Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 Reduce PepT1 Levels in IL-1ß-Treated Caco-2 Cells via Upregulation of miR-193a-3p.

PepT1, a proton-coupled oligopeptide transporter, is crucial for intestinal homeostasis. It is mainly expressed in small intestine enterocytes, facilitating the absorption of di/tri-peptides from dietary proteins. In the colon, PepT1 expression is minimal to prevent excessive responses to proinflammatory peptides from the gut microbiota. However, increased colonic PepT1 is linked to chronic inflammatory diseases and colitis-associated cancer. Despite promising results from animal studies on the benefits of extracellular vesicles (EVs) from beneficial gut commensals in treating IBD, applying probiotic EVs as a postbiotic strategy in humans requires a thorough understanding of their mechanisms. Here, we investigate the potential of EVs of the probiotic Nissle 1917 (EcN) and the commensal EcoR12 in preventing altered PepT1 expression under inflammatory conditions, using an interleukin (IL)-1-induced inflammation model in Caco-2 cells. The effects are evaluated by analyzing the expression of PepT1 (mRNA and protein) and miR-193a-3p and miR-92b, which regulate, respectively, PepT1 mRNA translation and degradation. The influence of microbiota EVs on PepT1 expression is also analyzed in the presence of bacterial peptides that are natural substrates of colonic PepT1 to clarify how the regulatory mechanisms function under both physiological and pathological conditions. The main finding is that EcN EVs significantly decreases PepT1 protein via upregulation of miR-193a-3p. Importantly, this regulatory effect is strain-specific and only activates in cells exposed to IL-1ß, suggesting that EcN EVs does not control PepT1 expression under basal conditions but can play a pivotal role in response to inflammation as a stressor. By this mechanism, EcN EVs may reduce inflammation in response to microbiota in chronic intestinal disorders by limiting the uptake of bacterial proinflammatory peptides.

Epstein-Barr Virus and immune status imprint the immunogenomics of non-Hodgkin lymphomas occurring in immune-suppressed environments.

Non-Hodgkin lymphomas (NHL) commonly occur in immune-deficient (ID) patients, both HIV-infected and transplanted, and are often EBV-driven with cerebral localization, raising the question of tumor immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 ID (34 posttransplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were Large B Cells Lymphoma (LBCL) and 25% were primary central-nervous-system lymphoma (PCNSL) while 40% were EBV-positive. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden (TMB), tumor landscape and microenvironment (TME) and prediction of tumor neoepitopes. Both TMB (2.2 vs 3.4/Mb, p=0.001) and neoepitopes numbers (40 vs 200, p=0.00019) were lower in EBVpositive than in EBV-negative NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations exclusively observed in EBV-positive and ID NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV-negative cases but in only half EBV-positive ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The TME analysis showed higher CD8 T cell infiltrates in EBVpositive vs EBV-negative NHL, together with a more tolerogenic profile composed of Tregs, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV-negative patients, offering potential opportunities for future T cell-based immune therapies.

Microbiota-Derived Extracellular Vesicles as a Postbiotic Strategy to Alleviate Diarrhea and Enhance Immunity in Rotavirus-Infected Neonatal Rats.

Rotavirus (RV) infection is a major cause of acute gastroenteritis in children under 5 years old, resulting in elevated mortality rates in low-income countries. The efficacy of anti-RV vaccines is limited in underdeveloped countries, emphasizing the need for novel strategies to boost immunity and alleviate RV-induced diarrhea. This study explores the effectiveness of interventions involving extracellular vesicles (EVs) from probiotic and commensal E. coli in mitigating diarrhea and enhancing immunity in a preclinical model of RV infection in suckling rats. On days 8 and 16 of life, variables related to humoral and cellular immunity and intestinal function/architecture were assessed. Both interventions enhanced humoral (serum immunoglobulins) and cellular (splenic natural killer (NK), cytotoxic T (Tc) and positive T-cell receptor γδ (TCRγδ) cells) immunity against viral infections and downregulated the intestinal serotonin receptor-3 (HTR3). However, certain effects were strain-specific. EcoR12 EVs activated intestinal CD68, TLR2 and IL-12 expression, whereas EcN EVs improved intestinal maturation, barrier properties (goblet cell numbers/mucin 2 expression) and absorptive function (villus length). In conclusion, interventions involving probiotic/microbiota EVs may serve as a safe postbiotic strategy to improve clinical symptoms and immune responses during RV infection in the neonatal period. Furthermore, they could be used as adjuvants to enhance the immunogenicity and efficacy of anti-RV vaccines.

