Comprehensive morphometric assessment of deltoid muscle development in children: A cross-sectional study.

BACKGROUND: Normative values for different morphometric parameters of muscle fibres during paediatric development, i.e. from 0 to 18 years, are currently unavailable. They would be of major importance to accurately evaluate pathological changes and could be used as reference biomarkers for evaluating treatment response in clinical trials, or physiological adjustments in sports or ageing. METHODS: Data were derived from 482 images with a total of 33 094 fibres from 10 µm cross-sections of snap-frozen muscle from 83 deltoid muscle biopsies from patients, 0-18 years, without neuromuscular pathology stained with ATPase 9.4. Data was acquired and analysed with patented image analysis algorithms from "CARPACCIO.cloud". Several parameters were extracted or calculated, including cross-sectional area (CSA), fibre type, circularity, as well as the Minimum diameter of Feret (MinFeret). FINDINGS: This study illustrates changes in quantitative parameters for muscle morphology over the course of paediatric development and the pivotal changes occurring around puberty. Only fibre size parameters (MinFeret, CSA) are dependent on gender, and only after puberty. All other parameters vary in a similar manner for females and males. The proportion of type 1 fibres is essentially constant from birth to age 10, decreasing to ≈40% by age 18. Circularity decreases with age, to plateau after age 10 for both fibre types. INTERPRETATION: Normative values and reference charts for muscle fibre types in this age range have been generated to allow comparison of data from patients in pathology laboratories working on neuromuscular diseases. FUNDING: BPI FRANCE, PULSALYS, Association de l'Institut de Myologie, French National Research Agency (ANR), LABEX CORTEX of Université de Lyon.

Gene profiling reveals a contact allergy signature in most positive Amerchol L-101 patch test reactions.

BACKGROUND: Diagnosis of contact allergy (CA) to Amerchol L-101 (AL-101), a marker for lanolin allergy, is problematic. Positive patch test reactions are frequently doubtful or weakly positive and difficult to associate with clinical relevance. OBJECTIVE: To gain further insight on the allergic or irritant nature of skin reactions induced by AL-101 patch test. METHODS: We re-tested in a dose-response fashion, 10 subjects with AL-101 CA and performed comprehensive transcriptomic analysis (gene arrays, quantitative real-time polymerase chain reaction [qRT-PCR]) of samples of their skin reactions. RESULTS: Eight of the 10 CA subjects reacted positively upon re-test, whereas two did not react. Most of AL-101 positive patch tests expressed an allergy signature with strong activation of gene modules associated with adaptive immunity and downregulation of cornification pathway genes. In addition, the breadth of gene modulation correlated with the magnitude of patch test reactions and the concentration of AL-101 applied. However, we observed that some of the positive patch test reactions to AL-101 expressed no/few allergy biomarkers, suggesting the induction of an irritant skin inflammation in these samples. CONCLUSIONS: This study confirms that AL-101 is an allergen that can cause both contact allergy and contact irritation. Our results also highlight that molecular profiling might help to strengthen clinical diagnosis.

Unique molecular signatures typify skin inflammation induced by chemical allergens and irritants.

BACKGROUND: Skin exposure to chemicals may induce an inflammatory disease known as contact dermatitis (CD). Distinguishing the allergic and irritant forms of CD often proves challenging in the clinic. METHODS: To characterize the molecular signatures of chemical-induced skin inflammation, we conducted a comprehensive transcriptomic analysis on the skin lesions of 47 patients with positive patch tests to reference contact allergens and nonallergenic irritants. RESULTS: A clear segregation was observed between allergen- and irritant-induced gene profiles. Distinct modules pertaining to the epidermal compartment, metabolism, and proliferation were induced by both contact allergens and irritants; whereas only contact allergens prompted strong activation of adaptive immunity, notably of cytotoxic T-cell responses. Our results also confirmed that: (a) unique pathways characterize allergen- and irritant-induced dermatitis; (b) the intensity of the clinical reaction correlates with the magnitude of immune activation. Finally, using a machine-learning approach, we identified and validated several minimal combinations of biomarkers to distinguish contact allergy from irritation. CONCLUSION: These results highlight the value of molecular profiling of chemical-induced skin inflammation for improving the diagnosis of allergic versus irritant contact dermatitis.

Low-Level Light Therapy Downregulates Scalp Inflammatory Biomarkers in Men With Androgenetic Alopecia and Boosts Minoxidil 2% to Bring a Sustainable Hair Regrowth Activity.

BACKGROUND AND OBJECTIVES: Low-level light therapies using visible to infrared light are known to activate several cellular functions, such as adenosine triphosphate and nitric oxide synthesis. However, few clinical observations report its biological consequences for skin and scalp homeostasis. Since scalp inflammation was recognized as a potential physiological obstacle to the efficacy of the reference hair regrowth drug Minoxidil in vivo and since perifollicular inflammation is the hallmark of about 50%-70% follicular units in androgenetic alopecia, we decided to investigate whether the anti-inflammatory activity of LLLT/GentleWaves® device were assigned to L'Oréal by Light BioScience L.L.C., Virginia Beach, VA (US) could enhance hair regrowth activity of Minoxidil. STUDY DESIGN/MATERIALS AND METHODS: We conducted a first experimental clinical study on 64 men with androgenetic alopecia using LLLT/GentleWaves®, 590-nm predominant wavelength 70 seconds, specifically pulsed once per day, for 3 days, and we performed a whole-genome analysis of treated scalp biopsies. In a second clinical study, including 135 alopecic volunteers, we evaluated the hair regrowth activity in response to the upgraded LLLT/GentleWaves® device and Minoxidil. RESULTS: In the first clinical study, whole-genome analysis of treated scalp biopsies showed downregulation of scalp inflammatory biomarkers, such as AP1/FOSB messenger RNA (mRNA) and mir21, together with the disappearance of CD69 mRNA, specific to scalp-infiltrating T cells of about 50% of the studied volunteers prior to the LLLT/GentleWaves® treatment. In the second clinical study, we observed that LLLT/GentleWaves® was able to boost the hair regrowth activity of a Minoxidil 2% lotion to the extent of the highest concentration (5%) in terms of efficacy, number of responders, and perceived performance. CONCLUSIONS: Altogether, these observations suggest the potential benefit of LLLT/GentleWaves® as a noninvasive adjunctive technology for skin and scalp conditions, where a mild perifollicular inflammation is involved. Lasers Surg. Med. 2021. Copyright © 2021 Wiley Periodicals LLC.

Antigen-Induced but Not Innate Memory CD8 T Cells Express NKG2D and Are Recruited to the Lung Parenchyma upon Viral Infection.

The pool of memory-phenotype CD8 T cells is composed of Ag-induced (AI) and cytokine-induced innate (IN) cells. IN cells have been described as having properties similar to those of AI memory cells. However, we found that pathogen-induced AI memory cells can be distinguished in mice from naturally generated IN memory cells by surface expression of NKG2D. Using this marker, we described the increased functionalities of AI and IN memory CD8 T cells compared with naive cells, as shown by comprehensive analysis of cytokine secretion and gene expression. However, AI differed from IN memory CD8 T cells by their capacity to migrate to the lung parenchyma upon inflammation or infection, a process dependent on their expression of ITGA1/CD49a and ITGA4/CD49d integrins.

Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery.

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge.

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge.

Immune signatures of protective spleen memory CD8 T cells.

Memory CD8 T lymphocyte populations are remarkably heterogeneous and differ in their ability to protect the host. In order to identify the whole range of qualities uniquely associated with protective memory cells we compared the gene expression signatures of two qualities of memory CD8 T cells sharing the same antigenic-specificity: protective (Influenza-induced, Flu-TM) and non-protective (peptide-induced, TIM) spleen memory CD8 T cells. Although Flu-TM and TIM express classical phenotypic memory markers and are polyfunctional, only Flu-TM protects against a lethal viral challenge. Protective memory CD8 T cells express a unique set of genes involved in migration and survival that correlate with their unique capacity to rapidly migrate within the infected lung parenchyma in response to influenza infection. We also enlighten a new set of poised genes expressed by protective cells that is strongly enriched in cytokines and chemokines such as Ccl1, Ccl9 and Gm-csf. CCL1 and GM-CSF genes are also poised in human memory CD8 T cells. These immune signatures are also induced by two other pathogens (vaccinia virus and Listeria monocytogenes). The immune signatures associated with immune protection were identified on circulating cells, i.e. those that are easily accessible for immuno-monitoring and could help predict vaccines efficacy.

CD1d-restricted peripheral T cell lymphoma in mice and humans.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.

Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection.

Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2(-/-) NK cells closely resembled that of Tbx21(-/-) NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21(-/-) NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells.

T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.

Trail(+)DX5(-)Eomes(-) natural killer (NK) cells arise in the mouse fetal liver and persist in the adult liver. Their relationships with Trail(-)DX5(+) NK cells remain controversial. We generated a novel Eomes-GFP reporter murine model to address this question. We found that Eomes(-) NK cells are not precursors of classical Eomes(+) NK cells but rather constitute a distinct lineage of innate lymphoid cells. Eomes(-) NK cells are strictly dependent on both T-bet and IL-15, similarly to NKT cells. We observed that, in the liver, expression of T-bet in progenitors represses Eomes expression and the development of Eomes(+) NK cells. Reciprocally, the bone marrow (BM) microenvironment restricts T-bet expression in developing NK cells. Ectopic expression of T-bet forces the development of Eomes(-) NK cells, demonstrating that repression of T-bet is essential for the development of Eomes(+) NK cells. Gene profile analyses show that Eomes(-) NK cells share part of their transcriptional program with NKT cells, including genes involved in liver homing and NK cell receptors. Moreover, Eomes(-) NK cells produce a broad range of cytokines, including IL-2 and TNF in vitro and in vivo, during immune responses against vaccinia virus. Thus, mutually exclusive expression of T-bet and Eomes drives the development of different NK cell lineages with complementary functions.

Retinoic acid receptor subtype-specific transcriptotypes in the early zebrafish embryo.

Retinoic acid (RA) controls many aspects of embryonic development by binding to specific receptors (retinoic acid receptors [RARs]) that regulate complex transcriptional networks. Three different RAR subtypes are present in vertebrates and play both common and specific roles in transducing RA signaling. Specific activities of each receptor subtype can be correlated with its exclusive expression pattern, whereas shared activities between different subtypes are generally assimilated to functional redundancy. However, the question remains whether some subtype-specific activity still exists in regions or organs coexpressing multiple RAR subtypes. We tackled this issue at the transcriptional level using early zebrafish embryo as a model. Using morpholino knockdown, we specifically invalidated the zebrafish endogenous RAR subtypes in an in vivo context. After building up a list of RA-responsive genes in the zebrafish gastrula through a whole-transcriptome analysis, we compared this panel of genes with those that still respond to RA in embryos lacking one or another RAR subtype. Our work reveals that RAR subtypes do not have fully redundant functions at the transcriptional level but can transduce RA signal in a subtype-specific fashion. As a result, we define RAR subtype-specific transcriptotypes that correspond to repertoires of genes activated by different RAR subtypes. Finally, we found genes of the RA pathway (cyp26a1, raraa) the regulation of which by RA is highly robust and can even resist the knockdown of all RARs. This suggests that RA-responsive genes are differentially sensitive to alterations in the RA pathway and, in particular, cyp26a1 and raraa are under a high pressure to maintain signaling integrity.

Bacterial vectors for active immunotherapy reach clinical and industrial stages.

Active immunotherapy based on live attenuated bacterial vectors has matured in terms of industrial development and develops through a combination of three phenomena. First, active immunotherapy that stimulates an antigen-specific cytotoxic T-cell immune response has become a reality after several years of work. Second, there is still a need to identify vectors that can deliver antigens to the cytosol of antigen-presenting cells in vivo. Third, the recent progress in the understanding of bacterial lifestyle and in developing genetic engineering tools has enabled the design of bioengineered bugs that are capable of delivering antigens. Here, we review the mechanisms by which clinical bacterial vectors deliver antigens into the cytosol of antigen-presenting cells and summarize the development strategy of the three identified firms in this field.

Early diet-induced non-alcoholic steatohepatitis in APOE2 knock-in mice and its prevention by fibrates.

BACKGROUND/AIMS: The molecular mechanisms leading to Non-Alcoholic Steatohepatitis (NASH) are not fully understood. In mice, NASH can be inhibited by fenofibrate, a synthetic agonist for the nuclear receptor peroxisome proliferator activated receptor alpha, which regulates hepatic triglyceride metabolism. This study aimed to elucidate the relation between steatosis and inflammation in NASH in a human-like hyperlipidemic mouse model. METHODS: Liver phenotype and gene expression were assessed in APOE2 knock-in mice that were fed a western-type high fat diet with or without co-administration of fenofibrate. RESULTS: In response to a western diet, APOE2 knock-in mice developed NASH characterized by steatosis and inflammation. Strikingly, macrophage accumulation in the liver preceded the steatosis during progression of the disease. This phenotype was in line with gene expression patterns, which showed regulation of two major groups of genes, i.e. inflammatory and lipid genes. Fenofibrate treatment decreased hepatic macrophage accumulation and abolished steatosis. Moreover, a marked reduction in the expression of inflammatory genes occurred immediately after fenofibrate treatment. CONCLUSIONS: These data indicate that inflammation might play an instrumental role during the development of NASH in this mouse model. Inhibition of NASH by fenofibrate may be due, at least in part, to its inhibitory effect on pro-inflammatory genes.

Integrated transcriptome analysis of the cellular mechanisms associated with Ha-ras-dependent malignant transformation of the human breast epithelial MCF7 cell line.

To understand the cellular mechanisms of malignant transformation induced by constitutive activation of the ras oncogene (Ha-ras), we used a subtractive hybridization method (VGID) together with an integrative analytical procedure based upon literature databases in the form of extensive interaction graphs. We found 166 over- and under-expressed genes which, in the human MCF7-ras breast epithelial cell line, are involved in the different aspects of tumoral transformation such as defined signaling pathways, cellular growth, protection against apoptosis, extracellular matrix and cytoskeleton remodeling. Integrative analysis led to the construction of a physiological model defining cross-talk and signaling pathway alterations which explicitly suggested mechanisms directly involved in tumor progression. The model further suggested points and means of intervention which could induce cell death in Ha-ras-transformed cells specifically. These hypotheses were directly tested in vitro and found to be largely correct, hence indicating that these new analytical and technological approaches allow the discovery of pathology-associated cellular mechanisms and physiologically defined targets leading to phenotype-specific pharmacological interventions.