Effect of DON and ZEN and their metabolites DOM-1 and HZEN on B cell proliferation and antibody production.

Introduction: The mycotoxins deoxynivalenol (DON) and zearalenone (ZEN), produced by Fusarium fungi, are frequently found in the cereal-rich diet of pigs and can modulate the immune system. Some enzymes or bacteria present in the digestive tract can de-epoxydize DON to deepoxy-deoxynivalenol (DOM-1) and biotransform ZEN into hydrolyzed ZEN (HZEN). The effects of these metabolites on immune cells, particularly with respect to the vaccine responses, are poorly documented. The aim of this study was to address the impact of DON and ZEN and their respective derivatives, on proliferation, and antibody production of porcine B cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs), isolated from healthy pigs, were stimulated with the Toll-like receptor (TLR) 7/8-agonist Resiquimod (R848) or the TLR/1/2-agonist Pam3Cys-SKKKK in combination with DON [0.1-1.6 µM] or DOM-1 [1.6 µM and 16 µM] and ZEN [2.5-40 µM] or HZEN [40 µM]. Results: A strong decrease in B-cell proliferation was observed at DON concentrations equal to or exceeding 0.8 µM and at ZEN concentrations equal to or exceeding 20 µM. Treatment with 1.6 µM DON or 40 µM ZEN led to almost a complete loss of live CD79α+ B cells. Moreover, CD21 expression of proliferating IgG+ and IgM+ B-cell subsets was decreased at DON concentrations equal to and exceeding 0.4 µM and at ZEN concentrations equal to or exceeding 10 µM. ELISpot assays revealed a decrease of IgG-secreting B cells at concentrations of and exceeding 0.4 µM and at ZEN concentrations equal to and exceeding 10 µM. ELISA assays showed a decrease of IgM, IgG, and IgA secretion at concentrations equal to or exceeding 0.4 µM DON. ZEN reduced IgM secretion at 20-40 µM (both R848 and Pam3Cys-SKKKK), IgG secretion at 40 µM (both R848 and Pam3Cys-SKKKK) and IgA secretion at 20-40 µM. Discussion: Our in vitro experiments show that while DON and ZEN impair immunoglobulin production and B-cell proliferation, this effect is abrogated by HZEN and DOM-1.

Zymosan-Induced Murine Peritonitis Is Associated with an Increased Sphingolipid Synthesis without Changing the Long to Very Long Chain Ceramide Ratio.

Sphingolipids are key molecules in inflammation and defense against pathogens. Their role in dectin-1/TLR2-mediated responses is, however, poorly understood. This study investigated the sphingolipidome in the peritoneal fluid, peritoneal cells, plasma, and spleens of mice after intraperitoneal injection of 0.1 mg zymosan/mouse or PBS as a control. Samples were collected at 2, 4, 8, and 16 h post-injection, using a total of 36 mice. Flow cytometry analysis of peritoneal cells and measurement of IL-6, IL-1ß, and TNF-α levels in the peritoneal lavages confirmed zymosan-induced peritonitis. The concentrations of sphingoid bases, dihydroceramides, ceramides, dihydrosphingomyelins, sphingomyelins, monohexosylceramides, and lactosylceramides were increased after zymosan administration, and the effects varied with the time and the matrix measured. The greatest changes occurred in peritoneal cells, followed by peritoneal fluid, at 8 h and 4 h post-injection, respectively. Analysis of the sphingolipidome suggests that zymosan increased the de novo synthesis of sphingolipids without change in the C14-C18:C20-C26 ceramide ratio. At 16 h post-injection, glycosylceramides remained higher in treated than in control mice. A minor effect of zymosan was observed in plasma, whereas sphinganine, dihydrosphingomyelins, and monohexosylceramides were significantly increased in the spleen 16 h post-injection. The consequences of the observed changes in the sphingolipidome remain to be established.

Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection.

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4+ T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4+ T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects.

Asthma and Cacosmia Could Be Predictive Factors of Olfactory Dysfunction Persistence 9 Months after SARS-CoV-2 Infection: The ANOSVID Study.

