Managing systemic reactions and venom immunotherapy in vespid-venom allergy: observations from a retrospective study of Portuguese patients.

Summary: Background. Vespula spp. and Polistes spp. are relevant species in South Europe, with Vespa velutina nigrithorax (VVN) being considered a public health problem. We aimed to characterize a cohort of Portuguese patients referred for large local reaction (LLR) and/or systemic reaction (SR) to vespids. In patients treated with venom immunotherapy (VIT), induction protocol and frequency of adverse reactions were evaluated. Methods. Retrospective study including patients with LLR and SR to vespids referred to our Immunoallergology Department (2008-2022). Results. A total of 129 patients were evaluated, the majority were male adults (n = 77, 59.7%), from rural areas. From these, 51 patients had SR (Mueller classification: 7.8% grade I, 19.6% grade II, 37.3% grade III, 35.3% grade IV). We found no differences regarding the levels of total serum IgE, basal serum tryptase value, sIgE levels to the eliciting venom or their molecular components, regarding the severity of the SR that motivated the referral to our clinic. In the SR group, previous LLR Hymenoptera sting were reported in 15.7%. Thirty-eight patients (74.5%) initiated VIT: 22 with wasp venom, 14 with paper wasp venom and 2 with Vespa velutina venom. There was one mild systemic reaction, not requiring adrenaline and 4 LLR. Re-stings after VIT occurred in 16 patients, without any systemic or local reactions. Currently, eleven patients remain under VIT. Conclusions. Most vespid-venom allergic patients are male adults from rural areas. Sensitization to wasp venom was present in 52.9%, paper wasp in 33.3% and Vespa velutina in 13.7%. The frequency of adverse reactions during both induction and maintenance phases appears to be low. Despite a reduced sample size, our experience with VVN VIT, suggests its safety.

YAP activation inhibits inflammatory signalling and cartilage breakdown associated with reduced primary cilia expression.

OBJECTIVE: To clarify the role of YAP in modulating cartilage inflammation and degradation and the involvement of primary cilia and associated intraflagellar transport (IFT). METHODS: Isolated primary chondrocytes were cultured on substrates of different stiffness (6-1000 kPa) or treated with YAP agonist lysophosphatidic acid (LPA) or YAP antagonist verteporfin (VP), or genetically modified by YAP siRNA, all ± IL1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were measured to monitor IL1ß response. YAP activity was quantified by YAP nuclear/cytoplasmic ratio and percentage of YAP-positive cells. Mechanical properties of cartilage explants were tested to confirm cartilage degradation. The involvement of primary cilia and IFT was analysed using IFT88 siRNA and ORPK cells with hypomorphic mutation of IFT88. RESULTS: Treatment with LPA, or increasing polydimethylsiloxane (PDMS) substrate stiffness, activated YAP nuclear expression and inhibited IL1ß-induced release of NO and PGE2, in isolated chondrocytes. Treatment with LPA also inhibited IL1ß-mediated inflammatory signalling in cartilage explants and prevented matrix degradation and the loss of cartilage biomechanics. YAP activation reduced expression of primary cilia, knockdown of YAP in the absence of functional cilia/IFT failed to induce an inflammatory response. CONCLUSIONS: We demonstrate that both pharmaceutical and mechanical activation of YAP blocks pro-inflammatory signalling induced by IL1ß and prevents cartilage breakdown and the loss of biomechanical functionality. This is associated with reduced expression of primary cilia revealing a potential anti-inflammatory mechanism with novel therapeutic targets for treatment of osteoarthritis (OA).

Unsupervised clustering to differentiate rheumatoid arthritis patients based on proteomic signatures.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have different presentations and prognoses. Cluster analysis based on proteomic signatures creates independent phenogroups of patients with different pathophysiological backgrounds. We aimed to identify distinct pathophysiological clusters of RA patients based on circulating proteomic biomarkers. METHOD: This was a cohort study including 399 RA patients. Clustering was performed on 94 circulating proteins (92 CVDII Olink®, high-sensitivity troponin T, and C-reactive protein). Unsupervised clustering was performed using a partitioning cluster algorithm. RESULTS: The clustering algorithm identified two distinct clusters: cluster 1 (n = 223) and cluster 2 (n = 176). Compared with cluster 1, cluster 2 included older patients with a higher burden of comorbidities (cardiovascular and RA related), more erosive and longer RA duration, more dyspnoea and fatigue, walking a shorter distance in the Six-Minute Walk Test, with more severe diastolic dysfunction, and a 4.5-fold higher risk of death or hospitalization for cardiovascular reasons. Tumour necrosis factor (TNF) receptor superfamily-related pathways were mainly responsible for the model's discriminative ability. CONCLUSION: Using unsupervised cluster analysis based on proteomic phenotypes, we identified two clusters of RA patients with distinct biomarkers profiles, clinical characteristics, and different outcomes that could reflect different pathophysiological backgrounds. TNF receptor superfamily-related proteins may be used to distinguish subgroups.

Microbial ecology and predicted metabolic pathways in various oral environments from patients with acute endodontic infections.

AIM: To assess in a cross-sectional clinical study the effect of antibiotics on the diversity, structure and metabolic pathways of bacterial communities in various oral environments in patients with acute primary infections. METHODOLOGY: Samples of saliva (SA), supragingival biofilm (SB) and from the pulp cavity (PC) were collected from teeth with acute primary infections and then grouped according to previous use of antibiotics (NoAtb = no antibiotics [n = 6]; Atb = antibiotics [n = 6]). DNA sequencing was conducted using MiSeq (Illumina, San Diego, CA, USA). The V1-V3 hyper-variable region of the 16S rRNA gene was amplified. A custom Mothur pipeline was used for 16S rRNA processing. Subsequent analyses of the sequence dataset were performed in R (using vegan, phyloseq and ggplot2 packages) or QIIME. RESULTS: Twelve patients aged from 22 to 56 years were recruited. Participants in the Atb group had taken the beta-lactamics amoxicillin (5/6) or cephalexin (1/6) for 2-3 days. A total of 332 bacterial taxa (OTUs) were identified, belonging to 120 genera, 60 families and nine phyla. Firmicutes (41%) and Bacteroidetes (38%) were the most abundant phyla in all samples. Taxa clustered significantly by oral site (PCoA analysis; P < 0.05, ANOSIM). Use of antibiotics had little effect on this clustering. However, SA, SB and PC had different degrees of richness, diversity and evenness. The greatest diversity was observed in SB samples and the least diversity was observed in PC samples. Metabolic prediction identified 163 pathways and previous use of antibiotics had a major effect on the estimated functional clustering in SA and PC samples. CONCLUSION: The ecological niche had a strong influence on the bacterial content of samples from various oral sites. Previous exposure to antibiotics may exert an effect on the phylogenetic composition of SA. Metabolic pathways appear to be modulated by antimicrobial agents in SA and PC samples. The dynamics of host/microbial interactions in the apical region and the functional ecology of the infected pulp cavity should be revisited.

Prevotella strains and lactamic resistance gene distribution in different oral environments of children with pulp necrosis.

AIM: To determine the presence of Prevotella species, the cfxA/cfxA2, blaZ and blaTEM genes associated with resistance to lactamic agents in different oral niches of children with pulp necrosis. METHODOLOGY: Children with pulp necrosis in primary teeth had samples of saliva, supragingival, pulp chamber and root canal biofilms collected and tested for Prevotella species (P. intermedia, P. nigrescens, P. tannerae) and for beta-lactam resistance genes (cfxA/cfxA2, blaZ and blaTEM). The presence of bacterial DNA was examined through PCR, with a specific primer directed to the 16S rRNA gene. Specific primers were used to detect the Prevotella species and beta-lactam resistance genes. The chi-square test was used to analyse associations between the presence of bacteria and clinical variables. The Cochran's Q test was used to assess whether the proportion of gene detection is the same between different sites. RESULTS: Thirty-two teeth were sampled from 27 patients with a mean age of 5.5 years (±1.76). The total detection rate of Prevotella strains was 29.1%, 25%, 21.8% and 32.29% in saliva, supragingival, pulp chamber and root canal samples, respectively. P. nigrescens was the most commonly detected species in all oral niches. The previous use of antibiotics was associated with detection of P. nigrescens in saliva (P = 0.03). Pain was associated with the presence of P. nigrescens (P = 0.04) and P. tannerae (P = 0.01) in pulp chamber biofilm. blaTEM was detected in the four oral niches, being more frequent (23.8%) in supragingival biofilm (Cochran's Q test, P = 0.04). The presence of P. intermedia in SB and PC was associated with the detection of blaTEM in saliva (P = 0.04). The cfxA/cfxA2 and blaZ genes were not detected in any of the four oral niches. CONCLUSIONS: The oral cavity of children with pulp necrosis had a variable distribution of Prevotella strains in different niches. Saliva, supragingival biofilm, pulp chamber and root canals of primary teeth with necrotic pulps can harbour resistance genes to beta-lactams agents.

The maddening itch: an approach to chronic urticaria.

Chronic spontaneous urticaria (CSU) is defined as the presence of urticaria with daily or almost daily symptoms for 6 weeks or more. CSU affects 0.1%-0.8% of the population. Its pathogenesis involves autoimmunity, abnormalities in signal transduction, and the action of histamine on H1 receptors. Investigation of CSU should be guided by a thorough history and physical examination. A concise laboratory evaluation, including the CU index, is recommended. This index can provide useful data on severity and response to therapy. Initial treatment should involve increasing doses of nonsedating antihistamines until the intended effect is achieved. Only when a patient is unresponsive to high-dose nonsedating antihistamines (or sedating antihistamines) can we consider CSU refractory and consider immunomodulatory therapy. The most promising drugs are cyclosporine and, more recently, omalizumab.

Culicoides spp. found near Lusitano stud farms in mainland Portugal which may contribute for IBH studies.

Insect Bite Hypersensitivity (IBH) is a common cutaneous disease, affecting a large number of horses worldwide. Several studies have identified Culicoides spp. saliva as a clinically relevant allergen source. The prevalence of IBH in Portugal, particularly in Lusitano horses, is still not known. However, the environmental characteristics of the national territory are favorable to the activity of Culicoides, and several species of this genus can be found, namely C. imicola and C. obsoletus/C. scoticus. In this study we characterized the Culicoides population present in Lusitano stud farms with a history of IBH. Thirteen stud farms with Lusitano horses were selected in several regions of mainland Portugal for having a previous history of IBH-affected horses, with a minimum of 5 affected horses. Culicoides were collected in May and June 2016 using OVI traps, placed in these stud farms, and we were able to identify several Culicoides species. We could also verify that C. obsoletus/C. scoticus, and C. imicola were the ones most frequently found, but other species like C. pulicaris were also found.

The potential protective effect of low potassium dextran against lipid peroxidation in a rat lung transplantation model.

The overproduction of reactive oxygen species plays an important role in the cascade of events during lung ischemia-reperfusion leading to graft failure. An evaluation of the peripheral markers of oxidative stress and antioxidant enzyme activities was carried out after reperfusion in a rat lung transplant model. The decrease in lipid peroxidation immediately after transplantation ( P < 0.05) may suggest an adaptative response and/or a protective effect of low potassium dextran against lipid peroxidation through natural scavenging mechanisms.

Bos indicus or Bos taurus mitochondrial DNA - comparison of productive and reproductive breeding values in a Guzerat dairy herd.

The observation of bovine mitochondrial DNA (mtDNA) polymorphisms allows the separation of American zebu cattle, according to its maternal lineage ancestry, into two groups: one with Bos indicus mtDNA and other with Bos taurus mtDNA. The aim of the present study was to determine the productive and reproductive differences between these two groups, in a Guzerat dairy herd. The genotyping of a sample of 56 animals allowed the categorization of most of the 3835 animals in the pedigree file. The production file included 3528 calving and 3198 lactation records from 729 cows, born during the years 1947 to 2007. The traits considered were: lactation milk yield (LMY); days in milk (DIM); age at first calving (AFC), and calving interval (CI). Heritabilities and breeding values were estimated using an animal model. The regression of the average breeding values per year of birth indicated the genetic trends of the herd. The heritability coefficients estimated for LMY, DIM, AFC, and CI were 0.42, 0.43, 0.20, and 0.10, respectively. The genetic trends were similar for both groups, pointing to an improvement in the productive and a worsening in the reproductive traits. The two groups differed significantly regarding the average estimated breeding values for LMY, DIM and AFC, in the starting period, until 1970, but no differences were observed in the more recent years, after 1970. The segregation between the groups existed in the starting period, probably because the Bos taurus contributions to the herd had occurred more recently at that moment. The conclusion is that mtDNA has no significant effect on these traits.

Subclinical mastitis interferes with ovulation, oocyte and granulosa cell quality in dairy cows.

The objective was to evaluate the effect of mastitis by somatic cell count (SCC) on follicular growth, ovulation, oocytes and cumulus cells quality and on the concentration and size of exosomes in follicular fluid of dairy cows. In the study, crossbred cows (Bos taurus - Holstein x Bos indicus - Gir) were classified for analysis as Control (SCC<200.000 cells/mL) and Mastitis (SCC>400.000 cells/mL) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasound evaluations every 24 h, from progesterone-releasing-intravaginal-device (PRID) removal (D8) until 48 h later (D10). Thereafter, evaluations were performed every 12 h, until ovulation or up to 96 h after PRID removal. In experiment 2 (oocyte, cumulus complexes, and follicular fluid evaluation), cows (n = 26) were submitted to follicular aspiration (OPU) for oocyte quality and cumulus cells transcript evaluation. The amount of cumulus complexes transcripts (BCL2, BAX, PI3K, PTEN, FOXO3) was determined by Real-Time Polymerase Chain Reaction. Moreover, seven days after the OPU session, the dominant follicle was aspirated. Exosomes were isolated from the follicular fluid for evaluation of particle size and concentration. Ovulation rate [Control 77.4% (24/31) and Mastitis 57.7% (15/26); P = 0.09] and viable oocytes rate [Control 59.1% (130/220) and Mastitis 41.9% (125/298); P = 0.01] were higher in Control animals. Additionally, there was a greater number of degenerate oocytes [Control 6.7 ±â€¯1.2 and Mastitis 13.3 ±â€¯5.5; (P = 0.001)] in subclinical mastitis cows. There was greater abundance (P = 0.003) of BAX cumulus cell transcripts and exosome mean (P = 0.03) and mode (P = 0.02) was smaller in subclinical mastitis cows. In conclusion, ovulation rate, oocyte quality, and exosome diameter were smaller in cows with SCC>400.000 cells/mL.

Allergic reactions to vaccines.

Vaccines have had a very significant impact in Human Health. Except for public water supply and sanitation measure, no other single public health intervention induced such a significant reduction in disease incidence and child mortality. In Portugal, a National Vaccination Program (NVP) began in 1965 and more recently, in January 2006, the new NVP was implemented. However, it is important to recognize and understand adverse reaction to vaccines, since this therapy is intended to be administered to healthy individuals, a fact which considerably lowers the threshold of acceptability of adverse reactions by the general public. In such a way, better understanding of vaccine adverse reactions allows a correct diagnosis and a more appropriate selection of vaccines, increasing the acceptance of immunization in the community.

Prescribing practice for antibiotic prophylaxis for 3 commonly performed surgeries in a teaching hospital in Brazil.

BACKGROUND: The contribution of antibiotic prophylaxis to reduce surgical wound infection for some procedures is well known. In spite of extensive knowledge about the effectiveness of antibiotic prophylaxis, its administrative regimens are often inappropriate. METHODS: Antibiotic prophylaxis was evaluated in a university-affiliated community teaching hospital. Data were collected from medical records of patients undergoing cholecystectomy, hysterectomy, and herniorrhaphy during a 9-month observation period. A total of 598 procedures were observed. Data were collected about the patient, the surgery, and the antibiotic prophylaxis. RESULTS: Based on the existing guidelines, the choice to use the prophylactic antibiotic was correct in 75% of the cases. Of these cases, only 3% of the patients received the recommended regimen according to the proposed procedure. It was verified that antibiotic prophylaxis was performed more frequently in patients undergoing open cholecystectomy than laparoscopic cholecystectomy (P =.004). In 46% of the cholecystectomies, more than 4 doses were administered. In hysterectomy, a single dose was used in 54% of the patients, and the antibiotic was administered preoperatively in 80% of the cases. Prophylactic antibiotics were used in 47% of herniorrhaphies. CONCLUSION: The surgeons infrequently met the hospital guideline for antibiotic prophylaxis, and compliance varied by type of procedure.

NMDA receptor antagonism in the basolateral amygdala blocks enhancement of inhibitory avoidance learning in previously trained rats.

Extensive evidence suggests that N-methyl-D-aspartate (NMDA) glutamate receptor channels in the amygdala are involved in fear-motivated learning, and infusion of NMDA receptor antagonists into the amygdala blocks memory of fear-motivated tasks. Recent studies have shown that previous training can prevent the amnestic effects of NMDA receptor antagonists on spatial learning. In the present study, we evaluated whether infusion of the NMDA antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) impairs reinforcement of inhibitory avoidance learning in rats given previous training. Adult male Wistar rats (220-310 g) were bilaterally implanted under thionembutal anesthesia (30 mg/kg, i.p.) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Infusion of AP5 (5.0 microg) 10 min prior to training in a step-down inhibitory avoidance task (0.4 mA footshock) blocked retention measured 24 h after training. When infused 10 min prior to a second training session in animals given previous training (0.2 mA footshock), AP5 blocked the enhancement of retention induced by the second training. Control experiments showed that the effects were not due to alterations in motor activity or footshock sensitivity. The results suggest that NMDA receptors in the basolateral amygdala are involved in both formation of memory for inhibitory avoidance and enhancement of retention in rats given previous training.

Prognostic significance of intraperitoneal free cancer cells obtained by laparoscopic peritoneal lavage in patients with gastric cancer.

Laparoscopy is a safe and useful method for examining the local extent and regional spread of disease in patients with gastric cancer. Peritoneal dissemination remains a frequent type of recurrence after surgical treatment. The aim of this study was to determine the prognostic value of intraperitoneal free cancer cells (IFCCs) detected by laparoscopic peritoneal lavage. Forty-nine patients with advanced gastric cancer underwent laparoscopy with cytologic examination for staging. Peritoneal lavage was performed when ascites was not present. Aspirated fluid from the peritoneal cavity was centrifuged and subjected to cytologic examination using Giemsa and Papanicolaou staining methods. Patients were surgically treated and followed for a minimum of 5 years. IFCCs were detected in 41% of the patients. In eight cases (16.3%) laparoscopy revealed carcinomatosis and/or multiple liver metastases, so laparotomy was not performed. Patterns of recurrence after curative resection included the following: peritoneal (n = 3), local (n = 4), liver (n = 1), and other (n = 1). All patients who tested positive for IFCCs had peritoneal recurrence. The absence of IFCCs was associated with improved overall survival (21 months for a 95% confidence interval of 7.4 to 34.6 vs. 4 months for a 95% confidence interval of 2.4 to 5.6). Overall survival adjusted for type of resection also demonstrated a favorable outcome for patients who were negative for IFCCs. The following conclusions were drawn: (1) laparoscopic peritoneal lavage cytology may be useful in identifying patients at high risk for peritoneal relapses and may alter treatment, and (2) lFCCs provide additional prognostic information in patients with gastric cancer.

NMDA-receptor-dependent, muscimol-sensitive role of the entorhinal cortex in post-training memory processing.

The bilateral infusion into the entorhinal cortex of the NMDA receptor antagonist, AP5 (5.0µg) or of the GABA(A) agonist, muscimol (0.03µg) 90min after training but not 30min before training, 0min after training or 10min before testing, hindered retention test performance 24h after inhibitory avoidance in rats. Glutamate (5.0µg) or picrotoxin (0.08µg) infused 90min after training had no effect. In animals trained with a low level footshock a second training session, 120min after the first, was needed in order to obtain a good retention test performance. This was taken to reflect summation of the consecutive memory traces left by the two training sessions. In these animals, the infusion of AP5 or muscimol into the entorhinal cortex between the two training sessions impeded their summation. The present results suggest that the entorhinal cortex plays a late role in memory processing, that this role does not need a hyperactivation of the entorhinal cortex, and that it is important for the interaction between consecutive memory traces.

The effects of acute and repeated restraint stress on the nociceptive response in rats.

The effects of acute and repeated restraint stress on nociception, as measured by the tail-flick latency, were studied in adult male and female rats. After the exposure to a single restraint session, both male and female rats presented an increased latency in the tail-flick test. On the other hand, chronically stressed females presented a performance similar to the control group, whereas chronically stressed male rats responded to restraint with a decrease in the tail-flick latency. This response could be determined by the chronic treatment itself or by the restraint done just before the measurement. Thus, the effect of chronic stress upon basal tail-flick latency was evaluated. In male rats, this latency was significantly decreased in the stressed animals compared with the control group. In female rats, no difference between those groups was observed. Therefore, the results suggest that: (a) acute restraint stress induces an analgesic response in both male and female rats, and (b) there is a gender-specific nociceptive response induced by repeated restraint stress with a hyperalgesic effect in response to stress only in males.

Effect of restraint stress on feeding behavior of rats.

The expression of appetite reflects the complex functioning of a psychobiological system organized in different levels closely related to each other, in which emotional changes can influence feeding behavior. Benzodiazepines are widely used as anxiolytics and can change behaviors caused by stress. The aim of the present study was to verify the feeding behavior of rats, submitted or not to fasting, after acute and chronic restraint stress. We also evaluated the response to the ingestion of sweet food of chronically restrained animals after the administration of diazepam. Male adult Wistar rats were exposed to restraint 1 h/day for 50 days in the chronic model. In the acute model, there was a single exposure. Four hours after the stress, the animals were placed in a lightened area in the presence of 10 pellets of sweet food (Froot Loops). The number of ingested Froot Loops was measured during a period of 3 min in the presence or absence of fasting. The groups acutely stressed showed ingestion similar to that of the control group, whether they had been fasted or not. The chronically stressed animals showed increased ingestion of sweet food. Diazepam given 60 min before the test session of the stressed rats reduced the ingestion of these animals to control levels. Thus, the chronic stress increases appetite for sweet food, independently of hunger, and diazepam is able to reverse this behavior.

Late posttraining memory processing by entorhinal cortex: involvement of NMDA and GABAergic receptors.

The NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid (AP5) (5 micrograms) and the GABAA receptor agonist, muscimol (0.03 microgram) were infused bilaterally into the entorhinal cortex of rats 0, 90, 180, or 360 min after training in habituation to a novel environment or in step-down inhibitory avoidance. Animals were tested for retention 22 h after training in each task. AP5 and muscimol were amnestic for both tasks when given 90 or 180 min after training, but had no effect when given 0 or 360 min after training. In contrast, intraamygdala injections or AP5 or muscimol were amnestic when given 0 but not 90 min after inhibitory avoidance training. The results indicate that the entorhinal cortex plays a late but important role in posttraining memory processing; this role involves glutamatergic NMDA receptors and is inhibited by GABAA receptors. The intervention of the entorhinal cortex in posttraining memory processing is subsequent, and could be secondary, to that of the amygdala and other limbic structures.