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BackgroundWork on long COVID-19 has mainly focused on clinical care in hospitals. Thermal spa therapies represent a therapeutic offer outside of health care institutions that are nationally or even internationally attractive. Unlike local care (hospital care, general medicine, para-medical care), their integration in the care pathways of long COVID-19 patients seems little studied. The aim of this article is to determine what place french thermal spa therapies can take in the care pathway of long COVID-19 patients.MethodsBased on the case of France, we carry out a geographic mapping analysis of the potential care pathways for long COVID-19 patients by cross-referencing, over the period 2020-2022, the available official data on COVID-19 contamination, hospitalisations in intensive care units and the national offer of spa treatments. This first analysis allows us, by using the method for evaluating the attractiveness of an area defined by David Huff, to evaluate the accessibility of each French department to thermal spas.ResultsUsing dynamic geographical mapping, this study describes two essential criteria for the integration of the thermal spa therapies offer in the care pathways of long COVID-19 patients (attractiveness of spa areas and accessibility to thermal spas) and three fundamental elements for the success of these pathways (continuity of the care pathways; clinical collaborations; adaptation of the financing modalities to each patient). Using a spatial attractiveness method, we make this type of geographical analysis more dynamic by showing the extent to which a thermal spa is accessible to long COVID-19 patients.ConclusionBased on the example of the French spa offer, this study makes it possible to place the care pathways of long COVID-19 patients in a wider area (at least national), rather than limiting them to clinical and local management in a hospital setting. The identification and operationalization of two geographical criteria for integrating a type of treatment such as a spa cure into a care pathway contributes to a finer conceptualization of the construction of healthcare pathways.

Abstract Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent kidney cancers, which is often asymptomatic and thus discovered at a metastatic state (mRCC). mRCC are highly heterogeneous tumors composed of subclonal populations that lead to poor treatment response rate. Several recent works explored the potential of ccRCC tumoroids culture derived from patients. However, these models were produced following a scaffold-based method using collagen I or Matrigel that exhibit lot variability and whose complexity could induce treatment response modifications and phenotypic alterations. Following the observation that ccRCC tumoroids can create their own niche by secreting extracellular matrix components, we developed the first scaffold-free tumoroid model of ccRCC tumors. Tumoroids from mice as well as from human tumors were generated with high success rate (≥90%) using a magnetic suspension method and standard culture media. Immunofluorescence analysis revealed their self-organization capacities to maintain multiple tumor-resident cell types, including endothelial progenitor cells. Transcriptomic analysis showed the reproducibility of the method highlighting that the majority of gene expression patterns was conserved in tumoroids compared to their matching tumor tissue. Moreover, this model enables to evaluate drug effects and invasiveness of renal cancer cells in a 3D context, providing a robust preclinical tool for drug screening and biomarker assessment in line with alternative ex vivo methods like tumor tissue slice culture or in vivo xenograft models.

ABSTRACTThe third, “booster”, vaccination increases the overall immune response against SARS-CoV-2 variants. However, after the initial peak at around 3 weeks post-vaccination, anti-spike antibody levels decline. Post-booster kinetics of cellular response has been less investigated and there is no documented evidence of a true boosting effect. Furthermore, multiple studies underline the less effective immune responses against Omicron, the latest variant of concern, at both humoral and cellular levels. In this letter, we analyse humoral (anti-RBD IgG levels) and cellular (IFN-γ release assay) immune response in 205 health care workers 3 weeks and 3 months after administration of an mRNA-based booster dose, either mRNA-1273 or BNT162b2. Since all subjects were SARS-CoV-2 infection-naïve, we also looked at the incidence of Omicron infection between 3 and 6 months post-booster.At both timepoints, 3x mRNA-1273 vaccination had the highest overall antibody and IFN-γ levels, followed by 3x BNT162b2 vaccination and heterologous mRNA-based regimens. Heterologous ChAdOx1–mRNA-based regimen had the lowest antibody levels while cellular response equal to that of 3x BNT162b2 vaccination and heterologous mRNA-based regimens. Our results show that both humoral and cellular responses waned at 3 months for all vaccination regimens. However, we identified three trajectories of dosage variation. Interestingly, the subgroup of subjects with increasing anti-RBD IgG levels over time had a lower incidence of Omicron infection. Whether increasing humoral response at 3 months post-booster is more indicative of protection than a high initial peak remains to be confirmed in a larger cohort.

