[Management of immune-related toxicities associated with immune checkpoints inhibitors: Data from the multidisciplinary meeting « ToxImmun ¼ in Eastern Occitania]. / Gestion des toxicités induites par les inhibiteurs de checkpoint immunologique : données de la réunion de concertation pluridisciplinaire « ToxImmun ¼ en Occitanie Est.

Immune checkpoint inhibitors (ICIs) can cause numerous and complex immune-related adverse events whose management need a multidisciplinary approach. Herein, we investigated 114 requests, mostly concerning patients suffering from lung cancer, that were submitted to the « ToxImmun ¼ multidisciplinary meeting in Eastern Occitania between December the 17th 2018 and January the 20th 2020. The leading reasons for the request concerned the putative causal link between immunotherapy and immune-toxicity and its management, followed by possible retreatment after temporary withdrawn because of adverse event, and finally the possibility to initiate ICIs in patients with pre-existing autoimmunity. Colitis, hepatitis and myocarditis were the most frequent immune-related adverse events (IRAEs), both all grade and grade 3-4. Sicca syndrome (with or without Sjogren criteria) was also frequent (26% of cases) and seems to be associated with severe toxicity and multi-toxicity. The mean time to first IRAE was 3.8 months, a time shortened with the use of anti-PD-L1 agents or ICI combination. A majority of requests came from initial evaluation by the internist confirming the early and main role of this specialty in the management of immunotoxicity. Expansion of this regional multidisciplinary meeting, coordinated by internists and medical oncologists, could improve management of immune-related adverse events for the patients' benefits.

Haplaxius crudus (Hemiptera: Cixiidae) Transmits the Lethal Yellowing Phytoplasmas, 16SrIV, to Pritchardia pacifica Seem. & H.Wendl (Arecaceae) in Yucatan, Mexico.

Lethal yellowing (LY) affects several palm species in the Americas. It is caused by 16SrIV group phytoplasmas. In Florida (USA), LY was shown to be transmitted by the planthopper Haplaxius crudus ( Van Duzee ) (Hemiptera, Cixiidae) to different palm species, including Pritchardia pacifica Seem . & H. Wendl . (Arecaceae) in insect-proof cage experiments in the 1980s, a result that had never been reproduced later. LY has destroyed many coconut plantations as well as other palm species in the Caribbean and Mexico. In order to evaluate if H. crudus is a vector of LY phytoplasmas in Mexico, experiments were carried out in Yucatan (Mexico). Several H. crudus from palms infected by LY in the field were introduced into cages containing young P. pacifica palms. These insects were able to transmit 16SrIV group phytoplasmas to P. pacifica palms. According to DNA sequences comparative analysis, virtual restriction fragment length polymorphism, and phylogenetic analysis, the phytoplasmas detected in these infected P. pacifica were of subgroups A and D. All of ten P. pacifica palms infected with the subgroup D phytoplasmas developed symptoms of LY and died, whereas only one of two palms infected with subgroup A developed LY symptoms and died. This is the first time, more than 30 years later, that the role of H. crudus as a vector of LY is confirmed.

Genetic defects of ovarian TGF-ß-like factors and premature ovarian failure.

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles; POF affects approximately 1-2% of women under the age of 40 yr, thus representing one major cause of female infertility. POF relevance is continuously growing because women tend to conceive always more frequently beyond 30 yr. Frequently, POF is the end-stage of an occult process [primary ovarian insufficiency (POI)]. POI is a heterogeneous disease caused by a variety of mechanisms. Though the underlying cause remains unexplained in the majority of cases, several data indicate that POI has a strong genetic component. These data include the existence of several causal genetic defects in human, experimental, and natural models, as well as the frequent familiarity. The candidate genes are numerous, but POF remains unexplained in most of the cases. Several recent evidences have driven the attention of researchers on the possible involvement of various elements belonging to the transforming growth factor ß family, which includes bone morphogenetic proteins, growth/differentiation factors, and inhibins. These peptides are produced by either the oocyte or granulosa cells to constitute a complex paracrine network within the ovarian follicle. Here, we review the studies reporting the genetic alterations of these factors in human and animal defects of ovarian folliculogenesis which support the fundamental roles played by these signals in ovarian morphogenesis and function.

[Anti-Müllerian hormone, an endocrine predictor of the response to ovarian stimulation in the bovine species]. / L'hormone antimüllérienne, prédicteur endocrinien de la réponse à une stimulation ovarienne chez les bovins.