Background. Long-term evolution data of olfactory disorders (OD) in COVID-19 are limited. Method. ANOSVID is a retrospective study in Nord Franche-Comté Hospital (France) that included COVID-19 patients from the first wave. The aim was to describe OD evolution, especially in patients with persistent OD (p-OD group) in comparison with patients with resolved OD (r-OD group). Results. Among 354 COVID-19 patients, 229 reported OD were included. Eighty-five percent of patients (n = 195) recovered from their OD within 90 days. However, 9.5 months (in average) after symptoms onset, OD were persisting in 93 patients (40.6%) and resolved in 136 patients (59.4%). In the p-OD group (n = 93), the mean age was 51.4 years (19-98) ± 20.2, and 65 patients (69.9%) were female; the three main comorbidities in the p-OD group were: asthma (20.4%, n = 19), allergic rhinitis (19.4%, n = 18), and arterial hypertension (16.1%, n = 15). Eleven patients (12%) presented anosmia, and 82 patients (88%) presented hyposmia. Asthma was more described in p-OD group than r-OD group (19 (20.4%) versus 10 (7.4%), p = 0.006). Cacosmia was more described in p-OD group than r-OD group (27 (29.0%) versus 18 (13.2%), p = 0.005). There was no significant difference between the two groups concerning other comorbidities and symptoms, clinical, biological, and imaging findings, and outcome or about the impact of OD on the quality of life of the patients between the p-OD group and r-OD group. sQOD-NS brief version score was 10.7 ± 5.89 and 12.0 ± 6.03, respectively (p = 0.137). Conclusion. Forty-one percent of patients with OD reported OD persistence 9.5 months after COVID-19 (hyposmia in 88% of cases). Asthma and cacosmia could be predictive factors of OD persistence.

Olfactory dysfunction in COVID-19, new insights from a cohort of 353 patients: The ANOSVID study.

Olfactory disorders (OD) pathogenesis, underlying conditions, and prognostic in coronavirus disease 2019 (COVID-19) remain partially described. ANOSVID is a retrospective study in Nord Franche-Comté Hospital (France) that included COVID-19 patients from March 1 2020 to May 31 2020. The aim was to compare COVID-19 patients with OD (OD group) and patients without OD (no-OD group). A second analysis compared patients with anosmia (high OD group) and patients with hyposmia or no OD (low or no-OD group). The OD group presented less cardiovascular and other respiratory diseases compared to the no-OD group (odds ratio [OR] = 0.536 [0.293-0.981], p = 0.041 and OR = 0.222 [0.056-0.874], p = 0.037 respectively). Moreover, history of malignancy was less present in the high OD group compared with the low or no-OD group (OR = 0.170 [0.064-0.455], p < 0.001). The main associated symptoms (OR > 5) with OD were loss of taste (OR = 24.059 [13.474-42.959], p = 0.000) and cacosmia (OR = 5.821 [2.246-15.085], p < 0.001). Most of all ORs decreased in the second analysis, especially for general, digestive, and ENT symptoms. Only two ORs increased: headache (OR = 2.697 [1.746-4.167], p < 0.001) and facial pain (OR = 2.901 [1.441-5.842], p = 0.002). The high OD group had a higher creatinine clearance CKD than the low or no-OD group (89.0 ± 21.1 vs. 81.0 ± 20.5, p = 0.040). No significant difference was found concerning the virological, radiological, and severity criteria. OD patients seem to have less comorbidity, especially better cardiovascular and renal function. Associated symptoms with OD were mostly neurological symptoms. We did not find a significant relationship between OD and less severity in COVID-19 possibly due to methodological bias.

Intestinal toxicity of the new type A trichothecenes, NX and 3ANX.

NX and its acetylated form 3ANX are two new type A trichothecenes produced by Fusarium graminearum whose toxicity is poorly documented. The aim of this study was to obtain a general view of the intestinal toxicity of these toxins. Deoxynivalenol (DON), which differs from NX by the keto group at C8, served as a benchmark. The viability of human intestinal Caco-2 cells decreased after 24 h of exposure to 3 µM NX (-21.4%), 3 µM DON (-20.2%) or 10 µM 3ANX (-17.4%). Histological observations of porcine jejunal explants exposed for 4 h to 10 µM of the different toxins showed interstitial edema and cellular debris. Explants exposed to NX also displayed cell vacuolization, a broken epithelial barrier and high loss of villi. Whole transcriptome profiling revealed that NX, DON and 3ANX modulated 369, 146 and 55 genes, respectively. Functional analyses indicated that the three toxins regulate the same gene networks and signaling pathways mainly; cell proliferation, differentiation, apoptosis and growth, and particularly immune and pro-inflammatory responses. Greater transcriptional impacts were observed with NX than with DON. In conclusion, our data revealed that the three toxins have similar impacts on the intestine but of different magnitude: NX > DON ≫ 3ANX. NX and 3ANX should consequently be included in overall risk analysis linked to the presence of trichothecenes in our diet.

Vaccination coverage in hematological patients undergoing chemotherapy: Should we move towards personalized vaccination?

INTRODUCTION: Immunocompromised patients are at high-risk for severe influenza and invasive pneumococcal diseases (IPD). Despite the French Public Health Council (FPHC) and the 7th European Conference on Infections in Leukaemia (ECIL7) recommendations, vaccination coverage remains insufficient. This study aimed to estimate the coverage and determinants of influenza, pneumococcal and diphtheria-tetanus-poliomyelitis (dTP) vaccinations in hematological patients underlying chemotherapy. METHODS: A survey was distributed to all patients of the hematology day hospital assessing vaccine uptakes and general opinion about vaccination. Vaccine uptakes were collected from medical and vaccination records; knowledge of and attitudes towards vaccinations in immunocompromised patients were evaluated for each general practitioner (GP) by phone call. Adequacy between vaccine uptakes and indication or not to vaccinate according to ECIL7 guidelines was assessed. Factors associated with vaccine uptakes were assessed by multivariate logistic regression. RESULTS: Among 145 patients, 66 % were aged 65 years or older, 40 % were followed for lymphoma and 38 % for multiple myeloma, 39 % were treated with anti-CD20 antibodies. Vaccination coverage was suboptimal for influenza (45-56 %), dTP (44 %) and IPD (16-19 %) regardless of the guidelines followed, with a wide variation in rates by information source (19-76 %). Adequacy rate with ECIL7 recommendations were 63 % and 87 % for influenza and IPD respectively. Information of patients on specific vaccinations was positively associated with flu and IPD vaccinations, as well as favorable attitude toward vaccination and age ≥ 65 years for flu vaccination, and recommendation by hematologist for pneumococcal vaccination. CONCLUSION: Despite vaccination opportunities, the complexity of these specific recommendations and the lack of communication between the health actors could explain the suboptimal vaccination coverage in this high-risk population. A proactive attitude of all actors in the city and hospital, including better patient information and a personalized and evolving vaccination schedule to help GPs to coordinate vaccination would allow to improve vaccine coverage.

Leclercia adecarboxylata as Emerging Pathogen in Human Infections: Clinical Features and Antimicrobial Susceptibility Testing.

