Helios-Negative Regulatory T Cells as a Key Factor of Immune Tolerance in Nonallergic Beekeepers.

BACKGROUND AND OBJECTIVES: Although exposure to stings has been identified as the leading risk factor for anaphylaxis due to Hymenoptera venom allergy, professional beekeepers receive hundreds of stings yearly without developing systemic reactions. This study aims to analyze the mechanisms underlying bee venom tolerance in beekeepers. METHODS: A cross-sectional study was conducted. Participants were recruited and classified into 3 groups: allergic patients (APs), who experienced systemic reactions after bee stings, with a positive intradermal test and specific IgE (sIgE) to Apis mellifera venom (AmV); tolerant beekeepers (TBKs), who received ≥50 stings/year; and healthy nonexposed controls (HCs). We measured serum levels of sIgE and specific IgG4 (sIgG4) to AmV, rApi m 1, rApi m 2, rApi m 3, Api m 4, rApi m 5, and rApi m10, as well as AmV-induced basophil degranulation, percentage of T-cell subsets, regulatory T cells (Treg), and IL-10 production. RESULTS: Compared with TBKs, APs had high levels of sIgE to AmV and all its allergic components (P<.001), together with a high basophil activation rate (P<.001). Conversely, compared with APs, TBKs had higher levels of sIgG4 (P<.001) and IL-10 (P<.0001), as well as an enhanced CTLA-4+ Treg population (P=.001), expanded Helios- Treg (P<.003), and reduced type 1 helper T cells (TH1) (P=.008), TH2 (P=.004), and TH17 (P=.007) subsets. CONCLUSIONS: The profile of TBKs, which was strongly marked by Treg activity, differed from that of TBKs. This natural tolerance would be led by the expansion of inducible Helios- Treg cells at the peripheral level. The Helios- Treg population could be a novel candidate biomarker for monitoring tolerance.

Isolation and Selection of Streptomyces Species from Semi-arid Agricultural Soils and Their Potential as Producers of Xylanases and Cellulases.

The Mezquital Valley (MV), Mexico, is a semi-arid region whose main economic activity is agriculture, this zone is characterized by the use of wastewater for crop irrigation. This condition has increased the amount nutrients in soils, organic carbon content and native microorganisms. The Streptomyces species are a group of saprophytic bacteria that represent between 20 and 60% of the total microbial population in soils, capable of producing metabolites of commercial importance. In this work, Streptomyces species were isolated from agricultural soils of the MV and was evaluated the production of endoglucanases (CMCase) and xylanases (Xyl) in Solid-State Cultivation (SSC). From soil samples, 73 possible strains of Streptomyces species were isolated for their ability to produce CMCase and Xyl in SSC. The study also included its characterization by morphological characteristics. Of the isolated microorganisms, 38 strains were selected as strong enzyme producers according to the measurement of the halo generated in plate and by growth on barley straw as only carbon source. Two different sizes of barley straw particle were tested, finding that the greatest enzymatic activity was observed in particle size 12. Three strains of Streptomyces species were chosen which presented the best catalytic capacities, a maximum of 100.69 AU Xyl/gram dry matter (gdm), 82 AU Xyl/gdm and 26.02 AU CMCase/gdm for strains 30, 28 and 12, respectively. The strains were identified by ribosomal gen16s sequence and identified as S. flavogriseus, S. virginiae and S. griseoaurantiacus. It is the first report of endogluconase and xylanolytic activity by S. virginiae isolated from a semi-arid soil.

Gynecological pelvic pain as emergency pathology. / Dolor pélvico de origen ginecológico como patología urgente.

Acute pelvic pain is a common condition in emergency. The sources of acute pelvic pain are multifactorial, so it is important to be familiar with this type of pathologies. The purpose of this article is review the main causes of gynecological acute pelvic pain and their radiologic appearances to be able to make an accurate diagnosis and provide objective criteria for patient management.

Screening for thermotolerant ligninolytic fungi with laccase, lipase, and protease activity isolated in Mexico.

