Ana María Rivas-Estilla

Identification of viral infections in the prostate and evaluation of their association with cancer

BackgroundSeveral viruses with known oncogenic potential infect prostate tissue, among these are the polyomaviruses BKV, JCV, and SV40; human papillomaviruses (HPVs), and human cytomegalovirus (HCMV) infections. Recently, the Xenotropic Murine Leukemia Virus-related gammaretrovirus (XMRV) was identified in prostate tissue with a high prevalence observed in prostate cancer (PC) patients homozygous for the glutamine variant of the RNASEL protein (462Q/Q). Association studies with the R462Q allele and non-XMRV viruses have not been reported. We assessed associations between prostate cancer, prostate viral infections, and the RNASEL 462Q allele in Mexican cancer patients and controls.Methods130 subjects (55 prostate cancer cases and 75 controls) were enrolled in the study. DNA and RNA isolated from prostate tissues were screened for the presence of viral genomes. Genotyping of the RNASEL R462Q variant was performed by Taqman method.ResultsR/R, R/Q, and Q/Q frequencies for R462Q were 0.62, 0.38, and 0.0 for PC cases and 0.69, 0.24, and 0.07 for controls, respectively. HPV sequences were detected in 11 (20.0%) cases and 4 (5.3%) controls. XMRV and HCMV infections were detected in one and six control samples, respectively. The risk of PC was significantly increased (Odds Ratio = 3.98; 95% CI: 1.17-13.56, p = 0.027) by infection of the prostatic tissue with HPV. BKV, JCV, and SV40 sequences were not detected in any of the tissue samples examined.ConclusionsWe report a positive association between PC and HPV infection. The 462Q/Q RNASEL genotype was not represented in our PC cases; thus, its interaction with prostate viral infections and cancer could not be evaluated.

Acetylsalicylic acid inhibits hepatitis C virus RNA and protein expression through cyclooxygenase 2 signaling pathways

It has been reported that salicylates (sodium salicylate and aspirin) inhibit the replication of flaviviruses, such as Japanese encephalitis virus and dengue virus. Therefore, we considered it important to test whether acetylsalicylic acid (ASA) had anti–hepatitis C virus (HCV) activity. To this end, we examined the effects of ASA on viral replication and protein expression, using an HCV subgenomic replicon cell culture system. We incubated Huh7 replicon cells with 2‐8 mM ASA for different times and measured HCV‐RNA and protein levels by northern blot, real‐time polymerase chain reaction, and western analysis, respectively. We found that ASA had a suppressive effect on HCV‐RNA and protein levels (nearly 58%). ASA‐dependent inhibition of HCV expression was not mediated by the 5′‐internal ribosome entry site or 3′‐untranslated regions, as determined by transfection assays using bicistronic constructs containing these regulatory regions. However, we found that HCV‐induced cyclooxygenase 2 (COX‐2) messenger RNA and protein levels and activity and these effects were down‐regulated by ASA, possibly by a nuclear factor kappa B–independent mechanism. We also observed that the ASA‐dependent inhibition of viral replication was due in part to inhibition of COX‐2 and activation of p38 and mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase 1/2 (MEK1/2) mitogen‐activated protein kinases (MAPKs). Inhibition of these kinases by SB203580 and U0126, respectively, and by short interfering RNA silencing of p38 and MEK1 MAPK prevented the antiviral effect of ASA. Taken together, our findings suggest that the anti‐HCV effect of ASA in the Huh7 replicon cells is due to its inhibitory effect on COX‐2 expression, which is mediated in part by the activation of MEK1/2/p38 MAPK. Conclusion: These findings suggest the possibility that ASA could be an excellent adjuvant in the treatment of chronic HCV infection. (HEPATOLOGY 2008.)

