Immunohistochemical detection of nestin in pediatric brain tumors.

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.     

Pregnancy incidence and correlates during the HVTN 503 Phambili HIV vaccine trial conducted among South African women

Background

HIV prevention trials are increasingly being conducted in sub-Saharan Africa. Women at risk for HIV are also at risk of pregnancy. To maximize safety, women agree to avoid pregnancy during trials, yet pregnancies occur. Using data from the HVTN 503/“Phambili” vaccine trial, we report pregnancy incidence during and after the vaccination period and identify factors, measured at screening, associated with incident pregnancy.

Methods

To enrol in the trial, women agreed and were supported to avoid pregnancy until 1 month after their third and final vaccination (“vaccination period”), corresponding to the first 7 months of follow-up. Unsterilized women, pooled across study arms, were analyzed. Poisson regression compared pregnancy rates during and after the vaccination period. Cox proportional hazards regression identified associations with first pregnancy.

Results

Among 352 women (median age 23 yrs; median follow-up 1.5 yrs), pregnancy incidence was 9.6/100 women-years overall and 6.8/100 w-yrs and 11.3/100 w-yrs during and after the vaccination period, respectively [Rate Ratio = 0.60 (0.32–1.14), p = 0.10]. In multivariable analysis, pregnancy was reduced among women who: enrolled at sites providing contraception on-site [HR = 0.43, 95% CI (0.22–0.86)]; entered the trial as injectable contraceptive users [HR = 0.37 (0.21–0.67)] or as consistent condom users (trend) [HR = 0.54 (0.28–1.04)]. Compared with women with a single partner of HIV-unknown status, pregnancy rates were increased among women with: a single partner whose status was HIV-negative [HR = 2.34(1.16–4.73)] and; 2 partners both of HIV-unknown status [HR = 4.42(1.59–12.29)]. Women with 2 more of these risk factors: marijuana use, heavy drinking, or use of either during sex, had increased pregnancy incidence [HR = 2.66 (1.24–5.72)].

Conclusions

It is possible to screen South African women for pregnancy risk at trial entry. Providing injectable contraception for free on-site and supporting consistent condom use may reduce incident pregnancy. Screening should determine the substance use, partnering, and HIV status of both members of the couple for both pregnancy and HIV prevention.

Trial Registration

SA National Health Research Database DOH-27-0207-1539; Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00413725","term_id":"NCT00413725"}}NCT00413725     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 11 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Dihomo-γ-linolenate suppresses platelet aggregation when administered in vitro or in vivo

Dihomo-γ-linolenate effectively prevented the irreversible aggregation of human platelets induced by collagen or epinephrine. Also, platelets from rats which received daily oral doses of dihomo-γ-linolenate showed significant reductions in platelet aggregatory responses to collagen and ADP which were attributable to increases in plasma and platelet ratios of dihomo-γ-linolenate to arachidonate. Similar results were obtained in rabbits. This data, together with that of enzymatic studies supports a hypothesis that oral ingestion of dihomo-γ-linolenate may effectively prevent arterial thrombosis in man by causing a redirection of platelet prostaglandin biosynthesis. Thus PGE₁ and its non-aggregatory endoperoxide intermediate (PGR₁) are formed at the expense of PGE₂ and LASS endoperoxide which together may induce platelet thrombus formation.     

Large-scale polymorphism of heterochromatic repeats in the DNA of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

The composition of the individual eukaryote's genome and its variation within a species remain poorly defined. Even for a sequenced genome such as that of the model plant Arabidopsis thaliana accession Col-0, the large arrays of heterochromatic repeats are incompletely sequenced, with gaps of uncertain size persisting in them.