Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro

Different subsets of dorsal root ganglion (DRG) mechanoreceptors transduce low- and high-intensity mechanical stimuli. It was shown recently that, in vivo, neurotrophin-4 (NT-4)-dependent D-hair mechanoreceptors specifically express a voltage-activated T-type calcium channel (Ca(v)3.2) that may be required for their mechanoreceptive function. Here we show that D-hair mechanoreceptors can be identified in vitro by a rosette-like morphology in the presence of NT-4 and that these rosette neurons are almost all absent in DRG cultures taken from NT-4 knock-out mice. In vitro identification of the D-hair mechanoreceptor allowed us to explore the electrophysiological properties of these cells. We demonstrate that the T-type Ca(v)3.2 channel induced slow membrane depolarization that contributes to lower the voltage threshold for action potential generation and controls spike latency after stimulation of D-hair mechanoreceptors. Indeed, the properties of the T-type amplifier are particularly well suited to explain the high sensitivity of D-hair mechanoreceptors to slowly moving stimuli

Switching from a protease inhibitor-based regimen to a dolutegravir-based regimen : a randomized clinical trial to determine the effect on peripheral blood and ileum biopsies from antiretroviral therapy-suppressed human immunodeficiency virus-infected individuals

Background: Optimization of combination antiretroviral therapy (cART) can impact the human immunodeficiency virus (HIV) reservoir. We evaluated the effect on the HIV reservoir in peripheral blood and ileum biopsies in patients switching from boosted protease inhibitor (PI/r)-based therapy to dolutegravir (DTG)-based therapy. Methods: Impact of Integrase-inhibitor DOlutegravir On the viral Reservoir (INDOOR) is a phase 4 open-label clinical trial that randomly included 42 HIV type 1-infected individuals on effective cART: 20 who switched from PI/r-based to DTG-based cART (switch group), and 22 who remained in PI/r-based regimens (control group). We analyzed blood and ileum biopsies to quantify episomal, total, and integrated HIV DNA, cell-associated HIV RNA, residual plasma viremia, T-cell subsets, cell activation, and inflammation markers. Results: There were no related adverse events or treatment discontinuations due to drug intolerance. The HIV reservoir was consistently larger in ileal than in peripheral CD4(+) T cells in both groups (P <.01). Residual viremia in plasma decreased in the switch group (P =.03). However, we did not observe significant longitudinal changes in low-level viral replication, total and integrated HIV reservoir, HIV transcription, T-cell maturation subsets, immunoactivation markers, inflammatory soluble proteins, or cellular markers of latently infected cells. Conclusions: The INDOOR study is the first evaluation of changes in HIV reservoir size in ileum biopsies and in peripheral blood in individuals switched from PI/r- to DTG-based cART. Although this switch was safe and well tolerated, it had no impact on a large array of immunological and inflammatory markers or on HIV reservoir markers in peripheral or in ileal CD4(+) T cells

Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue

Background: The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay). Methods: Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR. Results: vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs. Conclusion: We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy

Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors

Hypertension, diabetes and obesity are cardiovascular risk factors closely associated to the development of renal and cardiovascular target organ damage. VAV2 and VAV3, members of the VAV family proto-oncogenes, are guanosine nucleotide exchange factors for the Rho and Rac GTPase family, which is related with cardiovascular homeostasis. We have analyzed the relationship between the presence of VAV2 rs602990 and VAV3 rs7528153 polymorphisms with cardiovascular risk factors and target organ damage (heart, vessels and kidney) in 411 subjects. Our results show that being carrier of the T allele in VAV2 rs602990 polymorphism is associated with an increased risk of obesity, reduced levels of ankle-brachial index and diastolic blood pressure and reduced retinal artery caliber. In addition, being carrier of T allele is associated with increased risk of target organ damage in males. On the other hand, being carrier of the T allele in VAV3 rs7528153 polymorphism is associated with a decreased susceptibility of developing a pathologic state composed by the presence of hypertension, diabetes, obesity or cardiovascular damage, and with an increased risk of developing altered basal glycaemia. This is the first report showing an association between VAV2 and VAV3 polymorphisms with cardiovascular risk factors and target organ damage

Long-Term Habitat Degradation Drives Neotropical Macrophyte Species Loss While Assisting the Spread of Invasive Plant Species

