Alejandro Gómez-Delgado

Molecular detection of respiratory syncytial virus in postmortem lung tissue samples from Mexican children deceased with pneumonia.

Background. Respiratory syncytial virus (RSV) is the major viral cause of severe respiratory infections in children younger than 2 years of age. Nevertheless there are not enough epidemiologic data about the role of RSV as a cause of infantile mortality from pneumonia, mainly in young children from developing countries Aim. To determine the frequency of RSV infection in lung tissue samples from Mexican children deceased with pneumonia, by reverse transcription (RT) and PCR. Methods. Postmortem lung tissue samples from 98 children younger than 2 years of age who died of pneumonia during the period of 1989 to 1997 were studied. Paraffin was removed with xylene from 10-&mgr;m lung sections, the total RNA was extracted and complementary DNA was obtained by RT reaction. A nested PCR with the use of oligonucleotides specific for the F glycoprotein gene was developed. Samples negatives for RSV were tested for the absence of polymerase inhibitors and for complementary DNA integrity. Results. Twenty-nine of the 98 (30%) children deceased with pneumonia were positive for RSV by RT-PCR; 8 were detected from 13 (62%) children with histopathologic diagnosis of viral pneumonia and 21 from 85 (25%) children with histopathologic diagnosis of bacterial pneumonia (P = 0.018 ). There was no significant difference in RSV infection according to age groups or seasonal pattern. Conclusions. RSV infection is frequent in Mexican children younger than 2 years of age who died of pneumonia. Although RSV was more common in viral pneumonia, mixed infections with RSV and bacterial pneumonia were also common.

Epstein Barr virus and Helicobacter pylori co-infection are positively associated with severe gastritis in pediatric patients.

Background H. pylori infection is acquired during childhood and causes a chronic inflammatory response in the gastric mucosa, which is considered the main risk factor to acquire gastric cancer (GC) later in life. More recently, infection by Epstein-Barr virus (EBV) have also been associated with GC. The role of EBV in early inflammatory responses and its relationship with H. pylori infection remains poorly studied. Here, we assessed whether EBV infection in children correlated with the stage of gastritis and whether co-infection with H. pylori affected the severity of inflammation. Methodology/Principal Findings 333 pediatric patients with chronic abdominal pain were studied. From them, gastric biopsies were taken and inflammation graded according to the Sydney system; peripheral blood was drawn and antibodies against EBV (IgG and IgM anti-VCA) and H. pylori (IgG anti-whole bacteria and anti-CagA) were measured in sera. We found that children infected only by EBV presented mild mononuclear (MN) and none polymorphonuclear (PMN) cell infiltration, while those infected by H. pylori presented moderate MN and mild PMN. In contrast, patients co-infected with both pathogens were significantly associated with severe gastritis. Importantly, co-infection of H. pylori CagA+/EBV+ had a stronger association with severe MN (PR 3.0) and PMN (PR 7.2) cells than cases with single H. pylori CagA+ infection. Conclusions/Significance Co-infection with EBV and H. pylori in pediatric patients is associated with severe gastritis. Even single infections with H. pylori CagA+ strains are associated with mild to moderate infiltration arguing for a cooperative effect of H. pylori and EBV in the gastric mucosa and revealing a critical role for EBV previously un-appreciated. This study points out the need to study both pathogens to understand the mechanism behind severe damage of the gastric mucosa, which could identified children with increased risk to present more serious lesions later in life.

Use of dried blood samples for monitoring hepatitis B virus infection

BackgroundHepatitis B virus (HBV) infection is a problem in several regions of the world with limited resources. Blood samples dried on filter paper (DBS) have been successfully used to diagnose and monitor several infectious diseases. In Mexico there is an urgent need for an affordable and easy sampling method for viral load (VL) testing and monitoring of chronic HBV infection. The purpose of this work was to validate the utility of DBS samples for monitoring HBV infection in patients from Mexico City.MethodsMatched samples of plasma and DBS on filter paper from 47 HBV infected patients from the Instituto Mexicano del Seguro Social (IMSS), were included. To evaluate the DNA stability and purity from DBS stored at different temperature conditions, samples from ten patients were stored at 4 degree, 25 degree, and 37 degree C for 7 days. After DBS elution and DNA extraction, the purity of these samples was determined measuring the O.D. rate 260/280. The DBS utility for molecular studies was assessed with PCR assays to amplify a 322 bp fragment from the a determinant region of the HBV S gene. The VL from all samples was determined to evaluate the correlation between plasma and DBS matched samples.ResultsThe quality of the DNA from DBS specimen is not adversely affected by storage at 4 degree, 25 degree and 37 degree C for up 7 days. Statistical ANOVA analyses did not show any significant difference. The same amplification efficiency was observed between DNA templates from samples stored at different temperatures. The Pearson correlation between the VL from DBS and plasma matched samples was 0.93 (p = 0.01). The SD was 1.48 for DBS vs.1.32 for Plasma, and an average of log10 copies/mL of 5.32 vs. 5.53. ANOVA analysis did not show any statistically significant difference between the analyzed groups (p = 0.92).ConclusionThe results provide strong evidence that the isolation and quantification of DNA-HBV from DBS is a viable alternative for patient monitoring, and molecular characterization of the virus variants circulating in Mexico.

Is human cytomegalovirus associated with breast cancer progression

BackgroundIt has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV).FindingsTwo carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative.ConclusionsSamples studied showed no association between HCMV infection and breast cancer progression.

Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women

BackgroundBreast cancer is a complex multifactorial genetic disease. Among other factors, race and, to an even greater extent, viruses are known to influence the development of this heterogeneous disease. It has been reported that MMTV-like (HMTV) gene sequences with a 90 to 98% homology to mouse mammary tumor virus are found in several populations with a prevalence range of 0 to 74%. In the Mexican population, 4.2% of patients with breast cancer exhibit the presence of HMTV (MMTV-like) sequences. The aim of this study was to evaluate the presence and current prevalence of retroviral HMTV (MMTV-like) sequences in breast cancer in Mexican women.MethodsWe used nested PCR and real-time PCR with a TaqMan probe. As a positive control, we used the C3H MMTV strain inserted into pBR322 plasmid. To confirm that we had identified the HMTV sequences, we sequenced the amplicons and compared these sequences with those of MMTV and HMTV (GenBank AF033807 and AF346816).ResultsA total of 12.4% of breast tumors were HMTV-positive, and 15.7% of the unaffected tissue samples from 458 patients were HMTV-positive. A total of 8.3% of the patients had both HMTV-positive tumor and adjacent tissues. The HMTV-positive samples presented 98% similarity to the reported HMTV sequence.ConclusionsThese results confirm that the HMTV sequence is present in breast tumors and non-affected tissues in the Mexican population. HMTV should be considered a prominent causative agent of breast cancer.

Preeclampsia Is Associated with Lower Production of Vascular Endothelial Growth Factor by Peripheral Blood Mononuclear Cells

BACKGROUNDnRecent studies show that vascular endothelial growth factor (VEGF) downregulation is implicated in preeclampsia (PE) pathophysiology. This study assessed the relationship between PE and VEGF levels produced by peripheral blood mononuclear cells (PBMCs) and their serum levels.nnnMETHODSnA cross-sectional design was performed in 36 patients who had hypertensive disorders during pregnancy. We also used a longitudinal design with 12 pregnant women with risk factors for PE development and/or abnormal uterine arteries by Doppler study. VEGF and soluble fms-like tyrosine kinase-1 (sFlt-1) levels were measured for all patients in both designs.nnnRESULTSnsFlt-1 serum was higher in preeclamptic patients (n = 26), whereas VEGF produced by stimulated PBMCs was lower than in healthy pregnant women and VEGF levels produced by stimulated PBMCs were even lower (p <0.003) in severe PE (n = 16). The receiver-operating characteristic curve analysis allowed establishing a cut-off value to identify patients with PE. VEGF production by PBMCs was 339.87 pg/mL. In addition, a robust linear regression model was performed to adjust the variance in VEGF levels. The patients age decreased VEGF levels and was adjusted by weeks of gestation (WG) in our model. In the longitudinal study, 7/12 patients developed PE. VEGF produced by PBMCs cells was significantly lower in PE at 24-26 WG.nnnCONCLUSIONSnVEGF production by PBMCs is inhibited during PE, creating a downregulation of the microenvironment; this deficiency may contribute to the pathogenesis of disease.

Human Mesenchymal Stem/Stromal Cells from Umbilical Cord Blood and Placenta Exhibit Similar Capacities to Promote Expansion of Hematopoietic Progenitor Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) from bone marrow (BM) have been used in coculture systems as a feeder layer for promoting the expansion of hematopoietic progenitor cells (HPCs) for hematopoietic cell transplantation. Because BM has some drawbacks, umbilical cord blood (UCB) and placenta (PL) have been proposed as possible alternative sources of MSCs. However, MSCs from UCB and PL sources have not been compared to determine which of these cell populations has the best capacity of promoting hematopoietic expansion. In this study, MSCs from UCB and PL were cultured under the same conditions to compare their capacities to support the expansion of HPCs in vitro. MSCs were cocultured with CD34+CD38−Lin− HPCs in the presence or absence of early acting cytokines. HPC expansion was analyzed through quantification of colony-forming cells (CFCs), long-term culture-initiating cells (LTC-ICs), and CD34+CD38−Lin− cells. MSCs from UCB and PL have similar capacities to increase HPC expansion, and this capacity is similar to that presented by BM-MSCs. Here, we are the first to determine that MSCs from UCB and PL have similar capacities to promote HPC expansion; however, PL is a better alternative source because MSCs can be obtained from a higher proportion of samples.

Elevated Levels of Interferon-γ Are Associated with High Levels of Epstein-Barr Virus Reactivation in Patients with the Intestinal Type of Gastric Cancer

Background The inflammatory response directed against Helicobacter pylori (HP) is believed to be one of the main triggers of the appearance of gastric lesions and their progression to gastric cancer (GC). Epstein-Barr virus (EBV) has been found responsible for about 10% of all GCs, but the inflammatory response has not been studied in GC patients with evidence of high levels of EBV reactivation. Objective To determine the relationship between inflammation and antibodies against EBV reactivation antigens, HP, and the bacterium virulence factor CagA in patients with GC. Methods 127 GC patients, 46 gastritis patients, and 197 healthy subjects were studied. IL-1β, IL-6, IL-8, IL-10, TNF-α, TGF-β, MCP-1, and IFN-γ levels were measured in serum or plasma and compared against the antibody titers of VCA-IgG, HP, and the HP virulence factor CagA. Statistical associations were estimated. Results Significant ORs and positive trends were found between VCA-IgG and IFN-γ, specifically for patients with GC of intestinal type (OR: 6.4, 95% C.I. 1.2–35.4) (p < 0.044). Conclusions We confirmed a positive association between a marker of EBV reactivation and intestinal gastric cancer and present evidence of a correlation with elevated serum levels of IFN-γ, but not with the other cytokines.