Phenotypic characterization of human prostatic stromal cells in primary cultures derived from human tissue samples

Emerging evidence has shown that the tumor microenvironment plays a crucial role in prostate cancer (PCa) development and progression. However, the mechanism(s) through which stromal cells regulate epithelial cells and the differences among prostatic stromal cells of different histological/pathological origin in PCa progression remain unclear. Therefore, it is necessary to characterize the stromal cell populations present in benign prostatic hyperplasia (BPH) and PCa. To this end, we used cultures from stromal cells obtained from BPH-derived (15 cases) and PCa-derived (30 cases) primary cultures. In culture, stromal cells are a mixture of fibroblasts, myofibroblasts (MFs) and muscle cells. Fibroblasts are characterized for the expression of vimentin, MFs for the co-expression of α-smooth muscle actin (α-SMA) and vimentin, whereas muscle cells for the expression of α-SMA and desmin. Fibroblasts were present in large amounts in the BPH-compared to the PCa-derived cultures, whereas MFs were more representative of PCa-as opposed to BPH-derived cultures. Some α-SMA-positive cells retained the expression of basal cytokeratin K14. This population was defined as myoepithelial cells and was associated with senescent cultures. The percentage of MFs was higher in high-grade compared to moderate-and low-grade PCa-derived cultures, whereas the number of myoepithelial cells was lower in high-grade compared to moderate-and low-grade PCa-derived cultures. In addition, we analyzed the expression of p75NTR, as well as the expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of MMPs (TIMPs). p75NTR expression was elevated in the stromal cultures derived from PCa compared to those derived from BPH and in cultures derived from cases with Gleason scores.7 compared to those derived from cases with Gleason scores <7, as well as in cultures with a high concentration of MFs compared to those with a high concentration of fibroblasts. MMP-2 was secreted by all primary cultures, whereas MMP-9 secretion was observed only in some PCa-derived stromal cells, when the percentage of MFs was significantly higher compared to BPH-derived cultures. TIMP1, TIMP2 and TIMP3 were secreted in elevated amounts in the BPH-compared to the PCa-derived stromal cultures, suggesting the differential regulation of extracellular matrix (ECM) degradation. When we used 22rv1 and PC3 PCa xenograft models for the isolation and characterization of murine cancer-associated fibroblasts (CAFs) we noted that the angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, vimentin, tenascin, calponin, desmin and Masson's trichrome. In conclusion, MF stromal cells from PCa participate in the progression and metastasis of PCa, modualting inflammation, angiogenesis and epithelial cancer cell proliferation

Lung volumes of extreme breath-hold divers

Achievements in breath-hold diving depend, amongst others, on body oxygen stores at start of dive. A diver with very high lung volumes could increase dive's duration, and attain deeper depths for a given speed. Thus, we hypothesized that extreme breath-hold divers have very high lung volumes. On eight extreme breathhold divers (age 35 + 4 years, height 179 + 7 cm, body mass 76 + 6 kg) and 9 non-diving controls (age 37 + 6 years, height 177 + 4 cm, body mass 81 + 9 kg) residual volume, vital capacity and total lung capacity (TLC) were measured with a body plethysmograph. Forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) were measured with a spirometer. Peak expiratory flow and flow-volume loops were measured with a pneumotachograph. In divers, but not in controls, volumes and capacities were systematically and significantly (p<0.01, paired t-test) higher than predicted from their body size. Consistently, volumes and capacities were significantly higher in divers than in controls, except for residual volume. Divers' TLC was 22% higher than predicted, and 21% higher than in controls. All divers' TLC was higher than 8 L, two had it higher than 9 L. FVC and FEV1 were significantly higher in divers than in controls. The FEV1/FVC ratio was the same in both groups. We conclude that extreme breath-hold divers may constitute a niche population with physiological characteristics different from those of normal individuals, facilitating the achievement of excellent diving performance

Increased levels of DNA methyltransferases are associated with the tumorigenic capacity of prostate cancer cells

