Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor alpha (EF1 alpha)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-alpha) and interferon (IFN)-gamma after efficient destruction of CD19(+) lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19(+) pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AWLVs for the generation of improved CAR-T products

Marginal Level Dystrophin Expression Improves Clinical Outcome in a Strain of Dystrophin/Utrophin Double Knockout Mice

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies

Urinary exosome miR-146a is a potential marker of albuminuria in essential hypertension

BACKGROUND: There is increasing interest in using extracellular vesicle-derived microRNAs (miRNAs) as biomarkers in renal dysfunction and injury. Preliminary evidence indicates that miRNAs regulate the progression of glomerular disease. Indeed, exosomes from the renal system have provided novel evidence in the clinical setting of albuminuria. Thus, the aim of this study was to quantify the urinary miRNAs present in exosome and microvesicles (MVs), and to assess their association with the presence of increased urinary albumin excretion in essential hypertension. METHODS: Exosomes were collected from urine specimens from a cohort of hypertensive patients with (n = 24) or without albuminuria (n = 28), and from 20 healthy volunteers as a control group. Urinary exosomes were phenotyped by Western blot, tunable resistive pulse sensing, and electronic microscopy. Expression of miR-146a and miR-335* was analysed by qRT-PCR and any associations between albuminuria and exosomal miRNAs were analysed. RESULTS: Urinary miRNAs are highly enriched in exosome subpopulations compared to MVs, both in patients with or without increased albuminuria (p < 0.001), but not in the control group. High albuminuria was associated with 2.5-fold less miR-146a in exosomes (p = 0.017), whereas miR-146a levels in MV did not change. In addition, exosome miR-146a levels were inversely associated with albuminuria (r = 0.65, p < 0.0001), and discriminated the presence of urinary albumin excretion presence [area under the curve = 0.80, 95% confidence interval: 0.66-0.95; p = 0.0013]. CONCLUSIONS: Our results indicate that miRNAs were enriched in the urinary exosome subpopulation in hypertensive patients and that low miR-146a expression in exosomes was associated with the presence of albuminuria. Thus, urinary exosome miR-146a may be a potentially useful tool for studying early renal injury in hypertension

Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat 3 wild emmer wheat RIL population

Mineral nutrient malnutrition, and particularly deficiency in zinc and iron, afflicts over 3 billion people worldwide. Wild emmer wheat, Triticum turgidum ssp. dicoccoides, genepool harbors a rich allelic repertoire for mineral nutrients in the grain. The genetic and physiological basis of grain protein, micronutrients (zinc, iron, copper and manganese) and macronutrients (calcium, magnesium, potassium, phosphorus and sulfur) concentration was studied in tetraploid wheat population of 152 recombinant inbred lines (RILs), derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Wide genetic variation was found among the RILs for all grain minerals, with considerable transgressive effect. A total of 82 QTLs were mapped for 10 minerals with LOD score range of 3.2–16.7. Most QTLs were in favor of the wild allele (50 QTLs). Fourteen pairs of QTLs for the same trait were mapped to seemingly homoeologous positions, reflecting synteny between the A and B genomes. Significant positive correlation was found between grain protein concentration (GPC), Zn, Fe and Cu, which was supported by significant overlap between the respective QTLs, suggesting common physiological and/or genetic factors controlling the concentrations of these mineral nutrients. Few genomic regions (chromosomes 2A, 5A, 6B and 7A) were found to harbor clusters of QTLs for GPC and other nutrients. These identified QTLs may facilitate the use of wild alleles for improving grain nutritional quality of elite wheat cultivars, especially in terms of protein, Zn and Fe

Abacavir, efavirenz, didanosine, with or without hydroxyurea, in HIV-infected adults failing initial nucleoside/protease inhibitor-containing regimens

BACKGROUND: Hydroxyurea (HU) is an immunomodulatory agent that has been documented to enhance the antiretroviral activity of nucleoside reverse transcriptase inhibitors, such as abacavir (ABC) and didanosine (ddI), and would be expected to improve virologic efficacy. METHODS: A 48-week, phase IV, multicenter, open-label, proof-of-concept clinical trial was conducted to evaluate second-line, protease inhibitor (PI)-sparing therapy with ABC/efavirenz (EFV)/ddI plus HU or without HU in HIV-infected subjects failing to achieve HIV-1 RNA ≤ 400 copies/mL after ≥ 16 weeks of treatment with lamivudine/zidovudine or lamivudine/stavudine, plus 1 or 2 PIs. Subjects were assigned to ABC (300 mg twice daily)/ EFV (600 mg once daily)/ ddI (400 mg once daily) plus HU (500 mg twice daily) (n = 30) or this regimen without HU (n = 24). RESULTS: Baseline mean HIV-1 RNA was 3.86 log(10 )copies/mL and CD4+ cell count was 345 cells/mm(3). A similar percentage of subjects in the non-HU arm (58%) and HU arm (53%) completed the study. Intent-to-treat: missing = failure analysis showed no differences in proportions of subjects in the non-HU and HU arms achieving undetectable plasma HIV-1 RNA levels at week 24 (<400 copies/mL: 58% [14/24] vs 57% [17/30], P = 0.899; <50 copies/mL (50% [12/24] vs 47% [14/30], P = 0.780). Median change from baseline in CD4+ cell count in the non-HU and HU arms at week 48 was +114 cells/mm(3 )and -63 cells/mm(3 )(P = 0.007), respectively. Both regimens were generally well tolerated, although more subjects in the HU arm withdrew prematurely from the study due to adverse events (23% vs 4%). Four cases of possible ABC-related hypersensitivity were observed. CONCLUSION: ABC/EFV/ddI was an effective and well-tolerated second-line regimen for nucleoside/PI-experienced HIV-infected subjects. The addition of HU blunted the CD4+ cell response, did not appear to enhance antiviral activity, and resulted in more treatment-limiting adverse events

