Background and Aims: Tissues archived for long-term storage as formalin-fixed, paraffin-embedded (FFPE) samples represent rich source of material for genomic studies, but the nucleic acid isolation and downstream analysis is technically difficult. This is especially true when conducting mRNA expression studies, which strongly depend on the quality and quantity of the starting material for successful data normalization. Our objective was to investigate the mRNA expression levels in testicular FFPE samples using real-time PCR arrays and to present our experience with some technical challenges.

Methods: Total RNA was extracted from FFPE samples from six patients with hypospermatogenesis and three controls using the Qiagen AllPrep DNA/RNA FFPE Kit. The integrity of the isolated RNA was assessed by an Agilent Bioanalyzer 2100. Qiagen Human Male Infertility RT2 Profiler PCR Arrays were used to study the expression of 84 genes.

Results: Our experience with the analysis of the FFPE testicular samples using the RT2 Profiler PCR Array showed difficulties with the data normalization, despite using the same amount of starting material and similar values for the RNA integrity numbers (RINs) across all the samples, as recommended by the manufacturer. By using the percentage of the relatively intact RNA (area between 150 bp and 4,000 bp on the electropherograms of the Bioanalyzer 2100), we observed a negative correlation between amount of the intact RNA and average Ct values of the analyzed genes. Our initial results using PCR Array analysis on FFPE testicular tissues revealed 38 differentially expressed genes that are enriched in interactions, as well as for Gene Ontology molecular and cellular function terms.

Conclusions: Stratification of the FFPE samples according to their percentage of intact RNA could improve the quantitative real-time PCR Array analysis.

Acute viral hepatitis is usually a self-limiting illness. However, it can lead to complications that can be life-threatening, such as acute liver failure. Glucose 6 phosphate dehydrogenase (G6PD) deficiency in the setting of acute viral hepatitis can lead to a massive hemolysis, manifesting as acute kidney injury and markedly raised bilirubin levels; although cases are rare. Here, we report such a case. The patient had a viral hepatitis E infection and presented with kidney injury requiring dialysis. Examination showed very high mixed hyperbilirubinemia due to massive intravascular hemolysis. The patient experienced a long, protracted course of illness, requiring renal replacement therapy with other supportive management, which led to improvement over a period of four weeks. This case highlights the importance of recognizing associated hemolysis in a patient with viral hepatitis who presents with very high bilirubin levels or associated kidney injury. Such patients will require aggressive supportive care with prompt fluid and electrolyte management.

With the introduction of antiretroviral therapy (ART), a dramatic reduction in HIV-related morbidity and mortality has been observed. However, it is now becoming increasingly clear that liver-related complications, particularly rapid fibrosis development from ART as well as from the chronic HIV infection itself, are of serious concern to HIV patients. The pathophysiology of liver fibrosis in patients with HIV is a multifactorial process whereby persistent viral replication, and bacterial translocation lead to chronic immune activation and inflammation, which ART is unable to fully suppress, promoting production of fibrinogenic mediators and fibrosis. In addition, mitochondrial toxicity, triggered by both ART and HIV, contributes to intrahepatic damage, which is even more severe in patients co-infected with viral hepatitis. In recent years, new insights into the mechanisms of accelerated fibrosis and liver disease progression in HIV has been obtained, and these are detailed and discussed in this review.

Prostate cancer (PCa) is one of the most diagnosed cancers in American men and is a leading cause of cancer deaths in advanced forms of disease. Seventy to ninety percent of men who die of PCa will have developed bone metastases during the course of the disease, and these bone metastases cause severe pain that can lead to further skeletal complications. The standard treatment for PCa generally includes androgen deprivation therapy, radiation, and chemotherapy, either singly or in combination, depending on disease progression. These treatments are able to slow disease progression slightly, but an urgent need remains for a more curative approach. In addition to the adverse effects resulting from these treatments, androgen deprivation therapy can often lead to a castration-resistant form of the disease, which is even more difficult to treat and often becomes metastatic. In the past several years, new drugs that target androgens, the bone microenvironment, and other mechanisms involved in PCa have shown promise in clinical trials for advanced PCa; these include abiraterone, enzalutamide, sipuleucel-T, radium-223, denosumab, and cabazitaxel, and will be described in further detail in this review. The continuous improvement upon current therapies and development of new drugs is promising for the future of advanced PCa, and the authors will give their perspective on these different treatment approaches.

