Search

The Istrian (IS) and the Pag sheep (PS) are local Croatian breeds which provide significant income for the regional economy and have a cultural and traditional importance for the inhabitants. The aim of this study was to estimate some important population specific genetic parameters in IS (N = 1293) and PS (N = 2637) based on genome wide SNPs. Estimates of linkage disequilibrium effective population size (N e ) evidenced more genetic variability in PS (N e = 838) compared to IS (N e = 197), regardless of historical time (both recent and ancient genetic variability). The discrepancy in the recent genetic variability between these breeds was additionally confirmed by the estimates of genomic inbreeding (F ROH ), which was estimated to be notably higher in IS (F ROH>2 = 0.062) than in PS (F ROH>2 = 0.029). The average F ROH2-4 , F ROH4-8 , F ROH8-16 , and F ROH>16 were 0.26, 1.65, 2.14, and 3.72 for IS and 0.22, 0.61, 0.75, and 1.58 for PS, thus evidencing a high contribution of recent inbreeding in the overall inbreeding. One ROH island with > 30% of SNP incidence in ROHs was detected in IS (OAR6; 34,253,440-38,238,124 bp) while there was no ROH islands detected in PS. Seven genes (CCSER1, HERC3, LCORL, NAP1L5, PKD2, PYURF, and SPP1) involved in growth, feed intake, milk production, immune responses, and resistance were associated with the found autozygosity. The results of this study represent the first comprehensive insight into genomic variability of these two Croatian local sheep breeds and will serve as a baseline for setting up the most promising strategy of genomic Optimum Contribution Selection.

Producing hydrogen peroxide (H 2 O 2 ) from H 2 O and O 2 under visible light irradiation is a promising solar-to-chemical energy conversion technology. Hydrogen peroxide has versatile applications as a green oxidant and liquid energy carrier but has been produced through energy-intensive and complex anthraquinone processes. Herein, we report the rational design of efficient and stable porous organic polymer (POP) containing redox centers, anthraquinone photocatalyst (ANQ-POP) for solar H 2 O 2 production. ANQ-POP is readily synthesized with stable dioxin-linkages via efficient one-pot, transition-metal-free nucleophilic aromatic substitution reactions between 1,2,3,4,5,6,7,8-octafluoro-9,10-anthraquinone (OFANQ) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP). Exhibiting a fibrillar morphology, ANQ-POP boasts a high surface area of 380 m 2 ∙g -1 and demonstrates thermal stability. With 10 % ethanol, ANQ-POP yields an H 2 O 2 production rate of 320 μmol g -1 under visible light irradiation. Moreover, ANQ-POP alone can efficiently produce H 2 O 2 without any photosensitizers and cocatalysts. Density functional theory calculations reveal that the quinone groups of the anthraquinone moieties can serve as redox centers for H 2 O 2 production under light irradiation. Furthermore, unlike most conventional photocatalysts, it can produce H 2 O 2 using only water and air by catalyzing both oxygen reduction and evolution reactions under light irradiation. Our findings provide an efficient, eco-friendly pathway for photocatalytic production of H 2 O 2 under mild reaction conditions using a dioxin-derived POP-based photocatalyst., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the concentrations of essential trace and toxic elements in the milk of Lacaune sheep during lactation in intensive rearing systems. This research was conducted with 30 Lacaune sheep that were monitored in the early (60 days of lactation), medium (120 days of lactation), and late (180 days of lactation) stages of lactation. The sheep were fed a pelleted feed mixture (1 kg/day), a cereal mixture (600 g/day), and alfalfa hay (ad libitum). The essential (Fe, Zn, Cu, Co, Mn, Mo, Se, Cr, and Ni) and toxic element (heavy metals: Cd, Pb, As, and Hg) concentrations in the feed and milk were determined using an inductively coupled plasma mass spectrometer. Significant variations in the main essential trace and toxic elements, except for the Mo, Se, Ni, As, and Hg concentrations, were found in the milk of Lacaune sheep during lactation. As lactation progressed, in the late stage of lactation, significantly higher concentrations of Co, Mn, Mo, Cr, and Pb were found, while Zn and Cu in the milk of Lacaune sheep decreased significantly (4.15 and 0.21 mg/kg) compared to their concentrations in the early stage of lactation (5.66 and 0.43 mg/kg). Significantly lower concentrations of Fe and higher concentrations of Cd were found in the medium stage (0.23 mg/kg and 1.08 µg/kg) of lactation compared to both the early and late stages of lactation. An analysis of the correlation coefficients between the essential trace and toxic elements in Lacaune sheep milk during lactation determined a significantly positive correlation between Fe:Cr, Fe:Mn, Fe:Co, Fe:Se, Zn:Ni, Zn:Se, Cr:Mn, Cr:Co, Cr:Se, Cr:Mo, Mn:Co, Mn:Pb, Co:Ni, Co:Se, Ni:Se, Se:Mo, Se:Pb, and Cd:Pb. A significantly negative correlation was also found between Cu:Mn, Zn:Mo, Cg:Hg, and Hg:Pb. Based on the obtained results, it is recommended that the influence of the stage of lactation, as well as the breed of sheep, should be included when designing experiments. In general, sheep milk is rich in essential trace elements, but it also contains a very low content of toxic elements, which provides justification for increasing the breeding of Lacaune sheep and indicates the convenience of consuming their milk without risking the consumer's health.

