Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells.

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Effects of heat-stress-reducing systems on blood constituents, milk production and milk quality of Holstein and Jersey cows and heifers on pasture.

The objective of this study was to evaluate the influence of different heat-stress-reducing systems, i.e., sprinkler + artificial shade, shower + artificial shade, and artificial shade, on serum mineral, hormonal, hematological, and metabolite profiles, on milk production, and milk composition in lactating cows and pubertal heifers of Holstein and Jersey breeds. For this purpose, 12 animals were used: 3 Holstein cows with an average (mean ± SD) body weight of 600 ± 30 kg, 53 ± 11 months of age, and milk yield of 27 ± 3.5 kg/day; 3 Jersey cows with an average body weight of 370 ± 11 kg, 40 ± 6 months of age, and milk production of 11 ± 1.5 kg/day; 3 Holstein heifers (325 ± 25 kg and 16 ± 0.6 months of age); and 3 Jersey heifers (250 ± 25 kg and 13 ± 0.6 months of age). Animals were used in a replicated 3 × 3 Latin square design with a 3 × 2 × 2 factorial arrangement of treatments that included three treatments (sprinkler + artificial shade, shower + artificial shade, and artificial shade), two breeds (Holstein, Jersey), and two physiological stages (lactating cows, heifers). The experimental treatments influenced (P < 0.05) the concentrations of triiodothyronine, with the shower and shade systems showing greater and similar concentrations (99.5 and 96.3 µg/dL, respectively) when compared with sprinkler treatment (89.2 µg/dL). There was an effect (P < 0.05) of breed on the concentrations of Na + , K + , hemoglobin, hematocrit and mean corpuscular volume levels with the Holsteins having lower levels of Na + , K + , hemoglobin, hematocrit and mean corpuscular volume (101.1, 4.0 ng/mL, 11.2 g/dL, 24.7%, and 42.3 µm3, respectively) than the Jerseys (106.5 and 4.3 ng/mL, 12.4 g/dL, 27.7%, and 46.3 µm3, respectively. Total cholesterol and high-density lipoproteins were influenced by physiological stage (P < 0.05). Concentrations of cholesterol and high-density lipoproteins were higher for cows (94.1, and 56.9 mg/dL, respectively) than for heifers (56.9 and 42,9 mf/dL, respectively). Milk production and fat content were affected (P < 0.05) by breed (P < 0.05), with Holstein cows producing more milk (23.9 kg/day) than Jersey cows (12.0 kg/day), but Jersey cows had higher fat concentration (4.6%) than Holstein cows (3.0%). Therefore, the three different thermal-stress-reducing-systems tested were able to maintain the serum biomarkers within normal physiological ranges. However, the most appropriate thermal-stress-reducing-systems would be a sprinkler systema because it uses less water compared with the shower system.

Executive orders or public fear: What caused transit ridership to drop in Chicago during COVID-19?

The COVID-19 pandemic has induced significant transit ridership losses worldwide. This paper conducts a quantitative analysis to reveal contributing factors to such losses, using data from the Chicago Transit Authority's bus and rail systems before and after the COVID-19 outbreak. It builds a sequential statistical modeling framework that integrates a Bayesian structural time-series model, a dynamics model, and a series of linear regression models, to fit the ridership loss with pandemic evolution and regulatory events, and to quantify how the impacts of those factors depend on socio-demographic characteristics. Results reveal that, for both bus and rail, remote learning/working answers for the majority of ridership loss, and their impacts depend highly on socio-demographic characteristics. Findings from this study cast insights into future evolution of transit ridership as well as recovery campaigns in the post-pandemic era.

Yerba mate (Ilex paraguariensis) as a source of antioxidants with soybean grain in supplementation of lactating ewes reared in tropical pastures.