Alternative Oxidase Mediates Pathogen Resistance in Paracoccidioides brasiliensis Infection

Thermally dimorphic pathogenic fungi are responsible for potentially life-threatening diseases of immunocompetent and immunocompromised individuals. These microorganisms grow as conidia-producing mycelia in the environment, which when inhaled by the host convert to the pathogenic yeast form at 37°C. During adaptation and growth, fungi interact with host immune cells and must cope with defense mechanisms such as imposed-oxidative stress (e.g., reactive oxygen species; ROS). Alternative oxidase (AOX) is an enzyme recently implicated in the reduction of ROS production by the mitochondria when triggered by external stimuli, such as temperature and ROS. During this work we have evaluated the relevance of AOX during infection with Paracoccidioides brasiliensis, the etiological agent of one of the most prevalent mycoses in Latin America, paracoccidioidomycosis. We show that PbAOX gene expression is stimulated after interaction with alveolar macrophages or in the presence of H2O2 and is essential for survival against fungicidal activity of both the immune cells and the ROS compound. Moreover, decreasing PbAOX gene expression in P. brasiliensis led to increased survival of infected mice. Altogether, our data supports a relevant role for AOX in the virulence of P. brasiliensis

Diversity in Expression of Phosphorus (P) Responsive Genes in Cucumis melo L

Phosphorus (P) is a major limiting nutrient for plant growth in many soils. Studies in model species have identified genes involved in plant adaptations to low soil P availability. However, little information is available on the genetic bases of these adaptations in vegetable crops. In this respect, sequence data for melon now makes it possible to identify melon orthologues of candidate P responsive genes, and the expression of these genes can be used to explain the diversity in the root system adaptation to low P availability, recently observed in this species

Ruptured appendiceal cystadenoma presenting as right inguinal hernia in a patient with left colon cancer: A case report and review of literature

BACKGROUND: Mucoceles resulting from cystadenomas of the appendix are uncommon. Although rare, rupture of the mucoceles can occur with or without causing any abdominal complaint. There are several reports associating colonic malignancy with cystadenomas of the appendix. Herein, we report an unusual and interesting case of right inguinal hernia associated with left colon cancer. CASE PRESENTATION: A case of ruptured mucocele resulting from cystadenoma of the appendix was presented as right inguinal hernia in a 70-year-old male. The patient underwent colonoscopy, x-ray, ultrasound and computed tomography. Localized pseudomyxoma peritonei associated with adenocarcinoma of the descending colon was diagnosed. The patient underwent segmental resection of the colon, appendectomy, debridement of pseudomyxoma and closure of the internal ring of right inguinal canal. He is free of symptoms in one year follow-up. CONCLUSION: Synchronous colon cancer may occur in patients with appendiceal mucoceles. In such patients, the colon should be investigated and colonoscopy can be performed meticulously in cases of ruptured mucoceles and localized pseudomyxoma peritonei. Surgical intervention is the current choice of management

A-002 (Varespladib), a phospholipase A2 inhibitor, reduces atherosclerosis in guinea pigs

<p>Abstract</p> <p>Background</p> <p>The association of elevated serum levels of secretory phospholipase A₂(sPLA₂) in patients with cardiovascular disease and their presence in atherosclerotic lesions suggest the participation of sPLA₂enzymes in this disease. The presence of more advanced atherosclerotic lesions in mice that overexpress sPLA₂enzymes suggest their involvement in the atherosclerotic process. Therefore, the sPLA₂family of enzymes could provide reasonable targets for the prevention and treatment of atherosclerosis. Thus, A-002 (varespladib), an inhibitor of sPLA₂enzymes, is proposed to modulate the development of atherosclerosis.</p> <p>Methods</p> <p>Twenty-four guinea pigs were fed a high saturated fat, high cholesterol diet (0.25%) for twelve weeks. Animals were treated daily with A-002 (n = 12) or vehicle (10% aqueous acacia; n = 12) by oral gavage. After twelve weeks, animals were sacrificed and plasma, heart and aorta were collected. Plasma lipids were measured by enzymatic methods, lipoprotein particles size by nuclear magnetic resonance, aortic cytokines by a colorimetric method, and aortic sinus by histological analyses.</p> <p>Results</p> <p>Plasma total cholesterol, HDL cholesterol and triglycerides were not different among groups. However, the levels of inflammatory cytokines interleukin (IL)-10, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly reduced in the treatment group. This group also had a significant 27% reduction in cholesterol accumulation in aorta compared with placebo group. Morphological analysis of aortic sinus revealed that the group treated with A-002 reduced atherosclerotic lesions by 24%.</p> <p>Conclusion</p> <p>The use of A-002 may have a beneficial effect in preventing diet-induced atherosclerosis in guinea pigs.</p