Summary: The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map demonstrating that lymphoid specification follows independent direct or stepwise hierarchic routes converging toward the emergence of newly characterized CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (NK/ILC/T) or CD127+ (B) lymphoid pathways. While the differentiation of CD127- early lymphoid progenitors is mainly driven by Flt3 signaling, emergence of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their upstream precursors, including hematopoietic stem cells. Further, transcriptional mapping of differentiation trajectories reveals that whereas myeloid granulomonocytic lineages follow continuous differentiation pathways, lymphoid trajectories are intrinsically discontinuous and characterized by sequential waves of cell proliferation allowing pre-commitment amplification of lymphoid progenitor pools. Besides identifying new lymphoid specification pathways and regulatory checkpoints, our results demonstrate that NK/ILC/T and B lineages are under fundamentally distinct modes of regulation. (149 words)

Summary: Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127−/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

There are two major clinically described forms of IgE-dependent soy allergy: (i) a primary dietary form, linked to sensitization against soy storage proteins Gly m 5 and Glym 6, and (ii) a form included in birch-soy syndromes linked to Gly m 4, a PR-10-like allergen. This second form sometimes causes severe systemic reactions, even anaphylaxis, especially on consuming certain forms of soy such as soymilks or smoothies. Skin prick tests and specific IgE assays against soy whole extracts lack sensitivity. Assays of anti-Gly m 4, Gly m 5 and Gly m 6 specific IgEs have been developed to overcome this obstacle, but they unfortunately lack specificity, especially for anti-Gly m 4. We hypothesized that the basophil activation test (BAT) using molecular soy allergens Gly m 4, Gly m 5 and Gly m 6 would both remedy the lack of sensitivity of other tests and offer, through its mechanistic contribution, greater specificity than the assay of anti-Gly m 4 specific IgEs. This would enable the two types of soy allergy to be separately identified. In a characteristic clinical example of PR-10-induced anaphylactic reaction after consuming soymilk, we report preliminary results of Gly m 4-exclusive positivity of BAT supporting our hypothesis. It will be necessary to confirm these results on more patients in subsequent studies, and to specify the place of the BAT in an overall diagnostic strategy. Meanwhile, soy BAT using molecular allergens is a promising diagnostic tool for soy allergy and probably also for follow-up in specific immunotherapies.

Background: The clinical significance of early-onset acute kidney injury (EO-AKI) and recovery in severe COVID-19 intensive care unit (ICU) patients is poorly documented. Objective: The aim of the study was to assess the epidemiology and outcome of EO-AKI and recovery in ICU patients admitted for SARS-CoV-2 pneumonia. Design: This was a retrospective single-centre study. Setting: The study was carried out at the medical ICU of the university hospital of Clermont-Ferrand, France. Patients: All consecutive adult patients aged ≥18 years admitted between 20 March 2020 and 31 August 2021 for SARS-CoV-2 pneumonia were enrolled. Patients with chronic kidney disease, referred from another ICU, and with an ICU length of stay (LOS) ≤72 h were excluded. Interventions: EO-AKI was defined on the basis of serum creatinine levels according to the Kidney Disease Improving Global Outcomes criteria, developing ≤7 days. Depending on renal recovery, defined by the normalization of serum creatinine levels, EO-AKI was transient (recovery within 48 h), persistent (recovery between 3 and 7 days) or AKD (no recovery within 7 days after EO-AKI onset). Measurements: Uni- and multivariate analyses were performed to determine factors associated with EO-AKI and EO-AKI recovery. Main Results: EO-AKI occurred in 84/266 (31.5%) study patients, of whom 42 (50%), 17 (20.2%) and 25 (29.7%) had EO-AKI stages 1, 2 and 3, respectively. EO-AKI was classified as transient, persistent and AKD in 40 (47.6%), 15 (17.8%) and 29 (34.6%) patients, respectively. The 90-day mortality was 87/244 (35.6%) and increased with EO-AKI occurrence and severity: no EO-AKI, 38/168 (22.6%); EO-AKI stage 1, 22/39 (56.4%); stage 2, 9/15 (60%); and stage 3, 18/22 (81.8%) (p < 0.01). The 90-day mortality in patients with transient or persistent AKI and AKD was 20/36 (55.6%), 8/14 (57.1%) and 21/26 (80.8%), respectively (p < 0.01). MAKE-90 occurred in 42.6% of all patients. Conclusions: In ICU patients admitted for SARS-CoV-2 pneumonia, the development of EO-AKI and time to recovery beyond day 7 of onset were associated with poor outcome.