Isocitrate dehydrogenase (IDH) is a metabolic enzyme responsible for the enzymatic conversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the IDH gene result in a novel gain-of-function, with development of neomorphic enzymatic activity determining the pathological reduction of α-KG to (R)-2-hydroxyglutarate. The accumulation of this pathological metabolite (onco-metabolite) in cancer cells is, to a large extent, responsible for the development of several cancers, including acute myeloid leukemia (AML), low-grade gliomas (LGGs) or chondrocytic tumors. Furthermore, various experimental studies have shown that IDH mutations represent an early, driver event, conserved during tumor progression in neoplasias such AML and LGG. Given all these observations, potent and selective IDH inhibitors have been developed and are currently under investigation in phase I/II clinical studies. In particular, AG-221, a first-in-class inhibitor of mutant IDH2, was tested in hematological patients with refractory/relapsing AML or myelodysplasia and showed an overall response rate of 59/159 (37%), as well as a good safety profile. Similarly, AG-120, an inhibitor of mutant IDH1, was tested in 66 relapsing/refractory AML patients and showed an overall response rate of 36%, with a complete response rate of 16%. A new IDH inhibitor, AG-811 displayed the capacity to inhibit both mutants IDH1/2 and to penetrate the blood: brain barrier, a property that would be suitable for treatment of glioma patients. On the other hand, additional observations have suggested that IDH-mutant AMLs are sensitive to treatment with BCL-2 inhibitors and to the differentiative induction with all-trans retinoic acid. In conclusion, the collective studies carried out in recent years on the characterization of IDH-mutant tumors highlight an admirable paradigm of the virtuosic transfer from basic research (with improvements in our understanding of the physio-pathological role played by IDH mutations in the development of some tumors) to clinical studies (with the development of selective, potent and clinically-active IDH inhibitors).

Hematopoietic stem cell (HSC) transplantation is the most effective therapeutic modality for certain malignant and non-malignant diseases. Although umbilical cord blood can be a source of HSCs, the harvest from a single cord is insufficient for transplantation. Protocols have been developed to overcome this problem, but in vivo expansion of transplanted HSCs would be most desirable. Serotonin is a monoamine neurotransmitter that can increase the ex vivo expansion of both HSCs and bone marrow stromal cells (BMSCs). Selective serotonin reuptake inhibitors (SSRIs) increase the free serotonin concentration by binding to the serotonin transporter (SERT), thereby preventing reuptake by pre-synaptic cells. Studies have shown that SSRIs also have immunosuppressive effects. We hypothesized that the immunosuppressive effects of SSRIs may be due to the expansion of BMSCs, which can result in a decrease in the incidence of graft versus host disease (GVHD). Finally SSRIs may promote efficacy of transplantation by increasing the in vivo expansion of HSCs and decreasing GVHD incidence.

The role of Helicobacter pylori in the pathogenesis of irritable bowel syndrome (IBS) was investigated in 38 subjects, including 16 males (42%) and 22 females (58%), all of who had been diagnosed with IBS but showed no improvement in response to treatment.

Colonoscopy, biopsy and rapid urease test (CLO-test).

Nineteen patients were H. pylori-positive (50%) and 19 were H. pylori-negative (50%). One patient in this study previously presented colonic carcinoma.

All H. pylori-positive patients received treatment and showed improvement, including the patient with colonic carcinoma. Initially, the carcinoma presented 99% of stenosis, but after one month of treatment the stenosis had decreased to 50%. As a result of this investigation, we consider that the presence of H. pylori in the colonic mucosa is another important co-factor in the pathogenesis of IBS that should also be considered in the development of colonic carcinoma.

E-PodoFavalin-15999 (Atremorine®) is a novel bioproduct obtained by means of non-denaturing biotechnological procedures from structural components of the Vicia faba L. plant, for the prevention and treatment of Parkinsonian disorders. Preclinical studies have revealed that Atremorine is a powerful neuroprotectant with specific activity on dopaminergic neurons, reversing neurodegeneration and improving motor function in animal models of Parkinson’s disease (PD). Clinical studies indicate that Atremorine is a powerful catecholaminergic enhancer with time- and genotype-dependent effects in PD. In the present study, we investigated the effects of Atremorine on the levels of neurotransmitters (dopamine, adrenaline, noradrenaline, serotonin) and hormones (adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin (PRL), follicle-stimulating hormone (FSH), luteinizing hormone (LH), cortisol, estrogens, testosterone) in Parkinsonian patients (n=119) 1 hour after Atremorine administration (5 g/day). These effects were also studied after stratification of PD patients according to their cardiovascular function. Atremorine induced a significant increase in the levels of dopamine (p<0.001), noradrenaline (p=0.004) and adrenaline (p=0.01), and a significant decrease in the levels of PRL (p<0.001), cortisol (p<0.001), and GH (p=0.002), with no apparent changes in the levels of serotonin, ACTH, FSH, LH, testosterone or estrogen. The levels of dopamine were significantly higher in patients with normal EKG than in patients with abnormal electrocardiogram (EKG); however, the levels of adrenaline, noradrenaline and serotonin tended to be lower in patients with normal EKG as compared to patients with abnormal EKG. There were also some differences in hormonal levels in patients with normal EKG, compared to abnormal EKG. These results clearly show that Atremorine is an effective enhancer of catecholaminergic neurotransmission, which contributes to optimization of hormonal regulation in PD.

