Expression of RFC/SLC19A1 is associated with tumor type in bladder cancer patients

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.     

Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs)

Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families.     

DNA methyltransferase 3B (DNMT3B − 579 G > T) promotor polymorphism and the susceptibility to pediatric immune thrombocytopenic purpura in Egypt

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction. Although the etiology of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. The present study aimed at exploring a novel molecular determinant that may influence the susceptibility and course of ITP in Egyptian children. To achieve our aim, genotyping of DNMT3B − 579 G > T promotor polymorphism by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The current study was conducted on 140 ITP patients and 150 age and gender matched healthy controls. The results obtained revealed that DNMT3B − 579 TT homotype was significantly higher in ITP patients and conferred almost three fold increased risk of ITP (OR = 3.16, 95%CI = 1.73–5.79). There was no statistically significant difference between ITP patients with wild or mutant genotypes as regards their clinical or laboratory data. Furthermore, there was no statistical difference in the distribution of DNMT3B − 579 G > T genotypes between acute and chronic ITP patients. In conclusion, DNMT3B − 579 G > T promotor polymorphism represents a novel genetic risk factor for ITP but not a predictor for tendency to chronicity in pediatric ITP in Egypt.     

Performance of biomarkers FibroTest, ActiTest, SteatoTest, and NashTest in patients with severe obesity: meta analysis of individual patient data

Background

Liver biopsy is considered as the gold standard for assessing non-alcoholic fatty liver disease (NAFLD) histologic lesions in patients with severe obesity. The aim of this study was to perform an overview of 3 studies which assessed the performance of non-invasive markers of fibrosis (FibroTest), steatosis (SteatoTest) and steato-hepatitis (NashTest, ActiTest) in these patients.

Methods

494 patients with interpretable biopsy and biomarkers using of three prospective cohorts of patients with severe obesity (BMI >35 kg/m2) were included. Histology (NAS score) and the biochemical measurements were blinded to any other characteristics. The area under the ROC curves (AUROC), sensitivity, specificity, positive and negative predictive values were assessed. Weighted AUROC (wAUROC Obuchowski method) was used to prevent multiple testing and spectrum effect. Two meta-analyses were performed; one used the individual patient, and the other a classical meta-analysis.

Results

Prevalence of advanced fibrosis (bridging) was 9.9%, advanced steatosis (>33%) 54.2%, and steato-hepatitis (NAS score >4) 17.2%. The mean wAUROCs were: FibroTest for advanced fibrosis (95%CI; significance)  =  0.85 (0.83–0.87; P<0.0001); SteatoTest for advanced steatosis = 0.80 (0.79–0.83); and ActiTest for steato-hepatitis = 0.84 (0.82–0.86; P<0.0001). Using the classical meta-analysis (random effect model) the mean AUROCs were: FibroTest = 0.72 (0.63–0.79; P<0.0001); SteatoTest = 0.71 (0.66–0.75; P<0.0001); and ActiTest = 0.74 (0.68–0.79; P<0.0001). Despite more metabolic risk factors in one cohort, results were similar according to gender, presence of diabetes and between the 3 cohorts.

Conclusion

In patients with severe obesity, a significant diagnostic performance of FibroTest, SteatoTest and ActiTest was observed for liver lesions.     

Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.     

Phenotypic characterization of cells participating in transport of prion protein aggregates across the intestinal mucosa of sheep

The oral route is considered to be the main entry site of several transmissible spongiform encephalopathies or prion diseases of animals and man. Following natural and experimental oral exposure to scrapie, sheep first accumulate disease associated prion protein (PrP^d) in Peyer’s patch (PP) lymphoid follicles. In this study, recombinant ovine prion protein (rPrP) was inoculated into gut loops of young lambs and the transportation across the intestinal wall studied. In particular, the immunohistochemical phenotypes of cells bearing the inoculated prion protein were investigated. The rPrP was shown to be transported across the villi of the gut, into the lacteals and submucosal lymphatics, mimicking the transport route of PrP^d from scrapie brain inoculum observed in a previous intestinal loop experiment. The cells bearing the inoculated rPrP were mainly mononuclear cells, and multicolor immunofluorescence procedures were used to show that the rPrP bearing cells were professional antigen presenting cells expressing Major histocompatibility complex II (MHCII). In addition, the rPrP bearing cells labeled with CD205, CD11b and the macrophage marker CD68, and not with the dendritic cell markers CD11c and CD209. Others have reported that cells expressing CD205 and CD11b in the absence of CD11c have been shown to induce T cell tolerance or regulatory T cells. Based on this association, it was speculated that the rPrP and by extension PrP^d and scrapie infective material may exploit the physiological process of macromolecular uptake across the gut, and that this route of entry may have implications for immune surveillance.     

Hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza A virus

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.     

Immunological and parasitological parameters after treatment with dexamethasone in murine Schistosoma mansoni

This work aimed to evaluate the effect of diphenyl dimethyl bicarboxylate (DDB) and dexamethasone alone and in combination with praziquantel on various parasitological, immunological and pathological parameters reflecting disease severity and morbidity in murine schistosomiasis. DDB and dexamethasone had no effect on worm burden but altered tissue egg distribution. This indicates that, under the schedule used, neither drug interfered with the development of adult worms or oviposition, but both can modulate liver pathology. Dexamethasone resulted in a greater reduction in granuloma size than did DDB. Dexamethasone-treated mice also showed lower levels of serum gamma interferon (IFN-γ), interleukin-12 (IL-12) and IL-4, together with higher IL-10 levels, than infected untreated control animals. These data suggest that dexamethasone is a convenient and promising coadjuvant agent that results in decreased morbidity in murine schistosomiasis.