The hypothesis tested is that the association of soybean grain and yerba mate as an antioxidant source can increase the concentration of polyunsaturated fatty acids (PUFA) with lower oxidation of milk from lactating ewes reared in tropical pastures. Sixteen ewes were randomly distributed in a 2x2 factorial scheme (with or without yerba mate at 110 g / kg of dry matter (DM); with or without soybean grain at 210 g/kg of DM). Yerba mate intake reduced DM intake, and concentrations of ether extract (EE), protein, lactose, defatted dry extract, density, omega-3 concentrations, and the ratio between saturated/monounsaturated fatty acids (FA), but increased the concentration of monounsaturated FA and the sequestering power of free radicals by DPPH without altering the concentrations of conjugated dienes and the concentrations of substances reactive to thiobarbituric acid in milk. Supplementation with soybean grain decreased the birth weight of the lambs and increased the concentrations of conjugated linoleic acid (CLA), PUFA, omega - 6, omega ratio - 6/omega - 3, and total blood cholesterol. The addition of yerba mate did not influence the oxidative profile of milk. Supplementation with soybean grain improved the FA profile of milk, increasing the concentrations of CLA and PUFA. The results suggest that the association of yerba mate and soybean grain helps to improve the fat quality of milk from ewes raised in tropical pastures, as shown by the enhanced antioxidant activity, although it does not help prevent oxidation of milk rich in PUFA.

CariesCare International adapted for the pandemic in children: Caries OUT multicentre single-group interventional study protocol.

BACKGROUND: Comprehensive caries care has shown effectiveness in controlling caries progression and improving health outcomes by controlling caries risk, preventing initial-caries lesions progression, and patient satisfaction. To date, the caries-progression control effectiveness of the patient-centred risk-based CariesCare International (CCI) system, derived from ICCMS™ for the practice (2019), remains unproven. With the onset of the COVID-19 pandemic a previously planned multi-centre RCT shifted to this "Caries OUT" study, aiming to assess in a single-intervention group in children, the caries-control effectiveness of CCI adapted for the pandemic with non-aerosols generating procedures (non-AGP) and reducing in-office time. METHODS: In this 1-year multi-centre single-group interventional trial the adapted-CCI effectiveness will be assessed in one single group in terms of tooth-surface level caries progression control, and secondarily, individual-level caries progression control, children's oral-health behaviour change, parents' and dentists' process acceptability, and costs exploration. A sample size of 258 3-5 and 6-8 years old patients was calculated after removing half from the previous RCT, allowing for a 25% dropout, including generally health children (27 per centre). The single-group intervention will be the adapted-CCI 4D-cycle caries care, with non-AGP and reduced in-office appointments' time. A trained examiner per centre will conduct examinations at baseline, at 5-5.5 months (3 months after basic management), 8.5 and 12 months, assessing the child's CCI caries risk and oral-health behaviour, visually staging and assessing caries-lesions severity and activity without air-drying (ICDAS-merged Epi); fillings/sealants; missing/dental-sepsis teeth, and tooth symptoms, synthetizing together with parent and external-trained dental practitioner (DP) the patient- and tooth-surface level diagnoses and personalised care plan. DP will deliver the adapted-CCI caries care. Parents' and dentists' process acceptability will be assessed via Treatment-Evaluation-Inventory questionnaires, and costs in terms of number of appointments and activities. Twenty-one centres in 13 countries will participate. DISCUSSION: The results of Caries OUT adapted for the pandemic will provide clinical data that could help support shifting the caries care in children towards individualised oral-health behaviour improvement and tooth-preserving care, improving health outcomes, and explore if the caries progression can be controlled during the pandemic by conducting non-AGP and reducing in-office time. TRIAL REGISTRATION: Retrospectively-registered-ClinicalTrials.gov-NCT04666597-07/12/2020: https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S000AGM4&selectaction=Edit&uid=U00019IE&ts=2&cx=uwje3h . Protocol-version 2: 27/01/2021.

Cryoablation does not significantly contribute to systemic effector immune responses in poorly immunogenic B16F10 melanoma model.