The State of Hidalgo (Mexico) has a large area of forests known as the Huasteca Hidalguense, with a large variety of microorganisms inhabiting it. They represent an important resource from the ecological and technological point of view because they can be used in a broad variety of industrial processes. Due to the climatic conditions of this region, fungi inhabiting it must be thermophile or, at least, thermotolerant, as temperatures can be higher than 45°C in the summer, declining to 20°C in the winter. Use of ligninolytic fungi relies on their capacity to produce enzymes of industrial interest, a topic that has been under continuous research by academic and industrial investigators. Among the most important enzymes are proteases that are widely used due to their biotechnological applications with a high economic impact. Other enzymes, laccases, peroxidases, and lipases are of interest for the industries of the state of Hidalgo, especially in the textile industry, specifically in effluent processing. Fungi (n=156) were collected in the Huasteca Hidalguense, of which 100 were isolated in potato-dextrose-agar covered plates and maintained in tilted tubes. Afterwards, enzymatic activity (laccase, protease and lipase) was determined in the plates. The purpose was to select those fungi with the highest potential for biotechnological applications. Fungi generally grew at either 30°C or 37°C, and for some isolates enzymatic activities were detected at this higher temperature. Results are presented as the relation between enzymatic activity and growth rate: 60 fungi presented laccase activity, 49 had lipase activity, and none had protease activity. In most cases, enzymatic activity was higher than the growth rate, indicating that the isolated fungi have a great biotechnological potential. Statistical analysis revealed that isolates 31 (Trametes) and 8.1 (unidentified) have a larger potential to be studied as laccase-producing fungi. On the other hand, isolates 144.2 (Fomes), 154 (Trametes), and 147.2 (Pycnoporus) are of interest as lipase activity producers, an activity scarcely studied in this type of microorganisms.

Protective effects of melatonin on changes occurring in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

BACKGROUND: Melatonin has been related to the pathophysiology of multiple sclerosis (MS), and its anti-inflammatory and immunomodulatory properties have been proved in numerous neurodegenerative diseases. This study aimed to find out whether a melatonin supplement in MS is able to act as a benefit to its clinical status, i.e. oxidative stress, inflammation and indirect biomarkers of bacterial dysbiosis, lipopolysaccharide (LPS) and LPS-binding protein (LBP), verifying its therapeutic potential and its possible clinical use in patients with MS. METHODS: The animal MS model, experimental autoimmune encephalomyelitis (EAE), was employed whereby 25 male Dark Agouti rats (5 animals per group) were divided into: a control group (not manipulated); a control+vehicle group; a control+melatonin group; an EAE group; an EAE+melatonin group. Melatonin was administered daily for 51 days, at a dose of 1 mg/kg body weight/i.p., once a day, five days a week. RESULTS: The results from the administration of melatonin demonstrated an improvement in clinical status, a diminution in oxidative stress and inflammation, as well as in bacterial dysbiosis. CONCLUSION: Melatonin could play an effective role against MS, either alone or as a therapy combined with traditional agents.

Review: Production and functionality of active peptides from milk.

In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.

3-Methyl-2-benzothiazolinone hydrazone and 3-dimethylamino benzoic acid as substrates for the development of polyphenoloxidase and phenoloxidase activity by zymograms.

In the present study, a sequential staining process of polyphenoloxidase and phenoloxidase enzymes was designed by the zymography technique. As a first step, electrophoresis was carried out under native conditions, and later, first staining was carried out with a revealing solution of 3-methyl-2-benzothiazoline hydrazone (MBTH)-3-dimethylamino benzoic acid (DMAB) that allowed the visualization of polyphenoloxidase enzymes, and later and using the same gel, we proceeded to the differential staining of phenoloxidase, adding a solution of H2O2. The technique was standardized using commercial enzymes of laccase (T. versicolor) and horseradish. The technique was used to identify polyphenoloxidases (laccases) and phenoloxidases (lignin peroxidase) of crude extracts obtained from the growth of the basidiomycete Lentinus strigosus on Pinus radiata. The technique showed great sensitivity to detect the different enzymatic activities (1.56 Activity Unit/mL minimum) in the same gel without interference between the enzymes and the solutions used. On the other hand, the efficiency of the technique was compared with the substrates that are commonly used for the detection of this type of activities such as 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol, observing greater sensitivity and minimal interference, so that the present method will allow in the same gel, and visualize polyphenoloxidase and phenoloxidase activities simultaneously facilitating expression studies.

[Midfemoral nerve block for foot surgery: is there an anatomical-clinical correlation between motor response and latency?]. / Bloqueo mediofemoral para cirugía de pie: existe relación entre la respuesta motora y la latencia? Correlación anatomoclínica.

