Detection of HPV and the role of p16INK4A overexpression as a surrogate marker for the presence of functional HPV oncoprotein E7 in colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Based on the well-recognized etiological role of human papillomavirus (HPV) in cervical, anogenital and oropharyngeal carcinogenesis, a potential role of HPV in colorectal carcinogenesis has been suggested. For that reason, the aim of the present study was to investigate the presence of HPV DNA in colorectal carcinomas (CRC) and to study overexpression of p16^INK4Aas a marker for the presence of an active HPV oncoprotein E7. These findings were correlated with clinical and pathological prognostic factors of CRC.</p> <p>Methods</p> <p>The presence of HPV was assessed using a multiplex PCR system of 10 non-biotinylated primers. The amplified fragments of HPV positive samples were further analyzed by a highly sensitive, broad spectrum SPF10 PCR and subsequently genotyped using reverse hybridization in a line probe assay.</p> <p>P16^INK4Aprotein expression was investigated in a subset of 90 (30 HPV positive and 60 HPV negative) CRC samples by immunohistochemistry.</p> <p>Results</p> <p>HPV DNA was found in 14.2% of the CRC samples with HPV16 as the most prevalent type. No significant differences in clinical and pathological variables were found between HPV positive and negative CRCs, except for age. HPV positive patients were significantly younger (p = 0.05). There was no significant correlation between the presence of HPV and overexpression of p16^INK4A(p = 0.325).</p> <p>Conclusions</p> <p>In conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples may suggest that HPV may be involved in the carcinogenesis of some CRC. However, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16^INK4Adoes not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the role of HPV in CRC.</p

The Heregulin/Human Epidermal Growth Factor Receptor as a New Growth Factor System in Melanoma with Multiple Ways of Deregulation

In a screening for new growth factors released by melanoma cells, we found that the p185-phosphorylating capacity of a medium conditioned by a melanoma cell line was due to the secretion of heregulin, a ligand for the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. Expression of heregulin, including a new isoform, and secretion of functionally active protein was found in several cell lines. Receptor activation by heregulin, either autocrine or paracrine, resulted in a potent growth stimulation of both melanocytes and melanoma cells. Heregulin receptor HER3 and coreceptor HER2 were the main receptors expressed by these cells. Nevertheless, none of the cell lines in our panel overexpressed HER2 or HER3. In contrast, loss of HER3 was found in two cell lines, whereas one cell line showed loss of functional HER2, both types of deregulations resulting in unresponsiveness to heregulin. This implies the heregulin/HER system as a possible important physiologic growth regulatory system in melanocytes in which multiple deregulations may occur during progression toward melanoma, all resulting in, or indicating, growth factor independence

Skap2 is required for β2 integrin-mediated neutrophil recruitment and functions.

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in β2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the β2 integrin cytoplasmic domain, thereby being indispensable for β2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott-Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the β2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice

Cellular infiltrates and injury evaluation in a rat model of warm pulmonary ischemia–reperfusion

INTRODUCTION: Beside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia–reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia–reperfusion. Therefore, a model of warm ischemia–reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage. METHODS: Fifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1–4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of ×500. Immunohistochemistry with antibody ED-1 (magnification ×250) and antibody 1F4 (magnification ×400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05. RESULTS: Neutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion. CONCLUSIONS: After warm ischemia–reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion

HIV-1 V3 envelope deep sequencing for clinical plasma specimens failing in phenotypic tropism assays

<p>Abstract</p> <p>Background</p> <p>HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain.</p> <p>Results</p> <p>A cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly.</p> <p>Conclusions</p> <p>Although only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.</p

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice

<p>Abstract</p> <p>Background</p> <p>Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.</p> <p>Results</p> <p>In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.</p> <p>Conclusion</p> <p>We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.</p

Atypical uterine leiomyoma: a rare variant of a common problem

A myomectomy was performed in a 35-year-old woman, and microscopic examination of the resulting specimen revealed an atypical myoma. Due to the rarity of this diagnosis, it is difficult to give clinical guidance for these patients, especially when they are considering pregnancy in the future. We advised 6 monthly vaginal ultrasound and did not advise against pregnancy. At 18 months there are no signs of recurrence, although the patient is not yet pregnant

Molecular targets of growth, differentiation, tissue integrity, and ectopic cell death in cancer cells

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