PURPOSE: Cryoablation is a minimally invasive procedure implemented to destroy solid tumors. It also results in the release of tumor antigens into the systemic circulation. Pre-clinical studies employing immunogenic tumor models have shown that cryoablation evokes anti-tumor immune responses. How cryoablation impacts immune responses in poorly immunogenic tumors has not been sufficiently explored. EXPERIMENTAL DESIGN: We employed bilateral, B16F10 melanoma model, devoid of strong immunogenic antigens. Cryoablation induced effector immune responses were investigated, also in combination with peritumoral STING agonist and systemic anti-PD1. Selective immune cell depletion, T-cell migration arrest, in vivo T-cell transplantation, and cryoablation vs surgical removal techniques were employed to determine the contribution of cryoablation and immunotherapies to systemic, anti-tumor, effector immune responses. RESULTS: Treatment of a tumor with cryoablation + STING agonist + anti-PD1 resulted in the rejection of unablated, contralateral tumor. Depletion studies demonstrated that the tumor rejection is essentially dependent on CD8+ T-cells. T-cell arrest in the lymph nodes had no effect on the rejection process. Splenic CD8+ T-cells isolated from cryoablation treated mice with B16F10 melanoma, upon transplantation into melanoma bearing recipients did not impact the recipient's tumor growth. Finally, comparison of cryoablation + STING agonist + anti-PD1 vs surgery + STING agonist + anti-PD1 in bilateral tumor model showed no difference in the rejection of contralateral tumor. CONCLUSION: Cryoablation does not significantly contribute to systemic, anti-tumor, effector immune responses in a B16F10 melanoma model. Cryoablation primarily performs tumor debulking, and immunotherapy functions independently of cryoablation in eliciting anti-tumor effector immune responses.

Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

Adrenal malakoplakia a rare lesion that mimics a neoplasm.

Background: Malakoplakia is a rare disorder 75% of the reported cases affect mainly the genitourinary tract, its occurrence in the adrenal gland is extremely rare. Case presentation: A 65-year-old female patient presented to the emergency department for chronic abdominal pain. Radiographic and biochemical studies revealed a left adrenal incidentaloma and left adrenalectomy was performed. Histological examination showed the presence of Michaelis-Gutmann bodies, compatible with a malakoplakia of the adrenal gland. Conclusions: Malakoplakia is a rare disorder, with non-standardized treatment, medical and surgical therapies appear to be effective in treating the condition.

Can we predict psychostimulant use in youths? A study with structural equation modeling analysis.

Knowing which factors predict the use of psychostimulant drugs among youths is important for designing preventive programs and the identification of youths with the highest probability of use, to avoid some of the problems that can be derived from it. The purpose of this study is to examine the relationship between perceived availability, risk perception, personality traits, antisocial personality patterns, and use of other substances in the prediction of psychostimulant use. The sample was composed by 1,177 youths (584 males and 593 females) aged 14 to 25 in Spain, recruited randomly, stratified by lifetime use- non use of psychostimulants (cocaine or ecstasy). The path which best predicts psychostimulant use is: Personality traits-legal substance use-cannabis use-psychostimulant use. The next path is: Personality traits-antisocial personality patterns-psychostimulant use. Risk perception and perceived availability were significant in the prediction, but made a smaller contribution. The results of this study suggest that the model evaluated can predict psychostimulant use in youths. This model can be seen as a useful tool for developing preventive strategies and for identifying those youths that are at risk for problems related to drug use.

Influence of Different Heat-Stress-Reducing Systems on Physiological and Behavioral Responses and Social Dominance of Holstein and Jersey Cows and Heifers on Pasture.