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS., Competing Interests: Declaration of interests J. Khan, R.J.O., D.S.D., and A.T.C. are inventors on international patent application no. PCT/US2016/052496. The 3A11 CAR sequence is in this patent application (see https://patents.justia.com/patent/11078286) filed on September 19, 2016, titled “Monoclonal antibodies specific for fibroblast growth factor receptor 4 (FGFR4) and methods of their use.”, (Published by Elsevier Inc.)

The physical properties of nanomagnetic particles are expected to be highly dependent on their size. In this study, besides the promising applications of nanocrystalline Ni-Zn spinel ferrites in the area of photocatalysis and free radical scavenging, we present a detailed study with appropriate scientific explanations on the role of size change in modifying and tuning the microstructural, optical and magnetic properties. Three nanostructured Zn 0.3 Ni 0.7 Fe 2 O 4 samples of different particle sizes were prepared via the chemical co-precipitation method. Crystallographic phase purity and formation of the spinel cubic phase for all the samples were tested by X-ray diffraction studies. The magnetic properties of the as-synthesized ferrite nanoparticles have been examined thoroughly at 5 K and 300 K. Emergence of superparamagnetic behavior has been observed for the sample with the smallest size ferrite nanoparticles (ZNF-1). The photocatalytic efficiency of all the nanocatalysts was tested on methylene blue (MB) dye and the smallest sized nanocatalyst (ZNF-1) was identified as the most efficient catalyst in degrading MB dye under light illumination. The degradation efficiency was found to decrease with increasing mean particle size of the prepared samples. The antioxidant properties of the prepared ferrite samples were also studied. Here, too, the ZNF-1 sample with the smallest sized nanoparticles exhibited maximum scavenging of free radicals compared to other samples. Hence, the present study clearly demonstrates that smaller-sized Ni-Zn spinel ferrites are efficient materials for tuning the physical properties as well as for use in photocatalytic and antioxidant applications., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

With greater accessibility and an increased number of patients being treated with CAR T cell therapy, real-world toxicity continues to remain a significant challenge to its widespread adoption. We have previously shown that allogeneic umbilical cord blood-derived (UCB) regulatory T cells (Tregs) can resolve inflammation and treat acute and immune-mediated lung injuries. Allogeneic, cryopreserved UCB Tregs have shown a clinical benefit in patients suffering from COVID-19 acute respiratory distress syndrome. The unique properties of UCB Treg cells include a lack of plasticity under inflammatory micro-environments, no requirement for HLA matching, a long shelf life of cryopreserved cells, and immediate product availability, which makes them attractive for treating acute inflammatory syndromes. Therefore, we hypothesized that adjunct therapy with UCB Tregs may resolve the undesirable inflammation responsible for CAR T cell therapy-associated toxicity. In in vitro analysis, no interference from the addition of UCB Tregs was observed on CD19 CAR T cells' ability to kill CD19 Raji cells at different CAR T: Raji cell ratios of 8:1 (80.4% vs. 81.5%); 4:1 (62.0% vs. 66.2%); 2:1 (50.1% vs. 54.7%); and 1:1 (35.4% vs. 44.1%). In the xenogeneic B-cell lymphoma model, multiple injections of UCB Tregs were administered 3 days after CD19 CAR T cell injection, and no detrimental effect of add-on Tregs was noted on the circulating CD8 + T effector cells. The distribution of CAR T cells in multiple organs remained unaffected by the addition of the UCB Tregs. Specifically, no difference in the overall tumor burden was detected between the UCB Treg + CAR T vs. CAR T alone recipients. No tumor was detected in the liver or bone marrow in CAR T cells + UCB Tregs recipients, with a notable corresponding decrease in multiple circulating inflammatory cytokines when compared to CART alone recipients. Here we show the proof of concept for adjunct therapy with UCB Tregs to mitigate the hyper-inflammatory state induced by CAR T cells without any interference in their on-target anti-tumor activity. Administration of UCB Tregs after CAR T cells allows sufficient time for their synapse formation with tumor cells and exerts cytotoxicity, such that the UCB Tregs are diverted to interact with the antigen-presenting cells at the site of inflammation. Such a differential distribution of cells would allow for a two-pronged strategy of a UCB Treg "cooling blanket" effect and lay the groundwork for clinical study.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Background: Life satisfaction (LS), a useful construct in the study of psycho-social well-being, is an important indicator of healthy aging. With a view to investigate whether the improved longevity in India is accompanied by commensurate levels of well-being and contentment among the older adults , this study aimed to examine (1) the association between LS and sleep quality among older Indian adults aged 60 years and above (2) the mediating role of depression that accounts for the association and (3) the moderating role of functional limitation in this mediation., Methods: Cross-sectional data from the Longitudinal Ageing Study in India (LASI), Wave-1 (2017-18) was used. Pearson's correlation coefficients were calculated to investigate the pair-wise relationship between sleep quality, depressive symptoms, functional limitation, and LS. Structural Equation Model was employed to analyse the moderated-mediated association between sleep quality and the level of LS., Results: Sleep quality had a direct effect (β=-0.12) as well as an indirect effect (β=-0.024) via depressive symptoms on LS, accounting for 83.6 and 16.4 per cent of the total effects, respectively. Also, the interaction term between poor seep quality and functional limitation was positive (β = 0.03, p < 0.001) in determining depressive symptoms, suggesting that higher level of functional limitation aggravated the indirect effect of poor sleep quality on LS., Conclusion: The findings of the study suggested that ensuring both the physical as well as the mental well-being of the population during the life course may confer in later life the desired level of life satisfaction., (© 2022. The Author(s).)

Background: Although awareness and treatment rates of hypertension have significantly improved in recent years, the prevalence of undiagnosed and untreated hypertension remains a major public health concern for Indian policymakers. While the urban-rural variation in the prevalence, diagnosis, control, and treatment of hypertension is reasonably well-documented, the explanation behind such variation remains poorly understood given the dearth of studies conducted on exploring the determinants of the rural-urban gap in the prevalence of undiagnosed, untreated, and uncontrolled hypertension in India. In view of this research gap, our paper aims to decompose the inter-group differences between rural and urban areas in undiagnosed, untreated, and undertreated hypertension among older adults in India into the major contributing factors., Methods: Nationally representative data collected in the Longitudinal Ageing Study of India, Wave-1 (2017-18), was utilized for this study. Maximum-likelihood binary logistic-regression models were employed to capture the crude and adjusted associations between the place of residence and prevalence of undiagnosed, untreated, and undertreated hypertension. Fairlie's decomposition technique was used to decompose the inter-group differences between rural and urban residents in the prevalence of undiagnosed, untreated, and undertreated hypertension among the older population in India, into the major contributing factors, in order to explore the pathways through which these differences manifest., Results: The overall prevalence rates of undiagnosed, untreated, and undertreated hypertension among older adults were 42.3%, 6%, and 18.7%, respectively. However, the prevalence of undiagnosed and untreated hypertension was higher in rural areas, by 12.4 and 1.7 percentage-points, respectively, while undertreated hypertension was more prevalent in the urban areas (by 7.2 percentage-points). The decomposition analysis explained roughly 41% and 34% of the urban advantage over rural areas in the case of undiagnosed and untreated hypertension, while it explained 51% of the urban disadvantage in respect of undertreated hypertension. The rural-urban differentials in education and comorbidities accounted for the majority of the explained rural disadvantage in the prevalence of undiagnosed hypertension, explaining 13.51% and 13.27% of the gap, respectively. The regional factor was found to be the major driver behind urban advantage in the prevalence of untreated hypertension, contributing 37.47% to the overall gap. In the case of undertreated hypertension, education, comorbidities, and tobacco consumption were the major contributors to the urban-rural inequality, which accounted for 12.3%, 10.6%, and 9.8% of the gap, respectively., Conclusion: Socio-economic and lifestyle factors seemed to contribute significantly to the urban-rural gap in undiagnosed, untreated and undertreated hypertension in India among older adults. There is an urgent need of creating awareness programmes for the early identification of hypertensive cases and regular treatment, particularly in under-serviced rural India. Interventions should be made targeting specific population groups to tackle inequality in healthcare utilization., (© 2022. The Author(s).)

