Parameters to Predict the Outcome of Severe and Critical COVID-19 Patients when Admitted to the Hospital.

Manifestations of COVID-19 are diverse and range from asymptomatic to severe, critical illness and death. Cases requiring hospital care (in severe and critical illnesses) are associated with comorbidities and hyperactivation of the immune system. Therefore, in this exploratory observational study, we analyzed which parameters are associated with mortality. We evaluated: demographic characteristics (age, sex and comorbidities), laboratory data (albumin, leukocytes, lymphocytes, platelets, ferritin), days of hospital stay, interleukins (IL-2, IL-6, IL-7, IL-10, IL-17) and sP-selectin in 40 Mexican patients admitted to medical emergencies with a confirmed diagnosis of COVID-19, a complete clinical record, and who signed the informed consent. Twenty severe (they required intermediate care with non-invasive ventilation) and twenty critically ill patients (they required mechanical ventilation) were classified, and these were subsequently compared with healthy and recovered subjects. A significant difference was found between the hospitalized groups in the parameters of age, ferritin, days of hospital stay and death with p values = 0.0145, p = 0.0441, p = 0.0001 and p = 0.0001, respectively. In the determination of cytokines and P-selectin, a significant difference was found between the following groups: recovered patients and healthy volunteers compared with hospitalized patients in severe and critical condition. Importantly, IL-7 remained elevated one year later in recovered patients. Taken together, these values determined at the time of hospital admission could be useful to monitor patients closely and evaluate in-hospital progress, hospital discharge, and out-of-hospital progress.

Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers.

Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.

In silico analysis of the association of hsa-miR-16 expression and cell survival in MDA-MB-231 breast cancer cells subjected to photodynamic therapy.

BACKGROUND: Breast cancer is the most common malignancy effecting women, and the triple-negative breast cancer (TNBC) subtype is particularly aggressive. This study aimed to evaluate the differential expression pattern of microRNAs (miRNAs) between untreated MDA-MB-231 cells (TNBC cell model) and those that survived photodynamic therapy (PDT) to gain insights into cell survival mechanisms. METHODS: Two PDT cycles were applied to MDA-MB-231 cells, using δ-aminolevulinic acid (ALA) followed by laser light at 635 nm. RNA was obtained from cells surviving PDT and untreated cells. The miRNAs expression profile was analyzed to detect the differences between the two groups. The potential target network of hsa-miR-16 was examined in silico with the integrative database Ingenuity® Pathway Analysis software. RESULTS: After the first and second PDT cycles, 17.8% and 49.6% of the MDA-MB-231 cells were viable. Microarray profiling of miRNAs showed decreased hsa-miR-16 expression (p < 0.05) in MDA-MB-231 cells surviving PDT when compared to the control cells. The predicted downstream targets of hsa-miR-16 were: 1) tumor suppressor protein 53; 2) molecules related to the cell cycle, such as cyclin D1, D3, and E1, and checkpoint kinase 1; 3) cell proliferation molecules, including fibroblast growth factor 1, 2 and 7 and fibroblast growth factor receptor 1; and 4) apoptosis-related molecules, consisting of BCL-2, B-cell leukemia/lymphoma 2, caspase 3, and cytochrome c. CONCLUSIONS: The differential expression of hsa-miR-16 between untreated MDA-MB-231 cells and those surviving PDT has not been previously reported. There was a lower expression of hsa-miR-16 in treated cells, which probably altered its downstream target network. In silico analysis predicted, a network related to the cell cycle, proliferation and apoptosis. These results are congruent with previous descriptions of hsa-miR-16 as a tumor suppressor and suggest that the treated population has increased their capacity to survive.

Neonatal adiposity may increase plasmatic cytokines.

