Ceramide Suppresses Influenza A Virus Replication In Vitro.

Annual influenza outbreaks are associated with significant morbidity and mortality worldwide despite the availability of seasonal vaccines. Influenza pathogenesis depends on the manipulation of host cell signaling to promote virus replication. Ceramide is a sphingosine-derived lipid that regulates diverse cellular processes. Studies highlighted the differential role of ceramide de novo biosynthesis on the propagation of various viruses. Whether ceramide plays, a role in influenza virus replication is not known. In this study, we assessed the potential interplay between the influenza A (IAV) and ceramide biosynthesis pathways. The accumulation of ceramide in human lung epithelial cells infected with influenza A/H1N1 virus strains was evaluated using thin-layer chromatography and/or confocal microscopy. Virus replication was assessed upon the regulation of the de novo ceramide biosynthesis pathway. A significant increase in ceramide accumulation was observed in cells infected with IAV in a dose- and time-dependent manner. Inoculating the cells with UV-inactivated IAV did not result in ceramide accumulation in the cells, suggesting that the induction of ceramide required an active virus replication. Inhibiting de novo ceramide significantly decreased ceramide accumulation and enhanced virus replication. The addition of exogenous C6-ceramide prior to infection mediated an increase in cellular ceramide levels and significantly attenuated IAV replication and reduced viral titers (≈1 log10 PFU/ml unit). Therefore, our data demonstrate that ceramide accumulation through de novo biosynthesis pathway plays a protective and antiviral role against IAV infection. These findings propose new avenues for development of antiviral molecules and strategies.IMPORTANCE Understanding the effect of sphingolipid metabolism on viral pathogenesis provide important insights into the development of therapeutic strategies against microbial infections. In this study, we demonstrate a critical role of ceramide during influenza A virus infection. We demonstrate that ceramide produced through de novo biosynthesis possess an antiviral role. These observations unlock new opportunities for the development of novel antiviral therapies against influenza.

The transcriptome of human mammary epithelial cells infected with the HCMV-DB strain displays oncogenic traits.

Increasing evidence indicates that human cytomegalovirus (HCMV) populations under the influence of host environment, can either be stable or rapidly differentiating, leading to tissue compartment colonization. We isolated previously from a 30-years old pregnant woman, a clinical isolate of HCMV, that we refered to as the HCMV-DB strain (accession number KT959235). The HCMV-DB clinical isolate demonstrated its ability to infect primary macrophages and to upregulate the proto-oncogene Bcl-3. We observed in this study that the genome of HCMV-DB strain is close to the genomes of other primary clinical isolates including the Toledo and the JP strains with the later having been isolated from a glandular tissue, the prostate. Using a phylogenetic analysis to compare the genes involved in virus entry, we observed that the HCMV-DB strain is close to the HCMV strain Merlin, the prototype HCMV strain. HCMV-DB infects human mammary epithelial cells (HMECs) which in turn display a ER-/PR-/HER2- phenotype, commonly refered to as triple negative. The transcriptome of HCMV-DB-infected HMECs presents the characteristics of a pro-oncogenic cellular environment with upregulated expression of numerous oncogenes, enhanced activation of pro-survival genes, and upregulated markers of cell proliferation, stemcellness and epithelial mesenchymal transition (EMT) that was confirmed by enhanced cellular proliferation and tumorsphere formation in vitro. Taken together our data indicate that some clinical isolates could be well adapted to the mammary tissue environment, as it is the case for the HCMV-DB strain. This could influence the viral fitness, ultimately leading to breast cancer development.

In vitro anticancer activity of new gold(III) porphyrin complexes in colon cancer cells.

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. The limitations of cisplatin-based chemotherapy have prompted intense interest among scientists to search for alternative metal-based anticancer medicines. Gold(III) complexes have been among the most widely investigated since they showed higher cytotoxicity than cisplatin and promising in vitro and in vivo anticancer activities in CRC but their clinical usefulness has been limited by their poor stability under physiological conditions. A novel gold(III) porphyrin complexes [gold(III) porphyrin-adamantane chloride (SN1) and gold(III) porphyrin mono-acetate chloride (SN2)] with improved aqueous stability were synthesized. SN1 and SN2 reduced the survival of human CRC HT-29 and HCT-116 cell lines, caused cell cycle arrest in G2/M phase, and we observed downregulation of the expression of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) along with up-regulation of the active form of p53, p21 and Bcl-2-associated X (Bax). Furthermore, SN1 and SN2 induced apoptosis by the intrinsic pathway, since they lead to the cleavage of caspase 9, caspase 3 and poly(ADP-ribose) polymerase (PARP), and up-regulating Bax. Phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), nuclear factor-κB (NF-κB) and extracellular signal-regulated kinases (ERK) are important for cell survival and proliferation. SN1 and SN2 lead to decrease in the activity of Akt where the phosphorylated form decreased with time as well as they caused an important decrease in the phosphorylation of ERK and activity of NF-κB. Finally, SN1 and SN2 complexes affected p38/mitogen-activated protein kinase (MAPK) pathway then we recorded an increase in the cyclooxygenase-2 expression and its enzymatic product prostaglandin E2.

Expression of eukaryotic initiation factor 4E and 4E binding protein 1 in colorectal carcinogenesis.