BACKGROUND AND OBJECTIVE: The latency times of midfemoral sciatic nerve blocks vary greatly. This study investigated the correlation between the type of motor response to nerve stimulation on the one hand and latency and block efficacy on the other. PATIENTS AND METHODS: We enrolled 215 consecutive patients (184 women) undergoing orthopedic foot surgery. A tourniquet was applied above the malleolus. The puncture location was found by palpating to locate the groove between the vastus lateralis and biceps femoris muscles, at the mid-point of the line between the posterior edge of the greater trochanter muscle and the insertion of the biceps femoris muscle in the popliteal fossa. A solution of equal proportions (1:1) of 1.5% mepivacaine (with bicarbonate 1:10) and 0.75% levobupivacaine was injected at a dose of 0.45 mL x kg(-1) (maximum 40 mL) using a 10-cm needle. Nerve stimulation was applied at 100-300 ms, 02-0.4 mA, and 2 Hz. Latency was classified as response in less than 15 minutes, in 15 to 30 minutes, or later than 30 minutes. RESULTS: The evoked motor response was inversion in 30 patients, flexion or extension in 38, plantar flexion in 101, dorsiflexion in 37, and eversion in 9. Shorter latencies (15 minutes) were observed in all patients with inversion or flexion/extension and in 84 (83%) of the 101 patients with plantar flexion. Mid-range latencies were observed in 13% of those with a plantar flexion response and in 29.7% of those with dorsiflexion. All 9 patients with eversion and 17 (45.9%) of the 37 patients with dorsiflexion had the longest latencies. The surgical block was complete for all patients. CONCLUSIONS: This approach provides an effective block with minimum latency in patients who have a flexion or extension motor response in the foot and/or fingers, inversion, or plantar flexion, which assumes that the injection has reached the common trunk of the sciatic or tibial nerve. However, a longer latency is associated with a peroneal motor response, particularly eversion.

Biological decolorization of Amaranth dye with Trametes polyzona in an airlift reactor under three airflow regimes.

In the present work, a strain of the basidiomycete fungus Trametes polyzona was used to decolorize the Amaranth dye. The decolorization was carried out in an Airlift reactor with three flow regimes: 1, 2, and 3 vvm. The results showed that the decolorization was a function of the flow regime. The decolorization times for the regimes of 1, 2, and 3 vvm were 30, 25, and 19 days, respectively. The COD (Chemical Oxygen Demand) decreased from 1600 to 72 mg COD/L. The enzymatic activity kinetics of laccase (Lcc), lignin peroxidase (LiP), and manganese peroxidase (MnP) were determined. In all the treatments, the enzyme LiP was expressed during the first 6 days, at which point 80% decolorization was observed, whereas Lcc and MnP enzymes were produced from day 6 until the end of the decolorization process. The effluent generated showed no inhibitory effects on the growth of the algae Nannochloropsis salina. T. polyzona showed great versatility in the decolorization of synthetic effluents containing the Amaranth dye, and the fungus was able to use this dye as its only carbon source starting at the beginning of the process. LiP was the enzyme that contributed the most to the decolorization process, and on average, 95% decreases in color and the COD were observed.

Use of barley straw as a support for the production of conidiospores of Trichoderma harzianum.

In this work was to evaluate the conidiospores production of Trichoderma harzianum using barley straw as substrate. Four growth conditions were used; washed and unwashed barley straw and washed and unwashed barley straw supplemented with mineral salts. The highest spore production was observed when washed barley straw supplemented with mineral salts with 1.56 × 1010 conidiospores/gram of dry matter (gdm) at 216 h of cultivation was used. The effect of substrate moisture on spore production was studied, three initial moisture levels of the substrate were tested and it was observed that a humidity of 80 % of the substrate improves the production of conidiospores reaching a concentration of 2.35 × 1010 conidiospores/gdm at 136 h. Finally, conidiospores viability was evaluated for 12 months by keeping them on the conidia and substrate, and viability of 71 % of the conidiospores was observed, so this maintenance method is an excellent means of conserving the conidiospores viability.

Prognostic value of perirenal and/or sinus fat infiltration in patients with pT3a renal cell carcinoma: A multicentre cohort study. LARCG Group. / Valor pronóstico de la invasión de grasa perirrenal y/o del seno en pacientes con carcinoma de células renales pT3a: estudio de cohorte multicéntrico. Grupo LARCG.

INTRODUCTION AND OBJECTIVES: The objective of this study is to evaluate overall survival (OS), cancer-specific survival (CSS), relapse-free survival, local and distant (LRFS and DRFS, respectively) rates in patients with pT3a renal cell carcinoma (RCC) considering the perirenal and/or sinus fat infiltration (FI) as prognostic factors. MATERIALS AND METHODS: Retrospective cohort of patients with pT3a RCC who underwent radical or partial nephrectomy. The data were extracted from the LARCG (Latin American Renal Cancer Group) database. The demographic, clinical, pathological and surgical variables were evaluated. FI was divided into 4 groups (vein, perirenal, sinus and both fats infiltration). The Kaplan Meier and Cox regression curves were performed. RESULTS: 293 patients were included in the study. The mean age was 61.4 years. The median follow-up was 21 months (r: 1-194). CSS, RFS, LRFS and DRFS estimated at 3 years in the group of both fats' infiltration were 53.1, 45.1, 58.7 and 51.6 months, respectively, and always statistically lower than the rest (P˂0.005). In the multivariate analysis, the infiltration of both fats significantly increased specific mortality, overall and local relapse with respect to vein infiltration (HR: 4.5, 2.42 and 8.08, respectively). The Fuhrman grade and renal pelvis infiltration were independent predictors of CSS and RFS. CONCLUSIONS: Infiltration of both fats increases the risk of overall and local relapse in pT3a RCC. In the same way, it is associated with a lower cancer-specific survival and should be considered as a factor of poor prognosis.

Acute abdominal pain in patients with Crohn's disease: what urgent imaging tests should be done? / Dolor abdominal agudo en la enfermedad de Crohn. ¿Qué prueba de imagen urgente se debe hacer?

Crohn's disease is an autoimmune disease that predominantly affects the gastrointestinal tract. Crohn's disease is diagnosed at a young age and runs a chronic course with acute flare-ups. When patients with Crohn's disease present with flare-ups at the emergency department, they are usually managed in a way similar to patients with acute abdomen; there is no consensus about the most appropriate imaging work-up for patients with flare-ups of Crohn's disease. Thus, we decided to review the literature about the imaging tests indicated (whether related to their diagnostic performance or to lower exposure to ionizing radiation) for acute flare-ups in patients with Crohn's disease.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Quantitative analysis of pork dry-cured sausages to quality control by NIR spectroscopy.

Near infrared spectroscopy technology (diode array instrument) was used to study the feasibility of applying quality controls to typical Spanish sausages by performing a proximate analysis (fat, moisture and protein) on the finished product (intact and homogenized). This could be used to provide quality controls at various stages once the finished product was obtained: finished product, storage, distribution and marketing. The selected models were calibrated and evaluated by cross and external validation. For intact products, coefficients of determination for calibration (R(2)) for fat, moisture and protein were 0.98, 0.93 and 0.97, respectively. These values for homogenised products were 0.99, 0.98 and 0.97, respectively. The standard errors of prediction (SEP) for external validation in intact products were 1.47%, 0.97% and 1.08% for fat, moisture and protein, respectively. In homogenised products, these values were lower: 0.71%, 0.41% and 0.95%, respectively.

Effects of agricultural land use on fluvial carbon dioxide, methane and nitrous oxide concentrations in a large European river, the Meuse (Belgium).

We report a data-set of CO2, CH4, and N2O concentrations in the surface waters of the Meuse river network in Belgium, obtained during four surveys covering 50 stations (summer 2013 and late winter 2013, 2014 and 2015), from yearly cycles in four rivers of variable size and catchment land cover, and from 111 groundwater samples. Surface waters of the Meuse river network were over-saturated in CO2, CH4, N2O with respect to atmospheric equilibrium, acting as sources of these greenhouse gases to the atmosphere, although the dissolved gases also showed marked seasonal and spatial variations. Seasonal variations were related to changes in freshwater discharge following the hydrological cycle, with highest concentrations of CO2, CH4, N2O during low water owing to a longer water residence time and lower currents (i.e. lower gas transfer velocities), both contributing to the accumulation of gases in the water column, combined with higher temperatures favourable to microbial processes. Inter-annual differences of discharge also led to differences in CH4 and N2O that were higher in years with prolonged low water periods. Spatial variations were mostly due to differences in land cover over the catchments, with systems dominated by agriculture (croplands and pastures) having higher CO2, CH4, N2O levels than forested systems. This seemed to be related to higher levels of dissolved and particulate organic matter, as well as dissolved inorganic nitrogen in agriculture dominated systems compared to forested ones. Groundwater had very low CH4 concentrations in the shallow and unconfined aquifers (mostly fractured limestones) of the Meuse basin, hence, should not contribute significantly to the high CH4 levels in surface riverine waters. Owing to high dissolved concentrations, groundwater could potentially transfer important quantities of CO2 and N2O to surface waters of the Meuse basin, although this hypothesis remains to be tested.

