Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1's specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Role of Arf-like proteins (Arl1 and Arl2) of Mucor circinelloides in virulence and antifungal susceptibility.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Control of morphology and virulence by ADP-ribosylation factors (Arf) in Mucor circinelloides.

Mucor circinelloides is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor (Arf)-encoding genes (arf1-arf4) and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in M. circinelloides. Transcription analysis revealed that certain arf genes are affected by dimorphism of M. circinelloides, such as the arf2 transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four arf genes to evaluate their function in dimorphism and virulence. We found that both arf1 and arf2 are required for sporulation, but these genes also perform distinct functions; arf2 participates in yeast development, whereas arf1 is involved in aerobic growth. Conversely, arf3 and arf4 play only minor roles during aerobic growth. Moreover, we observed that all single arf-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the arf3 mutant being most virulent. Lastly, arf1/arf2 and arf3/arf4 double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in M. circinelloides such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.

Polysorbate 80 coated chitosan nanoparticles for delivery of α-melanocyte stimulating hormone analog (NDP-MSH) to the brain reverse cognitive impairment related to neuroinflammation produced by a high-fat diet (HFD).

This study aimed to develop polysorbate 80-coated chitosan nanoparticles (PS80/CS NPs) as a delivery system for improved brain targeting of α-Melanocyte Stimulating Hormone analog (NDP-MSH). Chitosan nanoparticles loaded with NDP-MSH were surface-modified with polysorbate 80 ([NDP-MSH]-PS80/CS NP), which formed a flattened layer on their surface. Nanoparticle preparation involved ionic gelation, followed by characterization using scanning electron microscopy (SEM) for morphology, dynamic light scattering (DLS) for colloidal properties, and ATR-FTIR spectroscopy for structure. Intraperitoneal injection of FITC-PS80/CS NPs and [NDP-MSH]-PS80/CS NP in rats demonstrated their ability to cross the blood-brain barrier, reach the brain, and accumulate in CA1 neurons of the dorsal hippocampus within 2 h. Two experimental models of neuroinflammation were employed with Male Wistar rats: a short-term model involving high-fat diet (HFD) consumption for 5 days followed by an immune stimulus with LPS, and a long-term model involving HFD consumption for 8 weeks. In both models, [NDP-MSH]-PS80/CS NPs could reverse the decreased expression of contextual fear memory induced by the diets. These findings suggest that [NDP-MSH]-PS80/CS NPs offer a promising strategy to overcome the limitations of NDP-MSH regarding pharmacokinetics and enzymatic stability. By facilitating NDP-MSH delivery to the hippocampus, these nanoparticles can potentially mitigate the cognitive impairments associated with HFD consumption and neuroinflammation.

Arthralgia and myalgia associated with aromatase inhibitors: frequency and characterization in real-life patients.

Introduction: Adjuvant treatment with aromatase inhibitors (AI) in oestrogen receptor-positive and/or progesterone receptor-positive breast cancer (BC) has been shown to increase overall survival. However, arthralgias and myalgias are common adverse effects in patients treated with AI. Objective: To evaluate the frequency and characteristics of arthralgias and myalgias in patients with early BC-treated adjuvantly with AI in the Mastology Unit of the Oncology Service of the Hospital de Clínicas and the Departmental Hospital of Soriano. Materials and methods: A prospective, cross-sectional and descriptive study was performed. A questionnaire was administered to patients to assess the presence and characteristics of arthralgias and myalgias associated with AI. Statistical analysis: 'Age' was described with measures of central tendency and dispersion. Qualitative variables were presented in absolute and relative frequencies. Logistic models were used to evaluate the association between patient characteristics, tumour characteristics, treatment characteristics and the presence of pain. Results were presented by odds ratio and p-value, using R software (version 4.1.2) with a significance threshold of 5%. Results: 83 patients were included, with a median age of 69 years. 75.9% presented arthralgias and/or myalgias related to treatment, with an average intensity of 5-7. 80.9% received non-steroidal anti-inflammatory drugs (NSAIDs), achieving satisfactory analgesia. The presence of arthralgias and myalgias was significantly associated with age and time since the last menstrual period (LMP), being more frequent in patients older than 50 years and those with more than 5 years since the LMP. Conclusion: Approximately 70% of the patients presented arthralgias or myalgias. These findings suggest a possible role of oestrogen withdrawal in its mechanism of development. Multidisciplinary and translational research is crucial to evaluate the ethology and therapeutic options for patients with AI-related arthralgia.