This study deals with the biomonitoring of cadmium (Cd) heavy metal in the three selected Tricholoma mushroom species collected from wild habitats of central and coastal Croatia. For this, mushroom (T. columbetta: n = 38, T. portentosum: n = 35, and T. terreum: n = 34) and surface soil samples were collected from nine forest localities of Croatia and analyzed for Cd concentration using inductively coupled plasma−optical emission spectrometry (ICP−OES) through the acid digestion method. The findings revealed that Cd was present in Tricholoma spp. and surface soil. However, the maximum mean Cd concentration (mg/kg dry weight) was recorded in T. portentosum (cap: 0.98; stipe: 0.72), followed by T. columbetta (cap: 0.96; stipe: 0.73) and T. terreum (cap: 0.81; stipe: 0.63). The bioconcentration factor (BCF) value (>1) revealed that the selected Tricholoma spp. had the potential for Cd accumulation. Moreover, the principal component (PC) and hierarchical cluster (HC) analyses were used to derive the interactions and similarities between Cd levels Tricholoma spp. and sampling localities. The multivariate analysis suggested that central sampling localities had higher Cd levels as compared to coastal localities. However, the daily intake of metals (DIM < 0.426) and health risk index (HRI < 1) showed that there was no potential health risk associated with the consumption of selected Tricholoma spp. The findings of this study are helpful to understand the Cd accumulation behavior of wild edible Tricholoma spp. collected from Croatia.

Objective: To examine the association of multimorbidity and physical activity among older adults in India., Design: A cross-sectional study was conducted using large representative survey data., Setting and Participants: The study used data from the nationally representative 'Longitudinal Ageing Study in India (LASI),' conducted during 2017-2018. The study included a total sample of 65 336 older adults aged 45 years and above in India., Methods: Moderate and vigorous physical activities were measured separately by self-reported questionnaires. Physical activity was calculated as minutes of metabolic equivalent tasks per week. The outcome variable was a categorical variable where 2=the prevalence of more than one morbidity, 1=presence of one morbidity and 0=none. Bivariate analysis and multinomial logistic regression were applied to fulfil the objectives., Results: 27.39% of older adults in India had multimorbidity. 31.02% of older adults did not engage in any moderate physical activities. Also, 59.39% of older adults did not engage in any vigorous physical activities. Older adults reporting low (adjusted relative risk ratio (A RRR): 1.10, 95% CI 1.03 to 1.18) and moderate (A RRR): 1.05, 95% CI 0.98 to 1.13) level of moderate physical activity were significantly more likely to suffer from multimorbidity compared with no involvement in moderate physical activity. However, older adults who reported high (A RRR: 0.79, 95% CI 0.75 to 0.84), moderate (A RRR: 0.88, 95% CI 0.80 to 0.98) and low level of vigorous physical activity (A RRR: 0.94, 95% CI 0.86 to 1.02) had significantly less multimorbidity in comparison to those who never engaged in vigorous physical activity., Conclusion: Lack of physical activity is associated with multimorbidity among older adults. Physical activity promotion should be adopted as a primary strategy in reducing the burden of morbidity and multimorbidity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: With the rising number of chimeric antigen receptor (CAR) T cell treated patients, it is increasingly important to understand the treatment's impact on patient-reported outcomes (PROs) and, ideally, identify biomarkers of central nervous system (CNS) adverse effects., Methods: The purpose of this exploratory study was to assess short-term PROs and serum kynurenine metabolites for associated neurotoxicity among patients treated in an anti-CD20, anti-CD19 (LV20.19) CAR T cell phase I clinical trial (NCT03019055). Fifteen CAR T treated patients from the parent trial provided serum samples and self-report surveys 15 days before and 14, 28, and 90 days after treatment., Results: Blood kynurenine concentrations increased over time in patients with evidence of neurotoxicity ( p  = 0.004) and were increased in self-reported depression ( r  = 0.52, p  = 0.002). Depression improved after CAR T infusion ( p  = 0.035). Elevated 3-hydroxyanthranilic acid (3HAA) concentrations prior to cell infusion were also predictive of neurotoxicity onset ( p  = 0.031), suggesting it is a biomarker of neurotoxicity following CAR T cell therapy., Conclusions: Elevated levels of kynurenine pathway metabolites among CAR T cell recipients are associated with depressed mood and neurotoxicity. Findings from this exploratory study are preliminary and warrant validation in a larger cohort., Competing Interests: Competing interestsThe authors declare the following competing interests. P.H. reports receiving honoraria from Incyte, BMS, Legend, Jannsen, Takeda, Amgen, Karyopharm, GSK, Pfizer. C.J.H. is a member of the Scientific Advisory Boards of Phytecs, Inc, and has equity in Formulate Biosciences. B.J. reports receiving research support and honoraria and travel support from Miltenyi Biotec. D.S. and B.D. are authors on a patent for the 20.19 CAR. N.N.S. reports receiving honoraria and/or travel support from Incyte, Celgene, Lily, and Miltenyi Biotec; serving on scientific advisory boards for Lily, Kite, Celgene, Legend, Epizyme, Seattle Genetics, and TG therapeutics; equity ownership in Exelixis, Geron; receiving institutional research support for clinical trials from Miltenyi Biotec. The remaining authors declare no competing interests., (© The Author(s) 2022.)

