Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

Intraoperative predictors of appendiceal abnormalities in patients with mucinous ovarian neoplasms.

OBJECTIVE: To evaluate intraoperative factors predicting appendiceal pathology during gynecologic oncology surgery for suspected mucinous ovarian neoplasms. METHODS: We conducted a retrospective study on 225 patients with mucinous ovarian neoplasms who underwent surgery for an adnexal mass with concurrent appendectomy between 2000 and 2018. Regression analyses were used to evaluate intraoperative factors, such as frozen section of the ovarian mass and surgeon's impression of the appendix in predicting appendiceal pathology. RESULTS: Most patients (77.8%) had a normal appendix on final pathology. Abnormal appendix cases (n = 26) included: metastasis from high-grade adenocarcinoma of the ovary (n = 1), neuroendocrine tumor of the appendix (n = 4), and low-grade appendiceal mucinous neoplasms (n = 26; 23 associated with a mucinous ovarian adenocarcinoma, 2 with a benign mucinous ovarian cystadenoma, and 1 with a borderline mucinous ovarian tumor). Combining normal intraoperative appearance of the appendix with benign or borderline frozen section yielded a negative predictive value of 85.1%, with 14.9% of patients being misclassified, and 6.0% having a neuroendocrine tumor or low-grade appendiceal neoplasm. CONCLUSION: Benign or borderline frozen section of an ovarian mucinous neoplasm and normal appearing appendix have limited predictive value for appendiceal pathology. Appendectomy with removal of the mesoappendix should be considered in all cases of mucinous ovarian neoplasm, regardless of intraoperative findings.

Clinical outcome and biomarker assessments of a multi-centre phase II trial assessing niraparib with or without dostarlimab in recurrent endometrial carcinoma.

This multi-centre, non-randomized, open-label, phase II trial (NCT03016338), assessed niraparib monotherapy (cohort 1, C1), or niraparib and dostarlimab (cohort 2, C2) in patients with recurrent serous or endometrioid endometrial carcinoma. The primary endpoint was clinical benefit rate (CBR), with ≥5/22 overall considered of interest. Secondary outcomes were safety, objective response rate (ORR), duration of response, progression free survival and overall survival. Translational research was an exploratory outcome. Potential biomarkers were evaluated in archival tissue by immunohistochemistry and next generation sequencing panel. In C1, 25 patients were enrolled, and CBR was 20% (95% CI: 9-39) with median clinical benefit duration of 5.3 months. The ORR was 4% (95% CI: 0-20). In C2, 22 patients were enrolled, and the CBR was 31.8% (95% CI: 16-53) with median clinical benefit duration of 6.8 months. The ORR was 14% (95% CI: 3-35). No new safety signals were detected. No significant association was detected between clinical benefit and IHC markers (PTEN, p53, MMR, PD-L1), or molecular profiling (PTEN, TP53, homologous recombination repair genes). In conclusion, niraparib monotherapy did not meet the efficacy threshold. Niraparib in combination with dostarlimab showed modest activity.

A double-blinded, randomized trial comparing surgeon-administered transversus abdominis plane block with placebo after midline laparotomy in gynecologic oncology surgery.

