Blanca Estela Bastidas-Ramirez

TH1/TH2 cytokine profile, metalloprotease-9 activity and hormonal status in pregnant rheumatoid arthritis and systemic lupus erythematosus patients

During the course of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), several immune and neuroendocrine changes associated with pregnancy may exert positive (amelioration) or negative (exacerbation) effects on the clinical outcome. In order to shed light on the mechanisms underlying these responses, we performed a prospective longitudinal study in RA and SLE pregnant women, including healthy pregnant women as a control group. Cytokine messenger RNA (mRNA) expression assessed by quantitative competitive polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMC), cytokine levels and lymphocyte proliferation responses (LPR) following phytohaemagglutinin (PHA) stimulation of PBMC, plasma metalloprotease‐9 activity (MMP‐9) and hormonal status during pregnancy were determined. TNFa was the most abundant cytokine mRNA expressed in PBMC in all groups studied (healthy pregnant women, RA and SLE pregnant patients). However, a general TH2 response reflected by high IL‐10 levels was found in RA, as well as SLE, patients. A significant change in IFN‐γ was observed in RA patients but only during the first trimester of pregnancy. This compared with a major TH1 response in healthy pregnant women. Interestingly, our study showed a homogeneous hormonal pattern in RA and SLE patients. Although decreased cortisol levels were observed in all patients studied, this is possibly related to the remission of disease activity status brought about by steroid treatment before and during pregnancy. In summary, we suggest that complex immune and hormonal networks are involved in pregnancy and that rheumatic diseases are very dynamic immune processes that cannot be described with a clear‐cut cytokine profile. Furthermore, the observations in this study may reflect treatment‐related immune effects more than those associated with disease.

Genetic Polymorphisms of Genes Coding to Alcohol-Metabolizing Enzymes in Western Mexicans: Association of CYP2E1*c2/CYP2E1*5B Allele with Cirrhosis and Liver Function

BACKGROUND Alcoholic cirrhosis constitutes a major public health problem in the world where ADH1B, ALDH2, and CYP2E1 polymorphisms could be playing an important role. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy control individuals (C) and patients with alcoholic cirrhosis (AC) from western Mexico. METHODS Ninety C and 41 patients with AC were studied. Genotype and allele frequency were determined through polymerase chain reaction-restriction fragment length polymorphisms. RESULTS Polymorphic allele distribution in AC was 1.6%ADH1B*2, 0.0%ALDH2*2, and 19.5%CYP2E1*c2; in C: 6.1%ADH1B*2, 0%ALDH2*2, and 10.6%CYP2E1*c2. CYP2E1*c2 polymorphic allele and c1/c2 genotype frequency were significantly higher (p < 0.05 and p < 0.01, respectively) in patients with AC when compared to C. Patients with AC, carrying the CYP2E1*c2 allele, exhibited more decompensated liver functioning evaluated by total bilirubin and prothrombin time, than c1 allele carrying patients (p < 0.05). Cirrhosis severity, assessed by Childs Pugh score and mortality, was higher in patients carrying the c2 allele, although not statistically significant. CONCLUSIONS In this study, CYP2E1*c2 allele was associated with susceptibility to AC; meanwhile, ADH1B*2 and ALDH2*2 alleles were not. CYP2E1*c2 allele was associated with AC severity, which could probably be attributed to the oxidative stress promoted by this polymorphic form. Further studies to clearly establish CYP2E1*c2 clinical relevance in the development of alcohol-induced liver damage and its usefulness as a probable prognostic marker, should be performed. Also, increasing the number of patients and including a control group conformed by alcoholic patients free of liver damage may render more conclusive results. These findings contribute to the understanding of the influence of gene variations in AC development among populations, alcohol metabolism, and pharmacogenetics.

Polymorphisms of Alcohol Metabolizing Enzymes in Indigenous Mexican Population: Unusual High Frequency of CYP2E1*c2 Allele