Additive effect of ethanol and HCV subgenomic replicon expression on COX‐2 protein levels and activity

Summary.  The mechanisms by which alcohol exacerbates liver injury in patients with hepatitis C are unknown. We used the hepatitis C virus (HCV) subgenomic replicon cell system to evaluate the effect of ethanol on HCV replication and viral protein synthesis. Our results demonstrate that alcohol stimulates HCV replicon expression at both HCV‐RNA and protein levels. Furthermore, we observed that ethanol treatment showed an additive effect in cyclooxygenase‐2 (COX‐2) protein expression and activity already induced by HCV viral proteins, and in turn increased HCV viral expression. Our results suggest that COX‐2 activity is involved in ethanol‐induced HCV‐RNA and NS5A protein expression, because acetylsalicylic acid (ASA), a COX‐1/2 inhibitor, blocked this induction and downregulated COX‐2 protein expression and activity. Therefore, we suggest that ethanol increases HCV replication expression, at least in part, by upregulating a key cellular regulator of oxidative stress pathway known as COX‐2 or its products.

Serologic Surveillance for West Nile Virus and Other Flaviviruses in Febrile Patients, Encephalitic Patients, and Asymptomatic Blood Donors in Northern Mexico

A clinical and serological investigation was performed to determine the presence of West Nile virus (WNV) among febrile and encephalitic patients in northern Mexico. In addition, asymptomatic blood donors were serologically assayed for WNV to determine the seroprevalence of WNV in the general population. The study cohort consisted of 1432 individuals (588 febrile patients, 44 encephalitic patients, and 800 asymptomatic blood donors). All subjects were negative for WNV IgM. Sixty subjects were reactive for dengue virus (DENV) IgM (16 blood donors and 44 febrile patients). A subset (n = 425) of individuals was also screened by ELISA for flavivirus IgG. The prevalence of flavivirus IgG in febrile patients, encephalitic patients, and blood donors ranged from 40% to 59%. A subset (n = 147) of sera reactive for flavivirus IgG was further tested by plaque reduction neutralization test. Six individuals with no history of travel during the preceding 12 months were seropositive for WNV. Another 65 individuals were seropositive for DENV1 and 24 were seropositive for DENV2. The high prevalence of dengue antibodies in northern Mexico appears to limit the incidence of WNV infection in this region. Article Summary Line: Antibodies to WNV, DENV-1, and DENV-2 were identified in humans in northern Mexico.

Oxidative stress modulation in hepatitis C virus infected cells

Hepatitis C virus (HCV) replication is associated with the endoplasmic reticulum, where the virus can induce cellular stress. Oxidative cell damage plays an important role in HCV physiopathology. Oxidative stress is triggered when the concentration of oxygen species in the extracellular or intracellular environment exceeds antioxidant defenses. Cells are protected and modulate oxidative stress through the interplay of intracellular antioxidant agents, mainly glutathione system (GSH) and thioredoxin; and antioxidant enzyme systems such as superoxide dismutase, catalase, GSH peroxidase, and heme oxygenase-1. Also, the use of natural and synthetic antioxidants (vitamin C and E, N-acetylcysteine, glycyrrhizin, polyenylphosphatidyl choline, mitoquinone, quercetin, S-adenosylmethionine and silymarin) has already shown promising results as co-adjuvants in HCV therapy. Despite all the available information, it is not known how different agents with antiviral activity can interfere with the modulation of the cell redox state induced by HCV and decrease viral replication. This review describes an evidence-based consensus on molecular mechanisms involved in HCV replication and their relationship with cell damage induced by oxidative stress generated by the virus itself and cell antiviral machinery. It also describes some molecules that modify the levels of oxidative stress in HCV-infected cells.

Dengue virus antibodies in blood donors from an endemic area

We evaluated the incidence of anti‐Dengue virus (DENV) antibodies and dengue viremia in a region of Mexico with a high prevalence of dengue. DENV is the most important arthropod‐borne virus in terms of human morbidity and mortality in America We tested 800 blood donors from a tertiary care teaching hospital that provides care in Northeast Mexico, to identify anti‐DENV IgM and IgG antibodies by enzyme‐linked immunosorbent assay (ELISA) and DENV genome by reverse transcription polymerase chain reaction (RT‐PCR). In addition, routine tests for donors including Brucella, Hepatitis C virus (HCV), Venereal Disease Research Laboratory (VDRL), HIV‐1 and HBsAg identification were performed. We found that 59% of donors were reactive for anti‐DENV IgG and none of them had reported recent DENV infection; however, 16 (2%) were reactive for anti‐DENV IgM antibodies. None of them were viremic at the time of donation. Routine tests showed that the prevalence of anti‐Brucella was 0.71%, anti‐HCV 0.71%, anti‐HIV‐1‐2 0.14%, HBsAg 0.14% and VDRL test 0.57%. Although DENV transmission by blood transfusion had not been confirmed in Mexico, the finding of a high prevalence of anti‐DENV IgM‐positive donors with asymptomatic manifestations and the recent viremia reported in blood donors suggests that this route of transmission might be possible.