DNA methylation might be the earliest somatic genome changes in prostate cancer that also play an important role in the process of tumor invasion, growth and metastasis. In recent years, several inhibitors of DNA methyltransferases (DNMTis) have been developed and evaluated in pre-clinical models and in clinical trials. While these compounds are effective in the treatment of hematological conditions, clinical trials in solid tumors and in prostate cancer have shown limited or no efficacy. This may be attributed to inappropriate dose regimens leading to toxicity-related adverse events. As with other anti-target compounds, one of the obstacles encountered with DNMTis in prostate cancer could be the inability to select patients for the clinical studies as well as the inability to monitor the efficacy of the drug if not the conclusion of the study. Primary cultures derived from human prostatic tissues harvested from patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) as well as neoplastic and non-neoplastic prostate cell lines were tested for DNMT expression/activity and to monitor azacitidine molecular efficacy. We observed that in primary cultures the levels of DNMT activity as well as the protein levels of DNMT1, DNMT3a and DNMT3b were higher in cultures derived from PCa compared to BPH tissue samples and significantly higher in cultures derived from PCa with Gleason scores ≥7 compared to those observed in cultures derived from Gleason scores <7. In addition, DNMT activity as well as DNMT1, DNMT3a and DNMT3b levels were higher in PCa cell lines compared to their non-neoplastic counterparts. Although DNMT activity was higher in high tumorigenic/aggressive PCa cell lines compared to low tumorigenic/aggressive cell lines, only the levels of DNMT3a and DNMT3b were significantly higher in the first group of cells, suggesting that DNMT1 activity is related to the transition to non-neoplastic versus neoplastic phenotype whereas the de novo methylation enzymes were mainly related to progression. Nevertheless, the comparison in the more aggressive PC3 cell derivatives (PC3-LN4 cells) also possessed higher levels of DNMT1 compared to PC3 and PC3M from which these cells were derived. Collectively, our results confirm previous data on the increased methylation in more aggressive tumors supporting the use of DNMTis in advanced prostate cancer. In addition, since glutathione S-transferase-π (GSTP1) was re-expressed or its protein levels were increased after treatment with non-toxic azacitidine doses and since GSTP1 can easily be measured in patient sera, the monitoring of this protein may aide in the evaluation of therapy in future clinical trials

Epididymal adrenal rest in an orchiectomy specimen with seminoma

Ectopic adrenal tissue is rare but is reported in various locations within the urogenital tract and abdominal structures. The vast majority of adrenal rests represent incidental findings in surgical specimens; thus, their incidence is unknown.1&nbsp;Notwithstanding, the results of reports on their higher frequency in infants than adults and sex distribution are conflicting.1,2&nbsp;In male subjects, the paratesticular and inguinal regions represent common sites of ectopic adrenal tissue, given the intimate embryologic relationship between the gonad and the adrenal cortex.3&nbsp;In testis and paratestis, they are also known as Marchand rest and are most commonly found in the spermatic cord,3&nbsp;followed by testis4&nbsp;and epididymis.5,6&nbsp;In these anatomic locations, ectopic adrenals may be associated with undescended testis, inguinal hernia, epididymal abnormality, and spermatic cord torsion, but none represent a predisposing factor. Also, the association with malignant testicular neoplasms merely represents a matter of chance. As a rule, adrenal rests do not show significant clinical implications. However, they may undergo hyperplasia when the function of the main adrenals is deficient or in congenital adrenal hyperplasia (CAH), an autosomal recessive disease with increased ACTH levels.5,6&nbsp;Also, adrenal rests may be accidentally removed during surgery, leading to adrenal insufficiency. Finally, ectopic adrenal may harbor benign or malignant tumors resulting in clinically evident dysfunctions.3&nbsp;The adrenal rests comprise nodules ranging between 1 mm and 1 cm, appearing as encapsulated or well-circumscribed round yellowish masses that may be multiple or bilateral. Microscopic appearance reminds normal adrenal cortex, often arranged in different zones, without medullary tissue.Figure 1&nbsp;refers to a case of a tiny adrenal rest nodule incidentally observed in an orchiectomy specimen obtained from a 40-year-old man affected by a suspect germ cell tumor of the right testis. The surgical specimen´s gross examination depicted a yellowish-brown nodule measuring 2 mm in its longest axis, located under the visceral mesothelium of the tunica vaginalis near the head of the epididymis (Figure 1A).Figure 1A -&nbsp;Gross orchiectomy specimen displaying a tiny nodule yellowish-brown in color (arrowhead) below the visceral layer of the tunica vaginalis and close to the head of the epididymis (scale bar= 3 cm);&nbsp;B -&nbsp;Encapsulated adrenal rest located between epididymis and rete testis;&nbsp;C -&nbsp;Encapsulated adrenal cortical tissue;&nbsp;D -&nbsp;Immunohistochemical positivity for Melan-A.:&nbsp;Microscopical examination diagnosed a pure testicular seminoma infiltrating the albuginea and the visceral part of the tunica vaginalis (pT2). Histology showed a well-encapsulated nodule between the epididymis head and the rete testis (Figure 1B). The nodule was composed of epithelial cells arranged into an organoid pattern consistent with the adrenal cortex (Figure 1C). No adrenal medullary tissue was present. The cells were immunohistochemically positive for Melan-A monoclonal antibody (clone A103) (Figure 1D). No immunostaining was observed for inhibin α (clone R1), calretinin (clone DAK-Calret 1), and BCL2 oncoprotein (clone 124)