Redox Imbalance in Nasal Epithelial Cells of Primary Ciliary Dyskinesia Patients.

Background: Primary Ciliary Dyskinesia (PCD) represents a rare condition marked by an abnormal mobility pattern of cilia and flagella, resulting in impaired mucociliary clearance. This deficiency leads to recurrent infections and persistent inflammation of the airways. While previous studies have indicated heightened oxidative stress levels in the exhaled breath condensate of pediatric PCD patients, the assessment of oxidative stress within the affected respiratory tissue remains unexplored. Aims: To assess the oxidative status of human nasal epithelial cells (NECs) in PCD patients. Methods: Thirty-five PCD patients and thirty-five healthy control subjects were prospectively included in the study. Levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione (GSH), intracellular Ca2+, plasma membrane potential, and oxidative damage in lipids and proteins were measured. In addition, apoptosis and mitochondrial function were analyzed by flow cytometry in NECs. Results: NECs from PCD patients showed reduced levels of apoptosis (p = 0.004), superoxide anion (O2-, p = 0.018), peroxynitrite (ONOO-, p = 0.007), nitric oxide (NO, p = 0.007), mitochondrial hydrogen peroxide (mtH2O2, p < 0.0001), and mitochondrial superoxide anion (mtO2-, p = 0.0004) and increased mitochondrial mass (p = 0.009) compared to those from healthy individuals. No significant differences were observed in oxidized proteins (p = 0.137) and the oxidized/reduced lipid ratio (p = 0.7973). The oxidative profile of NEC cells in PCD patients, according to their ciliary motility, recurrent otitis, recurrent pneumonia, atelectasis, bronchiectasis, and situs inversus, showed no statistically significant differences in the parameters studied. Conversely, patients with chronic rhinosinusitis exhibited lower levels of ONOO- than PCD patients without this condition, with no significant differences related to other symptoms. Conclusions: Our findings strongly suggest the presence of a redox imbalance, specifically leaning toward a reductive state, in PCD patients.

Efficacy of First-Line Treatment With Pertuzumab and Trastuzumab in Advanced Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer in Routine Clinical Practice.

Background: The first-line treatment for human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) involves a combination of trastuzumab, pertuzumab, and a taxane (TPH). This study assessed the efficacy of trastuzumab and pertuzumab (PH) in routine practice, following the treatment protocols of Uruguay's National Resources Fund (FNR), akin to clinical trials. Methods: Patients with advanced MBC treated with PH between 2008 and 2022 per FNR protocols were evaluated. The Kaplan-Meyer method and log-rank test were utilized for analyzing overall survival (OS). Demographic and clinical variables, including age, menopausal status, and hormone receptors (HR), were analyzed. Results: The study included 318 PH-treated patients. The median age was 56 years, with 63.2% being postmenopausal and 60.4% HR and HER-2 positive. With a median follow-up of 17.2 months, the median OS was 29 months. OS varied based on HR status and the presence of metastases at different sites, significantly lower in patients with brain, cutaneous/subcutaneous, and pulmonary metastases. Additionally, OS was higher in patients treated at private institutions compared to public ones. Conclusions: This study demonstrates the disparity in oncological treatment efficacy between clinical trials and clinical reality in Uruguay, emphasizing the importance of authentic environment research for more representative and effective medicine in Latin America.

What can Mendelian randomization contribute to biological anthropology?

Uncovering causal relationships between exposures and outcomes can be difficult in observational studies because of the potential for confounding and reverse causation to produce biased estimates. Conversely, randomized controlled trials (RCTs) provide the strongest evidence for causality but they are not always feasible. Mendelian randomization (MR) is a method that aims to strengthen causal inference using genetic variants as proxies or instrumental variables (IVs) for exposures, to overcome the above-mentioned biases. Since allele segregation occurs at random from parents to offspring, and alleles for a trait assort independently from those for other traits, MR studies have frequently been compared to "natural" RCTs. In biological anthropology (BA) relationships between variables of interest are usually evaluated using observational data, often remaining descriptive, and other approaches to causal inference have seldom been implemented. Here, we propose the use of MR to investigate cause and effect relationships in BA studies and provide examples to show how that can be done across areas of BA relevance, such as adaptation to the environment, nutrition and life history theory. While we consider MR a useful addition to the biological anthropologist's toolbox, we advocate the adoption of a wide range of methods, affected by different types of biases, in order to better answer the important causal questions for the discipline.

Modulatory role of α-MSH in hippocampal-dependent memory impairment, synaptic plasticity changes, oxidative stress, and astrocyte reactivity induced by short-term high-fat diet intake.

High-fat diet (HFD) consumption is associated with cognitive deficits and neurodegenerative diseases. Since the hippocampus is extremely sensitive to pathophysiological changes, neuroinflammation and the concomitant oxidative stress induced by HFD can significantly interfere with hippocampal-dependent functions related to learning and memory. The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) mediates neuroprotective actions in the central nervous system and can reverse the effects of neuroinflammation in cognitive functions that depend on the hippocampus. In this study, we used male Wistar rats to evaluate the effect of short-term HFD intake (5 days) plus a mild immune challenge, Lipopolysaccharide (LPS 10 µg/kg) on contextual fear, changes in structural plasticity, oxidative stress, and astrocyte reactivation in the hippocampus. We also determined the possible modulatory role of α-MSH. HFD consumption was associated with an increase in markers of oxidative stress (Advanced oxidation protein products and Malondialdehyde) in the dorsal hippocampus (DH). We also found changes in hippocampal structural synaptic plasticity, observing a decrease in total spine in the DH after HFD plus LPS. We observed astrocyte proliferation and a significant increase in the percentage of the area occupied by GFAP. Treatment with α-MSH (0.1 µg/0.25 µl) in the DH reversed the effect of short-term HFD plus LPS on contextual fear memory, oxidative stress, and spine density. α-MSH also reduced astrocyte proliferation. Our present results indicate that HFD consumption for a short period sensitizes the central nervous system (CNS) to a subsequent immune challenge and impairs contextual fear memory and that α-MSH could have a modulatory protective effect.

Assessing Adherence to Adjuvant Hormone Therapy in Breast Cancer Patients in Routine Clinical Practice.

Background: Adjuvant hormone therapy (HT) in patients with hormone receptor-positive breast cancer (BC) increases overall survival (OS). A lack of adherence to adjuvant endocrine therapy is common, 31.0-73.0% of women discontinue endocrine treatment before 5 years. The aim of the study was to assess adherence to HT in routine clinical practice in patients assisted at the Clinical Oncology Department of the Hospital de Clinicas - Universidad de la Republica, Uruguay. Methods: Patients treated with HT for stage 0-III BC between 2017 and 2019 were included. The medication possession (MPR) rate was calculated using pharmacy records, and the Morisky-Green Scale was applied to assess adherence. Adherent patients were those with MPR ≥ 0.80 and who correctly answered the Morisky-Green treatment adherence questionnaire. The association of adherence with polypharmacy, treatment, and patient characteristics was assessed using simple logistic models. The associations between qualitative variables and adherence were assessed using simple logistic regression model or Fisher's exact test. The association between quantitative variables and adherence was assessed using the Student's t-test. The odds ratio (OR) for non-adherence to treatment and its 95% confidence interval were estimated. Results: Totally, 118 patients were included; 65.2% were treated with aromatase inhibitors (AIs), 36.0% presenting polypharmacy. The adherence rate at the end of 2 years was 81.0 %; and it was associated with age (P = 0.03, OR = 0.96 for non-adherence), with adherent and non-adherent patients having a mean age of 65.0 and 60.3 years, respectively; however, adherence was not associated with polypharmacy, territory of origin, marital status, living alone, level of education, occupation, or stage. The adherence profile was similar for both drugs, but homemakers and retired women showed greater adherence to AI. Conclusions: Adherence to HT was assessed in real life, with 19.0% of the patients not adhering to the treatment, despite the known benefit for OS, being a well-tolerated treatment, and being provided free of charge. Older patients were associated with being more adherent. The results show the need of the Pharmacy Service and Department of Clinical Oncology Medical Oncology combining efforts to develop coordinated strategies and interventions to increase adherence, given the impact that this may have on patients' OS.

Trastuzumab-induced cardiotoxicity in early breast cancer over a 10-year period in Uruguay.

This srudy aimed to estimate the prevalence of trastuzumab-induced cardiotoxicity in Uruguayan women diagnosed with human epidermal growth factor receptor 2 (HER2)-positive breast cancer over a 10-year period, who were treated under the financial coverage of the National Resources Fund (Fondo Nacional de Recursos). This was an observational, descriptive study based on the analysis of an anonymized database of Uruguayan women diagnosed with HER2-positive breast cancer who received adjuvant trastuzumab treatment from to 2006 to 2016, provided by the Fondo Nacional de Recursos. Statistical analysis was performed using SPSS Statistics version 25, and variables were assessed using measures of central tendency, dispersion, contingency tables, and proportions. The chi-square test was used to analyze the association between the different variables. The study included 1401 patients diagnosed with stage I to III HER2-positive breast cancer. The mean age at diagnosis was 52 years. The prevalence of cardiotoxicity was 20.3%. Most patients who discontinued treatment owing to cardiotoxicity eventually resumed treatment (92.6%). Moreover, the prevalence of cardiotoxicity was similar among patients who received regimens with and without anthracyclines. No association was observed between prior cardiovascular events or trastuzumab administration (concurrent vs sequential) and the development of cardiotoxicity. In the present study, the prevalence of cardiotoxicity was similar to that reported nationally and internationally. Most patients did not develop cardiotoxicity, while the ones who developed it remained asymptomatic and cardiotoxicity was reversible.

Characterization of Early Peripheral Immune Responses in Patients with Sepsis and Septic Shock.

(1) Background: Sepsis is a life-threatening condition caused by an abnormal host response to infection that produces altered physiological responses causing tissue damage and can result in organ dysfunction and, in some cases, death. Although sepsis is characterized by a malfunction of the immune system leading to an altered immune response and immunosuppression, the high complexity of the pathophysiology of sepsis requires further investigation to characterize the immune response in sepsis and septic shock. (2) Methods: This study analyzes the immune-related responses occurring during the early stages of sepsis by comparing the amounts of cytokines, immune modulators and other endothelial mediators of a control group and three types of severe patients: critically ill non-septic patients, septic and septic shock patients. (3) Results: We showed that in the early stages of sepsis the innate immune system attempts to counteract infection, probably via neutrophils. Conversely, the adaptive immune system is not yet fully activated, either in septic or in septic shock patients. In addition, immunosuppressive responses and pro-coagulation signals are active in patients with septic shock. (4) Conclusions: The highest levels of IL-6 and pyroptosis-related cytokines (IL-18 and IL-1α) were found in septic shock patients, which correlated with D-dimer. Moreover, endothelial function may be affected as shown by the overexpression of adhesion molecules such as s-ICAM1 and E-Selectin during septic shock.

Survival and Time to Initiation of Adjuvant Chemotherapy Among Breast Cancer Patients in Uruguay.