BACKGROUND Alcohol abuse represents the major identified etiological factor of cirrhosis in México. ADH1B, ALDH2, and CYP2E1 have been considered candidate genes in alcohol-related diseases. Controversial results probably due to ethnic differences, among other factors, have been reported. Mexican Mestizos (MES) derive from the combination of indigenous, Spaniard, and African genes. Huichols (HUI) constitute an indigenous group from western Mexico with no racial admixture. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy HUI and MES from western Mexico. Lipid and hepatic profile were also carried out. METHODS One hundred and one HUI and 331 MES subjects were studied. Genotype and allele frequency were assessed through polymerase chain reaction-restriction fragment length polymorphism after DNA isolation from peripheral leukocytes. Commercial kits for lipid and hepatic determinations were used. RESULTS Polymorphic allele distribution in HUI was: 0%ADH1B*2, 0.5%ALDH2*2, 51.5%CYP2E1*c2; in MES: 3.4%ADH1B*2, 0%ALDH2*2, 16.1%CYP2E1*c2. Frequency of ADH1B*2 was statistically (p < 0.001) lower in HUI than MES. CYP2E1*c2 polymorphic allele was significantly higher (p < 0.0001) in HUI than MES. Hepatic profile was normal in both groups. HUI showed a better lipid profile than MES independently of genotype. CONCLUSIONS Huichols exhibited the highest CYP2E1*c2 allele frequency of the world documented up to this date; meanwhile, ADH1B*2 and ALDH2*2 were practically absent. This feature could be useful in the understanding of Mexican population gene composition, alcohol metabolism, and alcoholic liver disease development. However, further association studies are necessary. The heterogeneity of Mexican population was evidenced by the significantly different distribution of CYP2E1*c2 allele observed among different regions of the country. Lipid and hepatic values were not associated to genotype. This report constitutes the first study dealing with gene polymorphisms of alcohol metabolizing enzymes conducted in HUI.

Polymorphism of the β3-adrenergic receptor and lipid profile in patients with rheumatoid arthritis and systemic lupus erythematosus treated with chloroquine

We investigated the effect of beta 3-adrenergic receptor (β3AR) polymorphism on lipid profiles in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) treated with chloroquine. One hundred sixty-eight subjects were classified into three groups: 61 RA patients, 57 SLE patients, and 50 healthy subjects. All patients fulfilled the 1987 and 1982 classification criteria for RA and SLE, respectively, of the American College of Rheumatology. Demographic data and clinical characteristics of the patients were registered. Fasting lipid profile determination and leukocyte genomic DNA isolation from peripheral blood was performed in all the participants. Screening of the β3-AR gene polymorphic region (exon 1) was done by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Quantitative and qualitative variables were analyzed using analysis of variance (ANOVA) with the LSD and χ2 tests, respectively. An association between the arg64/arg64 β3-AR genotype and high levels of triglycerides (TG) and very low-density lipoprotein cholesterol (VLDL-c) was found in three RA patients (P=0.01), two of them taking chloroquine. Arg64/arg64 β3-AR polymorphism may contribute to increased TG and VLDL-c in RA patients, independently of chloroquine treatment.

Establishment of a cut‐point value of serum TNF‐α levels in the metabolic syndrome

Cardiovascular diseases and type 2 diabetes are the major causes of mortality in Mexico. Metabolic syndrome (MS) is a cluster of factors that increase the risk to develop such diseases. Previous studies have shown that MS is associated with high tumor necrosis factor (TNF‐α) levels. In fact, TNF‐α has been proposed to be a useful marker for clinical diagnosis of inflammation at an early stage. Therefore, we analyzed TNF‐α concentrations in Mexican individuals with or without MS and related these levels to the associated MS components. Clinical, anthropometric, and biochemical data were analyzed in 41 healthy and 39 MS individuals. Individuals were similarly grouped by age and gender.The serum TNF‐α levels measured bya highly sensitive enzyme‐linked immunosorbent assay (ELISA) kit were increased significantly in MS subjects compared with healthy individuals (P<0.001). The assay showed 78.1% sensitivity and 61.5% specificity with a cut‐point level of 1.36 pg/mL. TNF‐α levels higher than the cut‐point value were correlated with insulin resistance indices. These findings support the hypothesis that serum TNF‐α concentration could be a useful marker for early MS diagnosis. Nevertheless, we suggest the establishment of specific cut‐point values in each studied population to evaluate potential clinical applications. J. Clin. Lab. Anal. 23:51–56, 2009.

A cut-point value of uncarboxylated to carboxylated index is associated with glycemic status markers in type 2 diabetes.

Background The uncarboxylated osteocalcin (ucOC) has been described as a regulator of glucose metabolism in mice, and it is decreased in human type 2 diabetes mellitus (T2D). Although inversely correlated with serum glucose, insulin, and glycated hemoglobin, it is unclear if ucOC decrement is caused by diabetes or plays a role in the pathogenesis and/or progression of the disease. Whatever the case may be, diabetes affects osteoblast gene expression, and possibly the proportion of ucOC over carboxylated OC (cOC). The association of ucOC/cOC index with glycemic status markers in patients with T2D has not been described before. Objective The objective of this study was to assess the ucOC/cOC index and its relationship with glycemic status markers in patients with T2D. Methods The ucOC/cOC index was determined by the quotient of ucOC and cOC serum levels in 80 T2D patients and 160 healthy subjects. The relationship between the ucOC/cOC index and glycemic status markers was evaluated. Results The ucOC/cOC index was low and negatively correlated to fasting plasma glucose and homeostasis assessment-insulin resistance model in T2D patients. The odds ratio for T2D patients with an ucOC/cOC index below the cut-point obtained by receiver operating characteristic analysis was 12.64 (confidence interval, 5.75–27.77; P < 0.001). Conclusions A value of ucOC/cOC index less than 0.3 is associated with markers of poor metabolic control in patients with T2D.