The spread of invasive macrophyte species is a pressing threat to neotropical shallow lakes. Yet there are few studies addressing the full extent of biotic and abiotic changes that may occur in response to invasive species. Less is known of how other human-induced stressors such as eutrophication and lake draining may interact over time with invasive macrophytes to influence biodiversity. We combined limnological observations with paleoecological data from Fúquene Lake, Colombia, a eutrophic neotropical shallow lake, to provide information on the current and long-term (decades-centuries) dynamics of the spread of two well-established invasive plants Eichhornia crassipes and Egeria densa. We found a unique in macrophyte species composition in areas currently dominated by Egeria and Eichhornia. Eichhornia-dominated areas had 14 macrophyte species, turbid (secchi=19 ± 6 cm) and poorly oxygenated (3.94 ± 2.61 ppm) waters whereas Egeria-rich areas supported 5 species and had clearer (secchi=51 ± 12 cm) and better-oxygenated (6.06 ± 2.4 ppm) waters. Historical macrophyte community shifts were linked to eutrophication and lake level variation and characterized by the loss of charophytes and bryophytes before 1500 CE and subsequent reductions in Nymphaea sp., Potamogeton illinoensis and Najas guadalupensis in the early 1900s (lake draining). Eichhornia crassipes (since 1500 CE) and E. densa (early 1900s) occurred well before proposed dates of introduction (1950s and 1990 respectively). Both species have rapidly expanded since the 1990s along with Azolla filiculoides in response to an inflow water diversion scheme and heavy nutrient loads. Our results suggest that the spread of Eichhornia and Egeria was not responsible for native macrophyte species loss, but that their current dominance is exerting synergistic and antagonistic secondary effects on plant assemblages through habitat modification, competitive exclusion and promotion of habitat heterogeneity across the lake. It could therefore be misleading to suggest that invasive plants causes macrophyte species loss in degraded lakes. We suggest that aggressive species like Eichhornia, Azolla, and Egeria require hydrologically stable and eutrophic environments to spread; thus, management actions should focus on controlling these two factors. Our study demonstrates the need to use a long-term approach to fully-understand the effects of invasive macrophytes

Association of Salivary Human Papillomavirus Infection and Oral and Oropharyngeal Cancer: A Meta-Analysis

BACKGROUND: Human papillomavirus (HPV) infection has been recognized as an important risk factor in cancer. The purpose of this systematic review and meta-analysis was to determine the prevalence and effect size of association between salivary HPV DNA and the risk of developing oral and oropharyngeal cancer. METHODS: A systematic literature search of PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library was performed, without language restrictions or specified start date. Pooled data were analyzed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). Quality assessment was performed using the Newcastle-Ottawa Scale (NOS). RESULTS: A total of 1672 studies were screened and 14 met inclusion criteria for the meta-analysis. The overall prevalence of salivary HPV DNA for oral and oropharyngeal carcinoma was 43.2%, and the prevalence of salivary HPV16 genotype was 27.5%. Pooled results showed a significant association between salivary HPV and oral and oropharyngeal cancer (OR = 4.94; 2.82-8.67), oral cancer (OR = 2.58; 1.67-3.99) and oropharyngeal cancer (OR = 17.71; 6.42-48.84). Significant associations were also found between salivary HPV16 and oral and oropharyngeal cancer (OR = 10.07; 3.65-27.82), oral cancer (OR = 2.95; 1.23-7.08) and oropharyngeal cancer (OR = 38.50; 22.43-66.07). CONCLUSIONS: Our meta-analysis demonstrated the association between salivary HPV infection and the incidence of oral and oropharyngeal cancer indicating its value as a predictive indicator

Measurement of the (90,91,92,93,94,96)Zr(n,gamma) and (139)La(n,gamma) cross sections at n_TOF

Open AccessNeutron capture cross sections of Zr and La isotopes have important implications in the field of nuclear astrophysics as well as in the nuclear technology. In particular the Zr isotopes play a key role for the determination of the neutron density in the He burning zone of the Red Giant star, while the (139)La is important to monitor the s-process abundances from Ba up to Ph. Zr is also largely used as structural materials of traditional and advanced nuclear reactors. The nuclear resonance parameters and the cross section of (90,91,92,93,94,96)Zr and (139)La have been measured at the n_TOF facility at CERN. Based on these data the capture resonance strength and the Maxwellian-averaged cross section were calculated

Intraoperative radiotherapy electron boost followed by moderate doses of external beam radiotherapy in resected soft-tissue sarcoma of the extremities

To analyze the patterns of failure and the toxicity profile of intraoperative electron beam radiotherapy (IOERT) after resection of soft tissue sarcomas of the extremities (STS). PATIENTS AND METHODS: Forty-five patients with extremity STS were treated with IOERT and moderate-dose postoperative radiotherapy (45-50 Gy). Twenty-six patients were treated for primary disease (PD) and 19 patients for an isolated recurrence (ILR). Tumor size was >5 cm (maximum diameter) in 36 patients (80%), and high-grade histology in PD patients was present in 14 patients (54%). In nine patients, IOERT was used alone, due to previous irradiation or patient refusal. Chemotherapy (neoadjuvant and/or adjuvant) was mainly given to high-grade tumors. RESULTS: Nine patients relapsed in the extremity (20%), and 12 patients in distant sites (28%). Actuarial local control at 5 years was 88% for patients with negative/close margins and 57% for patients presenting positive margins (P=0.04). Five patients (11%) developed neuropathy associated with the treatment. Extremity preservation was achieved in 40 patients (88%). With a median follow-up of 93 months (range: 27-143 months) for the patients at risk, 25 patients remain alive (a 7-year actuarial survival rate of 75% for PD and 47% for ILR; P=0.01). CONCLUSIONS: IOERT combined with moderate doses of external beam irradiation yields high local control and extremity preservation rates in resected extremity STS. Peripheral nerves in the IOERT field are dose-limiting structures requiring a dose compromise in the IOERT component to avoid severe neurological damage

Evidence for HIV-1 cure after CCR5Δ32/Δ32 allogeneic haemopoietic stem-cell transplantation 30 months post analytical treatment interruption: a case report

BACKGROUND: The London patient (participant 36 in the IciStem cohort) underwent allogeneic stem-cell transplantation with cells that did not express CCR5 (CCR5Δ32/Δ32); remission was reported at 18 months after analytical treatment interruption (ATI). Here, we present longer term data for this patient (up to 30 months after ATI), including sampling from diverse HIV-1 reservoir sites. METHODS: We used ultrasensitive viral load assays of plasma, semen, and cerebrospinal fluid (CSF) samples to detect HIV-1 RNA. In gut biopsy samples and lymph-node tissue, cell-copy number and total HIV-1 DNA levels were quantified in multiple replicates, using droplet digital PCR (ddPCR) and quantitative real-time PCR. We also analysed the presence of intact proviral DNA using multiplex ddPCR targeting the packaging signal (ψ) and envelope (env). We did intracellular cytokine staining to measure HIV-1-specific T-cell responses. We used low-sensitive and low-avidity antibody assays to measure the humoral response to HIV-1. We predicted the probability of rebound using a mathematical model and inference approach. FINDINGS: HIV-1 viral load in plasma remained undetectable in the London patient up to 30 months (last tested on March 4, 2020), using an assay with a detection limit of 1 copy per mL. The patient's CD4 count was 430 cells per μL (23·5% of total T cells) at 28 months. A very low-level positive signal for HIV-1 DNA was recorded in peripheral CD4 memory cells at 28 months. The viral load in semen was undetectable in both plasma (lower limit of detection [LLD] <12 copies per mL) and cells (LLD 10 copies per 106 cells) at 21 months. CSF was within normal parameters at 25 months, with HIV-1 RNA below the detection limit (LLD 1 copy per mL). HIV-1 DNA by ddPCR was negative in rectum, caecum, and sigmoid colon and terminal ileum tissue samples at 22 months. Lymph-node tissue from axilla was positive for the long-terminal repeat (33 copies per 106 cells) and env (26·1 copies per 106 cells), negative for ψ and integrase, and negative by the intact proviral DNA assay, at 27 months. HIV-1-specific CD4 and CD8 T-cell responses have remained absent at 27 months. Low-avidity Env antibodies have continued to decline. Mathematical modelling suggests that the probability of remission for life (cure) is 98% in the context of 80% donor chimerism in total HIV target cells and greater than 99% probability of remission for life with 90% donor chimerism. INTERPRETATION: The London patient has been in HIV-1 remission for 30 months with no detectable replication-competent virus in blood, CSF, intestinal tissue, or lymphoid tissue. Donor chimerism has been maintained at 99% in peripheral T cells. We propose that these findings represent HIV-1 cure. FUNDING: Wellcome Trust and amfAR (American Foundation for AIDS Research)