BACKGROUND: Surgeon-administered transversus abdominis plane block is a contemporary approach to providing postoperative analgesia, and this approach is performed by transperitoneally administering local anesthetic in the plane between the internal oblique and transversus abdominis muscles to target the sensory nerves of the anterolateral abdominal wall. Although this technique is used in many centers, it has not been studied prospectively in patients undergoing a midline laparotomy. OBJECTIVE: This study aimed to evaluate whether surgeon-administered transversus abdominis plane block reduces postoperative opioid requirements and improves clinical outcomes. STUDY DESIGN: In this double-blind, randomized, placebo-controlled trial, patients with a suspected or proven gynecologic malignancy undergoing surgery through a midline laparotomy at 1 Canadian tertiary academic center were randomized to either the bupivacaine group (surgeon-administered transversus abdominis plane blocks with 40 mL of 0.25% bupivacaine) or the placebo group (surgeon-administered transversus abdominis plane blocks with 40 mL of normal saline solution) before fascial closure. The primary outcome was the total dose of opioids (in morphine milligram equivalents) received in the first 24 hours after surgery. The secondary outcomes included opioid doses between 24 and 48 hours, pain scores, postoperative nausea and vomiting, incidence of clinical ileus, time to flatus, and hospital length of stay. The exclusion criteria included contraindications to study medication, history of chronic opioid use, significant adhesions on the anterior abdominal wall preventing access to the injection site, concurrent nonabdominal surgical procedure, and the planned use of neuraxial anesthesia or analgesia. To detect a 20% decrease in opioid requirements with a 2-sided type 1 error of 5% and power of 80%, a sample size of 36 patients per group was calculated. RESULTS: From October 2020 to November 2021, 38 patients were randomized to the bupivacaine arm, and 41 patients were randomized to the placebo arm. The mean age was 60 years, and the mean body mass index was 29.3. A supraumbilical incision was used in 30 of 79 cases (38.0%), and bowel resection was performed in 10 of 79 cases (12.7%). Patient and surgical characteristics were evenly distributed. The patients in the bupivacaine group required 98.0±59.2 morphine milligram equivalents in the first 24 hours after surgery, whereas the patients in the placebo group required 100.8±44.0 morphine milligram equivalents (P=.85). The mean pain score at 4 hours after surgery was 3.1±2.4 (0-10 scale) in the intervention group vs 3.1±2.0 in the placebo group (P=.93). Clinically significant nausea or vomiting was reported in 1 of 38 patients (2.6%) in the intervention group vs 1 of 41 patients (2.4%) in the placebo group (P=.95). Time to first flatus, rates of clinical ileus, and length of stay were similar between groups. Subgroup analysis of patients with a body mass index of <25 and patients who received an infraumbilical incision showed similarly comparable outcomes. CONCLUSION: Surgeon-administered transversus abdominis plane block with bupivacaine was not found to be superior to the placebo intervention in reducing postoperative opioid requirements or improving other postoperative outcomes for patients undergoing a midline laparotomy. These results differed from previous reports evaluating the ultrasound-guided transversus abdominis plane block approach. Surgeon-administered transversus abdominis plane block should not be considered standard of care in postoperative multimodal analgesia.

The five-factor modified frailty index predicts adverse postoperative and chemotherapy outcomes in gynecologic oncology.

INTRODUCTION: Frailty is increasingly recognized as a predictor of postoperative morbidity and oncologic outcomes. Evidence of the predictive value of frailty assessment in gynecologic oncology remains sparse. OBJECTIVES: To evaluate the National Surgical Quality Improvement Program (NSQIP) comorbidity-based modified Frailty Index-5 (mFI-5) as predictor of severe postoperative complications, non-completion of chemotherapy and other patient-centered outcomes in gynecologic oncology patients >70 years-old undergoing surgery. METHODS: Prospectively-collected NSQIP data and retrospective chart review of patients undergoing elective laparotomies for gynecologic malignances at a tertiary academic center in Ontario, Canada, between 01/2016-09/2020 were reviewed. Primary outcome was rate of 30-day Clavien-Dindo (Clavien) grade III-V complications. Secondary outcomes included Clavien II-V complications, postoperative length of stay (LOS), non-home discharge and non-completion of chemotherapy. Logistic regression analyses and receiver-operator curves were performed. RESULTS: Two-hundred and fifty-nine patients were included; 103 were planned to receive adjuvant chemotherapy. Fifty-three patients (20.5%) had an mFI ≥ 2 and were categorized as frail. On multivariable analyses, frailty independently predicted grade III-V complications (OR 24.49, 95%CI 9.72-70.67, p < 0.0001), grade II-V complications (OR 4.64, 95%CI 2.31-9.94, p < 0.0001), non-home discharge (OR 7.37, 95%CI 2.81-20.46, p < 0.0001), LOS ≥ 7d (OR 3.6, 95% CI 1.54-8.6, p = 0.003) and non-completion of chemotherapy (OR 8.42, 95%CI 2.46-32.79, p = 0.001). Adjusted C-statistics demonstrated strong predictive value of the mFI-5 for grade III-V (0.92, 95%CI 0.86-0.97) and grade II-V (0.74, 95%CI 0.68-0.8) complications as well as non-home discharge (0.86, 95%CI 0.78-0.95) and chemotherapy non-completion (0.87, 95%CI 0.8-0.95). CONCLUSION: Frailty as assessed with the mFI-5 predicted adverse postoperative and chemotherapy outcomes in gynecologic oncology patients aged ≥70 undergoing a laparotomy. The mFI-5 is a concise tool that can be used for routine frailty screening and risk stratification.

Wildlife Symbiotic Bacteria Are Indicators of the Health Status of the Host and Its Ecosystem.

Lactic acid bacteria (LAB) are gut symbionts that can be used as a model to understand the host-microbiota cross talk under unpredictable environmental conditions, such as wildlife ecosystems. The aim of this study was to determine whether viable LAB can be informative of the health status of wild boar populations. We monitored the genotype and phenotype of LAB based on markers that included safety and phylogenetic origin, antibacterial activity, and immunomodulatory properties. A LAB profile dominated by lactobacilli appears to stimulate protective immune responses and relates to strains widely used as probiotics, resulting in a potentially healthy wildlife population, whereas microbiota overpopulated by enterococci was observed in a hostile environment. These enterococci were closely related to pathogenic strains that have developed mechanisms to evade innate immune systems, posing a potential risk for host health. Furthermore, our LAB isolates displayed antibacterial properties in a species-dependent manner. Nearly all of them were able to inhibit bacterial pathogens, raising the possibility of using them as an a la carte antibiotic alternative in the unexplored field of wildlife disease mitigation. Our study highlights that microbiological characterization of LAB is a useful indicator of wildlife health status and the ecological origin from which they derive. IMPORTANCE The wildlife symbiotic microbiota is an important component for the greater diversity and functionality of their bacterial populations, influencing host health and adaptability to its ecosystem. Although many microbes are partly responsible for the development of multiple physiological processes, only certain bacterial groups, such as lactic acid bacteria (LAB), have the capacity to overpopulate the gut, promoting health (or disease) when specific genetic and environmental conditions are present. LAB have been exploited in many ways due to their probiotic properties, particularly lactobacilli; however, their relationship with wildlife gut-associated microbiota hosts remains to be elucidated. On the other hand, it is unclear whether LAB such as enterococci, which have been associated with detrimental health effects, could lead to disease. These important questions have not been properly considered in the field of wildlife and, therefore, should be clearly addressed.

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

A pneumococcal pneumonia and influenza vaccination quality improvement program for women receiving chemotherapy for gynecologic cancers at a major tertiary cancer Centre.

OBJECTIVE: International guidelines recommend pneumococcal pneumonia and influenza vaccination for all patients with solid organ malignancies prior to initiating chemotherapy. Baseline vaccination rates (March 2019) for pneumococcal pneumonia and influenza at our tertiary cancer centre were 8% and 40%, respectively. The aim of this study was to increase the number of gynecologic chemotherapy patients receiving pneumococcal and influenza vaccinations to 80% by March 2020. METHODS: We performed an interrupted time series study using structured quality improvement methodology. Three interventions were introduced to address vaccination barriers: an in-house vaccination program, a staff education campaign, and a patient care bundle (pre-printed prescription, information brochure, vaccine record booklet). Process and outcome data were collected by patient survey and pharmacy audit and analyzed on statistical process control charts. RESULTS: We identified 195 eligible patients. Pneumococcal and influenza vaccination rates rose significantly from 5% to a monthly mean of 61% and from 36% to a monthly mean of 67%, respectively. The 80% target was reached for both vaccines during one or more months of study. The in-house vaccination and staff education programs were major contributors to the improvement, whereas the information brochure and record booklet were minor contributors. CONCLUSIONS: Three interventions to promote pneumococcal and influenza vaccination among chemotherapy patients resulted in significantly improved vaccination rates. Lessons learned about promoting vaccine uptake may be generalizable to different populations and vaccine types. In response to the global COVID-19 pandemic, initiatives to expand the program to all chemotherapy patients at our centre are underway.

Thirty-day outcomes after gynecologic oncology surgery: A single-center experience of enhanced recovery after surgery pathways.

INTRODUCTION: The purpose of the study is to evaluate the impact of an enhanced recovery after surgery (ERAS) program implemented in a Gynecologic Oncology population undergoing a laparotomy at a Canadian tertiary care center. MATERIAL AND METHODS: Prospectively collected data, using the American College of Surgeons' National Surgical Quality Improvement Program dataset (ACS NSQIP), was used to compare 30-day postoperative outcomes of gynecologic oncology patients undergoing a laparotomy before and after the 2018 implementation of an ERAS program in a Canadian regional cancer center. Patient demographics, surgical variables and postoperative outcomes of 187 patients undergoing surgery in 2019 were compared with those of 441 patients undergoing surgery between January 2016 and December 2017. Student's t, Mann-Whitney U and Chi-square tests, as well as multivariate linear and logistic regressions were used to evaluate baseline characteristics and 30-day postoperative complications. RESULTS: Length of stay was significantly shortened in the study population after introducing the ERAS protocol, from a mean of 4.7 (SD = 3.8) days to a mean of 3.8 (SD = 3.2) days (P = .0001). The overall complication rate decreased from 24.3% to 16% (P = .02). Significant decreases in the rates of postoperative infections (adjusted odds ratio [OR] 0.56, 95% confidence interval [CI] 0.31-0.99) and cardiovascular complications (adjusted OR 0.27, 95% CI 0.09-0.79) were noted, without a significant increase in readmission rate (adjusted OR 0.50, 95% CI 0.21-1.07). CONCLUSIONS: Introducing an ERAS program for gynecologic oncology patients undergoing laparotomy was effective in shortening length of stay and the overall complication rate without a significant increase in readmission. Advocacy for broader implementation of ERAS among gynecologic oncology services and ongoing discussion on challenges and opportunities in the implementation process are warranted to improve patient outcomes and experiences.

Spread of Antimicrobial Resistance by Salmonella enterica Serovar Choleraesuis between Close Domestic and Wild Environments.

The Salmonellaenterica serovar Choleraesuis affects domestic pig and wild boar (WB), causing clinical salmonellosis. Iberian swine production is based on a free-range production system where WB and Iberian pig (IP) share ecosystems. This study focuses on the negative impact on the pork industry of infections due to this serotype, its role in the spread of antibiotic resistance, and its zoonotic potential. Antibiotic resistance (AR) and genetic relationships were analyzed among 20 strains of S. Choleraesuis isolated from diseased WB and IP sampled in the southwest region of the Iberian Peninsula. AR was studied using the Kirby-Bauer method with the exception of colistin resistance, which was measured using the broth microdilution reference method. Resistance and Class 1 integrase genes were measured using PCR, and the genetic relationship between isolates and plasmid content by pulsed field gel electrophoresis. The results show a higher incidence of AR in isolates from IP. Phylogenetic analysis revealed seven profiles with two groups containing isolates from IP and WB, which indicates circulation of the same clone between species. Most pulsotypes presented with one plasmid of the same size, indicating vertical transmission. AR determinants blaTEM and tetA were routinely found in IP and WB, respectively. One isolate from IP expressed colistin resistance and presented the mcr-1 gene carried by a plasmid. This study suggests that S. Choleraesuis circulates between WB and IP living in proximity, and also that the mobilization of AR genes by plasmids is low. Furthermore, the detection of plasmid-mediated colistin resistance in bacteria from IP is alarming and should be monitored.

Phase II Trial of Cabozantinib in Recurrent/Metastatic Endometrial Cancer: A Study of the Princess Margaret, Chicago, and California Consortia (NCI9322/PHL86).

PURPOSE: The relevance of the MET/hepatocyte growth factor pathway in endometrial cancer tumor biology supports the clinical evaluation of cabozantinib in this disease. PATIENTS AND METHODS: PHL86/NCI#9322 (NCT01935934) is a single arm study that evaluated cabozantinib (60 mg once daily) in women with endometrial cancer with progression after chemotherapy. Coprimary endpoints were response rate and 12-week progression-free-survival (PFS). Patients with uncommon histology endometrial cancer (eg, carcinosarcoma and clear cell) were enrolled in a parallel exploratory cohort. RESULTS: A total of 102 patients were accrued. Among 36 endometrioid histology patients, response rate was 14%, 12-week PFS rate was 67%, and median PFS was 4.8 months. In serous cohort of 34 patients, response rate was 12%, 12-week PFS was 56%, and median PFS was 4.0 months. In a separate cohort of 32 patients with uncommon histology endometrial cancer (including carcinosarcoma), response rate was 6% and 12-week PFS was 47%. Six patients were on treatment for >12 months, including two for >30 months. Common cabozantinib-related toxicities (>30% patients) included hypertension, fatigue, diarrhea, nausea, and hand-foot syndrome. Gastrointestinal fistula/perforation occurred in four of 70 (6%) patients with serous/endometrioid cancer and five of 32 (16%) patients in exploratory cohort. We observed increased frequency of responses with somatic CTNNB1 mutation [four partial responses (PRs) in 10 patients, median PFS 7.6 months] and concurrent KRAS and PTEN/PIK3CA mutations (three PRs in 12 patients, median PFS 5.9 months). CONCLUSIONS: Cabozantinib has activity in serous and endometrioid histology endometrial cancer. These results support further evaluation in genomically characterized patient cohorts.

Detection of live M. bovis BCG in tissues and IFN-γ responses in European badgers (Meles meles) vaccinated by oropharyngeal instillation or directly in the ileum.

BACKGROUND: Oral vaccination with Mycobacterium bovis Bacille of Calmette and Guerin (BCG) has provided protection against M. bovis to badgers both experimentally and in the field. There is also evidence suggesting that the persistence of live BCG within the host is important for maintaining protection against TB. Here we investigated the capacity of badger inductive mucosal sites to absorb and maintain live BCG. The targeted mucosae were the oropharyngeal cavity (tonsils and sublingual area) and the small intestine (ileum). RESULTS: We showed that significant quantities of live BCG persisted within badger in tissues of vaccinated badgers for at least 8 weeks following oral vaccination with only very mild pathological features and induced the circulation of IFNγ-producing mononuclear cells. The uptake of live BCG by tonsils and drainage to retro-pharyngeal lymph nodes was repeatable in the animal group vaccinated by oropharyngeal instillation whereas those vaccinated directly in the ileum displayed a lower frequency of BCG detection in the enteric wall or draining mesenteric lymph nodes. No faecal excretion of live BCG was observed, including when BCG was delivered directly in the ileum. CONCLUSIONS: The apparent local loss of BCG viability suggests an unfavorable gastro-enteric environment for BCG in badgers, which should be taken in consideration when developing an oral vaccine for use in this species.

Nasal shedding of Mycobacterium tuberculosis in wild boar is related to generalised tuberculosis and concomitant infections.

BACKGROUND: Wild boar is an important reservoir of Mycobacterium tuberculosis variant bovis, the main causative agent of bovine tuberculosis (bTB). A proportion of tuberculosis (TB)-affected wild boars shed M tuberculosis by nasal route, favouring the maintenance of bTB in a multihost scenario. The aim of this work was to assess if M tuberculosis nasal excretion is influenced by factors commonly associated with high TB prevalence in wild boar. METHODS: TB diagnosis and M tuberculosis isolation were carried out in 112 hunted wild boars from mid-western Spain. The association between the presence of M tuberculosis DNA in nasal secretions and explanatory factors was explored using partial least squares regression (PLSR) approaches. RESULTS: DNA from M tuberculosis was detected in 40.8 per cent nasal secretions of the TB-affected animals. Explanatory factors provided a first significant PLSR X's component, explaining 25.70 per cent of the variability observed in M tuberculosis nasal shedding. The presence of M tuberculosis in nasal secretions is more probable in animals suffering from generalised TB and mainly coinfected with Metastrongylus species and porcine circovirus type 2, explaining nearly 90 per cent of the total variance of this model. CONCLUSION: Measures aiming to control these factors could be useful to reduce M tuberculosis shedding in wild boar.

Lactobacilli Isolated From Wild Boar (Sus scrofa) Antagonize Mycobacterium bovis Bacille Calmette-Guerin (BCG) in a Species-Dependent Manner.

Background: Wildlife poses a significant burden for the complete eradication of bovine tuberculosis (bTB). In particular, wild boar (Sus scrofa) is one of the most important reservoirs of Mycobacterium bovis, the causal agent of bTB. Wild boar can display from mild TB lesions, usually found in head lymph nodes, to generalized TB lesions distributed in different anatomical regions; but rarely clinical signs, which complicates the diagnosis of Mycobacterium bovis infection and bTB control. Among the possibilities for this variability in lesion distribution is the influence of the host-beneficial commensal-primed immune barrier. In this respect, beneficial microbes may delay bTB dissemination as a consequence of an antagonistic competition for nutrients and phagocytes. In order to explore this possibility, we have tested whether typical commensals such as lactobacilli have the capacity to reduce the survival rate of the surrogate M. bovis strain Bacillus Calmette-Guerin (BCG); and to modulate its phagocyte intake. Results: Three Lactobacillus species, L. casei, L. plantarum, and L. salivarius, isolated from wild boar feces displayed a pH-dependent inhibitory activity against BCG and influenced its intake by porcine blood phagocytes in a species-dependent manner. All lactobacilli showed a very significant bactericidal effect against BCG at low pH, but only isolates of L. plantarum and L. casei displayed such antimycobacterial activity at neutral pH. The genomes of these isolates revealed the presence of two-peptide bacteriocins whose precursor genes up-regulate in the presence of BCG cells. Furthermore, L. plantarum reduced significantly the BCG phagocytic intake, whereas L. casei had the opposite effect. L. salivarius had no significant influence on the phagocytic response to BCG. Conclusions: Our in vitro results show that lactobacilli isolated from wild boar antagonize BCG as a consequence of their antimycobacterial activity and a competitive phagocytic response. These findings suggest that commensal bacteria could play a beneficial role in influencing the outcome of bTB dissemination. Further work with lactobacilli as a potential competitive pressure to control bTB will need to take into account the complex nature of the commensal microbiome, the specific immunity of the wild boar and the in vivo infection context with pathogenic strains of M. bovis.

Seasonal and dietary effects on Vitamin D deficiencies detected in wild boar from mid-western Spain.

Vitamin D (VitD) is involved in important mammalian physiological mechanisms, such as Ca-P metabolism, bone development and immunological response. VitD deficiencies are frequently detected in domestic animals and related to various health problems (e.g., rickets, bone deformation). However, knowledge about the status of VitD in wildlife species, such as the wild boar, is scarce. The aims of this work were to explore VitD status in wild boar populations from mid-western Spain and to elucidate the influence of daylight exposure and food supplementation in levels of VitD. Serum concentration of VitD (measured as 25-hydroxivitaminD) was assessed in 276 wild boar from 27 game estates located in mid-western Spain using a commercial ELISA kit. In 19 out of 27 estates, the staff supplied a specific VitD-enriched food (2,000 UI/Kg) ad libitum throughout the year, while in the remaining estates (8), no food was supplied. Blood samples were extracted from hunted animals (198) between October and February of hunting seasons 2016/2017 and 2017/2018, and from live wild boar (78) that were captured, sampled and released (March-September of 2017). The percentage of animals with VitD deficiency (<20 ng/ml), VitD insufficiency (20-30 ng/ml) and VitD sufficiency (>30 ng/ml) was estimated, and the relationship of these levels to factors like sex, age and season was assessed using chi-square tests. Furthermore, associations between daylight exposure and supplemental food with VitD levels were explored using linear models. Of the studied wild boar population, 82.2% showed a VitD deficiency or insufficiency. VitD deficiencies were more frequent in animals sampled in winter and spring. Furthermore, levels of VitD positively correlated with daylight exposure and supplemental food intake. Ad libitum supplementation with VitD-enriched food was insufficient to prevent VitD deficiencies in wild boar from November to April, probably because food consumption is lower during this period.

Outcomes of Adjuvant Therapy for Stage IA Serous Endometrial Cancer.

PURPOSE: Serous adenocarcinoma is a rare, aggressive histologic subtype of endometrial cancer with a high rate of recurrence and a poor prognosis. The optimal adjuvant treatment for early-stage patients is unclear. Our objective was to evaluate the outcomes of stage IA serous endometrial cancers only treated at a single institution and determine whether our current approach of chemotherapy plus vaginal brachytherapy (VBT) is sufficient. METHODS: A retrospective chart review of our institution's pathology database, including all cases of stage IA serous endometrial carcinoma from 2000-2014 was completed. Kaplan-Meier estimates were calculated for Overall and Recurrence-Free Survival (OS and RFS); hazard ratios were calculated using Cox proportional hazards modeling for independent prognostic factors. RESULTS: There were 63 patients with stage IA serous endometrial cancer of whom 79.4% were surgically staged. Percent RFS was 76.5% at five years while OS was 84.7% for the whole cohort. One of the 23 patients receiving VBT and chemotherapy recurred at the vagina versus four of 32 patients who were observed. Two patients in the observation group recurred in the pelvis while there were no first pelvic recurrences in the VBT and chemotherapy group (non- significant). Overall survival was 95% in the brachytherapy and chemotherapy group versus 79.6% in the observation group (non-significant). Post-operative management included observation (n=33), combination VBT and chemotherapy (n=21), or chemotherapy with or without external beam radiation therapy (EBRT) (n=9). DISCUSSION: We report one of the largest cohorts of serous endometrial cancer stage IA patients. Our results emphasize the inferior RFS and OS of stage IA serous versus endometrioid endometrial cancer patients. While some centers continue to use EBRT for these patients, our results demonstrate low pelvic recurrence rates with radiotherapy limited to VBT, as well as the high systemic risk regardless of treatment. We advocate for combination chemotherapy and brachytherapy given the poor outcomes in these patients.

The expression of Indoleamine 2, 3-dioxygenase (IDO) is reduced in granulomas from BCG vaccinated cattle compared to granulomas from unvaccinated controls after experimental challenge with Mycobacterium bovis.

Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a major economic disease of livestock worldwide. Vaccination is considered as a potentially sustainable adjunct to the current control strategy. Cattle vaccination with the live attenuated M. bovis bacillus Calmette-Guerin (BCG) confers variable protection; the reasons for this variability are not understood. Indoleamine 2, 3-dioxygenase (IDO), through the catalysis of tryptophan, is thought to have an immunoregulatory role in the immune response to Mycobacterium tuberculosis (M. tuberculosis). In this work, we used immunohistochemistry and digital image analysis to evaluate the presence of IDO in granulomas at different stages of development in cattle that had been BCG-vaccinated or not and then challenged with M. bovis. Our results show that the expression of IDO in granulomas from non-vaccinated M. bovis challenged animals is higher than in granulomas from BCG-vaccinated M. bovis challenged animals. Thus, it is possible that vaccination with BCG prevents the induction of what are thought to be host immunosuppressive pathways by M. bovis, which contribute to pathology during the disease.

Vaccination Against Porcine Circovirus-2 Reduces Severity of Tuberculosis in Wild Boar.

Tuberculosis (TB) in wild boar (Sus scrofa) may be affected by coinfections with other pathogens, such as porcine circovirus type 2 (PCV2). Therefore, sanitary measures focused on controlling PCV2 could be useful in reducing the impact of TB in this wild suid. The aim of this study was to explore whether vaccination against PCV2 targeting young animals affects TB prevalence and TB severity in wild boar. The study was conducted on a game estate in mid-western Spain. Seventy animals of ages ranging from 4 to 8 months were captured, individually identified, vaccinated against PCV2 and released, forming a vaccinated group. Not-captured animals cohabiting with the vaccinated wild boar constituted the control group. Animals from both groups were hunted between 2013 and 2016 and a TB diagnosis based on pathological assessment and microbiological culture was made in all of them. The effect of PCV2 vaccination on TB prevalence and severity was explored using generalized lineal models. Whereas TB prevalence was similar in vaccinated and control groups (54.55 vs. 57.78%), vaccinated animals showed less probabilities to develop generalized TB lesions. Furthermore, mean TB severity score was significantly lower in vaccinated animals (1.55 vs. 2.42) suggesting a positive effect of PCV2 vaccination.

Histopathological and immunohistochemical characterisation of hepatic granulomas in Leishmania donovani-infected BALB/c mice: a time-course study.

BACKGROUND: Visceral leishmaniasis (VL) is a neglected tropical disease (NTD), caused by the intracellular protozoan parasites Leishmania donovani and Leishmania infantum. Symptomatic VL is considered fatal when left untreated. At present, there is no effective vaccine licensed for human use and available chemotherapies have limitations. Understanding the local immune mechanisms required for the control of infection is a key factor for developing effective vaccines and therapeutics. METHODS: We have investigated the development of the typical granulomatous lesions in the liver in experimental VL over time, together with the local immune responses. BALB/c mice were infected intravenously with a dose of 2 × 107 L. donovani amastigotes (MHOM/ET/67/HU3) and sacrificed at 15, 35 and 63 days post-infection (dpi). Histopathology and immunohistochemical techniques were used for the detection of Leishmania antigen, selected cell types including B and T lymphocytes, macrophages and neutrophils (CD45R-B220+, CD3+, F4/80+ and Ly-6G+) and iNOS. RESULTS: Granulomatous lesions were identified as early as 15 dpi in the livers of all infected animals. Three categories were used to classify liver granulomas (immature, mature and clear). Clear granulomas were exclusively detected from 35 dpi onwards. Kupffer cells (F4/80+) were predominant in immature granulomas, regardless of the dpi. Nonetheless, the highest expression was found 63 dpi. Positive staining for iNOS was mainly observed in the cytoplasm of fused Kupffer cells and the highest expression observed at 35 dpi. T cells (CD3+) and B cells (CD45R-B220+) were predominant in more advanced granuloma stages, probably related to the establishment of acquired immunity. Neutrophils (Ly-6G+) were predominantly observed in mature granulomas with the highest expression at 15 dpi. Neutrophils were lower in numbers compared to other cell types, particularly at later time points. CONCLUSIONS: Our results reflect the role of macrophages during the early stage of infection and the establishment of a lymphocytic response to control the infection in more advanced stages.

Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα.

An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.