Cu/Zn superoxide dismutase (SOD1) induction is implicated in the antioxidative and antiviral activity of acetylsalicylic acid in HCV-expressing cells

We evaluated the participation of oxidative stress in the negative regulation of hepatitis C virus (HCV)-RNA induced by acetylsalicylic acid (ASA). We used the HCV subgenomic replicon cell system that stably expresses HCV-nonstructural proteins (Huh7 HCV replicon cells) and the parental cell line. Cells were exposed to 4 mM ASA at different times (12-72 h), and pyrrolidine dithiocarbamate (PDTC) was used as an antioxidant control. Reactive oxygen species (ROS) production, oxidized protein levels, cytosolic superoxide dismutase (Cu/Zn-SOD), and glutathione peroxidase (GPx) activity were measured to evaluate oxidative stress. In addition, viral RNA and prostaglandin (PGE(2)) levels were determined. We observed that ASA treatment decreased ROS production and oxidized protein levels in a time-dependent fashion in both parental and HCV replicon cells with a greater extent in the latter. Similar results were found with PDTC exposure. Average GPx activity was decreased, whereas a striking increase was observed in average cytosolic SOD activity at 48 and 72 h in both cells exposed to ASA, compared with untreated cells. HCV replicon cells showed higher levels of Cu/Zn-SOD expression (mRNA and protein) with ASA treatment (48 and 72 h), whereas NS5A protein levels showed decreased expression. In addition, we found that inhibition of SOD1 expression reversed the effect of ASA. Interestingly, PDTC downregulated HCV-RNA expression (55%) and PGE(2) (60%) levels, imitating ASA exposure. These results suggest that ASA treatment could reduce cellular oxidative stress markers and modify Cu/Zn-SOD expression, a phenomenon that may contribute to the mechanisms involved in HCV downregulation.

Fatal Human Case of West Nile Disease, Mexico, 2009

To the Editor: West Nile virus (WNV; family Flaviviridae, genus Flavivirus) was first recognized in the Western Hemisphere in 1999 during an outbreak of human, equine, and avian encephalitis in New York (1). The virus has since spread across the United States and Canada, where it has caused ≈30,000 human infections and ≈1,000 deaths. Serologic evidence has demonstrated that WNV is present throughout Mexico, Central America, South America, and the Caribbean region (2–8). However, WNV illness in humans and vertebrate animals in these regions has been only sparsely reported. For instance, 7 human cases of WNV infection have occurred in Mexico (excluding the case described here), 3 of which were severe. All patients survived. To our knowledge, no fatal human cases of WNV infection have occurred in Central America, South America, or the Caribbean region. We describe a fatal case of WNV infection in a human in Central America. The patient, a man 40 years of age, lived in Monterrey, Nuevo Leon State, in northern Mexico. He had not traveled outside of the metropolitan area in the 6 months before illness onset. On June 11, 2009, influenza-like signs and symptoms (i.e., fever, malaise, fatigue, arthralgia, headache, and dizziness) developed in the patient. On June 26, the signs and symptoms had not resolved, and the man was admitted to University Hospital “Dr. Jose E. Gonzalez” at the Universidad Autonoma de Nuevo Leon (UANL). At the time of admission, cerebrospinal fluid (CSF) was collected, and laboratory analysis indicated a markedly elevated leukocyte count (182 cells/mm3; reference range 0–5 cells/mm3) and slightly elevated protein and glucose levels. Several days later, serious neurologic signs that included loss of consciousness developed in the patient. On July 6, he lapsed into a coma and was transferred to the intensive care unit and treated for intracranial hypertension. Another CSF specimen was collected, and laboratory findings demonstrated that the leukocyte count had increased to 495 cells/mm3. CSF cytologic examination showed atypical lymphocytes, some of which resembled plasma cells. Brain magnetic resonance imaging showed hydrocephalus with no brain parenchymal lesions. Because the patient was suspected to have a herpes simplex virus infection, intravenous acyclovir was administered. Several days later, the patient showed signs of improvement; on July 15, he was discharged. Eleven days later, he experienced severe headaches and, on July 29, was readmitted to the UANL Hospital for intracranial hypertension. On July 30, a ventriculoperitoneal shunt was implanted; however, the patient’s condition continued to decline, and he died on August 1. Personnel in the Laboratory of Molecular Infectology at the UANL were informed of the patient and were provided with the remainder of the second CSF specimen several days before his death. Total RNA and DNA were extracted from the CSF by using the QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA, USA) and DNAzol (Invitrogen, Carlsbad, CA, USA) and tested for nucleic acid to various pathogens associated with human central nervous system infections, specifically herpes simplex virus types 1 and 2, human enterovirus A–D, dengue virus types 1–4, WNV, and Mycobacterium tuberculosis. Complementary DNA samples were generated by using Superscript III reverse transcription (Invitrogen), and PCRs were performed by using Taq polymerase (Invitrogen) in accordance with the manufacturer’s instructions. PCR amplifications were conducted by using the following reaction conditions: 94oC for 3 min; 30 cycles of 94oC for 1 min, 50oC for 1 min, and 72oC for 2 min; followed by a final extension at 72oC for 8 min. Reverse transcription–PCRs performed with diethyl pyrocarbonate–treated distilled water in place of nucleic acid were included as negative controls. All test and control reactions were performed in duplicate. PCR products were examined by 2% agarose gel electrophoresis and visualized with ethidium bromide. A PCR product of the expected size was observed when the WNV-specific primers WN-cap-F (5′-CAGTGCTGGATCGATGGAGAG-3′) and WN-cap-R (5′-CCGCCGATTGATAGCACT GGT-3′) were used. These primers amplify a 104-nt region of the capsid gene. All other assay results were negative. Subsequent reactions were performed by using a second set of WNV-specific primers, WN-env-F (5′-GATGTGAAGATGATGAATATGG-3′) and WN-env-R (5′-AATGCTTCCTTTGCCAAATAG-3′), which amplify a 216-nt region of the envelope gene. A PCR product of the expected size was again observed. PCR products were purified by using the Purelink Gel Extraction Kit (Invitrogen) and sequenced by using a 3730×1 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Because of the small volume of CSF obtained, a comprehensive laboratory analysis (virus isolation, plaque reduction neutralization test) could not be performed. Nevertheless, detection of WNV in the CSF of a patient with encephalitis meets the Centers for Disease Control and Prevention established criteria for a case of West Nile neuroinvasive disease (9). Our findings highlight the fact that the low number of WNV cases in Mexico and elsewhere in Latin America should not deter healthcare personnel from performing WNV diagnostic testing and the public from using personal protective measures in these regions.

Synthesization, Characterization, and in Vitro Evaluation of Cytotoxicity of Biomaterials Based on Halloysite Nanotubes

Halloysite is an aluminosilicate clay that has been widely used for controlled drug delivery, immobilization of enzymes, and for the capture of circulating tumor cells (CTCs). Surface modification of halloysite by organosilanes has been explored to improve their properties. In this study halloysite clay nanotubes (HNTs) were functionalized by two different organosilanes: Trimethoxy(propyl)silane (TMPS), and Triethoxy(octyl)silane (EOS). Untreated and modified samples were characterized by scanning electron microscopy (SEM), X-ray diffractometry (XRD), thermogravimetrical analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Results showed a strong interaction of organosilanes with the chemical groups present in HNTs. Biocompatibility and cytotoxicity of these nanomaterials were determined using C6 rat glioblastoma cells. Our results indicate that prior to functionalization, HNTs show a high biocompatibility and low cytotoxicity. However, HNTs functionalized with EOS and TMPS showed high cytotoxicity by inducing apoptosis. These results allow the identification of potential applications in biomedical areas for HNTs.

Hepatitis C virus infection among HIV‐1 infected individuals from northern Mexico

Aims:  The prevalence of hepatitis C virus (HCV) infection, risk factors and HCV genotypes in 140 HIV‐1 infected individuals from northern Mexico was determined.