Background: Blood tryptase and fecal calprotectin levels may serve as biomarkers of necrotizing enterocolitis. However, their interpretation may be hindered by the little-known effects of perinatal factors. The aim of this study was to compare the tryptase and calprotectin levels in newborns according to their term, trophicity, and sex. Method: One hundred and fifty-seven premature newborns and 157 full-term newborns were included. Blood tryptase and fecal calprotectin were assayed. Results: Blood tryptase levels were higher in premature than in full-term newborns (6.4 vs. 5.2 µg/L; p < 0.001). In situations of antenatal use of corticosteroids (p = 0.007) and non-exclusive use of human milk (p = 0.02), these levels were also higher. However, in multiple linear regression analyses, only prematurity significantly influenced tryptase levels. Fecal calprotectin levels were extremely wide-ranging and were much higher in female than in male newborns (300.5 vs. 110.5 µg/g; p < 0.001). Conclusions: The differences in tryptase levels according to term could be linked to early aggression of the still-immature digestive wall in premature newborns, in particular, by enteral feeding started early. The unexpected influence of sex on fecal calprotectin levels remains unexplained.

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

Clinical trials and real-world evidence on COVID-19 vaccines have shown their effectiveness against severe disease and death but the durability of protection remains unknown. We analysed the humoral and T-cell immune responses in 110 healthcare workers (HCWs) vaccinated according to the manufacturer’s recommended schedule of dose 2 three weeks after dose 1 from a prospective on-going cohort in early 2021, 3 and 6 months after full vaccination with the BNT162b2 mRNA vaccine. Anti-RBD IgG titres were lower in HCWs over 60 years old 3 months after the second dose (p=0.03) and declined in all the subjects between 3 and 6 months with a median percentage change of -58.5%, irrespective of age and baseline comorbidities. Specific T-cell response measured by IGRA declined over time by at least 42% (median) in 91 HCWs and increased by 33% (median) in 17 others. Six HCWs had a negative T-cell response at 6 months. Ongoing follow-up should provide correlates of long-term protection according to the different immune response profiles observed. COVIDIM study was registered under the number NCT04896788 on clinicaltrials.gov.

ObjectiveCOVID 19 is often associated with hypercoagulability and thromboembolic (TE) events. The aim of this study was to assess the characteristics of hypercoagulability and its relationship with new-onset TE events and the composite outcome of need for intubation and/or death in intensive care unit (ICU) patients admitted for COVID.DesignProspective observational study.SettingMonocentric, intensive care, University Hospital of Clermont Ferrand, France.PatientsPatients admitted to intensive care from January 2020 to May 2021 for COVID-19 pneumonia.InterventionsStandard hemostatic tests and rotational thromboelastometry (ROTEM) were performed on admission and on day 4. Hypercoagulability was defined by at least one of the following criteria: D-dimers > 3000 μg/dL, fibrinogen > 8 g/L, EXTEM CFT below the normal range, EXTEM A5, MCF, Li 60 above the normal range, and EXTEM G-score ((5000 x MCF) / (100-MCF)) ≥ 11 dyne/cm2.Measurements and main resultsOf the 133 patients included, 17 (12.7%) developed new-onset TE events, and 59 (44.3%) required intubation and/or died in the ICU. ROTEM was performed in 133 patients on day 1 and in 67 on day 4. Hypercoagulability was present on day 1 in 115 (86.4%) patients. None of the hypercoagulability indices were associated with subsequent new-onset TE events on days 1 and 4 nor with the need for intubation and/or ICU death. Hyperfibrinogenemia > 8g/dL, higher D-dimers and higher EXTEM Li 60 on day 4 were predictive of need for intubation and/or of ICU death.ConclusionsOur study confirmed that most COVID-19 ICU patients have hypercoagulability on admission and almost all on day 4. Hyperfibrinogenemia or fibrinolysis shutdown on day 4 were associated with unfavorable outcome.

