Diversification and historical demography of Rhampholeon spectrum in West-Central Africa.

Pygmy Chameleons of the genus Rhampholeon represent a moderately diverse, geographically circumscribed radiation, with most species (18 out of 19 extant taxa) limited to East Africa. The one exception is Rhampholeon spectrum, a species restricted to West-Central African rainforests. We set out to characterize the geographic basis of genetic variation in this disjunctly distributed Rhampholeon species using a combination of multilocus Sanger data and genomic sequences to explore population structure and range-wide phylogeographic patterns. We also employed demographic analyses and niche modeling to distinguish between alternate explanations to contextualize the impact of past geological and climatic events on the present-day distribution of intraspecific genetic variation. Phylogenetic analyses suggest that R. spectrum is a complex of five geographically delimited populations grouped into two major clades (montane vs. lowland). We found pronounced population structure suggesting that divergence and, potentially, speciation began between the late Miocene and the Pleistocene. Sea level changes during the Pleistocene climatic oscillations resulted in allopatric divergence associated with dispersal over an ocean channel barrier and colonization of Bioko Island. Demographic inferences and range stability mapping each support diversification models with secondary contact due to population contraction in lowland and montane refugia during the interglacial period. Allopatric divergence, congruent with isolation caused by geologic uplift of the East African rift system, the "descent into the Icehouse," and aridification of sub-Saharan Africa during the Eocene-Oligocene are identified as the key events explaining the population divergence between R. spectrum and its closely related sister clade from the Eastern Arc Mountains. Our results unveil cryptic genetic diversity in R. spectrum, suggesting the possibility of a species complex distributed across the Lower Guinean Forest and the Island of Bioko. We highlight the major element of species diversification that modelled today's diversity and distributions in most West-Central African vertebrates.

Systematics of the Central African Spiny Reed Frog Afrixalus laevis (Anura: Hyperoliidae), with the description of two new species from the Albertine Rift.

The geographically widespread species Afrixalus laevis (Anura: Hyperoliidae) currently has a disjunct distribution in western Central Africa (Cameroon, Equatorial Guinea, Gabon, and possibly adjacent countries) and the area in and near the Albertine Rift in eastern Democratic Republic of the Congo and neighboring countries. At least two herpetologists have previously suggested that these disjunct populations represent distinct species, and herein, we utilize an integrative taxonomic approach with molecular and morphological data to reconcile the taxonomy of these spiny reed frogs. We sequenced 1554 base pairs of the 16S and RAG1 genes from 34 samples of A. laevis and one sample of A. orophilus (sympatric with eastern populations of A. laevis), and combined these data with previously sequenced GenBank Afrixalus samples via the bioinformatics toolkit SuperCRUNCH. Phylogenetic trees, dated phylogenetic analyses, and species-delimitation analyses were generated with RAxML, BEAST, and BPP, respectively. Eleven mensural characters were taken from multiple specimens of A. laevis and A. orophilus, and compared with paired t-tests and analyses of covariance. These combined results suggested populations of A. laevis in western Central Africa (Cameroon and Bioko Island, Equatorial Guinea) represent one species, whereas populations from the Albertine Rift and nearby forests represent two undescribed taxa that are sister to A. dorsimaculatus. The two new species (A. lacustris sp. nov. and A. phantasma sp. nov.) are distinguished by our phylogenetic and species-delimitation analyses, significant differences in several mensural characters, qualitative morphological differences, and by their non-overlapping elevational distribution.

Influence of HLA-C environment on the spontaneous clearance of hepatitis C in European HIV-HCV co-infected individuals.

Natural killer (NK) cell functions are regulated by diverse inhibitory and activating receptors, including killer cell immunoglobulin-like receptors (KIR), which interact with human leukocyte antigen (HLA) class I molecules. Some KIR/HLA genetic combinations were reported associated with spontaneous clearance (SC) of hepatitis C virus (HCV) but with discordant results, possibly reflecting KIR and/or HLA gene polymorphism according to populations. KIR/HLA genetic combinations associated with both an exhaustive NK and T cell repertoire were investigated in a cohort of HIV-HCV co-infected individuals with either SC (n = 68) or chronic infection (CI, n = 163) compared to uninfected blood donors [controls (Ctrl), n = 100]. Multivariate analysis showed that the HLA C2C2 environment was associated with SC only in European HIV-HCV co-infected individuals [odds ratio (OR) = 4·30, 95% confidence interval = 1·57-12·25, P = 0·005]. KIR2D+ NK cell repertoire and potential of degranulation of KIR2DL1/S1+ NK cells were similar in the SC European cohort compared to uninfected individuals. In contrast, decreased frequencies of KIR2DS1+ and KIR2DL2+ NK cells were detected in the CI group of Europeans compared to SC and a decreased frequency of KIR2DL1/S1+ NK cells compared to controls. Regarding T cells, higher frequencies of DNAX accessory molecule-1 (DNAM-1)+ and CD57+ T cells were observed in SC in comparison to controls. Interestingly, SC subjects emphasized increased frequencies of KIR2DL2/L3/S2+ T cells compared to CI subjects. Our study underlines that the C2 environment may activate efficient KIR2DL1+ NK cells in a viral context and maintain a KIR2DL2/L3/S2+ mature T cell response in the absence of KIR2DL2 engagement with its cognate ligands in SC group of HCV-HIV co-infected European patients.

10th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals.

Therapeutic proteins and emerging gene and cell-based therapies are attractive therapeutic tools for addressing unmet medical needs or when earlier conventional treatment approaches failed. However, the development of an immune response directed against therapeutic agents is a significant concern as it occurs in a substantial number of cases across products and indications. The specific anti-drug antibodies that develop can lead to safety adverse events as well as inhibition of drug activity or accelerated clearance, both phenomena resulting in loss of treatment efficacy. The European Immunogenicity Platform (EIP) is a meeting place for experts and newcomers to the immunogenicity field, designed to stimulate discussion amongst scientists across industry and academia, encourage interactions with regulatory agencies and share knowledge and the state-of-the-art of immunogenicity sciences with the broader scientific community. Here we report on the main topics covered during the EIP 10th Open Symposium on Immunogenicity of Biopharmaceuticals held in Lisbon, 26-27 February 2019, and the 1-d training course on practical and regulatory aspects of immunogenicity held ahead of the conference. These main topics included immunogenicity testing, clinical relevance of immunogenicity, immunogenicity prediction, regulatory aspects, tolerance induction as a mean to mitigate immunogenicity and immunogenicity in the context of gene therapy.

TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

Health-related quality of life in patients with melanoma expressed as utilities and disability weights.

BACKGROUND: Few studies about health-related quality of life (HRQoL) in patients with melanoma have expressed their results in terms of utilities or disability weights (DWs). Utilities are required for calculating quality-adjusted life years and therefore for cost-effectiveness analyses. DWs are useful to assess the burden of diseases through disability-adjusted life years. OBJECTIVES: To provide utilities and DWs regarding patients with melanoma. METHODS: The patients were classified into eight groups using four stages based on the 2009 American Joint Committee on Cancer stages, with each stage subdivided into treatment and remission phases. The EuroQoL Five Dimensions Five Levels (EQ-5D-5L) questionnaire was completed by the patients with melanoma to provide a mean utility for each group. In addition to this, the EuroQoL visual analogue scale (VAS) and a validated quality-of-life questionnaire dedicated to patients with melanoma [Functional Assessment of Cancer Therapy Melanoma (FACT-M)] were completed by the same patients in order to compare their results with the obtained utilities. DWs were obtained by calculating, for each patient, the difference between his/her utility and the corresponding sex- and age-specific population norm. RESULTS: A total of 395 questionnaire sets were completed. Utilities and DWs showed significant differences between follow-up groups. Treatment groups had similar utilities and DWs but these results were obtained during different treatment durations and therefore have different weights. The VAS and the FACT-M were found to be less sensitive. Nevertheless, the FACT-M identified some problems not found by the EQ-5D-5L questionnaire. CONCLUSIONS: The EQ-5D-5L questionnaire seems adequate to provide utilities and DWs in patients with melanoma. Lower HRQoL in female patients with melanoma is probably linked to lower HRQoL in the general population.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo.

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-)IL-2Rgamma(c)(-/-) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model.

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

CD8+ T lymphocytes in double alpha beta TCR transgenic mice. I. TCR expression and thymus selection in the absence or in the presence of self-antigen.

We derived Rag2-deficient mice bearing two rearranged alphabeta TCR transgenes, one specific for the HY male Ag and the second specific for the gp33-41 peptide of lymphocytic choriomeningitis virus, both restricted to the MHC H-2D(b) class I molecule. We found that, in female double transgenic (DTg) mice, most CD8 T cells express only the TCRbeta chain from the aHY transgene. By comparing the mRNA species for both beta-chains, we observed that in T cells from DTg mice the aHY TCRbeta chain transcripts are abundant, whereas the anti-lymphocytic choriomeningitis virus TCRbeta chain transcripts are rare. In contrast to TCRbeta chain expression, most of the T cells from DTg mice express two TCRalpha chains. We examined the thymus selection of the dual-receptor CD8 T cells in the presence of self-Ag. We found that the presence of a second TCRalpha chain allows a significant number of CD8 T cells expressing a self-reactive receptor to escape central deletion and migrate to the peripheral pools of male mice. Differences in TCR and coreceptor expression between female and male MoaHY and DTg mice suggest that peripheral T cell survival requires an optimal level of signaling, which implies a process of "adaptation" of lymphocyte populations to the host environment.

CD8+ T lymphocytes in double alpha beta TCR transgenic mice. II. Competitive fitness of dual alpha beta TCR CD8+ T lymphocytes in the peripheral pools.

We studied Rag2-deficient mice bearing two rearranged alphabeta TCR transgenes, both restricted to the MHC H-2D(b) class I molecule. We have previously shown that, in these DTg mice, most peripheral CD8 T cells express one TCRbeta chain associated with two TCRalpha chains, as in one-third of the mature T cells from normal mice. We examined the functional behavior of the dual-receptor CD8 T cells developing either in the absence or in the presence of self-Ag. The dual-receptor CD8 T cells, which develop in absence of self-Ag, show efficient responses to immunization and remain sensitive to induction of peripheral tolerance. In contrast to single TCR T cells, the dual-TCR cells, when tolerized upon exposure to high levels of self-Ag, are not deleted and therefore may exert important regulatory functions. When developing in the presence of self-Ag, the dual-receptor-expressing CD8 T cells escape central deletion, but are not fully competent to respond to cognate stimuli. Overall, we found that the dual-TCR CD8 T cells show a poor competitive value and can be out-competed by single-TCR cells, both in the course of immune responses and in reconstitution experiments. The decreased fitness of the dual-receptor cells may contribute to diminishing the autoimmune hazard that they could represent.

Substrate interactions in cytochrome P-450: correlation between carbon-13 nuclear magnetic resonance chemical shifts and C-O vibrational frequencies.

13CO NMR chemical shifts and 12CO infrared stretching frequencies have been measured for cytochrome P-450cam-CO in the presence of D-camphor and of various camphor analogues. A linear correlation between both parameters delta (13C) and v(CO) was found indicating that the steric and electrostatic interactions acting on the CO ligand are influenced by the substrate. It has been proved that P-450 complexes are on another line in this correlation than hemoglobins which is explained by the different proximal ligand.

Induction of long-term but reversible unresponsiveness after activation of murine T cell hybridomas.

T cell receptor (TCR)-mediated stimulation of murine T cell hybridomas induces cell activation and lymphokine production. We have observed that following productive activation, T cell hybridomas become refractory to a subsequent stimulation. Hyporesponsiveness is long-lasting but reversible, and is not due to reduced receptor expression. It is noteworthy that hyporesponsiveness is cell autonomous, persists in spite of cell division and does not require continuous exposure to ligands. Hyporesponsive cells retain the ability to produce lymphokines in response to pharmacological agents bypassing receptor triggering, indicating that they possess a functional enzymatic machinery for interleukin 2 synthesis and secretion. Reduced phosphatidylinositol hydrolysis was observed in these cells in response to TCR stimulation, suggesting that hyporesponsiveness result from defective transduction of activation signals. The development of unresponsiveness following receptor stimulation resembles desensitization, and may thus play an important role in the regulation of an immune response and in the induction of immune tolerance.

[X chromosome fragility and effects of trimethoprim]. / Fragilité du chromosome X et effets de la triméthoprime.

Severe clinical regression was observed in a patient carrier of a fragile X after treatment trimethoprime. This prompted us to examine the effect of this antibiotic in lymphocyte cultures: a dose ranging from 13 mg/l to 53 mg/l increases considerably the frequency of the Xq27 gap in four fragile-X patients; a dose of 13 mg/l allows a normal growth, without appearance of the Xq27 gap, in 19 normal, non-carrier subjects; a dose of 82 mg/l totally inhibits cell division in 10 normal, non carrier subjects. The reversibility of the blockade was demonstrated, either by washing out the trimethoprime before the 50th hour of incubation or by adding 5-formyl-tetrahydrofolate (0.125 mg/l). It is concluded that one of the steps of monocarbon metabolism is inhibited by trimethoprime. This antibiotic, which must be avoided when treating patients carrier of the fragile X can be utilized in vitro for cytogenetic investigations.