Maternal health and nutritional status before and during gestation may affect neonates' immune system and energy balance as they develop. The objective of this study was to associate certain clinical markers of maternal adiposity (body mass index and gestational weight gain) and neonatal adiposity (birth weight, abdominal circumference, and waist/height index) with the levels of pro- and anti-inflammatory cytokines in umbilical cord blood at birth: IL-1ß, IL-1Rα, IL-4, IL-6, IL-10, IFN-γ, and TNF-α. An exploratory cross-sectional study was conducted with a convenience sample of women from one hospital recruited shortly before giving birth through scheduled cesarean section. Of 31 the pregnant women who agreed to participate and met the inclusion criteria, twenty-nine newborns from these women were analyzed. Three cases of tobacco smoking during pregnancy were identified as an unexpected maternal risk factor and were included in the analysis. Typical of the population treated at this hospital, ten of our participants had diabetes during pregnancy, and nine of them had a pre-pregnancy BMI> 25. Non-parametric statistical analyses and a generalized linear model with gamma scale response with a log link were performed. Results: Correlation analyses, differences in medians, and a prediction model all showed positive and significant results between cytokine levels in cord blood and neonatal abdominal circumference, birth weight, and waist-height index. For maternal variables, smoking during pregnancy showed significant associations with cytokine levels in cord blood. Conclusion: This study found a variety of associations suggesting that increased neonatal adiposity increases pro-inflammatory cytokine levels at birth.

BRAF 1799T>A Mutation Frequency in Mexican Mestizo Patients with Papillary Thyroid Cancer.

Thyroid cancer is the most frequent endocrine malignancy, and its incidence and prevalence are increasing worldwide. Despite its generally good prognosis, the observed mortality rates are higher in the less-developed regions. This indicates that timely diagnosis and appropriate initial management of this disease are important to achieve a positive outcome. We performed an observational study in order to describe the frequency of the BRAF 1799T>A mutation in Mexican mestizo patients with thyroid nodules, a scarcely studied ethnic group with large populations. Competitive allele-specific Taqman PCR was performed in 147 samples of thyroid tissue DNA obtained from patients histologically diagnosed with papillary thyroid cancer (PTC), colloid goiters, and follicular adenomas. The BRAF 1799T>A mutation frequency was 61.1% in PTC samples (p = 4.99 × 10-11). Potential diagnostic values were as follows: sensitivity, 61.1%; specificity, 96%; PPV, 94.2%; NPV, 69.5%; accuracy, 77.9%. Taking into account the fact that this mutation is not frequently found in cytologically indeterminate nodules, we suggest that the BRAF mutational analysis should be implemented in the clinical setting along with other diagnostic criteria such as USG, in order to contribute to diagnosis and to surgical decision-making during the initial management of thyroid nodules in Mexican public hospitals.

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains.

BACKGROUND: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton's Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations. METHODS: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient. RESULTS: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes. CONCLUSIONS: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.

Polymorphism rs2275913 of Interleukin-17A is related to more intensive therapy with disease-modifying anti rheumatic drugs in Mexican patients with Rheumatoid Arthritis.

AIM: The study has two aims: 1) to evaluate the association of IL-17 polymorphism rs2275913 with RA severity and 2) to evaluate if this particular SNP is associated with susceptibility for RA in Mexican patients. METHODS: Seventy-six RA patients and ninety-four healthy controls were included in the study. RA patients were evaluated according to DAS 28. Treatment with DMARD'S was prescribed and radiological damage was evaluated according to the Larsen method. A case-control study was used. Oral epithelial cells were obtained as source for genetic material. DNA was amplified using PCR. Subsequently, a RFLP was carried out. Finally, in order to confirm the IL-17 SNP rs2275913 presence, direct sequencing of the DNA was performed. RESULTS: A significant difference was observed between the RA patients and controls when the prevalence of IL-17 SNP rs2275913 was compared. There was a statistically significant disparity among the two groups with an OR of 5.6 (95%CI 1.5 - 20.9, P=<0.01). In this study was observed that the RA patients who were positive for the IL-17 polymorphism rs2275913 required 3 DMARDs to control the disease compared to 32% of the patients who were negative for the IL-17 polymorphism rs2275913, OR 6.6 (95%CI 1.6 - 27.0, P<0.01). CONCLUSION: This study draws two main conclusions: 1) The presence of IL-17 polymorphism rs2275913 is closely related to a more severe form of the disease and as a result, a higher number of DMARDs required to control it, 2) The presence of IL-17 polymorphism rs2275913 may confer a risk of developing RA in Mexican carriers.