Multicentric Molecular and Pathologic Study On Canine Adenovirus Type 1 in Red Foxes (Vulpes vulpes) in Three European Countries

Canine adenovirus type 1 (CAdV-1) is the agent of infectious canine hepatitis, a severe frequently fatal disease affecting primarily dogs (Canis lupus familiaris). The virus has been detected in many wild carnivore species. Our aim was to evaluate the prevalence and genetic and histopathologic features of CAdV-1 in wild red foxes (Vulpes vulpes). Kidney and liver samples were obtained from 86 subjects, coming from the UK (n\ubc21), Italy (n\ubc36), and Germany (n\ubc29). We used PCR, targeting the viral E3 gene and flanked regions, to detect the presence of the virus; viral E3, fiber, and E4 genes were sequenced and their sequences were compared with published sequences. Kidneys and liver from foxes in Italy and Great Britain (n\ubc57) were prepared for histologic and immunohistochemical examination for CAdV-1. Viral DNA was detected in 22% (19 of 86) kidney samples, with E3 and E4 genes showing reported and unreported single nucleotide changes. No pathologic changes or viral immunopositive signals were detected in the examined tissues. Our study suggests that red foxes could be considered potential shedders of CAdV-1, as they showed a relatively high prevalence without related pathologic changes in the organs examined

Towards a Framework for a New Research Ecosystem

A major gap exists between the conceptual suggestion of how much a nation should invest in science, innovation, and technology, and the practical implementation of what is done. We identify 4 critical challenges that must be address in order to develop an environment conducive to collaboration across organizations and governments, while also preserving commercial rewards for investors and innovators, in order to move towards a new Research Ecosystem.Comment: 20 pages, 1 table, 2 figure

Hepatitis E virus infection in wild rabbit (Oryctolagus cuniculus) in Italy and in the UK: a serological, molecular, and pathological study

A novel animal strain of HEV demonstrating zoonotic potential rabbit HEV (rHEV) has been recently identified in farmed rabbits in China. To investigate the presence of rHEV in wild rabbit population, serum and tissue samples were taken from 65 rabbits, 35 Italian and 30 from UK. Sera were submitted to a double antigen sandwich ELISA, whereas hepatic tissues and other organs to molecular and pathological investigations. Sixteen serum samples (24.6%) scored positive for anti-HEV antibodies, and six samples (9%) of hepatic tissue were positive for HEV RT-PCR, while the other organs were negative. Sequencing and phylogenetic analysis of HEV RNA-positive samples indicated that while two Italian rabbits were infected with strains closely related to wild boar and swine strains (HEV-3), the other four (2 Italian and 2 English) were clustered within rHEV clade. Megalocytosis and multifocal areas of hepatocytes degeneration and necrosis with a pericentrilobular pattern were observed in rHEV-infected rabbits. In those infected by the strain analogous to HEV-3, the lesions were mainly localized in the periportal areas and were less severe. In both cases, inflammatory infiltrates were predominantly composed of CD3-positive lymphocytes and a reduced number of macrophages. By immunohistochemistry, only liver samples taken from HEV RNA-positive rabbits scored positive for viral antigen. Our results indicated that HEV infection is present in rabbit population with different clades and is endemic in the Italian and English wild rabbit population, suggesting the possibility that this species may be infected with rHEV or swine HEV-3 strains

Molecular detection of tick-borne pathogens in wild red foxes (Vulpes vulpes) from Central Italy

Spleen samples from 153 red foxes, shot during regular hunting season in the province of Pisa (Central Italy), were examined to detect DNA of Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii, Francisella tularensis, Hepatozoon canis and Babesia sp./Theileria sp. DNA of vector-borne pathogens was detected in 120 (78.43%; 95% CI: 71.06–84.66%) foxes. Specifically, 75 (49%; 95% CI: 40.86–57.22%) animals scored PCR-positive per H. canis, 68 (44.44%; 95% CI: 36.42–52.69%) for E. canis, 35 (22.88%; 95% CI: 16.48–30.35%) for piroplasms (Theileria annae), 3 (1.96%; 95% CI: 0.41–5.62%) for C. burnetii and 1 (0.65%; 95% CI: 0.02–3.59%) for A. phagocytophilum. No positive reaction was observed for F. tularensis. Fifty-six animals (36.6%; 95% CI: 28.97–44.76%) were positive for two or three pathogens. Red foxes result to be involved in the cycle of vector-borne pathogens that are associated to disease in dogs and humans

Immunological signatures unveiled by integrative systems vaccinology characterization of dengue vaccination trials and natural infection

Introduction: Dengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs). Methods and results: We analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response. Conclusion: This work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5) for anti-dengue-specific therapies and effective vaccination development