Background: HIV cure research involving cell and gene therapy has intensified in recent years. There is a growing need to identify ethical standards and safeguards to ensure cell and gene therapy (CGT) HIV cure research remains valued and acceptable to as many stakeholders as possible as it advances on a global scale., Methods: To elicit preliminary ethical and practical considerations to guide CGT HIV cure research, we implemented a qualitative, in-depth interview study with three key stakeholder groups in the United States: (1) biomedical HIV cure researchers, (2) bioethicists, and (3) community stakeholders. Interviews permitted evaluation of informants' perspectives on how CGT HIV cure research should ethically occur, and were transcribed verbatim. We applied conventional content analysis focused on inductive reasoning to analyze the rich qualitative data and derive key ethical and practical considerations related to CGT towards an HIV cure., Results: We interviewed 13 biomedical researchers, 5 community members, and 1 bioethicist. Informants generated considerations related to: perceived benefits of CGT towards an HIV cure, perceived risks, considerations necessary to ensure an acceptable benefit/risk balance, CGT strategies considered unacceptable, additional ethical considerations, and considerations for first-in-human CGT HIV cure trials. Informants also proposed important safeguards to developing CGT approaches towards an HIV cure, such as the importance of mitigating off-target effects, mitigating risks associated with long-term duration of CGT interventions, and mitigating risks of immune overreactions., Conclusion: Our study identified preliminary considerations for CGT-based HIV cure across three key stakeholder groups. Respondents identified an ideal cure strategy as one which would durably control HIV infection, protect the individual from re-acquisition, and eliminate transmission to others. Known and unknown risks should be anticipated and perceived as learning opportunities to preserve and honor the altruism of participants. Preclinical studies should support these considerations and be transparently reviewed by regulatory experts and peers prior to first-in-human studies. To protect the public trust in CGT HIV cure research, ethical and practical considerations should be periodically revisited and updated as the science continues to evolve. Additional ethics studies are required to expand stakeholder participation to include traditionally marginalized groups and clinical care providers., (© 2022. The Author(s).)

Background: Co-existence of multiple chronic diseases is increasingly becoming a norm among ageing population. The study aims to investigate the prevalence of multimorbidity and the association between anthropometric measures of obesity and multimorbidity among men and women aged 60 years and above in India., Methods: The present study is based on the first wave of the Longitudinal Aging Study in India. The analytical sample size for the study was 28,050 older adults aged 60 years and above. Descriptive statistics and multivariable analysis using logistic regression models were conducted., Results: Body Mass Index (BMI) based-obesity is more prevalent among older women than men (26.3% vs. 17.6%). Similarly, higher proportion of older women was at high-risk waist circumference (37.1% vs 8.9%) and waist-hip ratio (78.5 vs 75.4%) than men respectively. In Model-I, after controlling for several covariates, older adults with overweight/obesity were 1.6 times more likely to have multi-morbidity than non-obese older adults (Adjusted OR = 1.61; 95% CI: 1.48-1.74). Similarly, older adults with high-risk waist circumference [Adjusted OR: 1.66; 95% CI: 1.52-1.80] and waist-hip ratio [Adjusted OR: 1.45; 95% CI: 1.33-1.59] also had higher odds of having multi-morbidity in reference to their counterparts. In model-3 it was found that females with high-risk waist-hip ratio had 14% lower odds of multimorbidity than males with high-risk waist-hip ratio [Adjusted OR: 0.86; 95%CI: 0.78-0.94]., Conclusion: The findings of the study show significant gender difference in the prevalence of multimorbidity, men being at increased risk in the multivariate analysis which is uncommon in the existing epidemiological research. Interactive effect of male gender with anthropometric measures on multimorbidity reported in our study probably due to increased unhealthy behaviours among men requires further research., (© 2022. The Author(s).)