Helios-Negative Regulatory T Cells as a Key Factor of Immune Tolerance in Nonallergic Beekeepers

Background: Although exposure to stings has been identified as the leading risk factor for anaphylaxis due to Hymenoptera venom allergy, professional beekeepers receive hundreds of stings yearly without developing systemic reactions. Objective: This study aims to analyze the mechanisms underlying bee venom tolerance in beekeepers. Methods: A cross-sectional study was conducted. Participants were recruited and classified into 3 groups: allergic patients (APs), who experienced systemic reactions after bee stings, with a positive intradermal test and specific IgE (sIgE) to Apis mellifera venom (AmV); tolerant beekeepers (TBKs), who received ≥50 stings/year; and healthy nonexposed controls (HCs). We measured serum levels of sIgE and specific IgG4 (sIgG4) to AmV, rApi m 1, rApi m 2, rApi m 3, Api m 4, rApi m 5, and rApi m10, as well as AmV-induced basophil degranulation, percentage of T-cell subsets, regulatory T cells (Treg), and IL-10 production. Results: Compared with TBKs, APs had high levels of sIgE to AmV and all its allergic components (P<.001), together with a high basophil activation rate (P<.001). Conversely, compared with APs, TBKs had higher levels of sIgG4 (P<.001) and IL-10 (P<.0001), as well as an enhanced CTLA-4+ Treg population (P=.001), expanded Helios– Treg (P<.003), and reduced type 1 helper T cells (TH1) (P=.008), TH2 (P=.004), and TH17 (P=.007) subsets. Conclusions: The profile of TBKs, which was strongly marked by Treg activity, differed from that of TBKs. This natural tolerance would be led by the expansion of inducible Helios– Treg cells at the peripheral level. The Helios– Treg population could be a novel candidate biomarker for monitoring tolerance (AU)

Herbal medicine use, Epstein-Barr virus, and risk of nasopharyngeal carcinoma.

Herbal medicine use is thought to be linked to nasopharyngeal carcinoma (NPC) either through its ability to reactivate the Epstein-Barr virus (EBV) or through a direct promoting effect on EBV-transformed cells. To investigate this, 104 histologically confirmed NPC cases and 205 matched controls were studied in The Philippines. Blood was collected to assess antibody titers against EBV, and an interview was administered which elicited information concerning herbal medicine use and other risk factors for NPC. Subjects strongly positive for anti-EBV antibodies (Epstein-Barr nuclear antigen [EBNA]) (titers greater than or equal to 1:80) were at a 21-fold excess risk of disease (95% confidence interval, 8.4, 51.8). Herbal medicine use was also associated with NPC (relative risk, 2.5; 95% confidence interval, 1.4, 4.5). Associations persisted after adjustment for education, smoking, Chinese ancestry, and consumption of salted fish. Exposure to herbal medicines among subjects testing negative/weakly positive for anti-EBNA antibodies was not associated with an elevation in risk (relative risk, 0.6), strong positivity to anti-EBNA antibodies in the absence of herbal medicine use was associated with a significant 16-fold excess risk of disease, and exposure to herbal medicines among subjects testing strongly positive for anti-EBNA antibodies was associated with a significant 49-fold excess risk of NPC when cases were compared to controls. Similar results were obtained when other serological measures of EBV exposure were used. Anti-EBV antibody titers were elevated in herbal medicine users compared to nonusers among cases but not among control subjects. This suggests that, if herbal medicines interact with EBV in the development of NPC, they do not do so by reactivating EBV infection but rather through a direct proliferative effect on EBV-transformed cells. Although the interaction between EBV and herbal medicines is biologically plausible, larger, more detailed studies need to be conducted to validate this preliminary finding.