Low frequency of human papillomavirus detection in prostate tissue from individuals from Northern Brazil

The presence of human papillomavirus (HPV) was evaluated in 65 samples of prostate tumours and six samples of prostates with benign prostatic hyperplasia from individuals from Northern Brazil. We used a highly sensitive test, the Linear Array HPV Genotyping Test, to detect 37 high and low-risk HPV types. In this study, only 3% of tumour samples showed HPV infection. Our findings support the conclusion that, despite the high incidence of HPV infection in the geographic regions studied, HPV was not associated with a higher risk of prostate cancer. To our knowledge, this is the first study evaluating the frequency of HPV detection in prostatic tissue of individuals from Brazil.

“Unmixing” Tissue Gene Expression Signatures from Tumor Biopsies

Emergent molecular measurement methods, such as DNA microarray, qRTPCR, andmany others, offer tremendous promise for the personalized treatment of cancer. Thesetechnologies measure the amount of specific proteins, RNA, DNA or other moleculartargets from tumor specimens with the goal of “fingerprinting” individual cancers. Tumorspecimens are heterogeneous; an individual specimen typically contains unknownamounts of multiple tissues types. Thus, the measured molecular concentrations resultfrom an unknown mixture of tissue types, and must be normalized to account for thecomposition of the mixture.For example, a breast tumor biopsy may contain normal, dysplastic and cancerousepithelial cells, as well as stromal components (fatty and connective tissue) and bloodand lymphatic vessels. Our diagnostic interest focuses solely on the dysplastic andcancerous epithelial cells. The remaining tissue components serve to “contaminate”the signal of interest. The proportion of each of the tissue components changes asa function of patient characteristics (e.g., age), and varies spatially across the tumorregion. Because each of the tissue components produces a different molecular signature,and the amount of each tissue type is specimen dependent, we must estimate the tissuecomposition of the specimen, and adjust the molecular signal for this composition.Using the idea of a chemical mass balance, we consider the total measured concentrationsto be a weighted sum of the individual tissue signatures, where weightsare determined by the relative amounts of the different tissue types. We develop acompositional source apportionment model to estimate the relative amounts of tissuecomponents in a tumor specimen. We then use these estimates to infer the tissuespecificconcentrations of key molecular targets for sub-typing individual tumors. Weanticipate these specific measurements will greatly improve our ability to discriminatebetween different classes of tumors, and allow more precise matching of each patient tothe appropriate treatment

Assessment of adipose tissue metabolism by means of subcutaneous microdialysis in patients with sepsis or circulatory failure.

To evaluate the role of adipose tissue in the metabolic stress response of critically ill patients, the release of glycerol and lactate by subcutaneous adipose tissue was assessed by means of microdialysis in patients with sepsis or circulatory failure and in healthy subjects. Patients with sepsis had lower plasma free fatty acid concentrations and non-significant elevations of plasma glycerol concentrations, but higher adipose-systemic glycerol concentrations gradients than healthy subjects or patients with circulatory failure, indicating a stimulation of subcutaneous adipose lipolysis. They also had a higher lipid oxidation. Lipid metabolism (adipose-systemic glycerol gradients, lipid oxidation) was not altered in patients with circulatory failure. These observations highlight major differences in lipolysis and lipid utilization between patients with sepsis and circulatory failure. Hyperlactataemia was present in both groups of patients, but the adipose-systemic lactate concentration gradient was not increased, indicating that lactate production by adipose tissue was not involved. This speaks against a role of adipose tissue in the development of hyperlactataemia in critically ill patients.

Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair.

OBJECTIVES/HYPOTHESIS: Facial nerve regeneration is limited in some clinical situations: in long grafts, by aged patients, and when the delay between nerve lesion and repair is prolonged. This deficient regeneration is due to the limited number of regenerating nerve fibers, their immaturity and the unresponsiveness of Schwann cells after a long period of denervation. This study proposes to apply glial cell line-derived neurotrophic factor (GDNF) on facial nerve grafts via nerve guidance channels to improve the regeneration. METHODS: Two situations were evaluated: immediate and delayed grafts (repair 7 months after the lesion). Each group contained three subgroups: a) graft without channel, b) graft with a channel without neurotrophic factor; and c) graft with a GDNF-releasing channel. A functional analysis was performed with clinical observation of facial nerve function, and nerve conduction study at 6 weeks. Histological analysis was performed with the count of number of myelinated fibers within the graft, and distally to the graft. Central evaluation was assessed with Fluoro-Ruby retrograde labeling and Nissl staining. RESULTS: This study showed that GDNF allowed an increase in the number and the maturation of nerve fibers, as well as the number of retrogradely labeled neurons in delayed anastomoses. On the contrary, after immediate repair, the regenerated nerves in the presence of GDNF showed inferior results compared to the other groups. CONCLUSIONS: GDNF is a potent neurotrophic factor to improve facial nerve regeneration in grafts performed several months after the nerve lesion. However, GDNF should not be used for immediate repair, as it possibly inhibits the nerve regeneration.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

Chagas disease, adipose tissue and the metabolic syndrome

Trypanosoma cruzi infection of the adipose tissue of mice triggers the local expression of inflammatory mediators and a reduction in the expression of the adipokine adiponectin. T. cruzi can be detected in adipose tissue by PCR 300 days post-infection. Infection of cultured adipocytes results in increased expression of cytokines and chemokines and a reduction in the expression of adiponectin and the peroxisome proliferator-activated receptor ³, both of which are negative regulators of inflammation. Infection also results in the upregulation of cyclin D1, the Notch pathway, and extracellular signal-regulated kinase and a reduction in the expression of caveolin-1. Thus, T. cruzi infection of cultured adipocytes leads to an upregulation of the inflammatory process. Since adiponectin null mice have a cardiomyopathic phenotype, it is possible that the reduction in adiponectin contributes to the pathogenesis of chagasic cardiomyopathy. Adipose tissue may serve as a reservoir for T. cruzi from which parasites can become reactivated during periods of immunosuppression. T. cruzi infection of mice often results in hypoglycemia. In contrast, hyperglycemia as observed in diabetes results in increased parasitemia and mortality. Adipose tissue is an important target tissue of T. cruzi and the infection of this tissue is associated with a profound impact on systemic metabolism, increasing the risk of metabolic syndrome.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-α, TGF-β and CD4+ and acinar IFN-γ, TNF-α, IL-1β and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-β. Significant positive correlations were found between portal CD4+ and TNF-α, portal CD8+ and TGF-β, portal CD45+RO and TNF-α, acinar CD45+RO and IFN-γ and acinar CD57+ and TGF-β. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Suture repair of umbilical hernia during caesarean section: a case-control study.

PURPOSE: The objective of this study was to investigate the additional burdens in terms of pain, prolongation of surgery and morbidity which is added to elective caesarean section if umbilical hernia suture repair is performed simultaneously. Secondly, patient's satisfaction and hernia recurrence rate were assessed. METHODS: Consecutive women with symptomatic umbilical hernia undergoing internal or external suture repair during elective caesarean were included in this retrospective cohort-control study. Data on post-operative pain, duration of surgery and morbidity of a combined procedure were collected. These patients were matched 1:10 to women undergoing caesarean section only. Additionally, two subgroups were assessed separately: external and internal suture hernia repair. These subgroups were compared for patient's satisfaction, cosmesis, body image and recurrence rate. RESULTS: Fourteen patients with a mean age of 37 years were analysed. Internal suture repair (n = 7) prolonged caesarean section by 20 min (p = 0.001) and external suture repair (n = 7) by 34 min (p < 0.0001). Suture repair did not increase morphine use (0.38 ± 0.2 vs. 0.4 ± 02 mg/kg body weight), had no procedure-related morbidity and prolonged hospitalization by 0.5 days (p = 0.01). At a median follow-up of 37 (5-125) months, two recurrences in each surgical technique, internal and external suture repair, occurred (28 %). Body image and cosmesis score showed a higher level of functioning in internal suture repair (p = 0.02; p = 0.04). DISCUSSION: Despite a high recurrence rate, internal suture repair of a symptomatic umbilical hernia during elective caesarean section should be offered to women if requested. No additional morbidity or scar is added to caesarean section. Internal repair is faster, and cosmetic results are better, additional skin or fascia dissection is avoided, and it seems to be as effective as an external approach. Yet, women must be informed on the high recurrence rate.

Angiogenesis and schistosomiasis

Angiogenesis has been recognised as a precursor of fibrosis in several pathologic conditions. Its participation has been demonstrated in schistosomiasis, both during periovular granuloma formation and in the genesis of schistosomal periportal fibrosis. Paradoxically, proliferation of new blood vessels, accompanied by production of vascular-endothelial growth factor, appeared prominent during fibrosis regression months after curative treatment of schistosomiasis. Thus, angiogenesis in schistosomiasis seems to have a two-way mode of action, participating both in fibrogenesis and in fibrosis degradation. Morphological observations presented here are in keeping with the possibility that, in the first case, angiogenesis allows pericytes to come in great numbers to the site of lesions and be detached from capillary walls and transformed into myofibroblasts, which are important extra-cellular matrix forming cells. During post-curative fibrosis regression, actin-containing pericytes appeared at various foci of tissue remodelling, especially at sites of repair of vascular lesions. The molecular and cell factors involved in both situations seem to be important subjects in need of further investigations and the schistosomiasis model certainly will be of great avail in this regard.

Angiogenesis and experimental hepatic fibrosis

Angiogenesis is a basic change occurring during repair by granulation tissue. This process seems to precede fibrosis formation in most types of chronic liver disease. To examine its presence and significance in different types of hepatic insults, this paper sought to identify the presence, evolution and peculiarities of angiogenesis in the most common experimental models of hepatic fibrosis. The characterization of cells, vessels and extracellular matrix and the identification of factors associated with endothelium (factor VIII RA), vascular basement membrane, other components of the vascular walls (actin, elastin) and the presence of the vascular-endothelial growth factor were investigated. The models examined included Capillaria hepatica septal fibrosis, whole pig serum injections, carbon tetrachloride administration, main bile duct ligation and Schistosoma mansoni infection. The first four models were performed in rats, while the last used mice. All models studied exhibited prominent angiogenesis. The most evident relationship between angiogenesis and fibrosis occurred with the C. hepatica model due to circumstances to be discussed. Special attention was paid to the presence of pericytes and to their tendency to become detached from the vascular wall and be transformed into myofibroblasts, which is a sequence of events that explains the decisive role angiogenesis plays in fibrosis.

Differential tissue tropism of Trypanosoma cruzi strains: an in vitro study

We have previously demonstrated selection favoring the JG strain of Trypanosoma cruziin hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.