Effectiveness of tipranavir versus darunavir as a salvage therapy in HIV-1 treatment-experienced patients.

INTRODUCTION: Although both tipranavir (TPV) and darunavir (DRV) represent important options for the management of patients with multi-protease inhibitor (PI)-resistant human immunodeficiency virus (HIV), currently there are no studies comparing the effectiveness and safety of these two drugs in the Mexican population. The aim of this study was to compare the effectiveness of TPV versus DRV as a salvage therapy in HIV-1 treatment-experienced patients. METHODOLOGY: This was a comparative, prospective, cohort study. Patients with HIV and triple-class drug resistance evaluated at the Hospital de Infectología "La Raza", National Medical Center, were included. All patients had the protease and retrotranscriptase genotype; resistance mutation interpretation was done using the Stanford database. RESULTS: A total of 35 HIV-1 triple-class drug-resistant patients were analyzed. All of them received tenofovir and raltegravir, 22 received darunavir/ritonavir (DRV/r), and 13 received tipranavir/ritonavir (TPV/r) therapies. The median baseline RNA HIV-1 viral load and CD4+ cell count were 4.34 log (interquartile range [IQR], 4.15-4.72) and 267 cells/mm3 (IQR, 177-320) for the DRV/r group, and 4.14 log (IQR, 3.51-4.85) and 445 cells/mm3 (IQR, 252-558) for the TPV/r group. At week 24 of treatment, 91% of patients receiving DRV/r and 100% of patients receiving TPV/r had an RNA HIV-1 viral load < 50 copies/mL and a CD4+ cell count of 339 cells/mm3 (IQR, 252-447) and 556 cells/mm3 (IQR, 364-659), respectively. CONCLUSIONS: No significant difference was observed between DRV/r and TPV/r in terms of virological suppression in HIV-1 patients who were highly experienced in antiretroviral therapy.

Molecular Analysis by Gene Expression of Mitochondrial ATPase Subunits in Papillary Thyroid Cancer: Is ATP5E Transcript a Possible Early Tumor Marker?

BACKGROUND: Cancer development involves an "injury" to the respiratory machinery (Warburg effect) due to decreased or impaired mitochondrial function. This circumstance results in a down regulation of some of the ATPase subunits of the malignant tissue. The objective of this work was to assess and compare the relative expression of mRNA of mitochondrial ATPase subunits between samples of thyroid cancer and benign nodules. MATERIAL AND METHODS: Samples from 31 patients who had an operation for PTC at the General Hospital of Mexico were snap-frozen and stored at -70°C. Thirty-five patients who had an operation for benign tumors were also included in the study. mRNA expression levels of alpha, beta, gamma, and epsilon subunits of F1 and "c12" of subunit Fo were determined by real-time RT-PCR (by duplicate), in order to determine if abnormal expression of these genes could partially explain the Warburg effect in papillary thyroid cancer (PTC). RESULTS: ATP5E transcript alteration (down-expression) was highly associated to PTC diagnosis OR=11.76 (95% confidence interval, 1.245-237.98; p=0.04). CONCLUSIONS: Relative down-expression of ATP5E transcript was highly associated with PTC diagnosis. This transcript alteration may be used as a tumoral marker in papillary thyroid cancer.

[Serological evidence of infection by three species of Chlamydia in pregnant women in Mexico]. / Evidencia serológica de infección por tres especies de Chlamydia en mujeres embarazadas.

UNLABELLED: Background: Chlamydiales are obligate intracellular pathogens causing a wide range of diseases in humans and animals. BACKGROUND: Chlamydiales are obligate intracellular pathogens causing a wide range of diseases in humans and animals. OBJECTIVE: To evaluate the seroprevalence of IgG antibodies to Chlamydophila psittaci, Chlamydophila pneumoniae and Chlamydia trachomatis in a group of pregnant women few hours before resolution of gestation, thus allowing for assessment of the potential effects in their pregnancy outcomes. MATERIAL AND METHODS: A cross-sectional study was done involving 110 hospitalized women for pregnancy resolution in two secondary care hospitals, one of Mexico City and other of Cuernavaca, Mor. Obstetric outcomes were followed in every case. Serum antibody measures were performed by microimmunofluorescence. Possible associations between seropositivity and pregnancy outcomes were analyzed. RESULTS: 110 pregnant women were studied, 85 were seropositive for any of the species (77.3%), of which 39 (46%) had antibodies to the three species, 27 against two (32%) and 19 (22%) against one. No associations of serologic results with obstetric complications were observed in these pregnant women. High socioeconomic status was found as a risk factor for seropositivity against C trachomatis. CONCLUSIONS: This study reveals high exposure to three species of Chlamydiaceae in Mexican pregnant women. Demonstrating complex interactions in two different places where all species are present. No correlation was found between seropositivity towards these Chlamydia species and pregnancy complications.

The molecular fingerprint of human papillomavirus infection and its effect on the Langerhans cell population in squamous cell carcinomas of the genital skin.

BACKGROUND: Information is scarce about the presence of molecular alterations related to human papillomavirus (HPV) infection in squamous cell carcinomas of the genital skin and about the effect of this infection in the number of Langerhans cells present in these tumors. AIMS: To determine the presence of HPV in genital skin squamous cell carcinomas and to see the relationship between HPV infection and changes in the expression of Ki-67 antigen (Ki-67), p53 protein (p53), retinoblastoma protein (pRb) and E-cadherin and to alterations in Langerhans cell density, if any. METHODS: A descriptive, comparative, retrospective and cross-sectional study was performed with all the cases diagnosed as squamous cell carcinomas of the genital skin at the Dermatopathology Service from 2001 to 2011. The diagnosis was verified by histopathological examination. The presence of HPV was examined using chromogenic in situ hybridization, and protein expression was studied via immunohistochemical analysis. RESULTS: The 34 cases studied were verified as squamous cell carcinomas and 44.1% were HPV positive. The degree of expression of pRb was 17.50% ±14.11% (mean ± SD) in HPV-positive cases and 29.74% ±20.38% in HPV-negative cases (P = 0.0236). The degree of expression of Ki-67 was 47.67% ±30.64% in HPV-positive cases and 29.87% ±15.95% in HPV-negative cases (P = 0.0273). CONCLUSION: HPV infection was related to lower pRb expression and higher Ki-67 expression in comparison with HPV negative samples. We could not find a relationship between HPV infection and the degree of expression of p53 and E-cadherin or with Langerhans cell density.

Reporting detection of Chlamydia trachomatis DNA in tissues of neonatal death cases.

OBJECTIVE: to determine whether C. trachomatis was present in neonates with infection, but without an isolated pathogen, who died during the first week of life. METHODS: early neonatal death cases whose causes of death had been previously adjudicated by the institutional mortality committee were randomly selected. End-point and real-time polymerase chain reaction of the C. trachomatis omp1 gene was used to blindly identify the presence of chlamydial DNA in the paraffinized samples of five organs (from authorized autopsies) of each of the dead neonates. Additionally, differential diagnoses were conducted by amplifying a fragment of the 16S rRNA of Mycoplasma spp. RESULTS: in five cases (35.7%), C. trachomatis DNA was found in one or more organs. Severe neonatal infection was present in three cases; one of them corresponded to genotype D of C. trachomatis. Interestingly, another case fulfilled the same criteria but had a positive polymerase chain reaction for Mycoplasma hominis, a pathogen known to produce sepsis in newborns. CONCLUSION: the use of molecular biology techniques in these cases of early infant mortality demonstrated that C. trachomatis could play a role in the development of severe infection and in early neonatal death, similarly to that observed with Mycoplasma hominis. Further study is required to determine the pathogenesis of this perinatal infection.

Skin biopsy: a pillar in the identification of cutaneous Mycobacterium tuberculosis infection.

INTRODUCTION: The present study aimed to establish the frequency and clinical characteristics of cutaneous tuberculosis among Mexican adult patients. METHODOLOGY: Ninety-five patients with clinically compatible lesions to cutaneous tuberculosis participated in the study. All patients were HIV negative and none of them had previous anti-TB treatment. A skin biopsy was taken from every patient suspected of having tuberculosis, and a histopathologic examination was performed as follows: Ziehl-Neelsen staining; culturing of mycobacteria by Löwenstein-Jensen (L-J) medium; Mycobacteria Growth Indicator Tube detection via BACTEC (MGIT-360); and polymerase chain reaction (PCR) with the sequence of insertion IS6110 for Mycobacterium tuberculosis complex. RESULTS: Tuberculosis was confirmed in 65 out of 95 cases (68.4%). Identified lesions were scrofuloderma (42 cases, 64.6%); lupus vulgaris (12 cases, 18.4%); warty tuberculosis (six cases, 9.2%); and papulonecrotic tuberculoid (five cases; 7.7%). The Ziehl-Neelsen staining was positive for acid fast bacilli in nine cases (13.8%) and 48 patients were positive for the PCR amplification (73.8%). All skin biopsies resulted positive for tuberculosis. A positive clinical response to the specific treatment was considered a confirmation for tuberculosis. The noninfectious etiology corresponded to 30 cases (31.6%). CONCLUSIONS: Tuberculosis in developing countries is still an important cause of skin lesions which must be studied via histopathological examination and culture due to their low bacillary load. A PCR test is necessary to obtain faster confirmation of the disease and to establish an early, specific and effective treatment.

Comparative effects of levamisole, Staphylococcus, and Freund's adjuvant on rat immunization with excretory and secretory antigens of Trichinella spiralis muscle larvae.

A comparison was made of the effects of levamisole, the bacterial fractions of Staphylococcus, and Freund's adjuvant on the immunization of rats with the excretory and secretory antigens of Trichinella spiralis muscle larvae. Wistar rats were immunized with the antigen and a saline solution, levamisole (LV), Staphylococcus (ST), or Freund's adjuvant (FA). After immunization, rats were infected, and the parasite burden at muscular phase was calculated for each group. Levels of IgG1 and IgG2 antibodies, as well as levels of two cytokines, IL-4 and IFN-γ, were evaluated during the immunization and postinfection periods. Differences were found in the kinetics of antibody production between groups (p < 0.01). In all cases, there was reactivity with the main 45-, 50-, and 55-kDa antigens of Trichinella muscle larvae. Immunization with FA and ST enhanced the production of IgG1, but only FA showed a significant increase in the production of IFN-γ (p < 0.01), resulting in 86% protection against the infection. In contrast, only 60-70% protection was attained in the ST and LV groups (p < 0.01). These data support the idea that levamisole and Staphylococcus can be used as adjuvant to enhance the humoral response and, at the same time, demonstrate that IFN-γ could be involved in protection against Trichinella.

Impact of CYP1A1 and COMT genotypes on breast cancer risk in Mexican women: a pilot study.

BACKGROUND: Data suggest that estrogen-metabolizing genes may be involved in breast cancer carcinogenesis. OBJECTIVE: The aim of this study was to determine the association of CYP1A1 and COMT polymorphisms with this disease. MATERIAL AND METHODS: A pilot case-control study was conducted with Mexican women. Ninety-one breast cancer patients and 94 healthy controls were selected. Epidemiological and clinical questionnaires were answered by all participants, and genotyping data were obtained. CYP1A1 3801 T>C (rs4646903), CYP1A1 4889 A>G (rs1048943) and COMT 1947 G>A (rs4680) polymorphisms were analyzed by PCR-RFLP. RESULTS: The results showed a high risk of breast cancer in women carrying the CYP1A1 (3801 T>C) m2/m2 genotype (OR=2.52; 95%CI=1.04-6.08). The risk was higher in postmenopausal women (OR=3.38; 95%CI=1.05-10.87). No association between COMT 1947 G>A (rs4680) or CYP1A1 4889 A>G (rs1048943) and breast cancer was found. CONCLUSIONS: This study suggests that the CYP1A1 (3801 T>C) m2/m2 genotype may contribute to breast cancer susceptibility in Mexican women.

Differential CD4(+) T-cell memory responses induced by two subsets of human monocyte-derived dendritic cells.

Dendritic cells (DC) are powerful inducers of primary T-cell responses, but their role in secondary responses has not been extensively analysed. Here, we address the role of two DC subsets derived from human CD16(+) (16(+) mDC) or CD16(-) (16(-) mDC) monocytes on the reactivation of memory responses. CD4(+) CD45RA(-) memory T cells were obtained from adult blood donors, and central (T(CM)) and effector (T(EM)) memory T cells were isolated by fluorescence-activated cell sorting with anti-CCR7 antibodies. The 16(+) mDC and 16(-) mDC were cocultured with autologous lymphocytes, either unpulsed or loaded with purified protein derivatives of Mycobacterium tuberculosis (PPD) or tetanus toxoid (TT), and were analysed for up to 8 days. Over a range of doses, 16(+) mDC drove stronger T-cell proliferative responses against both antigens. Overall, antigen-specific memory cells tended to acquire a phenotype of T(EM) at later time-points in the culture, whereas cells that had completed fewer cycles of division were similar to T(CM). The 16(+) mDC induced higher rates of proliferation on both T(CM) and T(EM) lymphocytes than 16(-) mDC. This phenomenon was not related to the ability of both DC to induce CD25 expression on T cells, to lower secretion of interleukin-2, or to raise production of interleukin-10 during T-cell/16(-) mDC cocultures. The induction of T(CM) effector capacity in terms of interferon-gamma production was faster and more pronounced with 16(+) mDC, whereas both DC had similar abilities with T(EM). In conclusion, these data might reveal new potentials in vaccination protocols with 16(+) mDC aimed at inducing strong responses on central memory T cells.

Ozone exposure induces iNOS expression and tyrosine nitration in rat aorta.

The aim was to study whether ozone affects vascular endothelium by causing inducible nitric oxide synthase (iNOS) expression and tyrosine nitration. We also studied biomarkers of endothelial function. Male Wistar rats were exposed to ozone (0.25ppm, 4h/day) or filtered air (control, ozone <0.05ppm). After ozone exposure, blood samples were taken to measure 6-keto prostaglandin F1α (6-keto PGF1α), dehydro-thromboxane B(2) (DH-TxB(2)), endothelin-1 and NO(2)(-)/NO(3)(-) (NO(x)(-)). iNOS and nitrotyrosine were detected in aorta by immunohistochemistry. Nitrotyrosine was also detected by immunoelectromicroscopy. Control aortae failed to show either iNOS or nitrotyrosine. Time-dependent positive iNOS and nitrotyrosine cells were observed in exposed animals. Except for NO(x)(-), endothelial markers decreased after 14 days of ozone exposure (P<0.05). After 28 days of ozone, 6-keto PGF1α remained low (P<0.05) while DH-TxB(2) increased (P<0.05). It is concluded that ozone causes endothelial dysfunction manifested early with peroxynitrite formation and lately with changes in endothelial markers.