Production of first generation adenoviral vectors for preclinical protocols: Amplification, purification and functional titration

Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture.

Albumin mRNA in peripheral white blood cells of cirrhotic patients with a superimposed alcoholic hepatitis is associated to fatal outcome.

The aim of this study was to analyze the relationship of plasma colloid osmotic pressure (COP), relative viscosity (eta) and overall outcome on the expression of albumin (ALB) mRNA in peripheral white blood cells (PWBC) of cirrhotic patients with superimposed alcoholic hepatitis (LC+AH). ALB messanger was detected in PWBC by RT-nPCR in control individuals (C), patients with liver cirrhosis (LC) and LC+AH. A higher number of LC+AH patients were positive to ALB mRNA (67%), compared to C (30%) and LC (28%). COP was decreased in LC and LC+AH groups compared to C group. No statistically significant changes were detected in eta in the different populations studied. Most of the LC+AH patients positive to peripheral ALB expression (87%) had a fatal outcome, compared to survivors (25%). Such difference was not observed with the conventional liver function tests or Maddreys discriminant function.

An approach to the immunophenotypic features of circulating CD4⁺NKG2D⁺ T cells in invasive cervical carcinoma.

BackgroundNKG2D, an activating immunoreceptor, is primarily restricted to NK cells and CD8+ T cells. The existence of an atypical cytotoxic CD4+NKG2D+ T cell population has also been found in patients with autoimmune dysfunctions. Nonetheless, contradictory evidence has categorized this population with a regulatory rather than cytotoxic role in other situations. These confounding data have led to the proposal that two distinct CD4+NKG2D+ T cell subsets might exist. The immune response elicited in cervical cancer has been characterized by apparent contradictions concerning the role that T cells, in particular T-helper cells, might be playing in the control of the tumor growth. Interestingly, we recently reported a substantial increase in the frequency of CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade-1. However, whether this particular population is also found in patients with more advanced cervical lesions or whether they express a distinctive phenotype remains still to be clarified. In this urgent study, we focused our attention on the immunophenotypic characterization of CD4+NKG2D+ T cells in patients with well-established cervical carcinoma and revealed the existence of at least two separate CD4+NKG2D+ T cell subsets defined by the co-expression or absence of CD28.ResultsPatients with diagnosis of invasive cervical carcinoma were enrolled in the study. A group of healthy individuals was also included. Multicolor flow cytometry was used for exploration of TCR alpha/beta, CD28, CD158b, CD45RO, HLA-DR, CD161, and CD107a. A Luminex-based cytokine kit was used to quantify the levels of pro- and anti-inflammatory cytokines. We found an increased percentage of CD4+NKG2D+ T cells in patients with cervical cancer when compared with controls. Accordingly with an increase of CD4+NKG2D+ T cells, we found decreased CD28 expression. The activating or degranulation markers HLA-DR, CD161, and CD107a were heterogeneously expressed. The levels of IL-1beta, IL-2, TNF-alpha, and IL-10 were negatively correlated with the percentages of CD4+NKG2D+ T cells in patients with cervical carcinoma.ConclusionsTaken together, our results reveal the existence of two separate CD4+NKG2D+ T cell subsets defined by the co-expression or absence of CD28, the latter more likely to be present in patients with cervical cancer.

Hepatocellular carcinoma is rarely present in Western Mexico

Abstract Hepatocellular carcinoma (HCC) is considered uncommon in most countries of Latin-America. The aim of this study was to analyze the frequency of HCC in Western Mexico and to evaluate the use of molecular markers—albumin and AFP mRNA in peripheral blood cells—in its diagnosis. The frequency of HCC was determined in new patients with possible diagnosis of cancer, from a reference hospital from 1996 to 1998. Albumin and AFP mRNA were determined by RT-PCR. Twenty five patients with HCC were identified with an incidence of 2.5 cases per 1000 new patients with cancer per year and general incidence of 0.22 per 100 000 per year. Albumin mRNA was positive in peripheral blood cells of patients with HCC, chronic liver disease and healthy control individuals, whereas AFP mRNA was positive in HCC and embrionary carcinoma patients. However, the simultaneous detection of albumin and AFP mRNA was only present in HCC patients. The HCC incidence is extremely rare in Western Mexico and both molecular markers were detected only in these patients.