B‐cell capacity for differentiation changes with age

BackgroundAge‐related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin.AimsTo compare the intrinsic B‐cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T‐cell help or activation via TLR engagement.MethodsB cells were isolated from healthy individuals, in younger (30–38 years) and older (60–64 years) donors. An in vitro model system of B‐cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T‐dependent (TD: CD40/BCR stimulation) or T‐independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR.ResultsTI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B‐cell‐executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age‐related differences in B‐cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient.ConclusionAltogether, B‐cell differentiation into PC appeared similar between age groups when provided with T‐cell help, in contrast to TI differentiation, where multiple age‐related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

Systemic Down-Regulation of Delta-9 Desaturase Promotes Muscle Oxidative Metabolism and Accelerates Muscle Function Recovery following Nerve Injury

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles.     

Short-Chain Fructo-Oligosaccharides Modulate Intestinal Microbiota and Metabolic Parameters of Humanized Gnotobiotic Diet Induced Obesity Mice

Prebiotic fibres like short-chain fructo-oligosaccharides (scFOS) are known to selectively modulate the composition of the intestinal microbiota and especially to stimulate Bifidobacteria. In parallel, the involvement of intestinal microbiota in host metabolic regulation has been recently highlighted. The objective of the study was to evaluate the effect of scFOS on the composition of the faecal microbiota and on metabolic parameters in an animal model of diet-induced obesity harbouring a human-type microbiota. Forty eight axenic C57BL/6J mice were inoculated with a sample of faecal human microbiota and randomly assigned to one of 3 diets for 7 weeks: a control diet, a high fat diet (HF, 60% of energy derived from fat)) or an isocaloric HF diet containing 10% of scFOS (HF-scFOS). Mice fed with the two HF gained at least 21% more weight than mice from the control group. Addition of scFOS partially abolished the deposition of fat mass but significantly increased the weight of the caecum. The analysis of the taxonomic composition of the faecal microbiota by FISH technique revealed that the addition of scFOS induced a significant increase of faecal Bifidobacteria and the Clostridium coccoides group whereas it decreased the Clostridium leptum group. In addition to modifying the composition of the faecal microbiota, scFOS most prominently affected the faecal metabolome (e.g. bile acids derivatives, hydroxyl monoenoic fatty acids) as well as urine, plasma hydrophilic and plasma lipid metabolomes. The increase in C. coccoides and the decrease in C. leptum, were highly correlated to these metabolic changes, including insulinaemia, as well as to the weight of the caecum (empty and full) but not the increase in Bifidobacteria. In conclusion scFOS induce profound metabolic changes by modulating the composition and the activity of the intestinal microbiota, that may partly explain their effect on the reduction of insulinaemia.     

An Evaluation of the Impact of PD‐1 Pathway Blockade on Reproductive Safety of Therapeutic PD‐1 Inhibitors

This report discusses the principles of reproductive toxicity risk assessment for biopharmaceuticals blocking the PD‐1/programmed cell death ligand 1 (PD‐L1) pathway, which have been developed for the treatment of patients with advanced malignancies. The PD‐1/PD‐L1 pathway is a T‐cell co‐inhibitory pathway that normally maintains immune tolerance to self. Its role in pregnancy is to maintain immune tolerance to the fetal allograft. In cancer patients, this signaling pathway is hijacked by some neoplasms to avoid immune destruction. PD‐1/PD‐L1‐blocking agents enhance functional activity of the target lymphocytes to eventually cause immune rejection of the tumor. A therapeutic blockade of PD‐1/PD‐L1 pathway that occurs at full target engagement provides a unique challenge to address the risk to pregnancy because disruption of the same pathway may also reduce or abrogate maternal immune tolerance to the fetal alloantigens inherited through the father. Typically, nonclinical reproductive and developmental toxicity (DART) studies in animals (rats and rabbits) with clinical drug candidates are conducted to identify potential risk in humans and to determine exposure margin for the effects on reproduction as part of the risk assessment. However, for biopharmaceuticals for which the desired mechanism of action cannot be separated from potential deleterious effects to the fetus and when the only relevant toxicology species is nonhuman primate (NHP), the risk to reproduction can be predicted by a mechanism‐based assessment using data generated from murine surrogate models as supportive information without conducting DART in NHPs. Such an approach has been used in the evaluation of pregnancy risk of anti‐PD‐1 agent, pembrolizumab, and has been demonstrated as an important alternative to performing DART studies in NHPs     

A brain-specific isoform of small glutamine-rich tetratricopeptide repeat-containing protein binds to Hsc70 and the cysteine string protein

Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is a ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70, a site used by several tetratricopeptide repeat-containing binding partners to recruit Hsc70 into complexes of diverse function. We describe here an isoform of SGT with 60% amino acid sequence identity that we name betaSGT. In contrast to the previously published alphaSGT, betaSGT is almost exclusively expressed in brain. Both isoforms of SGT possess similar binding properties toward Hsc70 and cysteine string protein, a synaptic vesicle-associated J-domain-containing protein. In addition, SGTs oligomerize without preferences among isoforms. The distribution of protein binding motifs on SGTs reveals a modular structure. The N-terminal domains mediate oligomerization. Binding to Hsc70 is impaired by mutations of basic residues within the central tetratricopeptide repeat domain of betaSGT, indicating a two-carboxylate clamp as the binding mode. The tetratricopeptide repeats are also necessary for binding to the cysteine string protein. However, this binding mode is distinct from the two-carboxylate clamp that is involved in Hsc70 binding. The C-terminal regions of SGTs might constitute independent protein interaction domains. We conclude that betaSGT is likely to cooperate with alphaSGT as co-chaperone of Hsc70 in the brain. The modular structure of SGTs allows them to recruit client proteins to Hsc70 and to direct the resulting complex toward downstream proteins that take over the respective client proteins.     

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.     

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.