INTRODUCTION: Increases in disease-free survival and overall survival (OS) with the use of adjuvant chemotherapy in early breast cancer (BC) are widely known; however, the optimal time to initiate treatment with adjuvant chemotherapy remains controversial. OBJECTIVE: To evaluate the time elapsed between surgery and the initiation of adjuvant chemotherapy and its possible impact on OS in patients diagnosed with BC stages I-III. MATERIALS AND METHODS: This retrospective study included 112 patients diagnosed with BC stages I-III who received adjuvant chemotherapy at the Mastology Unit of the Hospital de Clínicas in Uruguay from 2009 to 2019. OS was estimated using the Kaplan-Meier method, and a Cox proportional hazards model was used to estimate hazard ratios (HRs) and 95% confidence intervals. RESULTS: No statistically significant association was found between the time from surgery to the initiation of chemotherapy and the described variables. OS was worse for patients initiating chemotherapy more than 90 days after breast surgery (n = 19) (HR 7.63; p = 0.004) and between 61 and 90 days after surgery (n = 46) (HR 4.58; p = 0.040) compared to those who started before 30 days (n = 23). Controlling by type of surgery and stage, the prognosis of patients who started chemotherapy between 61 and 90 days after surgery was similar to that of patients who underwent chemotherapy within the first 30 days, controlling for surgery (HR 4.10; p = 0.056) and controlling for stage (HR 3.76; p = 0.075). Prognosis was worse for patients with stage III disease (p = 0.022) who underwent a mastectomy and/or axillary lymph node dissection (p = 0.025). CONCLUSION: Patients who started chemotherapy more than 90 days following surgery and those with stage III disease or underwent mastectomy and/or axillary lymph node dissection who initiated it between 61 and 90 days had a worse OS. Multiple factors are involved in the time between surgery and the initiation of chemotherapy, and further studies are needed to evaluate which of these factors influence the delay of chemotherapy in order to design strategies to avoid such delays and their negative impact on survival.

New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry.

Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.

Synthesis and in vitro cytotoxicity profile of the R-enantiomer of 3,4-dihydroxymethamphetamine (R-(-)-HHMA): comparison with related catecholamines.

(+/-)-3,4-Methylenedioxymethamphetamine (MDMA, also known as "ecstasy") is a chiral drug that is essentially metabolized in humans through O-demethylenation into 3,4-dihydroxymethamphetamine (HHMA). There has recently been a resurgence of interest in the possibility that MDMA metabolites, especially 5-(N-acetylcystein-S-yl)-N-methyl-alpha-methyldopamine (designated as 5-NAC-HHMA), might play a role in MDMA neurotoxicity. However, the chirality of MDMA was not considered in previously reported in vivo studies because HHMA, the precursor of the 5-NAC-HHMA metabolite, was used as the racemate. Since the stereochemistry of this chiral drug needs to be considered, the first total synthesis of R-(-)-HHMA is reported. Using L-DOPA as the chiral source, the preparation of R-(-)-HHMA is achieved through seven steps, in 30% overall yield and 99.5% enantiomeric excess. The cytotoxicity of R-(-)-HHMA and related catecholamines has been further determined by flow cytometric analysis of propidium iodide uptake in human dopaminergic neuroblastoma SH-SY5Y cells and by an Escherichia coli plate assay, specific for the detection of oxidative toxicity. The good correlation between the toxicities observed in both systems suggests that SH-SY5Y cells are sensitive to oxidative toxicity and that cell death (necrosis) would be mediated by reactive oxygen species mainly generated from redox active quinonoid centers. In contrast, apoptosis was detected for 3,4-dimethoxymethamphetamine (MMMA), the synthetic precursor of HHMA possessing a protected catechol group. MMMA was not toxic in the bacterial assay, indicating that its toxicity is not related to increased oxidative stress. Finally, we can conclude that there is a need to distinguish the toxicity ascribed to MDMA itself, also bearing a protected catechol moiety, from that depending on MDMA biotransformation leading to catechol metabolites such as HHMA and the thioether conjugates.

Oxidative and Nitrosative Pattern in Circulating Leukocytes of Very Early/Early Hepatocellular Carcinoma Patients.

BACKGROUND/AIM: In chronic liver disease, various immune cell subsets exert pro or anti-tumour effects by releasing reactive oxygen and nitrogen species (ROS, RNS). Here, we evaluated the oxidative and nitrosative pattern in peripheral blood leukocyte subpopulations of early hepatocellular carcinoma (HCC) patients compared with HCC-free cirrhotic patients. MATERIALS AND METHODS: Venous blood samples from 18 HCC-free cirrhotic patients and 17 early stage HCC patients were collected to determine ROS, RNS and reduced glutathione levels in isolated leukocytes analyzed by flow cytometry. RESULTS: Intracellular levels of ROS and glutathione were higher in lymphocytes, monocytes, and neutrophils from HCC patients as well as mitochondrial superoxide in neutrophils and monocytes whereas intracellular levels of nitric oxide were lower in lymphocytes, monocytes, and neutrophils. CONCLUSION: Early HCC alters intracellular levels of ROS and RNS of some circulating leukocytes subsets. This finding may represent a potential area of interest concerning the development of new treatments and prognostic markers.

Early hospital discharge and early puerperal complications.

OBJECTIVE: To evaluate the association between time of postpartum discharge and symptoms indicative of complications during the first postpartum week. MATERIALS AND METHODS: Women with vaginal delivery at a Mexico City public hospital, without complications before the hospital discharge, were interviewed seven days after delivery. Time of postpartum discharge was classified as early (<24 hours) or late (>25 hours). The dependent variable was defined as the occurrence and severity of puerperal complication symptoms. RESULTS: Out of 303 women, 208 (68%) were discharged early. However, women with early discharge and satisfactory prenatal care had lower odds of presenting symptoms in early puerperium than women without early discharge and inadequate prenatal care (OR 0.36; 95% confidence intervals = 0.17-0.76). CONCLUSIONS: There was no association between early discharge and symptoms of complications during the first postpartum week; the odds of complications were lower for mothers with early discharge and satisfactory prenatal care.

Memory consolidation impairment induced by Interleukin-1ß is associated with changes in hippocampal structural plasticity.

Pro-inflammatory cytokines, particularly Interleukin-1ß (IL-1ß), can affect cognitive processes such as learning and memory. The aim of this study was to establish whether the effect of IL-1ß on contextual fear memory is associated with changes in hippocampal structural plasticity. We also studied the effect of α-melanocyte-stimulating hormone (α-MSH), a potent anti-inflammatory and neuro-protective peptide. Different groups of animals were implanted bilaterally in dorsal hippocampus (DH). After recovery they were conditioned for contextual fear memory and received the different treatments (vehicle, IL-1ß, α-MSH or IL-1ß + α-MSH). Memory was assessed 24 hs after conditioning and immediately after rats were perfused for dendritic spine analysis. Our results show that local hippocampal administration of IL-1ß just after memory encoding induced impairment in contextual memory and a reduction in the total density of CA1 hippocampal dendritic spines, particularly the mature ones. α-MSH administration reversed the IL-1ß induced changes. The results suggest that neuro-inflammation induced by IL-1ß interferes with experience-dependent structural plasticity in DH whereas α-MSH has a beneficial modulatory role in preventing this effect.

Multi-centre validation of a flow cytometry method to identify optimal responders to interferon-beta in multiple sclerosis.

BACKGROUND AND OBJECTIVES: Percentages of blood CD19+CD5+ B cells and CD8+perforin+ T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. METHODS: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. RESULTS: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19+CD5+ B cell and 0.89 (CI: 0.85-0.93) for CD8+ perforin+ T cell quantification; Spearman r was 0.92 (0.89-0.95; p <0.0001) and 0.92 (0.88-0.95; p <0.0001) respectively. All centres obtained z-scores≤0.5 for both biomarkers. CONCLUSION: Homogenous percentages of CD19+CD5+ B cells and CD8 perforin+ T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.