Hepatitis C virus (HCV) infection remains a significant medical concern in the United States and around the world. It is still one of the leading causes of chronic liver disease, and, for more than 20 years, there has been little progress in the treatment of HCV infection. The advent of direct-acting antivirals (DAAs) initiated the era of high efficacy and well-tolerated medications with high cure rates. The efficacy of these medications has prompted many professional societies around the world to update their treatment guidelines to include DAAs as first-line treatment. Guidelines by the American Association for the Study of Liver Disease/Infectious Disease Society of America, World Health Organization, Asian-Pacific Association for the Study of Liver and the European Association for the Study of Liver have all incorporated DAAs into their treatment guidelines. Despite the promising data supporting these medications, however, their cost represents a limiting factor to their use, even though studies have shown DAAs to be cost-effective. In addition to the expense of these medications and limited resources, there are many barriers preventing patients from receiving this potentially life-saving treatment. In order to overcome these barriers, these issues need to be recognized and addressed.

Hepatitis C virus (HCV) infection is a growing public health concern, with 184 million people infected worldwide. During the past decade, interferon has been the backbone of HCV treatment, even though it remains far from ideal. The latest development of the new direct antivirals has drastically changed the treatment approach for chronic hepatitis C (CHC). Inhibitors of the HCV NS5A region have garnered remarkable interest among treating physicians, due to their high potency and favourable safety profile. In particular, treatment with daclatasvir (DCV) has yielded high rates of vriologic response in patients infected with genotype (Gt) 1 and Gt 3, when used in combination with other antivirals of a different class, such as sofosbuvir. Although few data are available for DCV treatment of the other Gts, the results in patients with Gt 2 and Gt 4 infection appear promising, as do those for unique patient populations. NS5A-resistant viral variants can pre-exist or emerge after treatment failure for the HCV NS5A inhibitors. Nonetheless, DCV-resistant viral variants continue to be sensitive to interferon and other classes of antivirals such as NS3/4A and NS5B inhibitors. Herein, we aimed to provide an overview of the current knowledge about DCV in the treatment of CHC.

Use of nucleoside and nucleotide analogues to treat patients infected with hepatitis B virus (HBV) has been found to be associated with mutations in the polymerase gene of the virus. The current study was carried out in HBV-related hepatocellular carcinoma (HCC) cases to trace the presence of drug-related mutants. A total of 75 HBV-related HCC cases were included for the study as per Bruix et al., 2001 EASL guidelines. HBV viral DNA was isolated by the previously standardized manual phenol-chloroform methods. The 3.2 kb genome of HBV was amplified by six sets of overlapping primers. The amplicons were sequenced commercially [Macrogen, South Korea (ABI PRISM)]. Sequences for the polymerase gene were analyzed using commercial bioinformatics software (http://www.hepseq.org/Public/Tool/annotator_tool.php ). The different drug-resistant mutations detected were confirmed twice, ahead of reporting. Four drug-resistant mutations were detected in total: L80I (lamivudine), N236T (adefovir), I169T (entecavir) and A181V (lamivudine/adefovir). Interestingly, all four of the drug-resistant mutants were found in genotype D of HBV. The low number (only four) of drug-resistant mutations detected in this study population can be attributed to the fact that most of the cases were not treated and presented late. This study’s findings confirm the presence of previously reported drug-resistant mutations in the HBV genome infecting Indian patients; however, its associations with late stage disease and with the virus genotype D, in particular, need to be further studied in a larger population.

Therapy for human immunodeficiency virus (HIV) and chronic hepatitis C has evolved over the past decade, resulting in better control of infection and clinical outcomes; however, drug-drug interactions remain a significant hazard. Joint recommendations from the American Association for the Study of Liver Diseases and the Infectious Diseases Society of America regarding drug-drug interactions between HIV antiretroviral agents and direct-acting antiviral agents for treatment of hepatitis C virus (HCV) infection are reviewed here. This review is oriented to facilitate appropriate selection of an antiviral therapy regimen for HCV infection based on the choice of antiretroviral therapy being administered and, if necessary, switching antiretroviral regimens.

Chronic hepatitis B is a worldwide disease, with significant burden on health care systems. While universal vaccination programs have led to an overall decrease in incidence of transmission of hepatitis B, unfortunately, there remain large areas in the world where vaccination against hepatitis B is not practiced. In addition, vertical transmission of hepatitis B persists as a major concern. Hepatitis B treatment of the pregnant patient requires a thorough assessment of disease activity and close monitoring for flares, regardless of initiation of antiviral therapy. We discuss, in this article, the current and emergent strategies which aim to reduce the rate of transmission of hepatitis B from the pregnant mother to the infant and we review the updated guidelines regarding management of liver disease in pregnant women with hepatitis B.

Viral hepatitis remains a significant worldwide threat, in spite of the availability of several successful therapeutic and vaccination strategies. Complications associated with acute and chronic infections, such as liver failure, cirrhosis and hepatocellular carcinoma, are the cause of considerable morbidity and mortality. Given the significant burden on the healthcare system caused by viral hepatitis, it is essential that novel, more effective therapeutics be developed. The present review attempts to summarize the current treatments against viral hepatitis, and provides an outline for upcoming, promising new therapeutics. Development of novel therapeutics requires an understanding of the viral life cycles and viral effectors in molecular detail. As such, this review also discusses virally-encoded effectors, found to be essential for virus survival and replication in the host milieu, which may be utilized as potential candidates for development of alternative therapies in the future.

Background and Aims: Utilization of living donor liver transplantation (LDLT) and its relationship with recipient Model for End-Stage Liver Disease (MELD) needs further evaluation in the United States (U.S.). We evaluated the association between recipient MELD score at the time of surgery and survival following LDLT.

Methods: All U.S. adult LDLT recipients with MELD < 25 were evaluated using the 1995–2012 United Network for Organ Sharing registry. Survival following LDLT was stratified into three MELD categories (MELD < 15 vs. MELD 15–19 vs. MELD 20–24) and evaluated using Kaplan-Meier methods and multivariate Cox proportional hazards models.

Results: Overall, 2,258 patients underwent LDLT. Compared to patients with MELD < 15, overall 5-year survival following LDLT was similar among patients with MELD 15–19 (80.9% vs. 80.3%, p = 0.77) and MELD 20–24 (81.2% vs. 80.3%, p = 0.73). When compared to patients with MELD < 15, there was no significant difference in long-term post-LDLT survival among those with MELD 15–19 (HR: 1.11, 95% CI: 0.85−1.45, p = 0.45) and a non-significant trend towards lower survival in patients with MELD 20–24 (HR: 1.28, 95% CI: 0.91−1.81, p = 0.16). Only 14% of LDLTs were performed in patients with MELD 20–24 and the remaining 86% in patients with MELD < 20.

Conclusion: LDLT is underutilized in patients with MELD 20 and higher.

Folate is a UV-labile vitamin, critical to maintenance of DNA synthesis and repair mechanisms. Evidence supports a number of folate-dependent enzymes as being UV-responsive, suggesting that the activity of pathways operating through such enzymes may differ between geographic areas of varied UV exposure. Therefore, we explored the possible association of global UV levels with the prevalence of common polymorphisms in folate-metabolism enzymes.

Data from the PubMed electronic database was collated for the polymorphisms MTHFR-C677T, MTHFR-A1298C, cSHMT-C1420T, TYMS 28bp 2R>3R, TYMS 3′UTR ins/del and DHFR 19bp deletion. Data extracted were analysed by the latitude of the study locations, as a surrogate measure of population UV exposure.

Frequency of the MTHFR-C677T and MTHFR-A1298C polymorphisms was positively associated with latitude, while a negative association was observed between latitude and frequency of the cSHMT-C1420T and TYMS 28bp 2R>3R variants.

These findings provide novel evidence suggestive of folate genotypes being selected to maintain homeostasis between folate-dependent de novo thymidylate synthesis and methylation pathways in environments of differing UV levels. To the authors’ knowledge, this is the first study to report significant associations between latitude and the occurrence of MTHFR-A1298C, TYMS 28bp 2R>3R and cSHMT-C1420T polymorphisms. On-going studies are required to further explore the biological significance of these findings.