(1) Background: Leclercia adecarboxylata (L. adecarboxylata) is a gram-negative bacillus of the Enterobacteriaceae family, which is uncommonly isolated from clinical specimens. L. adecarboxylata is considered as an aquatic opportunistic pathogen and most of the human infections are polymicrobial and usually occur in immunocompromised hosts. (2) Methods: In this retrospective study, we included all L. adecarboxylata strains since the introduction of MALDI-TOF MS in the Microbiology Department of Nord Franche-Comté Hospital, France (from 1 March 2015 to 31 July 2019). We studied demographic characteristics, comorbidities, characteristics of the current infection and outcome as well as antimicrobial susceptibility testing in all isolates. (3) Results: A total of 8 samples were identified (in 6 patients (4M/2F), with a recurrent L. adecarboxylata infection in 2 patients). The patients' mean age was 66.2 years (range: 19-84). All patients were considered as immunocompetent, except a peritoneal dialysis patient with kidney transplantation. An exposition to an aquatic environment was identified in one patient. The most prevalent clinical feature was catheter-associated male urinary tract infection (in 3 cases) followed by ventilator-associated pneumonia (in 2 cases). One of 6 patients presented L. adecarboxylata bacteremia. L. adecarboxylata was part of a polymicrobial infection in 4 patients. The isolates showed a high susceptibility to all tested antibiotics, except one strain, which was resistant to fosfomycin. All patients with L. adecarboxylata infection were treated with antibiotics with a favorable outcome. (4) Conclusion: This study confirms the pathogenicity of L. adecarboxylata, even in immunocompetent patients, with a high susceptibility to antibiotics.

COVEVOL: Natural Evolution at 6 Months of COVID-19.

Many studies have investigated post-COVID symptoms, but the predictors of symptom persistence remain unknown. The objective was to describe the natural course of the disease at 6 months and to identify possible factors favoring the resurgence or persistence of these symptoms. COVEVOL is a retrospective observational descriptive study of 74 patients. All patients with positive SARS-CoV-2 PCR from March 2020 were included. We compared a group with symptom persistence (PS group) with another group without symptom persistence (no-PS group). Fifty-three out of seventy-four patients (71.62%) described at least one persistent symptom at 6 months of SARS-CoV-2 infection. In the PS group, 56.6% were women and the average age was 54.7 years old [21-89.2] ± 16.9. The main symptoms were asthenia (56.6%, n = 30), dyspnea (34%, n = 18), anxiety (32.1% n = 17), anosmia (24.5%, n = 13) and agueusia (15.1% n = 8). Ten patients (13.51%) presented a resurgence in symptoms. Patients in the PS group were older (p = 0.0048), had a higher BMI (p = 0.0071), and were more frequently hospitalized (p = 0.0359) compared to the no-PS group. Odynophagia and nasal obstruction were less present in the inaugural symptoms of COVID-19 in the PS group (p = 0.0202 and p = 0.0332). Persistent post-COVID syndromes are common and identification of contributing factors is necessary for understanding this phenomenon and appropriate management.

Post-COVID-19 Syndrome: Nine Months after SARS-CoV-2 Infection in a Cohort of 354 Patients: Data from the First Wave of COVID-19 in Nord Franche-Comté Hospital, France.

(1) Background. Post-COVID-19 syndrome is defined as the persistence of symptoms after confirmed SARS-CoV-2 infection. (2) Methods. ANOSVID is an observational retrospective study in Nord Franche-Comté Hospital in France that included adult COVID-19 patients confirmed by RT-PCR from 1 March 2020 to 31 May 2020. The aim was to describe patients with post-COVID-19 syndrome with persistent symptoms (PS group) and to compare them with the patients without persistent symptoms (no-PS group). (3) Results. Of the 354 COVID-19 patients, 35.9% (n = 127) reported persistence of at least one symptom after a mean of 289.1 ± 24.5 days after symptom onset. Moreover, 115 patients reported a recurrence of symptoms after recovery, and only 12 patients reported continuous symptoms. The mean age of patients was 48.6 years (19-93) ± 19.4, and 81 patients (63.8%) were female. Patients in the PS group had a longer duration of symptoms of initial acute SARS-CoV-2 infection than patients in the no-PS group (respectively, 57.1 ± 82.1 days versus 29.7 ± 42.1 days, p < 0.001). A majority of patients (n = 104, 81.9%) reported three or more symptoms. The most prevalent persistent symptoms were loss of smell (74.0%, n = 94), fatigue (53.5%, n = 68), loss of taste (31.5%, n = 40), and dyspnea (30.7%, n = 39). These were followed by pain symptoms (26.8% (n = 34), 26.0% (n = 33), 24.4% (n = 31); headache, arthralgia, and myalgia, respectively). More than half of patients reporting persistent symptoms (58%, n = 73) were healthcare workers (HCWs). Among outpatients, this population was more present in the PS group than the no-PS group ((86.6%) n = 71/82 versus (72.2%) n = 109/151, p = 0.012). Post-COVID-19 syndrome was more frequent in patients with a past history of chronic rhinosinusitis (8.7% (n = 11%) versus 1.3% (n = 3), p < 0.001). No significant difference was found regarding clinical characteristics and outcome, laboratory, imaging findings, and treatment received in the two groups. (4) Conclusions. More than a third of our COVID-19 patients presented persistent symptoms after SARS-CoV-2 infection, particularly through loss of smell, loss of taste, fatigue, and dyspnea, with a high prevalence in HCWs among COVID-19 outpatients.

Shigella sonnei, an emerging multidrug-resistant sexually transmitted pathogen in Franche-Comté, France.

Shigella sonnei (S. sonnei) is sometimes sexually transmitted. Men, who have sex with men (MSM), may have sexual behaviours different from heterosexual population, and thus may be at risk for S. sonnei infection. We describe three cases of multidrug-resistant S. sonnei in MSM (one HIV-infected patient and two patients receiving pre-exposure prophylaxis against HIV). S. sonnei was isolated from stool specimens and all patients were successfully treated with parenteral third-generation cephalosporins following laboratory confirmation that the isolates were resistant to azithromycin. Two men (patients 2 and 3) were linked epidemiologically. These cases highlight the emergence of this pathogen and its association with some sexual behaviours among MSM in Franche-Comté, France.

Systematic Review and Subgroup Meta-analysis of Randomized Trials to Determine Tocilizumab's Place in COVID-19 Pneumonia.

INTRODUCTION: Tocilizumab randomized clinical trial results are heterogeneous because of the heterogenous population included in them. METHODS: We conducted a meta-analysis with subgroup meta-analysis (PRISMA guidelines) between severe and non-severe COVID-19. RESULTS: We included nine trials. Overall, the mortality rate was 24.5% (821/3357) in the tocilizumab group and 29.1% (908/3125) in the control group at day 28-30 (pooled OR, 0.85; 95% CI 0.76-0.96; p = 0.006). Considering the subgroup analysis, this benefit on mortality was confirmed and amplified in the severe COVID-19 group (pooled OR, 0.82; 95% CI 0.73-0.93; p = 0.001) but not in the non-severe COVID-19 group (pooled OR, 1.46; 95% CI 0.91-2.34; p = 0.12). For patients who were not mechanically ventilated at baseline (5523/6482), the pooled OR (0.74; 95% CI 0.64-0.85; p < 0.0001) for mechanical ventilation incidence at day 28-30 was in favor of tocilizumab (cumulative incidence of 14.8% versus 19.4% in tocilizumab and control arm, respectively). This benefit was confirmed in both subgroups, i.e., severe and non-severe COVID-19. CONCLUSION: Tocilizumab is an effective treatment in hospitalized patients with COVID-19 and hypoxemia by improving survival and decreasing mechanical ventilation requirement. The greatest benefit is observed in severe COVID-19.

Recurrence of COVID-19 after recovery ? A case series in health care workers, France.

We describe 3 cases of coronavirus disease 2019 in health care workers in France involving presumed clinical and microbiological recurrence after recovery. All patients were immunocompetent with clinical mild form. These cases highlight the possibility of coronavirus disease-recurrence.

Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells.

Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 µM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.

Reduced toxicity of 3-epi-deoxynivalenol and de-epoxy-deoxynivalenol through deoxynivalenol bacterial biotransformation: In vivo analysis in piglets.