High ambient temperatures and relative humidity affect the behavior and physiology of the animal. This study investigated the influence of different heat-stress-reducing systems on the physiological, behavioral, and preferential responses of Holstein and Jersey cows and heifers on pasture. Experimental treatments were: (1) three heat-stress-reducing systems (sprinklers + artificial shade; showers + artificial shade; and artificial shade); (2) two breeds (Holstein and Jersey); and (3) two physiological stages (lactating cows and pubertal heifers). Physiological and behavioral responses to treatments were measured every 30 min on collection days. The frequency and duration of the use of the systems were recorded continuously 24 h/day for 3 days in each period. The air temperature and the relative humidity were 26 ± 4.1 °C and 74 ± 11.3%, respectively. The experimental treatments affected (p = 0.0354) standing idle, grazing behavior (p = 0.0435), and the frequency and duration of use of the systems by the animals (p < 0.0001). For all treatments, the respiratory rate and the coat surface temperature were highly and significantly correlated (p < 0.05) with the temperature and humidity index. In conclusion, under ambient conditions, dairy cows preferred using sprinklers or showers over artificial shade. These systems were more efficient at reducing the heat load and led to better behavioral and physiological responses.

Feeding Calcium-Ammonium Nitrate to Lactating Dairy Goats: Milk Quality and Ruminal Fermentation Responses.

We aimed to investigate the effects of calcium-ammonium nitrate (CAN) fed to lactating dairy goats on dry matter (DM) intake, digestibility of nutrients, milk properties (composition, antioxidant capacity, fatty acid profile, and nitrate residues), and ruminal fermentation parameters. Twelve lactating Saanen goats averaging 98.5 ± 13.1 days in milk, 53.5 ± 3.3 kg of body weight, and 2.53 ± 0.34 kg of milk/day were randomly assigned in four 3 × 3 Latin squares to receive the following diets: a control group (without CAN) with 7.3 g/kg DM of urea (URE), 10 g/kg DM of CAN (CAN10), and 20 g/kg DM of CAN (CAN20). Each period lasted 21 days, with 14 days for diet adaptation and seven days for data and sample collection. The DM intake, digestibility of nutrients, yields of milk, 3.5% fat-corrected milk, and energy-corrected milk were not affected by treatments. Similarly, there were no treatment effects on the yields and concentrations of milk fat, true protein, and lactose, along with minor effects on milk fatty acid profile. Total antioxidant capacity in milk was unaffected by treatments; however, concentration of conjugated dienes increased, while thiobarbituric acid reactive substances in milk decreased linearly. Nitrate and nitrite residues in milk were elevated by treatments, while the total of volatile fatty acids and ammonia-N concentration in the rumen were unaffected. Collectively, feeding CAN (up to 20 g/kg of DM) to lactating dairy goats did not affect feed intake, nutrient digestibility, and milk composition; however, it may increase milk lipid oxidation, as evidenced by increased conjugated diene concentration.

Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell-infiltrated tumors. SIGNIFICANCE: Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1.

Interpretation of T cell states from single-cell transcriptomics data using reference atlases.

Single-cell RNA sequencing (scRNA-seq) has revealed an unprecedented degree of immune cell diversity. However, consistent definition of cell subtypes and cell states across studies and diseases remains a major challenge. Here we generate reference T cell atlases for cancer and viral infection by multi-study integration, and develop ProjecTILs, an algorithm for reference atlas projection. In contrast to other methods, ProjecTILs allows not only accurate embedding of new scRNA-seq data into a reference without altering its structure, but also characterizing previously unknown cell states that "deviate" from the reference. ProjecTILs accurately predicts the effects of cell perturbations and identifies gene programs that are altered in different conditions and tissues. A meta-analysis of tumor-infiltrating T cells from several cohorts reveals a strong conservation of T cell subtypes between human and mouse, providing a consistent basis to describe T cell heterogeneity across studies, diseases, and species.

Annatto seeds as Antioxidants Source with Linseed Oil for Dairy Cows.

