Relevant SARS-CoV-2 viremia is associated with COVID-19 severity: Prospective cohort study and validation cohort.

Early kinetics of SARS-CoV-2 viral load (VL) in plasma determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR) was evaluated as a predictor of poor clinical outcome in a prospective study and assessed in a retrospective validation cohort. Prospective observational single-center study including consecutive adult patients hospitalized with COVID-19 between November 2020 and January 2021. Serial plasma samples were obtained until discharge. Quantitative RT-PCR was performed to assess SARS-CoV-2 VL. The main outcomes were in-hospital mortality, admission to the Intensive Care Unit (ICU), and their combination (Poor Outcome). Relevant viremia (RV), established in the prospective study, was assessed in a retrospective cohort including hospitalized COVID-19 patients from April 2021 to May 2022, in which plasma samples were collected according to clinical criteria. Prospective cohort: 57 patients were included. RV was defined as at least a twofold increase in VL within ≤2 days or a VL > 300 copies/ml, in the first week. Patients with RV (N = 14; 24.6%) were more likely to die than those without RV (35.7% vs. 0%), needed ICU admission (57% vs. 0%) or had Poor Outcome (71.4% vs. 0%), (p < 0.001 for the three variables). Retrospective cohort: 326 patients were included, 18.7% presented RV. Patients with RV compared with patients without RV had higher rates of ICU-admission (odds ratio [OR]: 5.6 [95% confidence interval [CI]: 2.1-15.1); p = 0.001), mortality (OR: 13.5 [95% CI: 6.3-28.7]; p < 0.0001) and Poor Outcome (OR: 11.2 [95% CI: 5.8-22]; p < 0.0001). Relevant SARS-CoV-2 viremia in the first week of hospitalization was associated with higher in-hospital mortality, ICU admission, and Poor Outcome. Findings observed in the prospective cohort were confirmed in a larger validation cohort.

Frequent HLA class I alterations in human prostate cancer: molecular mechanisms and clinical relevance.

Reduced expression of HLA class I is an important immune escape mechanism from cytotoxic T cells described in various types of malignancy. It often correlates with poor prognosis and resistance to therapy. However, current knowledge about the frequency, underlying molecular mechanisms, and prognostic value of HLA class I and II alterations in prostate cancer (PC) is limited. Immunohistochemical analysis demonstrated that 88 % of the 42 studied cryopreserved prostate tumors have at least one type of HLA alteration as compared to adjacent normal prostate epithelium or benign hyperplasia. Total loss of HLA-I expression found in 50 % of tumors showed an association with increased incidence of tumor relapse, perineural invasion, and high D'Amico risk. The remaining HLA-I-positive tumors demonstrated locus and allelic losses detected in 26 and 12 % of samples, respectively. Loss of heterozygosity at chromosome 6 was detected in 32 % of the studied tumors. Molecular analysis revealed a reduced expression of B2M, TAP2, tapasin and NLRC5 mRNA in microdissected HLA-I-negative tumors. Analysis of twelve previously unreported cell lines derived from neoplastic and normal epithelium of cancerous prostate revealed different types of HLA-I aberration, ranging from locus and/or allelic downregulation to a total absence of HLA-I expression. The high incidence of HLA-I loss observed in PC, caused by both regulatory and structural defects, is associated with more aggressive disease development and may pose a real threat to patient health by increasing cancer progression and resistance to T-cell-based immunotherapy.

Removal of tetracyclines from swine manure at full-scale activated sludge treatment plants.

The purpose of this study was to investigate the fate of three tetracyclines (TCs), namely oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC) at two different full-scale swine manure-activated sludge treatment plants. Throughout treatment, OTC, CTC and DC were removed by 71-76%, 75-80% and 95%, respectively. Removal of these TCs under physical treatment was deniable. On the contrary, the flocculation-coagulation and the secondary clarification resulted in a relevant reduction of the concentration of these TCs.

Immune escape of cancer cells with beta2-microglobulin loss over the course of metastatic melanoma.

Cancer cells escape T-cell-mediated destruction by losing human leukocyte antigen (HLA) class I expression via various mechanisms, including loss of beta2-microglobulin (ß2m). Our study illustrates the immune escape of HLA class I-negative tumor cells and chronological sequence of appearance of tumor ß2m gene mutation in successive lesions obtained from a patient with metastatic melanoma. We observed a gradual decrease in HLA expression in consecutive lesions with few HLA-negative nodules in the primary tumor and the emergence of a totally negative lesion at later stages of the disease. We detected loss of ß2m in ß2m-negative nests of the primary tumor caused by a combination of two alterations: (i) a mutation (G to T substitution) in codon 67 in exon 2 of ß2m gene, producing a stop codon and (ii) loss of the second gene copy by loss of heterozygosity (LOH) in chromosome 15. The same ß2m mutation was found in a homogeneously ß2m-negative metastasis 10 months later and in a cell line established from a biopsy of a postvaccination lymph node. Microsatellite analysis revealed the presence of LOH in chromosomes 6 and 15 in tumor samples, showing an accumulation of chromosomal loss at specific short tandem repeats in successive metastases during disease progression. HLA loss correlated with decreased tumor CD8+ T-cell infiltration. Early incidence of ß2m defects can cause an immune selection and expansion of highly aggressive melanoma clones with irreversible genetic defects causing total loss of HLA class I expression and should be taken into consideration as a therapeutic target in the development of cancer immunotherapy protocols.

Sepsis Stewardship: The Puzzle of Antibiotic Therapy in the Context of Individualization of Decision Making.

The main recent change observed in the field of critical patient infection has been universal awareness of the need to make better use of antimicrobials, especially for the most serious cases, beyond the application of simple and effective formulas or rigid protocols. The increase in resistant microorganisms, the quantitative increase in major surgeries and interventional procedures in the highest risk patients, and the appearance of a significant number of new antibiotics in recent years (some very specifically directed against certain mechanisms of resistance and others with a broader spectrum of applications) have led us to shift our questions from "what to deal with" to "how to treat". There has been controversy about how best to approach antibiotic treatment of complex cases of sepsis. The individualized and adjusted dosage, the moment of its administration, the objective, and the selection of the regimen are pointed out as factors of special relevance in a critically ill patient where the frequency of resistant microorganisms, especially among the Enterobacterales group, and the emergence of multiple and diverse antibiotic treatment alternatives have made the appropriate choice of antibiotic treatment more complex, requiring a constant updating of knowledge and the creation of multidisciplinary teams to confront new infections that are difficult to treat. In this article, we have reviewed the phenomenon of the emergence of resistance to antibacterials and we have tried to share some of the ideas, such as stewardship, sparing carbapenems, and organizational, microbiological, pharmacological, and knowledge tools, that we have considered most useful and effective for individualized decision making that takes into account the current context of multidrug resistance. The greatest challenge, therefore, of decision making in this context lies in determining an effective, optimal, and balanced empirical antibiotic treatment.

Challenges in Septic Shock: From New Hemodynamics to Blood Purification Therapies.

Sepsis and septic shock are associated with high mortality, with diagnosis and treatment remaining a challenge for clinicians. Their management classically encompasses hemodynamic resuscitation, antibiotic treatment, life support, and focus control; however, there are aspects that have changed. This narrative review highlights current and avant-garde methods of handling patients experiencing septic shock based on the experience of its authors and the best available evidence in a context of uncertainty. Following the first recommendation of the Surviving Sepsis Campaign guidelines, it is recommended that specific sepsis care performance improvement programs are implemented in hospitals, i.e., "Sepsis Code" programs, designed ad hoc, to achieve this goal. Regarding hemodynamics, the importance of perfusion and hemodynamic coherence stand out, which allow for the recognition of different phenotypes, determination of the ideal time for commencing vasopressor treatment, and the appropriate fluid therapy dosage. At present, this is not only important for the initial timing, but also for de-resuscitation, which involves the early weaning of support therapies, directed elimination of fluids, and fluid tolerance concept. Finally, regarding blood purification therapies, those aimed at eliminating endotoxins and cytokines are attractive in the early management of patients in septic shock.

Decrease in Mortality after the Implementation of a Hospital Model to Improve Performance in Sepsis Care: Princess Sepsis Code.

Sepsis is a time-dependent disease whose prognosis is influenced by early diagnosis and therapeutic measures. Mortality from sepsis remains high, and for this reason, the guidelines of the Surviving Sepsis Campaign recommend establishing specific care programs aimed at patients with sepsis. We present the results of the application of a hospital model to improve performance in sepsis care, called Princess Sepsis Code, with the aim of reducing mortality. A retrospective study was conducted using clinical, epidemiological, and outcome variables in patients diagnosed with sepsis from 2015 to 2022. A total of 2676 patients were included, 32% of whom required admission to the intensive care unit, with the most frequent focus of the sepsis being abdominal. Mortality in 2015, at the beginning of the sepsis code program, was 24%, with a declining rate noted over the study period, with mortality reaching 17% in 2022. In the multivariate analysis, age > 70 years, respiratory rate > 22 rpm, deterioration in the level of consciousness, serum lactate > 2 mmol/L, creatinine > 1.6 mg/dL, and the focus of the sepsis were identified as variables independently related to mortality. The implementation of the Princess Sepsis Code care model reduces the mortality of patients exhibiting sepsis and septic shock.

Bone mineral density changes in lactating adolescent mothers during the first postpartum year.

OBJECTIVES: This study aimed to compare changes in whole body bone mineral density (wbBMD) during the first postpartum year in adolescent mothers with those of nulliparous adolescents. METHODS: The study included 21 adolescent mothers and 16 nulliparous adolescent non-indigenous Mexican women (State of Sonora) from a low income level. All mothers were assessed at 15 days (0.5 months), 3 months, and 6 months postpartum; 16 were measured at 12 months postpartum. Nulliparous adolescents were assessed in the same periods. Multiple regression models was used to assess adjusted associations of changes in wbBMD (by DPX-MD+ densitometer) with dietary calcium and physical activity assessments (estimated using pre-tested questionnaires), post menarche years, and number of breast feedings. RESULTS: At baseline, no differences were observed between nulliparous and adolescent mothers regarding age, post-menarche years, or BMD values. Changes in wbBMD of -0.56% and 0.77% were observed in mothers and nulliparous adolescents, respectively, after the first 3 months (P = 0.006). Changes in wbBMD in mothers were associated with number of breast feedings and changes in BMI. At 12 months postpartum, the BMD of adolescent mothers was similar to that of nulliparous adolescents. CONCLUSIONS: At 1 year postpartum, adolescent mothers exhibited BMD similar to those of nulliparous adolescents. This result is likely attributable to the breastfeeding practices adopted by mothers during late adolescence.

Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.

PURPOSE: To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). DESIGN: Transversal, comparative case series. PARTICIPANTS: We studied 59 eyes from 43 patients clinically diagnosed with LSCD. METHODS: Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. MAIN OUTCOME MEASURES: Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. RESULTS: Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. CONCLUSIONS: The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/µl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.

Effect of micronutrient-fortified milk on zinc intake and plasma concentration in adolescent girls.

OBJECTIVE: To assess the effect of dietary fortified milk with zinc and other micronutrients on zinc intake and plasma zinc content of adolescent girls. METHODS: The study included 108 schoolgirls (12-18 years old) from northwest Mexico, randomly assigned to either the control group (CG; n = 55) or the intervention group consuming a regular diet plus fortified milk (MG; n = 53). At the beginning of the study, age, weight, and height were measured. Food intake by the 24-hour recall method and plasma zinc levels assessed by absorption spectrophotometry were determined before and after 27 days of fortified milk intake. RESULTS: At baseline, no significant group-related differences were observed for energy, protein intake, zinc intake, and plasma zinc level (p > 0.05), and 35.2% of participant girls did not achieve their zinc requirement. After 27 days of treatment, there were no significant differences in energy and protein intake between groups (p > 0.05). Zinc intake was higher for MG than CG (16.7 ± 8.3 mg/d vs 10.5 ± 6.4 mg/d; p < 0.01), and there was a lower proportion of low zinc intake in MG than for CG (7 vs 16, respectively; p = 0.04). In addition, plasma zinc improved in the MG (116.6 ± 26.9 µg/dL, p < 0.01) compared with CG (98.5 ± 26.6 µg/dL), and it was mainly attributed to the fortified milk intake, as the main dietary zinc contributor. CONCLUSION: Fortified milk intake is effective in increasing both intake and plasma zinc levels of adolescent Mexican girls; therefore, it could be an adequate strategy for zinc deficiency prevention or correction among adolescent girls.

SARS-CoV-2 Viremia Precedes an IL6 Response in Severe COVID-19 Patients: Results of a Longitudinal Prospective Cohort.

Background: Interleukin 6 (IL6) levels and SARS-CoV-2 viremia have been correlated with COVID-19 severity. The association over time between them has not been assessed in a prospective cohort. Our aim was to evaluate the relationship between SARS-CoV-2 viremia and time evolution of IL6 levels in a COVID-19 prospective cohort. Methods: Secondary analysis from a prospective cohort including COVID-19 hospitalized patients from Hospital Universitario La Princesa between November 2020 and January 2021. Serial plasma samples were collected from admission until discharge. Viral load was quantified by Real-Time Polymerase Chain Reaction and IL6 levels with an enzyme immunoassay. To represent the evolution over time of both variables we used the graphic command twoway of Stata. Results: A total of 57 patients were recruited, with median age of 63 years (IQR [53-81]), 61.4% male and 68.4% Caucasian. The peak of viremia appeared shortly after symptom onset in patients with persistent viremia (more than 1 sample with > 1.3 log10 copies/ml) and also in those with at least one IL6 > 30 pg/ml, followed by a progressive increase in IL6 around 10 days later. Persistent viremia in the first week of hospitalization was associated with higher levels of IL6. Both IL6 and SARS-CoV-2 viral load were higher in males, with a quicker increase with age. Conclusion: In those patients with worse outcomes, an early peak of SARS-CoV-2 viral load precedes an increase in IL6 levels. Monitoring SARS-CoV-2 viral load during the first week after symptom onset may be helpful to predict disease severity in COVID-19 patients.

Analysis of HLA-ABC locus-specific transcription in normal tissues.

We developed a novel human leukocyte antigen HLA-ABC locus-specific quantitative real-time polymerase chain reaction (PCR) to determine the locus-specific gene expression of HLA-ABC in peripheral blood leukocytes (PBLs, n = 53), colon mucosa (n = 15), and larynx mucosa (n = 15). Laser-assisted tissue microdissection allowed us to study the selected cells without interference from surrounding stroma. We report evidence on the specificity of the technique, describing the HLA-ABC locus-specific gene expression patterns found in the PBLs and two solid tissues studied. PBLs showed a higher gene expression of HLA-B than of HLA-A or HLA-C (p = 4.7 × 10(-10) and p = 1.6 × 10(-6), respectively). In solid tissue, HLA-A and HLA-B gene expressions were similar and HLA-C expression lower. In particular, in larynx mucosa, significant differences were found between HLA-A and HLA-C expressions and between HLA-B and HLA-C expressions (p = 6.5 × 10(-4) and p = 8.1 × 10(-4), respectively). The same differences were observed in colon mucosa, but significance was not reached (p = 0.08 and p = 0.06, respectively). Differences in locus-specific regulation may be related to the control of cytotoxic responses of NK and CD8 positive T cells. Gene expression of HLA-ABC specific locus showed no intra-individual variability, but there was a high inter-individual variability. This may result from differences in the expression of common regulatory factors that control HLA-ABC constitutive expression.