Background: SARS-CoV-2 has been responsible for considerable mortality worldwide, owing in particular to pulmonary failures such as ARDS, but also to other visceral failures and secondary infections. Recent progress in the characterization of the immunological mechanisms that result in severe organ injury led to the emergence of two successive hypotheses simultaneously tested here: hyperinflammation with cytokine storm syndrome or dysregulation of protective immunity resulting in immunosuppression and unrestrained viral dissemination. Methods: In a prospective observational monocentric study of 134 patients, we analysed a panel of plasma inflammatory and anti-inflammatory cytokines and measured monocyte dysregulation via their membrane expression of HLA-DR. We first compared the results of patients with moderate forms hospitalized in an infectious disease unit with those of patients with severe forms hospitalized in an intensive care unit. In the latter group of patients, we then analysed the differences between the surviving and non-surviving groups and between the groups with or without secondary infections. Findings: Higher blood IL-6 levels, lower quantitative expression of HLA-DR on blood monocytes and higher IL-6/mHLA-DR ratios were statistically associated with the risk of severe forms of the disease and among the latter with death and the early onset of secondary infections. Interpretation: The unique immunological profile in patients with severe COVID-19 corresponds to a moderate cytokine inflammation associated with severe monocyte dysregulation. Individuals with major CSS were rare in our cohort of hospitalized patients, especially since the use of corticosteroids, but formed a very severe subgroup of the disease. Funding: None.

Background: Asthma is a chronic inflammatory disease of the airways common in children. Soluble advanced glycation end-product receptor (sRAGE) is a blood biomarker of lung damage and inflammation. We sought to determine whether it could also be a biomarker in childhood asthma. Methods: We conducted a prospective, observational, analytical study at Clermont-Ferrand University Hospital. We measured plasma sRAGE levels in asthmatic and healthy children aged 7 years. Results: Of the 68 children assessed, 15 (22.05%) presented asthma. All presented normal respiratory function. The mean plasma sRAGE level was 1875 pg/mL in the children with asthma and 1794 pg/mL in the healthy children (p = 0.525). The mean plasma sRAGE level was significantly decreased with tobacco exposure during pregnancy: 1478 pg/mL versus 1870 pg/mL without (p = 0.007). Lower levels were observed in children living in apartments (1557 pg/mL) than in those living in houses (1863 pg/mL) (p = 0.031). Conclusions: No difference was observed in plasma sRAGE levels in children with asthma in our well-treated and controlled population. Environmental exposure may affect these levels. Further studies are required to better characterize the role of sRAGE.

Background: We set out to assess the risk factors for asthma outcome in a cohort of infants who experienced their first episode of acute bronchiolitis. Methods: A cohort of 222 infants who were included during a first episode of acute bronchiolitis was prospectively followed. Herein, we present the results of their assessments (symptom history, skin prick tests, specific IgE assay, respiratory function tests) at age seven. Results: Of the 68/222 (30.6%) children assessed at age seven, 15 (22.05%) presented with asthma and were mainly males (p = 0.033), 14 (20%) had respiratory allergies, 17 (25%) presented atopic dermatitis and none had a food allergy. Family history of atopy was associated with asthma and sensitisation to aeroallergens at age seven (p = 0.003, p = 0.007). Rhinovirus (hRV) infection and rhinovirus/respiratory syncytial virus (RSV) co-infection were significantly associated with asthma at age seven (p = 0.035, p = 0.04), but not with the initial severity of bronchiolitis. Eosinophil counts at ages three and seven were significantly higher in the asthmatics (p = 0.01, p = 0.046). Conclusion: Any infant, especially male, presenting a first episode of acute bronchiolitis due to hRV with a family history of atopy should be closely monitored via follow-up due to a higher risk for asthma at school age.

Background: Immunosenescence contributes to reduced vaccine response in elderly persons, and is worsened by deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can both potentiate the innate immune response and inhibit the adaptive system, and so modulate vaccination response.Objective: This randomized placebo-controlled double-blind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response.Design: Deficient volunteers (Vit-D serum

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

Recent studies strongly support the use of the aryl hydrocarbon receptor (AhR) as a therapeutic target in breast cancer. Glyceollins, a group of soybean phytoalexins, are known to exert therapeutic effects in chronic human diseases and also in cancer. To investigate the interaction between glyceollin I (GI), glyceollin II (GII) and AhR, a computational docking analysis, luciferase assays, immunofluorescence and transcriptome analyses were performed with different cancer cell lines. The docking experiments predicted that GI and GII can enter into the AhR binding pocket, but their interactions with the amino acids of the binding site differ, in part, from those interacting with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Both GI and GII were able to weakly and partially activate AhR, with GII being more potent. The results from the transcriptome assays showed that approximately 10% of the genes regulated by TCDD were also modified by both GI and GII, which could have either antagonistic or synergistic effects upon TCDD activation. In addition, we report here, on the basis of phenotype, that GI and GII inhibit the migration of triple-negative (ER-, PgR-, HER2NEU-) MDA-MB-231 breast cancer cells, and that they inhibit the expression of genes which code for important regulators of cell migration and invasion in cancer tissues. In conclusion, GI and GII are AhR ligands that should be further investigated to determine their usefulness in cancer treatments.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, known to affect T lymphocytes. However, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, benzo[α]pyrene (B[α]P), using a microarray-based transcriptome analysis. After a 48 h exposure to B[α]P, we identified 158 genes differentially expressed in T lymphocytes, including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, but also others not previously shown to be targeted by B[α]P such as genes encoding the gap junction beta (GJB)-2 and 6 proteins. Functional enrichment analysis revealed that these candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signaling pathways; a marked alteration in T lymphocyte recruitment was also observed. Using functional tests in transwell migration experiments, B[α]P was then shown to significantly decrease the chemokine (C-X-C motif) ligand 12-induced chemotaxis and transendothelial migration of T lymphocytes. In total, this study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, thus providing a more thorough understanding of the molecular basis of the immunotoxicity of PAHs.

Bacterial vaginosis (BV), the most common genital infection in reproductive-aged women, is associated with increased risk of sexually transmitted infections. Its etiology remains unclear, especially the role of Gardnerella (G.) vaginalis, an anaerobic bacterium characteristic of the BV-alteration of the vaginal ecosystem. In the genital mucosa, dendritic cells (DCs) sense bacteria of the microenvironment via receptors and then orchestrate the immune response by induction of different T cell subtypes. We investigated the interactions between G. vaginalis and human monocyte-derived DCs using a wide range of bacterial concentrations (multiplicity of infection from 0.01 to 100), and the effects of this pathogen on PHA-induced lymphocyte proliferation. As observed by electron microscopy and cytometry, G. vaginalis reduced the internalization ability of DCs by forming extracellular clusters and induced neither DC maturation, nor DC secretion of cytokines, except at the highest dose with a very early DC maturation state. The same profile was observed on lymphocytes with significant increases of proliferation and cytokine secretion only at the highest bacterial concentration. Our findings indicate that G. vaginalis possesses slight immune-stimulating activities against DCs and T cells, reflecting thus a defective inflammatory response and giving rise to the atypical, non- or low-grade, inflammatory clinical disease profile.

The response of the immune system to probiotics remains controversial. Some strains modulate the cytokine production of dendritic cells (DCs) in vitro and induce a regulatory response, while others induce conversely a pro-inflammatory response. These strain-dependent effects are thought to be linked to specific interactions between bacteria and pattern recognition receptors. We investigated the effects of a well characterized probiotic strain, Lactobacillus rhamnosus Lcr35, on human monocyte-derived immature DCs, using a wide range of bacterial concentrations (multiplicity of infection, MOI, from 0.01 to 100). DNA microarray and qRT-PCR analysis showed that the probiotic induced a large-scale change in gene expression (nearly 1,700 modulated genes, with 3-fold changes), but only with high doses (MOI, 100). The upregulated genes were mainly involved in immune response and identified a molecular signature of inflammation according to the model of Torri. Flow cytometry analysis also revealed a dose-dependent maturation of the DC membrane phenotype, until DCs reached a semi-mature state, with an upregulation of the membrane expression of CD86, CD83, HLA-DR and TLR4, associated with a down-regulation of DC-SIGN, MR and CD14. Measurement of the DC-secreted cytokines showed that Lcr35 induced a strong dose-dependent increase of the pro-Th1/Th17 cytokine levels (TNFα, IL-1β, IL-12p70, IL-12p40 and IL-23), but only a low increase in IL-10 concentration. The probiotic L. rhamnosus Lcr35 therefore induce a dose-dependent immunomodulation of human DCs leading, at high doses, to the semi-maturation of the cells and to a strong pro-inflammatory effect. These results contribute to a fuller understanding of the mechanism of action of this probiotic, and thus of its potential clinical indications in the treatment of either infectious or IgE-dependent allergic diseases.