Cap dependent translation is mainly regulated at the level of the eukaryotic initiation factor 4E (eIF4E), the activity of which is controlled by phosphorylation and sequestration by its well established regulator, 4E binding protein 1 (4E-BP1). Both eIF4E and 4E-BP1 have been shown to be involved in the malignant progression of multiple human cancers, including colorectal cancer. However, the data on determining the expression of eIF4E, 4E-BP1 and their phosphorylated forms simultaneously in a single patient with colorectal cancer is lacking. Therefore the aim of our study was to explore the roles of these factors in colorectal carcinogenesis by immunohistostaining colorectal tissues (normal, low grade adenoma, high grade adenoma, and adenocarcinoma). Our results showed that the expression levels of eIF4E increased steadily as the cancer progressed from the case of benign dysplasia to an adenocarcinoma; all the while maintaining an unphosphorylated form. On the other hand, total expression levels of 4E-BP1 increased only in the premalignant state of the disease and decreased (but highly phosphorylated or inactivated) or abolished upon malignancy. Taken together, our findings suggest that strong correlations exist between the expression of eIF4E (not p-eIF4E) and tumor grade providing evidence that eIF4E expression plays a pivotal role in the malignant progression of colorectal cancer. Moreover, 4E-BP1 showed a bi-phasic level of expression during carcinogenesis, which is expressed only in hyperplasic or dysplastic tissues as an endogenous tumor suppressor molecule.

Mycobacterium tuberculosis spoligotypes circulating in the Syrian population: A retrospective study.

OBJECTIVES: To characterize by spoligotyping clinical isolates of Mycobacterium tuberculosis (MTB) collected between July 2003 and October 2005 from all Syrian provinces (muhafazat). METHODS: All isolates (n=96) were cultured and identified by biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. RESULTS: Twelve patterns were identified: 46.8% of the strains belonged to T 1 family; 20.8% to LAM 9; 10.4% to CAS; 9.3% to Haarlem 3; 4.1% to Haarlem 1; 2.1% to Family 34; and 1% to each of Family 36, EAI 5, LAM 1, LAM 8, T 3, and X 3 families. The noticeable absence of the Beijing family was not consistent with the patterns reported in most neighboring countries. CONCLUSION: A more inclusive study of the Syrian population is necessary to more accurately identify most of the prevailing families in the country.

High frequency of sex chromosomal disomy in spermatozoa of Lebanese infertile men.

In Lebanon, assisted reproductive techniques (ART) are widely used to overcome infertility, but the genetic risk associated with these techniques is still ignored. In this study, in order to estimate the transmission risk of paternal chromosomal anomalies to ART offspring, the meiotic segregation of chromosomes X, Y, 18, and 21 was analyzed by fluorescent in situ hybridization on the spermatozoa of 19 Lebanese infertile men. Our results show significantly higher frequencies of sex chromosome disomies in the group of patients with oligozoospermia compared with a control group of fertile males. Interestingly, the sex chromosome aneuploidy rates were highly variable between oligozoospermic patients, and ranged between 0.9% and 12.87%. No significant increase in aneuploidy rates was found for the group of nonoligozoospermic patients with asthenozoospermia and/or teratozoospermia. In addition, the disomy rate for chromosome 21 was analyzed in 8 patients, in whom higher disomy rates were shown as compared with the controls. Altogether, the results suggest that Lebanese oligozoospermic men undergoing ART may have an increased risk of transmitting sex chromosome anomalies to their offspring, as well as, in some cases, trisomy 21. Based on this work, genetic counseling programs for Lebanese infertile couples undergoing ART procedures should be developed, in order to improve the investigation and selection of Lebanese infertile couple candidates for ART procedures and optimize the choice of ART techniques.

Inflammatory bowel disease in rats: bacterial and chemical interaction.

AIM: To develop a novel model of colitis in rats, using a combination of iodoacetamide and enteropathogenic E. coli (EPEC), and to elucidate the pathophysiologic processes implicated in the development of ulcerative colitis (UC). METHODS: Male Sprague-Dawley rats (n = 158) were inoculated intrarectally on a weekly basis with 4 different combinations: (a) 1% methylcellulose (MC), (b) 100 microL of 6% iodoacetamide (IA) in 1% MC, (c) 200 microL containing 4 x 10(8) colony factor units (CFU) of EPEC, and (d) combined treatment of (IA) followed by bacteria (B) after 2 d. Thirty days post treatment, each of the four groups was divided into two subgroups; the inoculation was stopped for one subgroup and the other subgroup continued with biweekly inoculation until the end of the experiment. Colitis was evaluated by the clinical course of the disease, the macroscopic and microscopic alterations, activity of myeloperoxidase (MPO), and by TNF-alpha gene expression. RESULTS: Findings indicative of UC were seen in the combined treatment (IA + B) as well as the IA continued treatment groups: the animals showed slow rate of increase in body weight, diarrhea, bloody stools, high colonic ulcer score, as well as histological alterations characteristic of UC, with an extensive inflammatory reaction. During the course of the experiment, the MPO activity was consistently elevated and the TNF-alpha gene expression was upregulated compared to the control animals. CONCLUSION: The experimental ulcerative colitis model used in the present study resembles, to a great extent, the human disease. It is reproducible with characteristics indicative of chronicity.