Ingestion of deoxynivalenol (DON), one of the most common mycotoxin contaminants of cereals, leads to adverse effects for animal and human health. Bacterial biotransformation is a strategy to mitigate the toxicity of this mycotoxin. The present study aims to evaluate the toxicity of two bacterial biotranformation products of DON: 3-epi-deoxynivalenol (3-epi-DON) and de-epoxy-deoxynivalenol (DOM-1) through zootechnical, hematological, histological and immunological assays. Twenty-four 4-weeks-old piglets received a control diet or a diet contaminated with 3 mg kg-1 DON, DOM-1, or 3-epi-DON for 7 days. Sample tissues were collected for histomorphometrical analysis, expression of cytokines and cell protein junctions. The zootechnical and hematological parameters were not modulated by any treatment. Ingestion of DON induced histological alterations in the intestine, liver and lymphoid organs, as well as an overexpression of pro-inflammatory cytokines, E-cadherin and occludin. These changes were not observed in piglets receiving the DOM-1 and 3-epi-DON contaminated diets. Pigs fed 3-epi-DON contaminated diet showed an increase in IgM levels in comparison with other diets, while no change was observed in IgA and IgG levels among the diets. Our results indicate that DOM-1 and 3-epi-DON are not toxic for piglets; thus bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.

Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken.

The protozoan parasite Histomonas meleagridis is the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite's extracellular occurrence, a type-2 differentiation of H. meleagridis-specific T cells has been hypothesized. In contrast, a recent study suggested that IFN-γ mRNA+ cells are involved in protection against histomonosis. However, the phenotype and cytokine production profile of H. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain of H. meleagridis were used for infecting chickens to detect IFN-γ protein and IL-13 mRNA by intracellular cytokine staining and PrimeFlow™ RNA Assays, respectively, in CD4+ and CD8ß+ T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding with H. meleagridis detection by immunohistochemistry and H. meleagridis-specific antibodies in serum. In splenocytes stimulated either with H. meleagridis antigen or PMA/ionomycin, IFN-γ-producing CD4+ T cells from infected chickens increased in comparison to cells from non-infected birds 2 weeks and 5 weeks post-infection. Additionally, an increase of IFN-γ-producing CD4-CD8ß- cells upon H. meleagridis antigen and PMA/ionomycin stimulation was detected. Contrariwise, frequencies of IL-13 mRNA-expressing cells were low even after PMA/ionomycin stimulation and mainly had a CD4-CD8ß- phenotype. No clear increase of IL-13+ cells related to H. meleagridis infection could be found. In summary, these data suggest that H. meleagridis infection induces a type-1 differentiation of CD4+ T cells but also of non-CD4+ cells. This phenotype could include γδ T cells, which will be addressed in future studies.

Deoxynivalenol Affects Proliferation and Expression of Activation-Related Molecules in Major Porcine T-Cell Subsets.

The Fusarium mycotoxin deoxynivalenol (DON) contaminates animal feed worldwide. In vivo, DON modifies the cellular protein synthesis, thereby also affecting the immune system. However, the functional consequences of this are still ill-defined. In this study, peripheral blood mononuclear cells from healthy pigs were incubated with different DON concentrations in the presence of Concanavalin A (ConA), a plant-derived polyclonal T-cell stimulant. T-cell subsets were investigated for proliferation and expression of CD8α, CD27, and CD28, which are involved in activation and costimulation of porcine T cells. A clear decrease in proliferation of all ConA-stimulated major T-cell subsets (CD4+, CD8+, and γδ T cells) was observed in DON concentrations higher than 0.4 µM. This applied in particular to naïve CD4+ and CD8+ T cells. From 0.8 µM onwards, DON induced a reduction of CD8α (CD4+) and CD27 expression (CD4+ and CD8+ T cells). CD28 expression was diminished in CD4+ and CD8+ T cells at a concentration of 1.6 µM DON. None of these effects were observed with the DON-derivative deepoxy-deoxynivalenol (DOM-1) at 16 µM. These results indicate that DON reduces T-cell proliferation and the expression of molecules involved in T-cell activation, providing a molecular basis for some of the described immunosuppressive effects of DON.

Deepoxy-deoxynivalenol retains some immune-modulatory properties of the parent molecule deoxynivalenol in piglets.

Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.