Could giardiasis be a risk factor for low zinc status in schoolchildren from northwestern Mexico? A cross-sectional study with longitudinal follow-up.

BACKGROUND: Both giardiasis and zinc deficiency are serious health problems worldwide. In Mexico, the prevalence of G. intestinalis was estimated at 32% in 1994. It remains a health problem in northwestern Mexico. Recent surveys (1987, 1995, and 1999) reported zinc deficiency in the Mexican population. The association of giardiasis and malabsorption of micronutrients has been well documented, although the association with zinc remains controversial. This study investigated the association between giardiasis and zinc deficiency in schoolchildren from northwestern Mexico. METHODS: We combined a cross-sectional design with a longitudinal follow-up six months after parasite treatment. The baseline sample consisted of 114 schoolchildren (mean age 8.8 yr) from seven suburban public schools, grouped as Giardia-free (n = 65, 57%) and Giardia-infected (n = 49, 43%). Three stool analyses per child were done using Faust's method. Children with giardiasis received secnidazole. Serum zinc was determined by atomic absorption spectrophotometry. Height and weight were measured. Socioeconomic information was obtained in an oral questionnaire, and daily zinc intake was assessed using 24 hour-recalls. Pearson's correlation and ANCOVA and paired t-test analyses were used to determine the association between giardiasis and zinc status. RESULTS: Longitudinal analysis demonstrated a significant increase of the mean serum zinc levels in the Giardia-infected group six months after treatment (13.78 vs. 19.24 mumol/L mumol/L; p = 0.001), although no difference was found between the Giardia-free and the Giardia-infected groups (p = 0.86) in the baseline analysis. Z scores for W/A and H/A were lower in the Giardia-infected than in the Giardia-free group (p < 0.05). No difference was observed in the socioeconomic characteristics and mean daily intakes of zinc between the groups (p > 0.05). CONCLUSIONS: Giardiasis may be a risk factor for zinc deficiency in schoolchildren from northwestern Mexico.

[Estimation of body fat by DXA and the four compartment model in Mexican youth]. / Estimación de la masa grasa por DXA y el modelo de cuatro compartimentos en púiberes mexicanos de 9 a 14 años.

The objective of this study was to validate the estimation of body fat (%BF) by DXA (Dual-Energy X-Ray AbsorciomDPX-MD) against the four compartment model (4C) of body composition in 32 Mexican pubertal girls and boys (aged 9-14 y; F=16). The mean of the difference between DXA and 4C model was -3.5 %BF (p=0.171). The limits of agreement (95% = 2 SD) were +5% to -12%BF. The precision of estimated limits of y the confidence intervals were -1.9% to -5.1%BF (P = 0.050). The concordance correlation coefficient was p = 0.85. The test of accuracy for coincidence of slop intercepts between DXA and the 4C model showed no coincidence (p < 0.05). The precision by R2 explained 83% of the variance (SEE, 4.1%). The individual accuracy assess by the total error was 5.6%. The group mean accuracy by two way analysis of variance of body fat did not show interaction between method (DXA-4C model) and separate analysis of gender and overweight. However, there was an effect of method (p = 0.043) in the presence of overweight (p < 0.001). In conclusion, the estimation of percent of body fat by DXA was not precise and accurate in a group of Mexican children. However, results do not limit the utility of DXA for the measurements of body composition and its relation with health outcomes, especially in follow up studies.

HLA and melanoma: multiple alterations in HLA class I and II expression in human melanoma cell lines from ESTDAB cell bank.

Altered HLA class I and class II cell surface expression has been reported in many types of malignancy and represents one of the major mechanism by which tumour cells escape from T lymphocytes. In this report, we review the results obtained from the study of constitutive and IFN-gamma-induced expression of HLA class I and II molecules in 91 human melanoma cell lines from the European Searchable Tumour Cell Line Database, and compare them with published data on HLA expression in other types of cancer. Various types of alterations in HLA class I cell surface expression were found in a high percentage (67%) of the studied cell lines. These alterations range from total to selective HLA class I loss and are associated with beta2-microglobulin gene mutations, transcriptional downregulation of HLA class I genes and antigen processing machinery components, or with the loss of heterozygosity in chromosome 6. The most frequently observed phenotype is selective downregulation of HLA-B locus, reversible after treatment with IFN-gamma. The expression of constitutive- or IFN-gamma induced-surface expression of at least one HLA class II locus is positive in 71.5% of the analysed cell lines. Four different HLA class II expression phenotypes were defined, and a positive correlation between the expression of class I and II molecules is discussed. More detailed information on the HLA expression patterns and others immunological characteristics of these melanoma cell lines can be found on the following website http://www.ebi.ac.uk/ipd/estdab .

Regulation of clusterin expression in human cancer via DNA methylation.

BACKGROUND/AIMS: Clusterin has attracted much recent attention because of its association with tumorigenesis and the progression of human carcinomas. The present study was designed to examine the role of clusterin methylation as an indicator of clusterin expression in tumor cell lines and breast tissue samples. METHODS: For this purpose, we used methylation-sensitive restriction analysis followed by PCR. RESULTS: None of the non-tumoral breast samples showed expression of clusterin by immunohistochemistry, and a methylated state was found in the promoter region of the gene. However, a demethylated state was found in 5 of 6 analyzed carcinoma cell lines. Four of 5 demethylated cell lines presented moderate to strong expression of clusterin, while no expression was detected in the unmethylated cell line. The inverse correlation found in most cell lines between clusterin expression and promoter methylation was also found in most human tumors analyzed (p < 0.001). Thus, a methylated state was present in 14 carcinomas, 12 of them with a null expression of clusterin, while a demethylated state was detected in 7 breast tumor samples, with 5 of them presenting strong expression. CONCLUSIONS: We conclude that clusterin expression is under epigenetic control via methylation of its promoter.

Four-compartment model and validation of deuterium dilution technique to estimate fat-free mass in Mexican youth.

OBJECTIVE: To validate the measurement of fat-free mass (FFM) with the deuterium oxide (D(2)O) dilution technique (2C) against the four-compartment (4C) model in Mexican children. METHODS: This was designed as a cross-sectional, non-probabilistic study. Sixty subjects (30 male and 30 female) 6-14 y of age were recruited and completed the study during 5 mo. Total body water was measured using the D(2)O dilution technique and FFM was calculated using Fomon's (6-10 y) and Lohman's (11-14 y) hydration constants. Body composition using the 4C model was calculated with Lohman's equation. RESULTS: Group mean accuracy showed no differences in FFM determined by D(2)O dilution and the 4C model (1.24 kg, P > 0.4), by gender (2.1 kg, P > 0.2), or by method-by-gender interaction (P > 0.7). FFMs were 26.9 and 25.7 kg by the 4C and 2C models, respectively. The test for coincidence of slopes and intercepts between the 2C and 4C models and the line of identity were not different (P > 0.05). Precision by R(2) explained 98% of the variance (standard error of the estimate 1.2 kg). Bias for the difference in FFM was not significant (-1.27, 95% confidence interval -1.5 to -0.9) and no association between the mean of the differences and the magnitude of the measurements was found (P > 0.05). Mean bias was -1.27 kg for FFM (P > 0.05), and limits of agreement were -3.1 to 0.8 kg. CONCLUSION: The D(2)O dilution technique used with these hydration constants was accurate, precise, and free of bias in Mexican children and adolescents compared with the 4C model.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines.

BACKGROUND: The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS: Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS: Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION: We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Role of altered expression of HLA class I molecules in cancer progression.