Dual Chimeric Antigen Receptor T Cells Targeting CD38 and SLAMF7 with Independent Signaling Demonstrate Preclinical Efficacy and Safety in Multiple Myeloma.

Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (BCMA) induces high overall response rates. However, relapse still occurs and novel strategies for targeting multiple myeloma cells using CAR T-cell therapy are needed. SLAMF7 (also known as CS1) and CD38 on tumor plasma cells represent potential alternative targets for CAR T-cell therapy in multiple myeloma, but their expression on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such treatments. Here, we used CRISPR/Cas9 deletion of the CD38 gene in T cells and developed DCAR, a double CAR system targeting CD38 and CS1 through activation and costimulation receptors, respectively. Inactivation of CD38 enhanced the anti-multiple myeloma activity of DCAR T in vitro. Edited DCAR T cells showed strong in vitro and in vivo responses specifically against target cells expressing both CD38 and CS1. Furthermore, we provide evidence that, unlike anti-CD38 CAR T-cell therapy, which elicited a rapid immune reaction against hematopoietic cells in a humanized mouse model, DCAR T cells showed no signs of toxicity. Thus, DCAR T cells could provide a safe and efficient alternative to anti-BCMA CAR T-cell therapy to treat patients with multiple myeloma.

Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection.

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1.

The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.

Natural Killer Cells in Post-Transplant Lymphoproliferative Disorders.

Post-transplant lymphoproliferative disorders (PTLDs) are life-threatening complications arising after solid organ or hematopoietic stem cell transplantations. Although the majority of these lymphoproliferations are of B cell origin, and are frequently associated with primary Epstein-Barr virus (EBV) infection or reactivation in the post-transplant period, rare cases of T cell and natural killer (NK) cell-originated PTLDs have also been described. A general assumption is that PTLDs result from the impairment of anti-viral and anti-tumoral immunosurveillance due to the long-term use of immunosuppressants in transplant recipients. T cell impairment is known to play a critical role in the immune-pathogenesis of post-transplant EBV-linked complications, while the role of NK cells has been less investigated, and is probably different between EBV-positive and EBV-negative PTLDs. As a part of the innate immune response, NK cells are critical for protecting hosts during the early response to virus-induced tumors. The complexity of their function is modulated by a myriad of activating and inhibitory receptors expressed on cell surfaces. This review outlines our current understanding of NK cells in the pathogenesis of PTLD, and discusses their potential implications for current PTLD therapies and novel NK cell-based therapies for the containment of these disorders.

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease.

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

Distinct immunopathological mechanisms of EBV-positive and EBV-negative posttransplant lymphoproliferative disorders.

EBV-positive and EBV-negative posttransplant lymphoproliferative disorders (PTLDs) arise in different immunovirological contexts and might have distinct pathophysiologies. To examine this hypothesis, we conducted a multicentric prospective study with 56 EBV-positive and 39 EBV-negative PTLD patients of the K-VIROGREF cohort, recruited at PTLD diagnosis and before treatment (2013-2019), and compared them to PTLD-free Transplant Controls (TC, n = 21). We measured absolute lymphocyte counts (n = 108), analyzed NK- and T cell phenotypes (n = 49 and 94), and performed EBV-specific functional assays (n = 16 and 42) by multiparameter flow cytometry and ELISpot-IFNγ assays (n = 50). EBV-negative PTLD patients, NK cells overexpressed Tim-3; the 2-year progression-free survival (PFS) was poorer in patients with a CD4 lymphopenia (CD4+ <300 cells/mm3 , p <  .001). EBV-positive PTLD patients presented a profound NK-cell lymphopenia (median = 60 cells/mm3 ) and a high proportion of NK cells expressing PD-1 (vs. TC, p = .029) and apoptosis markers (vs. TC, p < .001). EBV-specific T cells of EBV-positive PTLD patients circulated in low proportions, showed immune exhaustion (p = .013 vs. TC) and poorly recognized the N-terminal portion of EBNA-3A viral protein. Altogether, this broad comparison of EBV-positive and EBV-negative PTLDs highlight distinct patterns of immunopathological mechanisms between these two diseases and provide new clues for immunotherapeutic strategies and PTLD prognosis.

Ethnobotanical Documentation of Medicinal Plants Used by the Indigenous Panay Bukidnon in Lambunao, Iloilo, Philippines.

The Panay Bukidnon is a group of indigenous peoples living in the interior highlands of Panay Island in Western Visayas, Philippines. Little is known about their ethnobotanical knowledge due to limited written records, and no recent research has been conducted on the medicinal plants they used in ethnomedicine. This study aims to document the medicinal plants used by the indigenous Panay Bukidnon in Lambunao, Iloilo, Panay Island. Semi-structured interviews were conducted with 75 key informants from June 2020 to September 2021 to determine the therapeutic use of medicinal plants in traditional medicine. A total of 131 medicinal plant species distributed in 121 genera and 57 families were used to address 91 diseases in 16 different uses or disease categories. The family Fabaceae was best represented with 13 species, followed by Lamiaceae with nine species and Poaceae with eight species. The leaf was the most frequently used plant part and decoction was the most preferred form of preparation. To evaluate the plant importance, use value (UV), relative frequency citation (RFC), relative important index (RI), informant consensus factor (ICF), and fidelity level (FL) were used. Curcuma longa L. had the highest UV (0.79), Artemisia vulgaris L. had the highest RFC value (0.57), and Annona muricata L. had the highest RI value (0.88). Diseases and symptoms or signs involving the respiratory system and injury, poisoning, and certain other consequences of external causes recorded the highest ICF value (0.80). Blumea balsamifera (L.) DC. and Chromolaena odorata (L.) R.M. King & H. Rob were the most relevant and agreed species for the former and latter disease categories, respectively. C. odorata had the highest FL value (100%) and was the most preferred medicinal plant used for cuts and wounds. The results of this study serve as a medium for preserving cultural heritage, ethnopharmacological bases for further drug research and discovery, and preserving biological diversity.

Skewed T cell responses to Epstein-Barr virus in long-term asymptomatic kidney transplant recipients.

Kidney transplant recipients (KTRs) abnormally replicate the Epstein Barr Virus (EBV). To better understand how long-term immunosuppression impacts the immune control of this EBV re-emergence, we systematically compared 10 clinically stable KTRs to 30 healthy controls (HCs). The EBV-specific T cell responses were determined in both groups by multiparameter flow cytometry with intra cellular cytokine staining (KTRs n = 10; HCs n = 15) and ELISpot-IFNγ assays (KTRs n = 7; HCs n = 7). The T/B/NK cell counts (KTRs n = 10; HCs n = 30) and the NK/T cell differentiation and activation phenotypes (KTRs n = 10; HCs n = 15/30) were also measured. We show that in KTRs, the Th1 effector CD4+ T cell responses against latent EBV proteins are weak (2/7 responders). Conversely, the frequencies total EBV-specific CD8+T cells are conserved in KTRs (n = 10) and span a wider range of EBNA-3A peptides (5/7responders) than in HCs (5/7responders). Those modifications of the EBV-specific T cell response were associated with a profound CD4+ T cell lymphopenia in KTRs compared to HCs, involving the naïve CD4+ T cell subset, and a persistent activation of highly-differentiated senescent CD8+ T cells. The proportion of total NK / CD8+ T cells expressing PD-1 was also increased in KTRs. Noteworthy, PD-1 expression on CD8+ T cells normalized with time after transplantation. In conclusion, we show modifications of the EBV-specific cellular immunity in long term transplant recipients. This may be the result of both persistent EBV antigenic stimulation and profound immunosuppression induced by anti-rejection treatments. These findings provide new insights into the immunopathology of EBV infection after renal transplantation.

Hydrogen peroxide induced phenylpropanoids pathway eliciting a defensive response in plants micropropagated in Temporary Immersion Bioreactors (TIBs).

The relation between the oxidative burst and phenylpropanoid pathways has been studied using the sugarcane cultivar C86-56, which does not release phenolics in agar-base micropropagation systems. In stationary liquid culture, a significant production of phenolic compounds and plant survival were determined in sugarcane plants treated with 5mM H(2)O(2). The spectrophotometer determinations and the gene expression analysis corroborated that releasing of phenolics and soluble θ-quinones was induced during the first 24h of treatment. In comparison with the control treatments, sugarcane plants treated with H(2)O(2) demonstrated differences in the micropropagation-related variables when multiplied in Temporary Immersion Bioreactors (TIBs) supplemented with polyethyleneglycol (PEG 20%). Expression of selected genes related to photosynthesis, ethylene, auxins, oxidative burst, and defense pathways were confirmed during the entire PEG 20% stress in the plants coming from the 5mM H(2)O(2) treatment; whereas, much more heterogeneous expression patterns were evidenced in plants stressed with PEG but not previously treated with H(2)O(2). RT-PCR expression analysis supports the hypothesis that while H(2)O(2) induces the oxidative burst, the phenylpropanoids pathways elicit and maintain the defensive response mechanism in micropropagated sugarcane plants.

Differential expression of candidate defense genes of poplars in response to aphid feeding.

Species belonging to the genus Populus (poplars) produce a series of defensive proteins in response to insect damage. Proteinase inhibitors, polyphenol oxidases, and chitinases are the most relevant and intensively studied proteins. Most of the knowledge about the relation between these proteins and herbivores has been obtained from studies with chewing insects. Nothing is known about whether phloem-feeder insects such as aphids are able to trigger a comparable response. In the current study, the expression of genes encoding a Kunitz trypsin inhibitor 3 (KTI3), a polyphenol oxidase 1 (PPO1), and a class I chitinase (CHI) was characterized in two poplar hybrids (one resistant hybrid and one susceptible hybrid, to aphids) attacked by the aphid Chaitophorus leucomelas Koch. The expression pattern was analyzed using a semiquantitative reverse transcription-polymerase chain reaction approach. The expression of KTI3 was increased by aphids only in the aphid-susceptible hybrid. Differently, PPO1 expression was increased by aphids in the aphid-resistant hybrid. The expression of CHI was down-regulated by aphids in the susceptible hybrid. This is the first study to report the differential expression of poplar defense genes in response to phloem-feeder insects such as aphids. The findings from the current study suggest that the expression levels of defensive proteins are affected by poplar genotype and by aphid infestation.

Phylogeographical analysis of neotropical Rhagoletis (Diptera: Tephritidae): did the Andes uplift contribute to current morphological differences? / Análisis filogeográfico de Rhagoletis neotropicales (Diptera: Tephritidae): ¿ha contribuido el levantamiento de los Andes a explicar las actuales diferencias morfológicas?

Neotropical Rhagoletis species are arranged in four groups: nova,psalida,striatella and ferruginea, which include 18 species. On both sides of the Andes, the evolution of morphological differences among these groups has been suggested to be related to the Andes uplift process. In order to test this hypothesis, a phylogenetic analysis of morphological and molecular data was performed. The results suggest that: 1) Neotropical species of Rhagoletis constitute a separate group from Paleartic and North American species, with the only exception being a member of the striatella group having a certain association with the northern species. 2) Neotropical species seem to form a monophyletic clade, although statistical support for this is weak. 3) The split of South American Rhagoletis from other groups was dated at 4.333 million years ago, which is before the emergence of a continuous landbridge between Central and South América. 4) Within species distributed in South América, morphological and molecular data were coincident, placing species of the ferruginea group separate from the other Neotropical Rhagoletis. 5) The divergence of the ferruginea group from the other groups was dated at 3.882 million years ago, which is before the last uplift of the Andes. These results suggest that diversification of the ferruginea,psalida and nova groups, on each side of the Andes, was the result of a vicariant separation followed by dispersal and isolation processes. Thus, these results support the hypothesis that the Andes uplift has played an important role in Neotropical Rhagoletis diversification.

Las especies de Rhagoletis neotropicales han sido agrupadas en cuatro grupos: nova,psalida,striatella y ferruginea, constituyendo 18 especies. Se han descrito diferencias morfológicas entre estas especies a ambos lados de la cordillera de los Andes que podrían relacionarse con el proceso de levantamiento cordillerano. En este trabajo se evalúa esta hipótesis usando análisis filogenético de atributos morfológicos y moleculares. Los resultados muestran que: a) las especies Neotropicales de Rhagoletis constituyen un grupo separado de las especies Palearticas y Norteamericanas, con la excepción de un miembro del grupo striatella el cual presenta cierta asociación con las especies Norteamericanas; 2) Las especies Neotropicales parece conformar un clado monofilético; 3) La separación de los grupos Sudamericanos de otros grupos fue estimada en 4.333 millones de años antes del presente, proceso anterior a la emergencia del puente de tierra entre América Central y Sudamérica; 4) Dentro de las especies con distribución Sudamericana, los caracteres morfológicos y moleculares coinciden en ubicar algunas especies del grupo ferruginea separadas de especies de Rhagoletis Neotropicales. 5) La separación del grupo ferruginea fue estimada en 3.882 millones de años antes del presente, evento que precede al último levantamiento de los Andes. La diversificación de los grupos ferruginea,psalida y nova a uno y otro lado de la cordillera de los Andes parece responder inicialmente a un proceso vicariante y posteriores eventos de dispersión y aislamiento. Estos resultados sugieren que el levantamiento de los Andes habría participado en los patrones de diversificación de las Rhagoletis Neotropicales.

Phylogeographical analysis of neotropical Rhagoletis (Diptera: Tephritidae): did the Andes uplift contribute to current morphological differences?

Neotropical Rhagoletis species are arranged in four groups: nova,psalida,striatella and ferruginea, which include 18 species. On both sides of the Andes, the evolution of morphological differences among these groups has been suggested to be related to the Andes uplift process. In order to test this hypothesis, a phylogenetic analysis of morphological and molecular data was performed. The results suggest that: 1) Neotropical species of Rhagoletis constitute a separate group from Paleartic and North American species, with the only exception being a member of the striatella group having a certain association with the northern species. 2) Neotropical species seem to form a monophyletic clade, although statistical support for this is weak. 3) The split of South American Rhagoletis from other groups was dated at 4.333 million years ago, which is before the emergence of a continuous landbridge between Central and South América. 4) Within species distributed in South América, morphological and molecular data were coincident, placing species of the ferruginea group separate from the other Neotropical Rhagoletis. 5) The divergence of the ferruginea group from the other groups was dated at 3.882 million years ago, which is before the last uplift of the Andes. These results suggest that diversification of the ferruginea,psalida and nova groups, on each side of the Andes, was the result of a vicariant separation followed by dispersal and isolation processes. Thus, these results support the hypothesis that the Andes uplift has played an important role in Neotropical Rhagoletis diversification.

Involvement of ethylene in stress-induced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun.

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5'-LTR region of this element can drive stress-induced transcriptional activation of the beta-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the beta-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense.