酶免疫染色法和ELISA一致率为96．36％,阳性检出率差异无统计学意义（χ2＝0,P＞0．05）。结论酶免疫染色法检测小鼠脏器鼠疫F1抗原特异敏感、简单快速。     

Role of polymorphonuclear neutrophils in the clearance of Enterococcus faecalis derived from saliva and infected root canals

Objectives

The goal of this study was to measure (1) the ability of polymorphonuclear neutrophil leukocytes (PMNs) to kill oral Enterococcus faecalis strains, (2) up-regulation of inflammatory mediators by PMNs in interaction with E. faecalis, and (3) the ability of E. faecalis to cause inflammation in mouse muscle tissue.

Methods

Fifteen endodontic and nine saliva strains of E. faecalis were isolated and identified by specific 16S ribosomal RNA (16S rRNA) primers. The bacteria were grown in BHI broth and incubated with mouse PMN in appropriate media to determine the ability of the PMNs to kill the bacteria. In other experiments up-regulation of interleukin (IL)-1α, tumor necrosis factor α (TNF-α), matrix metalloproteinase-8 (MMP-8), and cyclooxygenase (COX)-2 messenger RNA in the PMNs was measured after exposure of the leukocytes to the bacteria using real-time polymerase chain reaction. Finally, the inflammatory potential of and PMN response to E. faecalis suspension in mouse muscle tissue was examined from histological sections using hematoxylin-eosin staining and immunostaining.

Results

Murine PMNs killed about 80% of the E. faecalis cells in 1 hour, irrespective of the source of isolation of the strains. Quantitative PCR results showed that IL-1α, TNF-α, MMP-8, and COX-2 messenger RNA were markedly up-regulated in E. faecalis-stimulated PMNs or in E. faecalis-invaded muscular tissues. MMP-8 messenger RNA level was positively related to COX-2 messenger RNA level. Histological evaluation and immunostaining disclosed that all E. faecalis strains could recruit PMNs to the local infectious sites and cause abscess formation.

Conclusion

E. faecalis strains from saliva and infected root canals have the potential to recruit PMNs in the infectious sites leading to inflammation via up-regulation of PMN IL-1α, TNF-α, MMP-8, and COX-2. PMNs can play an important role in killing of E. faecalis.     

MicroCT for molecular imaging: quantitative visualization of complete three-dimensional distributions of gene products in embryonic limbs

We present a broadly applicable procedure for whole-mount imaging of antibody probes in embryonic tissues at microscopic resolutions based on combining a metal-based immunodetection scheme with x-ray microtomography (microCT). The method is generally accessible, relying on standard enzyme-conjugated secondary antibodies and other readily available reagents, and is demonstrated here with microCT visualizations of acetylated α-tubulin in the chick nervous system and of type II collagen in developing limbs. The tomographic images offer complete three-dimensional representations of molecular patterns obtained with immunostaining methods at the level of organ development, with added possibilities to quantify both spatial distributions and varying densities of gene products in situ. This imaging modality bridges a crucial gap in three-dimensional molecular imaging by combining the histological resolutions of confocal microscopy with a greater specimen size range than optical projection tomography, and thus enables a powerful new approach to long-standing issues of skeletogenic pattern formation in vertebrate limbs.     

Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome

In clinical practice it is possible to find patients with clinical signs suggestive of anti-phospholipid syndrome (APS) who are persistently negative for the routinely used anti-phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN-APS). We investigated the clinical usefulness of thin-layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN-APS. Sera from 36 patients with SN-APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti-hepatitis C virus (HCV)-positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti-β(2) -glycoprotein-I, anti-annexin II, anti-annexin V and anti-prothrombin antibodies were tested by enzyme-linked immunosorbent assays (ELISA). Eahy926, a human-derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN-APS patients and analysis of phospho-interleukin (IL)-1 receptor-associated kinase (IRAK) and phospho-nuclear factor (NF)-κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM-1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN-APS patients: anti-cardiolipin in 47·2%, anti-lyso(bis)phosphatidic acid in 41·7% and anti-phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti-annexin II. Incubation of Eahy926 cells with IgG from SN-APS induced IRAK phosphorylation, NF-κB activation, VCAM-1 surface expression and TF cell release. TLC immunostaining could identify the presence of aPL in patients with SN-APS. Moreover, the results suggest the proinflammatory and procoagulant effects in vitro of these antibodies.     

A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines

Neutralizing antibodies are considered an important component of protective immunity against congenital infection of human cytomegalovirus (HCMV), a frequently cited cause of birth defects. An effective HCMV vaccine is desired to induce potent neutralizing antibodies in seronegative females, so that the viral transmission to fetus would be blocked if the women contracted HCMV infections during their pregnancies. We describe a novel microneutralization assay to measure antiviral activities against HCMV in serum samples. The assay is based on detection of a dominant HCMV antigen expressed in cells, using near infrared dye-labeled immune reagents. Since the detection is independent of viral cytopathic effects, this assay format has the appeal of a short turn-around time and a read-out that is not subject to operator experience and judgment, making it a promising platform to support large scale clinical studies. In a serological survey of a cohort of 200 healthy females, we showed that the neutralizing titers measured in this assay are highly comparable to those from a neutralization assay based on an enzyme-linked immunostaining method. Lastly, to demonstrate the utility of this assay to support HCMV vaccine study, we presented the results of neutralizing titers from a rhesus macaque vaccination study.     

High Quality Tissue Miniarray Technique Using a Conventional TV/Radio Telescopic Antenna

Background: The tissue microarray (TMA) is widely accepted as a fast and cost-effective research tool forin situ tissue analysis in modern pathology. However, the current automated and manual TMA techniqueshave some drawbacks restricting their productivity. Our study aimed to introduce an improved manual tissueminiarray (TmA) technique that is simple and readily applicable to a broad range of tissue samples. Materialsand Methods: In this study, a conventional TV/radio telescopic antenna was used to punch tissue cores manuallyfrom donor paraffin embedded tissue blocks which were pre-incubated at 40oC. The cores were manuallytransferred, organized and attached to a standard block mould, and filled with liquid paraffin to construct TmAblocks without any use of recipient paraffin blocks. Results: By using a conventional TV/radio antenna, it waspossible to construct TmA paraffin blocks with variable formats of array size and number (2-mm x 42, 2.5-mmx 30, 3-mm x 24, 4-mm x 20 and 5-mm x 12 cores). Up to 2-mm x 84 cores could be mounted and stained on astandard microscopic slide by cutting two sections from two different blocks and mounting them beside eachother. The technique was simple and caused minimal damage to the donor blocks. H&E and immunostainedslides showed well-defined tissue morphology and array configuration. Conclusions: This technique is easy toreproduce, quick, inexpensive and creates uniform blocks with abundant tissues without specialized equipment.It was found to improve the stability of the cores within the paraffin block and facilitated no losses during cuttingand immunostaining.     

Lymphatic invasion according to D2-40 immunostaining is a predictor of nodal metastasis in endometrioid adenocarcinoma of the uterine corpus

In endometrioid adenocarcinoma of the uterine corpus, nodal metastasis is related to prognosis. D2-40 immunostaining has recently been used to detect lymphatic invasion, but a study of D2-40 immunostaining for endometrioid adenocarcinoma of the uterine corpus has not been published. Therefore, as a predictor of nodal metastasis in endometrioid adenocarcinoma of the uterine corpus, the detection of lymphatic invasion on D2-40 immunostaining and lymphovascular invasion on HE stain was compared. A total of 104 cases of invasive endometrioid adenocarcinoma of the uterine corpus, in which the tumor was located in the uterus, were examined on immunohistochemistry using D2-40. In 20 cases there was lymphatic invasion according to D2-40 immunostaining, and the lymphatic invasion was well detected on D2-40 immunostaining. Nodal metastasis was present in 11 cases. Both lymphatic invasion on D2-40 immunostaining and lymphovascular invasion on HE stain were statistically correlated with nodal metastasis, but the evaluation of lymphatic invasion on D2-40 immunostaining was more accurate than detection of lymphovascular invasion using HE stain, in the current and previous studies, for the prediction of nodal metastasis. In conclusion, lymphatic invasion demonstrated on D2-40 immunostaining is very useful as a predictor for nodal metastasis in endometrioid adenocarcinoma of uterine corpus.     

Immunohistochemical staining of human islet cells with region-specific antibodies against secretogranins II and III

Chromogranins and secretogranins belong to the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In earlier publications we have described the development of region-specific antibodies against CgA and CgB. In this study we describe antibodies to SgII and SgIII and their usefulness for immunohistochemical staining. Peptides homologous to defined parts of secretogranins II and III were selected and synthesized. Antibodies were raised and immunostainings were performed on normal human pancreas. The SgII 154-165 (N-terminal secretoneurin), SgII 172-186 (C-terminal secretoneurin) and SgIII antibodies immunostained all insulin-immunoreactive cells, most of the glucagon cells and some of the pancreatic polypeptide cells. The SgII 225-242 antibody immunostained only the insulin-containing cells. None of the antibodies immunostained the somatostatin cells. This study is the first observation of the expression of SgIII in human tissues, where we show expression of SgIII in three of the four major islet cell types in human pancreas.     

Polysialogangliosides Expressed by Amelanotic Melanoma: A Possible Explanation for the Poor Response to Anti-monosialoganglioside Antibody 202 in a Patient with Melanoma

A 52-year-old Japanese woman developed numerous amelanotic metastatic melanomas on the skin and in various organs three years after a surgical operation for primary melanoma on the right axilla. The patient was treated with monosialoganglioside specific monoclonal antibody 202; however, no apparent clinical effects were observed. Ganglioside analysis of a metastatic tumor demonstrated that it expressed GM3, GM2, GD3, GD2, and polysialogangliosides. Since polysialogangliosides rarely appear in melanomas, their expression may explain the patient's poor response to MAb 202. The relationship between ganglioside composition and the effect of anti-ganglioside monoclonal antibody is discussed.     

Endocrinology: The effect of high-dose medium- and long- term progestogen exposure on endometrial vessels

A total of 19 paraffin-embedded endometrial tissue blocks wereobtained from high-dose progestogen-exposed patients. A labelledstreptavidin—biotin—alkaline phosphatase methodwas used with antibodies against von Willebrand factor (vWF)and CD34. The density of CD34 and vWF positive (CD34⁺ and vWF⁺)vessels in progestogen-exposed endometria (103 ± 9.6/mm²and 106 ± 8.7/mm²) was significantly lower than in endometriafrom women with normal cycles (169 ± 9.3/mm² and 136± 8.0/mm²) (P < 0.05). In women with normal menstrualcycles the concentration of CD34⁺ vessels was significantlyhigher than the number of vWF⁺ vessels (P = 0.0001). By comparison,the concentration of CD34⁺ vessels was similar to the concentrationof vWF⁺ vessels in progestogen-exposed endometria. The ratiosof vascular density as determined by vWF⁺ and CD34⁺ stainingin the control and progestogen groups were 0.81 and 1.05 respectively(P = 0.0001). Dilated venules were seen in the progestogen group.This study has demonstrated firstly that CD34 antibody detectedthe endothelial cells in a higher proportion of small endometrialvessels than vWF, and secondly that high-dose progestogen exposuresignificantly decreased the density of microvessels and increasedthe number of dilated venules in endometrium.