CAR T-cell therapies targeting the B-cell maturation antigen eliminate tumors in relapsed/refractory multiple myeloma patients, however durable remissions remain difficult to attain. Transforming growth factor beta (TGF-β) is a multifunctional cytokine abundantly expressed in the multiple myeloma bone marrow niche, where it promotes an immunosuppressive tumor microenvironment. We hypothesized that BCMA CAR T-cells armored to resist the suppressive effects of TGF-β will provide an advantage in treating multiple myeloma. The armored B2ARM CAR T cells, co-expressing BCMA targeting CAR with TGF-β dominant-negative receptor II, were generated by lentiviral transduction of primary human CD4+ and CD8+ T cells. The B2ARM CAR T cells eliminated MM.1S multiple myeloma targets in long-term cytotoxicity assays, even under TGF-β-high conditions, whereas cytotoxic function of the non-armored B2 CAR -T cells was inhibited by TGF-β. Concordantly, after long-term exposure to targets in the presence of TGF-β, the B2ARM CAR T cells were enriched for Granzyme B, CD107a, Ki67 and polyfunctional cells T-cells (double or triple-positive for IFN-γ, IL-2 and/or TNF-α), as determined by flow cytometry. In addition, the B2ARM CAR T-cells, but not the conventional B2 CAR T-cells, resisted the TGF-β-mediated suppression of activation (CD25), exhaustion (PD-1, LAG3), and differentiation to T effectors (CD45RA+ CD45RO-CD62L-). In NSG mice bearing RPMI-8226 tumors overexpressing TGF-β, the B2ARM CAR mediated 100% tumor rejection and survival, superior infiltration of tumors on day 7 post CAR T treatment (%CD3+CAR+), and greater expression of IFN-γ, TNF-α, Ki67, Granzyme B, and PD-1, as compared to tumor-infiltrating non-armored B2 CAR T-cells. In NSG RPMI-8226 xenograft model in which tumors were additionally supplemented with TGF-β injections on days -1 through 11 of CAR T treatment, the B2ARM CAR T cells rejected tumors faster than the non-armored B2 CARs, and showed greater numbers of CD3+ and CD3+CAR+, central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) T cells in the peripheral blood 18 days after treatment. In summary, the armored B2ARM CAR T cells mediate superior persistence, proliferation, multi-functionality, effector differentiation and anti-tumor function in pre-clinical models of multiple myeloma, while abrogating TGF-β-mediated suppression., Competing Interests: DS, LA, YX, ZZ, BV, DW, PH, PD are employees of Lentigen Technology, a Miltenyi Biotec Company. BW is an employee of Miltenyi Biotec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alabanza, Xiong, Vu, Webster, Wu, Hu, Zhu, Dropulic, Dash and Schneider.)