This study aimed to evaluate the effects of annatto seeds, linseed oil and their combination on DMI, apparent total tract digestibility, antioxidant capacity and milk composition of dairy cows. Four lactating Holstein cows (120 ± 43 days in milk; 15.98 ± 2.02 kg of milk/day, mean ± SD) were allocated in a 4 × 4 Latin square with a 2 × 2 factorial arrangement (with or without annatto seeds at 15 g/kg of dry matter (DM); with or without linseed oil at 30 g/kg of DM) and provided four different diets: control (no annatto seeds or linseed oil); annatto seeds (15 g/kg of DM); linseed oil (30 g/kg of DM); and a combination of both annatto seeds and linseed oil. Annatto seeds reduced DM intake, and milk yield, protein and lactose, but increased content of fat, total solids and short chain fatty acid, with no effect on total antioxidant capacity of milk. Linseed oil supplementation decreased medium chain fatty acid proportion and n-6/n-3 ratio, conversely it increased long chain fatty acids and n-3 fatty acid content of milk, ether extract intake and total-tract digestibility. Thus, linseed oil supplementation in dairy cow diets improved the milk FA profile but decreased milk fat concentration, whereas annatto seeds did not influence antioxidant capacity and depressed feed intake and milk yield.

Modulation of CD4 T cell function via CD6-targeting.

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3+ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate.

Renal and Adrenal Minilaparoscopy: A Prospective Multicentric Study.

OBJECTIVE: To investigate the role of contemporary minilaparoscopy (ML; 3 mm instruments and laparoscope) and to identify predictive factors for complications in a prospective multicenter series for renal and adrenal surgeries. MATERIALS AND METHODS: From July 2013 to December 2014, 110 patients from 6 laparoscopic Spanish centers were enrolled. A common database was used and data were collected in a prospective manner. Standard approach was defined as 3 to 4 3-mm trocars with a 3-mm laparoscope and 3-mm instruments (Karl Storz, Tuttlingen, Germany). Descriptive variables were analyzed and statistical analysis was performed for predictive factors for complications. RESULTS: Patient mean age was 57.8 ± 14.6 years, with an average body mass index of 25.3 ± 3.6 kg/m(2). Median American Society of Anesthesiologists score was II and 32% (n = 35) of the patients had a previous surgery. A total of 59 nephrectomies, 20 partial nephrectomies, 9 nephroureterectomies, 13 pyeloplasties, 3 pyelolithotomies, and 6 adrenalectomies were performed. Overall operative time was 180 ± 64 minutes. There were 12 clampless partials and 8 with a mean warm ischemia time of 14 ± 7 min. There were 5% of intraoperative and 8% of postoperative complications (Clavien II-IV). Mean hospital stay was 5 ± 2.3 days, with optimal pain and cosmetic control. CONCLUSION: To our knowledge, this is one of the largest prospective series of ML for renal and adrenal surgeries. Despite a mean operative time possibly longer than in standard laparoscopy, clinical and safety outcomes are not compromised. Furthermore, ML results in excellent pain control and cosmetic outcomes.

First-time admissions for opioid treatment: cross-sectional and descriptive study of new opioid users seeking treatment.

BACKGROUND: The purpose of this study was to gain an understanding of the profiles of the new treatment demands posed by opioid addicts between 2005 and 2010 at the addictive disorders assistance units in Galicia, Spain. METHODS: A cluster analysis was performed using data from 1,655 treatment entrants. Clusters were constructed using sociodemographic and medicolegal variables. A cluster analysis was also conducted according to age. Once clusters were defined, their association with the following variables was analyzed: age at first use of opioids, years of use, frequency of opioid use in the previous month, psychiatric treatment, cocaine use, existence of a drug-dependent partner, and source of referral. RESULTS: Four clusters were obtained in the main analysis. Cluster 1 (34.01%) consisted of young males, cluster 2 (16.19%) consisted of not-so-young males, cluster 3 (32.62%) consisted mainly of older males and a small group of females, and cluster 4 (17.18%) was made up entirely of women. With regard to age-related clusters, two clusters were obtained in those under the age of 30 years: cluster 1 (73%) without medicolegal complications and cluster 2 (27%) with medicolegal complications. For those over the age of 30 years, two clusters were obtained: cluster 1 (53.92%) with hardly any medicolegal complications and cluster 2 (46.08%) with medicolegal complications. CONCLUSION: Cluster analysis suggests that there have been no substantial changes in variables indicating greater severity in this new group of patients. Women are likely to seek help earlier, which reduces their duration of opioid use. The younger the patient, the shorter the duration of opioid use and the greater the likelihood of cessation of intravenous use. Public health systems should use a two-pronged treatment strategy of short but intense cessation therapies for women and younger treatment entrants and longer maintenance and replacement therapies for older treatment entrants with more psychosocial and medical complications.

Producción de plásmidos recombinantes para su uso como controles de la técnica RT-PCR para el diagnósticode los virus Chikungunya y Zika / Production of recombinant plasmids as controls of diagnosis technique (RTPCR) of Chikungunya and Zika viruses

El diagnóstico molecular de arbovirus es indispensable para identificar agentes etiológicos, particularmente en zonas endémicas para al menos uno de ellos. Estas deben ser validadas con controles positivos, los cuales están clásicamente representados por virus vivos, cuya obtención puede ser riesgosa, laboriosa y costosa. El objetivo de este estudio fue producir plásmidos recombinantes para su uso como controles positivos en la validación de la técnica RT-PCR para el diagnóstico de los virus Chikungunya (CHIKV) y Zika (ZIKV). A partir de los ARN extraídos de los virus [CHIKV (LARD809-GC) y ZIKV (MR766)] se obtuvieron por RT-PCR fragmentos parciales de ADN correspondientes a secuencias nucleotídicas de los genes E1 y NS5 de los virus Chikungunya y Zika, respectivamente, para serclonados en el plásmido comercial pGEM®-T Easy. La clonación se confirmó mediante PCR de colonias y PCR de ADN plasmídicos extraídos a partir de las colonias recombinantes. Se logró la producción de dos plásmidos recombinantes CHIKV-E1/pGEM®-T Easy y ZIKV-NS5192/pGEM®-T Easy con cada una de las secuencias especificadas, para su uso en la validación y control de las técnicas moleculares descritas en este reporte, para el diagnóstico de agentes virales CHIKV y ZIKV, evitando la manipulación de cultivos celulares y garantizando una fuente confiable de controles positivos(AU)

The use of molecular techniques for the viral diagnosis requires the use of positive controls.Classically, the controls are live viruses, whose manipulation may be risky, laborious and expensive. The objective of this study was produced recombinant plasmids to obtain cloned sequences of Chikungunya (CHIKV) and Zika (ZIKV) virus for their use as controls in the specificdiagnostic by RT-PCR. DNA fragments were obtained fromRNA [CHIKV (LARD809-GC) and ZIKV (MR766)] using specific primers to amplify the nucleotide sequences from fragments of Envelope 1 protein (E1) of CHIKV and Non Structural 5 protein (NS5) of ZIKV genomes. The 548 bp (CHIKV) and 192 bp(ZIKV) bands were purified from agarose gel and ligations were performed with the cloning vector pGEM®-T Easy. The Escherichia coli XL1-Blue MRF` cells were transformed with the ligation mixture, the recombinant colonies were identified by colony PCR using the specific primers to the specific viral agent. One recombinant colony from CHIKV and six recombinant colonies from ZIKV were obtained from which plasmidic DNAs was extracted. The plasmidic DNAs were used as reaction controls in CHIKV and ZIKV RT-PCR, obtaining the characteristic bands. The cloning of the sequences was successful to produce the recombinant plasmids (CHIKV-E1/pGEM®-T Easy y ZIKV-NS5192/pGEM®-T Easy) to use in the validation of RT-PCR techniques(AU)

Dissemination of clonal groups of Brachyspira hyodysenteriae amongst pig farms in Spain, and their relationships to isolates from other countries.

BACKGROUND: Swine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. METHODOLOGY/PRINCIPAL FINDINGS: A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. CONCLUSIONS/SIGNIFICANCE: This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains.