Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection.

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

[French national standard for the treatment of squamous cell carcinoma of upper aero-digestive tract - General principles of treatment]. / Référentiel national de traitement des carcinomes épidermoïdes des voies aérodigestives supérieures ­ Principes généraux de traitement.

OBJECTIVES: The management of upper aerodigestive tract cancers is a complex specialty. It is essential to provide an update to establish optimal care. At the initiative of the INCa and under the auspices of the SFORL, the scientific committee, led by Professor Béatrix Barry, Dr. Gilles Dolivet, and Dr. Dominique De Raucourt, decided to develop a reference framework aimed at defining, in a scientific and consensus-based manner, the general principles of treatment for upper aerodigestive tract cancers applicable to all sub-locations. METHODOLOGY: To develop this framework, a multidisciplinary team of practitioners was formed. A systematic analysis of the literature was conducted to produce recommendations classified by grades, in accordance with the standards of the French National Authority for Health (HAS). RESULTS: The grading of recommendations according to HAS standards has allowed the establishment of a reference for patient care based on several criteria. In this framework, patients benefit from differentiated care based on prognostic factors they present (age, comorbidities, TNM status, HPV status, etc.), conditions of implementation, and quality criteria for indicated surgery (operability, resectability, margin quality, mutilation, salvage surgery), as well as quality criteria for radiotherapy (target volume, implementation time, etc.). The role of medical and postoperative treatments was also evaluated based on specific criteria. Finally, supportive care must be organized from the beginning and throughout the patients' care journey. CONCLUSION: All collected data have led to the development of a comprehensive framework aimed at harmonizing practices nationally, facilitating decision-making in multidisciplinary consultation meetings, promoting equality in practices, and providing a state-of-the-art and reference practices for assessing the quality of care. This new framework is intended to be updated every 5 years to best reflect the latest advances in the field.

Expanding Radical Chloropentafluorosulfanylation of Alkynes.

The chloropentafluorosulfanylation of alkynes is a delicate but crucial operation for accessing SF5-alkynes that serve as substrates in numerous transformations. Dolbier's procedure using Et3B/O2 was the most efficient approach, while recent efforts make use of other initiators and light activation. We found that THF, as a single stimulus, is sufficient to trigger the reaction of SF5Cl with alkynes. We determined the configuration of Cl/SF5 products and clarified the structure of side-products.

Palladium-Catalyzed Regioselective Synthesis of 2-SF5 -Indenols and Further Derivatizations.

A palladium-catalyzed synthesis of 2-SF5 -indenols has been developed by reacting commercially available boronic acid derivatives and readily accessible SF5 -alkynes. The present methodology is fully regioselective thanks to the intrinsic polarization of SF5 -alkynes. A selection of downstream functionalizations has been performed to highlight the versatility of 2-SF5 -indenols and indenones as platforms for the design of more complex SF5 -containing molecules.

Divergent Synthesis of 6- or 7-Aza-Indazoles via Intramolecular Diels-Alder Cascade of Pyrazines.

Pyrazines are reactive 4π partners in intermolecular Diels-Alder cycloaddition with exceptionally activated dienophiles or in an intermolecular version at elevated temperatures. Herein, it is shown that an intramolecular cascade could occur even at room temperature, delivering a collection of 6- or 7-aza-indazoles. An interesting substituent effect of the cycloaddition precursor on the product distribution was uncovered, and in situ NMR studies were conducted to gain insights into this unexpected selectivity.

Tracking SF5 I in the Iodopentafluorosulfanylation of Alkynes.

In the vibrant field of SF5 chemistry, SF5 X reagents (X=F, Cl, Br) are at the heart of current investigations in radical pentafluorosulfanylation reactions. SF5 I is the missing link whose existence has not been reported despite its potential as SF5 donor. This study reports the formal addition of the hitherto unknown SF5 I reagent to alkynes by means of a combination of SF5 Cl/KI/18-crown-6 ether. The exclusive regio- and stereoselective synthesis of unprecedented (E)-1-iodo-2-(pentafluoro-λ6 -sulfanyl) alkenes was achieved. A consensus was reached through computational and mechanistic studies for the realistic formation of SF5 - anion but not SF5 I in solution and the rational involvement of SF5 ⋅ and iodine radicals in the iodo pentafluorosulfanylation reaction.

Copper-Catalyzed, Ligand-Controlled N(sp3)- or N(sp)-Selective Arylation of Cyanamides.

Cyanamides possess both nucleophilic and electrophilic centers, and their arylation reactions are known to proceed at N(sp3) and C(sp) sites, leading to N-aryl cyanamides or amidines. N(sp) selectivity has also been reported only in the presence of amines, thus leading to guanidines. Herein, we report a general copper-catalyzed ligand-controlled Chan-Lam-Evans arylation of cyanamides proceeding regioselectively at the N(sp3) or N(sp) atoms and leading to either N-aryl cyanamides or dissymmetric carbodiimides. The nature of the ligand, either a bipyridine or a diamine, controls the product distribution and thus offers a divergent entry to useful building blocks from readily available cyanamides.

The apicoplast is important for the viability and persistence of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.

Antigen recognition detains CD8+ T cells at the blood-brain barrier and contributes to its breakdown.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8+ T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8+ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8+ T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8+ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8+ T cell entry into the CNS and triggers CD8+ T cell-mediated focal BBB breakdown.

A common mechanism of Sec61 translocon inhibition by small molecules.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.

Regio- and Stereoselective Hydroelementation of SF5 -Alkynes and Further Functionalizations.

Herein is described a fully regio- and stereoselective hydroelementation reaction of SF5 -alkynes with N, O and S-nucleophiles and further functionalization of the corresponding Z-(hetero)vinyl-SF5 intermediates, a suitable platform to access α-SF5 ketones and esters, ß-SF5 amines and alcohols under mild reaction conditions. Experimental and computational comparative studies between SF5 - and CF3 -alkynes have been performed to highlight and explain the difference of reactivity and selectivity observed between these two fluorinated motifs.

Efficient Cholesterol Transport in Dendritic Cells Defines Optimal Exogenous Antigen Presentation and Toxoplasma gondii Proliferation.

Dendritic cells are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway to stimulate CD4+ T cells, or with MHC-I to activate CD8+ T lymphocytes through the cross-presentation pathway. DCs represent one of the main cellular targets during infection by Toxoplasma gondii. This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, T. gondii modulates the host immune response through multiple interactions with proteins and lipids. Cholesterol is an important cellular component that regulates cellular physiology at the structural and functional levels. Although different studies describe the relevance of cholesterol transport for exogenous antigen presentation, the molecular mechanism underlying this process is not defined. Here, we focus our study on the inhibitor U18666A, a drug widely used to arrest multivesicular bodies biogenesis that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of T. gondii-associated antigens and parasite proliferation were hampered in treated cells. However, neither antigen uptake nor BMDC viability was significantly affected by the U18666A treatment. By contrast, this drug altered the transport of MHC-I and MHC-II molecules to the plasma membrane. Since U18666A impairs the formation of MVBs, we analyzed in T. gondii infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by T. gondii. Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. Altogether, our results highlight the importance of cholesterol trafficking and MVBs formation in DCs for optimal antigen presentation and T. gondii proliferation.

Microbial (co)infections: Powerful immune influencers.

It is well established that by modulating various immune functions, host infection may alter the course of concomitant inflammatory diseases, of both infectious and autoimmune etiologies. Beyond the major impact of commensal microbiota on the immune status, host exposure to viral, bacterial, and/or parasitic microorganisms also dramatically influences inflammatory diseases in the host, in a beneficial or harmful manner. Moreover, by modifying pathogen control and host tolerance to tissue damage, a coinfection can profoundly affect the development of a concomitant infectious disease. Here, we review the diverse mechanisms that underlie the impact of (co)infections on inflammatory disorders. We discuss epidemiological studies in the context of the hygiene hypothesis and shed light on the sometimes dual impact of germ exposure on human susceptibility to inflammatory disease. We then summarize the immunomodulatory mechanisms at play, which can involve pleiotropic effects of immune players and discuss the possibility to harness pathogen-derived compounds to the host benefit.

Molecular Mechanisms Underpinning the Circulation and Cellular Uptake of Mycobacterium ulcerans Toxin Mycolactone.

Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.

New Insights into Blood Circulating Lymphocytes in Human Pneumocystis Pneumonia.

The host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(-)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(-) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis.

Impact of Neck Dissection in Head and Neck Squamous Cell Carcinomas of Unknown Primary.

PURPOSE: Management of head and neck cancers of unknown primary (HNCUP) combines neck dissection (ND) and radiotherapy, with or without chemotherapy. The prognostic value of ND has hardly been studied in HNCUP. METHODS: A retrospective multicentric study assessed the impact of ND extent (adenectomy, selective ND, radical/radical-modified ND) on nodal relapse, progression-free survival (PFS) or survival, taking into account nodal stage. RESULTS: 53 patients (16.5%) had no ND, 33 (10.2%) had lymphadenectomy, 116 (36.0%) underwent selective ND and 120 underwent radical/radical-modified ND (37.3%), 15 of which received radical ND (4.7%). With a 34-month median follow-up, the 3-year incidence of nodal relapse was 12.5% and progression-free survival (PFS) 69.1%. In multivariate analysis after adjusting for nodal stage, the risk of nodal relapse or progression was reduced with lymphadenectomy, selective or radical/modified ND, but survival rates were similar. Patients undergoing lymphadenectomy or ND had a better PFS and lowered nodal relapse incidence in the N1 + N2a group, but the improvement was not significant for the N2b or N2 + N3c patients. Severe toxicity rates exceeded 40% with radical ND. CONCLUSION: In HNCUP, ND improves PFS, regardless of nodal stage. The magnitude of the benefit of ND does not appear to depend on ND extent and decreases with a more advanced nodal stage.

The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection.

The unfolded protein response (UPR) has emerged as a central regulator of immune cell responses in several pathologic contexts including infections. However, how intracellular residing pathogens modulate the UPR in dendritic cells (DCs) and thereby affect T cell-mediated immunity remains uncharacterized. Here, we demonstrate that infection of DCs with Toxoplasma gondii (T. gondii) triggers a unique UPR signature hallmarked by the MyD88-dependent activation of the IRE1α pathway and the inhibition of the ATF6 pathway. Induction of XBP1s controls pro-inflammatory cytokine secretion in infected DCs, while IRE1α promotes MHCI antigen presentation of secreted parasite antigens. In mice, infection leads to a specific activation of the IRE1α pathway, which is restricted to the cDC1 subset. Mice deficient for IRE1α and XBP1 in DCs display a severe susceptibility to T. gondii and succumb during the acute phase of the infection. This early mortality is correlated with increased parasite burden and a defect in splenic T-cell responses. Thus, we identify the IRE1α/XBP1s branch of the UPR as a key regulator of host defense upon T. gondii infection.

Synthesis and Physicochemical Properties of 2-SF5-(Aza)Indoles, a New Family of SF5 Heterocycles.

Structural diversity in heterocyclic chemistry is key to unlocking new properties and modes of action. In this regard, heterocycles embedding emerging fluorinated substituents hold great promise. Herein is described a strategy to access 2-SF5-(aza)indoles for the first time. The sequence relies on the radical addition of SF5Cl to the alkynyl π-system of 2-ethynyl anilines followed by a cyclization reaction. A telescoped sequence is proposed, making this strategy very appealing and reproducible on a gram scale. Downstream functionalizations are also demonstrated, allowing an easy diversification of N- and C3-positions. Ames test, pK a, log P, and differential scanning calorimetry measurements of several fluorinated 2-Rf-indoles are also disclosed. These studies highlight the strategic advantages that a C2-pentafluorosulfanylated motif impart to a privileged scaffold such as an indole.

Spatiotemporal analysis of mycolactone distribution in vivo reveals partial diffusion in the central nervous system.

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.