The strong between-animal variability in the number of ovulations and embryos produced after ovarian stimulation by gonadotropins is a major limit to the development of embryo biotechnologies in cattle. In reproductive medicine, anti-mullerian hormone (AMH) is now widely used as an endocrine marker of the ovarian follicular reserve. In the cow, as in the woman, AMH is secreted by the granulosa cells of growing follicles. We have shown recently that in the cow, AMH is a very good endocrine marker of the population of small antral follicles that constitute the direct target of ovarian stimulatory treatments. AMH concentration measured in plasma before treatment varies between animals and is positively correlated to the number of ovulations and transferable embryos produced after an ovarian stimulatory treatment. Interestingly, AMH concentrations can remain stable over several months for each animal. Moreover, the number of embryos produced after ovarian stimulation is highly repeatable and has a relatively good heritability. From these observations, we propose the determination of AMH concentration in the plasma of a potential donor cow as a simple predictive method to evaluate both its level of ovarian activity and its capacity to produce high or low numbers of embryos. Optimal conditions for implementing this diagnostic test in cattle remain to be defined considering the age, the breed, the physiological status and the environmental factors related to breeding conditions for each animal.

Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep.

In the Lacaune sheep population, two major loci influencing ovulation rate are segregating: FecX and FecL. The FecX(L) mutation is a non-conservative substitution (p.Cys53Tyr) in BMP15 that prevents the processing of the protein. Using a statistical approach, FecL has been shown to be an autosomal major gene. A full genome scan localized the FecL locus on sheep chromosome 11. Fine mapping reduced the interval containing FecL to markers BM17132 and FAM117A, corresponding to a synteny block of 1.1 megabases on human chromosome 17, which encompasses 20 genes. The expression of 16 genes from this interval was observed in tissues of the reproductive axis, but expression was not affected in homozygous FecL(L) females. In this interval, a unique haplotype was associated with the FecL(L) mutation. This particular haplotype could be predicted by the DLX3:c.*803A>G SNP in the 3' UTR sequence of the DLX3 gene. This SNP provided accurate classification of animals (99.5%) as carriers or non-carriers of the mutation and therefore maybe useful in marker assisted selection. A synergistic action of FecL(L) and FecX(L) mutations on both ovulation rate and litter size was demonstrated. Until now, all the Fec genes identified in sheep belong to the bone morphogenetic protein (BMP) system. Based on the human orthologous region, none of the 20 genes in the FecL region corresponds to known molecules in the BMP system. The identification of the FecL(L) mutation could lead to the discovery of a new pathway involved in the regulation of ovulation rate.

Protein biochip array technology to monitor rituximab in rheumatoid arthritis.

In rheumatoid arthritis (RA) there are currently no good indicators to predict a clinical response to rituximab. The purpose of this study was to monitor and determine the role of peripheral blood cytokine profiling in differentiating between a good versus poor response to rituximab in RA. Blood samples were collected at baseline and at 3 months from 46 RA patients who were treated with rituximab. Responders are defined by the presence of three of four American College of Rheumatology criteria: >or=20% decrease in C-reactive protein, visual analogical score of disease activity, erythrocyte sedimentation rate and improvement of the disease activity score (28) (four values) by >or=1.2 obtained at 3 months. Twelve cytokines were measured from serum collected on days 0 and 90 by proteomic array, including interleukin-6 (IL-6), tumour necrosis factor-alpha, IL-1a, IL-1b, IL-2, IL-8, interferon-gamma, IL-4, IL-10, monocyte chemoattractant protein-1, epidermal growth factor and vascular growth factor. We showed that C-reactive protein and IL-6 levels decrease significantly at 3 months in the responder group compared with baseline. At day 90 we identified a cytokine profile which differentiates responders and non-responders. High serum levels of two proinflammatory cytokines, monocyte chemoattractant protein-1 and epidermal growth factor, were significantly higher in the responder group at day 90 compared with non-responders. However, we were not able to identify a baseline cytokine profile predictive of a good response at 3 months. These findings suggest that cytokine profiling by proteomic analysis may be a promising tool for monitoring rituximab and may help in the future to identify responder RA patients.

Protein biochip array technology for cytokine profiling predicts etanercept responsiveness in rheumatoid arthritis.

In rheumatoid arthritis (RA) there are currently no useful indicators to predict a clinical response to tumour necrosis factor-alpha (TNF-alpha) blockade. The purpose of this study was to determine the role of peripheral blood cytokine profiling in differentiating between a good versus poor response to etanercept in RA. Peripheral blood samples were collected at baseline and at 3 months from 33 patients with active disease who were treated twice weekly by etanercept therapy. Responders are defined by the presence of three of four American College of Rheumatology criteria: > or =20% decrease in C-reactive protein (CRP), visual analogue score of disease activity, erythrocyte sedimentation rate and improvement of the disease activity score (28; four values) by > or =1.2 obtained at 3 months. Twelve cytokines were measured from serum collected on days 0 and 90 by proteomic array (protein biochip array, Investigator Evidence, Randox France), including interleukin (IL)-6, TNF-alpha, IL-1a, IL-1b, IL-2, IL-8, interferon-gamma, IL-4, IL-10, monocyte chemoattractant protein (MCP)-1, epidermal growth factor (EGF) and vascular endothelium growth factor. Our results showed that high serum levels of MCP-1 and EGF were associated with a response to etanercept. In addition, the increase of two combined parameters CRP and EGF was predictive of a response to etanercept treatment at 3 months (sensitivity: 87.5% and specificity: 75%, accuracy: 84.4%). These findings suggest that cytokine profiling by proteomic analysis before treatment initiation may help to identify a responder patient to TNF-alpha blocking agents in RA.

In vivo gene expression in granulosa cells during pig terminal follicular development.

Ovarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive view of these molecular mechanisms. First, we developed a specific micro-array using cDNAs from suppression subtractive hybridization libraries (345 contigs) obtained by comparison of three follicle size classes: small, medium and large antral healthy follicles. In a second step, a transcriptomic analysis using cDNA probes from these three follicle classes identified 79 differentially expressed transcripts along the terminal follicular growth and 26 predictive genes of size classes. The differential expression of 18 genes has been controlled using real-time PCR experiments validating the micro-array analysis. Finally, the integration of the data using Ingenuity Pathways Analysis identified five gene networks providing descriptive elements of the terminal follicular development. Specifically, we observed: (1) the down-expression of ribosomal protein genes, (2) the genes involved in lipid metabolism and (3) the down-expression of cell morphology and ion-binding genes. In conclusion, this study gives new insight into the gene expression during pig terminal follicular growth in vivo and suggested, in particular, a morphological change in pig granulosa cells accompanying terminal follicular growth.

Phenotypic and functional characterisation of ovine mesenchymal stem cells: application to a cartilage defect model.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are of particular interest for their potential clinical use in cartilage engineering, but a consistent model is missing in large animals. OBJECTIVE: In the absence of any detailed study reporting a complete characterisation of the mesenchymal cells isolated from sheep bone marrow, we fully characterised adherent stromal cells and developed a pre-clinical model of cartilage engineering by implantation of autologous MSC in the Merinos sheep. METHODS: Ovine MSC (oMSC) were isolated from bone marrow, expanded and further characterised according to the recently proposed definition of the MSC. The experimental model consists of partial-thickness lesions created in the inner part of the patellae of the posterior legs. Lesions were filled with oMSC with or without chitosan, with or without transforming growth factor (TGF)beta-3, in a fibrin clot. RESULTS: oMSC were shown to display the three main characteristics of MSC: adherence to plastic, phenotypic profile (positive for CD44, CD105, vimentin and negative for CD34 and CD45), and trilineage differentiation potential. We also report two other important functional characteristics of MSC: support of long-term haematopoiesis and immunosuppressive capacity. In vivo, 2 months after implantation the histological analysis revealed chondrocyte-like cells surrounded by a hyaline-like cartilaginous matrix that was integrated to the host cartilage when oMSC were combined with chitosan and TGFbeta-3. CONCLUSIONS: This study provides for the first time a strong characterisation of oMSC and establishes the basis for a model of cartilage engineering in a large animal.

BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.

Activins and inhibins, members of the transforming growth factor-beta family are able to stimulate and inhibit, respectively, FSH synthesis and release. Other members of this superfamily, the bone morphogenetic proteins (BMPs), may also affect FSH synthesis in the mouse. The aim of this work was to determine whether BMPs are expressed in the ovine pituitary and whether they play a role in the regulation of FSH release. The mRNAs encoding BMP-2, BMP-4, BMP-7 and the oocyte-derived growth factor, growth differentiation factor (GDF)-9 were detected in the pituitaries of cyclic ewes by reverse-transcriptase PCR, as well as the mRNAs encoding the BMP type I receptors, BMPR-IA (activin-receptor-like kinase (ALK)-3) and BMPR-IB (ALK-6), and type II receptors (BMPR-II). Immunolabeling of pituitary sections revealed the presence of BMPR-IA (ALK-3) and BMPR-II in gonadotrope cells. To investigate the potential effects of BMPs on FSH secretion, ewe pituitary cell cultures were treated with BMP-4 (10(-11) M to 10(-9) M) for 48 h. Interestingly, FSH release was decreased in a dose-dependent manner. At 10(-9) M BMP-4 both FSH concentration and FSHbeta mRNA expression were reduced by 40% of control values. In contrast, there was no inhibitory effect on either LH or LHbeta mRNA expression. A similar result was found with BMP-6. BMP-4 triggered the phosphorylation of Smad1, suggesting that the effect of BMP-4 on FSH secretion is due to the activation of the BMPs signaling pathway. Furthermore, BMP-4 blocked the stimulatory effect of activin on both FSH release and FSHbeta mRNA and amplified the suppression of FSH release and FSHbeta mRNA levels induced by 17beta-estradiol. These results indicate that a functional BMP system operates within the sheep pituitary, at least in vitro, to decrease FSH release and to modulate the effect of activin.

Visceral leishmaniasis infection in a rheumatoid arthritis patient treated with infliximab.

Anti-TNFalpha strategies can result in significant clinical benefits in rheumatoid arthritis (RA), but with an increased rate of opportunistic infections. Visceral leishmaniasis (VL) is a severe disease that can develop in immunocompromised hosts, principally in HIV patients. VL in RA patients treated with TNFalpha antagonists is an extremely rare event, and only one case has been described. Here we report a case of VL, occurring after 9 infusions of infliximab in association with azathioprine, in a patient who developed blood cytopenia, fluctuant fever, and splenomegaly.

Molecular basis of bone morphogenetic protein-4 inhibitory action on progesterone secretion by ovine granulosa cells.

We have recently reported that bone morphogenetic protein-4 (BMP-4) can inhibit progesterone production by ovine granulosa cells (GCs). Here, we have investigated the underlying mechanisms of this effect in basal as well as in FSH-induced conditions. We have confirmed that treatment with BMP-4 decreased basal GC progesterone secretion and totally abolished FSH-stimulating action. This inhibitory action was associated with a decrease in the expression of cAMP-regulated genes, steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage (P450 scc) at mRNA and protein levels. However, BMP-4 did not alter basal cAMP production by GCs. In contrast, BMP-4 decreased by half the FSH-induced cAMP production and strongly inhibited cAMP-induced progesterone production. Thus, the inhibitory effect of BMP-4 was exerted both upstream and downstream of cAMP signalling. We next examined the downstream effect, focusing on cAMP-dependent transcription factors, steroidogenic factor-1 (SF-1) and CREB, through the BMP factor signalling intermediary, Smad1. As expected, BMP-4 induced phosphorylation and transcriptional activity of Smad1 in ovine GCs. BMP-4-activated Smad1 did not affect CREB activity but inhibited the transcriptional activity of SF-1 on the canonical SF-1 responsive element. Interestingly, this transcriptional inhibitory mechanism occurred on transfected StAR and P450 scc promoter. Based on these results, we propose that SF-1 is a key target in the inhibitory mechanism exerted by BMP-4 on progesterone synthesis by ovine GCs in culture. Because SF-1 plays an essential role in the differentiation of GCs, our findings could have new implications in understanding the role of BMP family members in the control of ovarian folliculogenesis.

Prolificacy genes in sheep: the French genetic programmes.

It has been demonstrated that variations in litter size or ovulation rate in different breeds of sheep can be associated with the segregation of several major genes. This set of natural mutants constitutes a valuable resource to determine key points in the biochemical pathways controlling the development of ovarian follicles. The French genetic programmes were devised to identify two of these genes: the Booroola (FecB) and Lacaune genes. The FecB prolific mutation corresponds to a non-conservative mutation (Q249R) in the intracellular kinase-signalling domain of the bone morphogenetic protein receptor type IB (BMPR-IB) gene. The Lacaune gene is situated on ovine chromosome 11. Positional cloning is currently in progress to identify the relevant gene and mutation. A similar approach, limited to linkage testing of candidate genes, is proposed to classify the different prolificacy genes in sheep.

[Patients surviving 5 years after curative oesophagectomy for oesophageal cancer]. / Patients vivants à 5 ans après oesophagectomie curative pour cancer.

AIM OF THE STUDY: To analyse the clinical and pathological parameters of 5-year survival patients after curative oesophageal resection for cancer and to identify factors predictive of long-term survival. METHODS: The data of 370 patients who underwent oesophagectomy with curative intent from January 1982 for oesophageal squamous cell carcinoma (n = 320) or adenocarcinoma (n = 50) were reviewed. After excluding postoperative deaths (n = 20), these patients were surviving (S group, n = 113) or dead (NS group, n = 237) with a 60-month follow-up. Uni- and multivariate analysis allowed comparison between the two groups. RESULTS: Postoperative mortality and morbidity rates were 4.0% and 37.6%, respectively. Parameters related to 5-year survival were: absence of preoperative malnutrition or dysphagia, transhiatal resection, no reoperation, limited tumour, histological response to neoadjuvant treatment, absence of lymph node capsular invasion, number of invaded lymph nodes < or = 4, invaded lymph node ratio < or = 0.1, absence of tumour recurrence or metachronous primary cancer. On multivariate analysis, factors predictive of 5-year survival were: absence of preoperative dysphagia (P < 0.001), stage 0-I-IIA tumour (P<0.001) and absence of metachronous cancer (P = 0.016). CONCLUSION: Complete surgical resection allows 5-year survival. Factors predictive of long-term survival assessed in preoperative evaluation, dysphagia and tumour stage, should be useful to select patients for neoadjuvant treatment.

The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality.

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.