HLA class I antigens play a key role in immune recognition of transformed and virally infected cells via binding to the peptides of "non-self" or aberrantly expressed proteins and subsequent presentation of the newly formed "HLA-I-peptide" complex to T lymphocytes. Consequently, a chain of immune reactions is initiated leading to tumor cell elimination by cytotoxic T cells. Altered tumor expression of HLA class I is frequently observed in various types of malignancies. It represents one of the main mechanisms used by cancer cells to evade immunosurveillance. Because of immune selection, HLA class I-negative variants escape and lead to tumor growth and metastatic colonization. Loss or downregulation of HLA class I antigens on tumor cell surface is a factor that limits clinical outcome of peptide-based cancer vaccines aimed to increasing specific anti-tumor activity of cytotoxic T lymphocytes. Thus, gaining more knowledge regarding frequency of HLA class I defect, its tissue specificity, and underlying molecular mechanisms may help designing appropriate therapeutic strategies in cancer treatment. Here, we describe various types of HLA class I alterations found in different malignancies and molecular mechanisms that underlie these defects. We also discuss a correlation between HLA class I defects cancer progression in melanoma patients with poor clinical response to autologous vaccination.

Código Sepsis: esquivando la mortalidad en un hospital terciario / Sepsis Code: dodging mortality in a tertiary hospital

Introducción. En el hospital de La Princesa comienza el “Código Sepsis” (CSP) en el año 2015, como un grupo multidisciplinar que dota al personal sanitario de herramientas clínicas, analíticas y organizativas, con el objetivo de la detección y el tratamiento precoz del paciente con sepsis. El objetivo de este estudio es evaluar el impacto de la implantación de CSP en la mortalidad y determinar las variables asociadas con un aumento de la misma.Material y métodos. Se realizó un estudio analítico retrospectivo de los pacientes con activación de la alerta CSP de 2015 a 2018. Se recogieron variables clínico-epidemiológicas, parámetros analíticos y factores de gravedad como el ingreso en Unidades de Cuidados Críticos (UCC) y la necesidad de aminas. La significación estadística se estableció en una p < 0,05. Resultados. Se incluyeron 1.121 pacientes. La estancia media fue de 16 días y un 32% requirieron ingreso en UCC. La mortalidad mostró una tendencia lineal descendente estadísticamente significativa del 24% en 2015 hasta el 15% en 2018. Las variables predictivas de mortalidad con asociación estadísticamente significativa fueron el lactato > 2 mmol/L, la creatinina > 1,6 mg/dL y la necesidad de aminas. Conclusiones. La implementación de Código Sepsis disminuye la mortalidad de los pacientes con sepsis y shock séptico. La presencia de una cifra de lactato > 2 mmol/L, los niveles de creatinina > 1,6 mg/dL y/o la necesidad de administrar aminas en las primeras 24 horas, se asocian con un aumento de la mortalidad en el paciente con sepsis. (AU)

Background. In the hospital of La Princesa, the “Sepsis Code” (CSP) began in 2015, as a multidisciplinary group that provides health personnel with clinical, analytical and organizational tools, with the aim of the detection and early treatment of patients with sepsis. The objective of this study is to evaluate the impact of CSP implantation on mortality and to determine the variables associated with an increase in it. Material and methods. A retrospective analytical study of patients with CSP alert activation from 2015 to 2018 was conducted. Clinical-epidemiological variables, analytical parameters, and severity factors such as admission to critical care units (UCC) and the need for amines were collected. Statistical significance was established at p < 0.05. Results. We included 1,121 patients. The length of stay was 16 days and 32% required admission to UCC. Mortality showed a statistically significant linear downward trend from 24% in 2015 to 15% in 2018. The predictive mortality variables with statistically significant association were lactate > 2 mmol/L, creatinine > 1.6 mg/dL and the need for amines. Conclusions. The implementation of Sepsis Code decreases the mortality of patients with sepsis and septic shock. The presence of a lactate > 2 mmol/L, creatinine > 1.6 mg/dL and/or the need to administer amines in the first 24 hours, are associated with an increase in mortality in the patient with sepsis. (AU)