Although Olympic athletes are celebrated for their sports achievements, they often face serious difficulties in their post-sport career employment. Factors of development that are affecting the quality of post-sport career transition of Olympic athletes are important to acknowledge in the dual career (DC) development perspective. Due to the side lining of academic activities, athletes are often not well prepared for the labor market. If they do not gain sufficient financial background in their careers, it can lead to a lack of proper economic inclusion of athletes in their post-sport career employment and further impact their lives. Career transitions of athletes have been the subject of research in different aspects of DC support (e.g., athletic, psychological, psychosocial, academic/vocational, financial), but most research is linked to the student-athlete DC perspective. Therefore, the aim of our research was to examine the impact of factors directly contributing to the quality of the post-sport career transition in Slovenian elite and Olympic athletes and the social class position and employment of these athletes after the termination of their sports career. From DC support practice, we learned that although athletes often have a proper level of education, their post-sport career transitions were not successful. To fill this gap, 168 elite athletes (M age = 33.34, SD = 13.1) from Slovenia were asked to complete online questionnaires. The results showed a significant contribution of education and DC support-related finances (e.g., employment of athletes in public administration) to the quality of post-sport career transition. Regarding developing a national DC model and based on empirical research, this study identifies the social class position and employment status of former elite athletes from Slovenia. It also identifies opportunities for further research on the quality of the post-sport career transitions and perspectives on DC support. Understanding how different factors contribute to the integrated development of individual athletes to reach their potential in sports, education, and their post-sport career employment is important for theorists, DC practitioners, and stakeholders working with DC athletes. To develop a sufficient mechanism, DC support providers should consider supporting education along with the financial support of athletes during their sports careers and recognizing study-training ecosystems, based on good practices to successfully transition to their post-sport careers. These findings can also be useful for athletes and their athletic triangle support network (e.g., coaches and parents) as a support in the decision-making., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Robnik, Kolar, Štrumbelj and Ferjan.)

The emergence of the COVID-19 pandemic imposed fundamental changes in the field of surgery. Reorganization was made in order to adequately treat the patients during the pandemic. WALANT (Wide Awake Local Anesthesia No Tourniquet) approach was found to be a very convenient method in facilitating continuity in hand surgery with limited staff. A retrospective comparative study was performed between period of April 2020 till September 2021 at our clinic to evaluate advantages of WALANT approach. This study included 136 patients, from which 72 (53%) were operated with WALANT, compared to the control group of 64 (47%) patients without WALANT. Average hospital stay for the WALANT group was 2.2 days vs. 4.7 days for the control group. Average operating room personnel were 3.8 for WALANT and 6.2 for the control non-WALANT group. Intraoperative and postoperative VAS (visual analogue scale) score was evaluated. Due to its diversity, low cost and low complication rate, we recommend WALANT approach in acute and elective hand surgery.

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5‒97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies., (© 2021. The Author(s).)

Gene expression signatures have proven their potential to characterize important cancer phenomena like oncogenic signaling pathway activities, cellular origins of tumors, or immune cell infiltration into tumor tissues. Large collections of expression signatures provide the basis for their application to data sets, but the applicability of each signature in a new experimental context must be reassessed. We apply a methodology that utilizes the previously developed concept of coherent expression of genes in signatures to identify translatable signatures before scoring their activity in single tumors. We present a web interface (www.rosettasx.com) that applies our methodology to expression data from the Cancer Cell Line Encyclopaedia and The Cancer Genome Atlas. Configurable heat maps visualize per-cancer signature scores for 293 hand-curated literature-derived gene sets representing a wide range of cancer-relevant transcriptional modules and phenomena. The platform allows users to complement heatmaps of signature scores with molecular information on SNVs, CNVs, gene expression, gene dependency, and protein abundance or to analyze own signatures. Clustered heatmaps and further plots to drill-down results support users in studying oncological processes in cancer subtypes, thereby providing a rich resource to explore how mechanisms of cancer interact with each other as demonstrated by exemplary analyses of 2 cancer types., (Copyright © 2021. Published by Elsevier Inc.)

Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and immune effector cell - associated neurotoxicity syndrome (ICANS) are well-known complications. Tocilizumab, a monoclonal antibody targeting the interleukin-6 (IL-6) receptor was administered 1 hour prior to infusion of anti-CD19 CAR-T cells with CD3ζ/4-1BB costimulatory signaling used to treat non-Hodgkin lymphoma patients. Relapsed/refractory lymphoma patients treated with anti-CD19 CAR-T cells were included in this analysis. Cytokine plasma levels were measured by electrochemiluminescence before lymphodepleting chemotherapy, prior to infusion and then on days 2, 4,6, and 14 days after treatment. Twenty patients were treated. Cell products included locally manufactured anti-CD19 CAR-T (n=18) and tisagenlecleucel (n=2). There were no adverse events attributed to tocilizumab. Ten patients had grade 1-2 CRS at a median of 4 (range 3-7) days. There were no cases of grade ≥3 CRS. Five patients had ICANS, grade 1 (n=4) and grade 4 (n=1). Laboratory studies obtained prior to lymphodepleting chemotherapy were comparable between patients with and without CRS, except for interleukin (IL)-15 plasma concentrations. patients with CRS had higher post-infusion ferritin and C reactive protein, with more marked increases in inflammatory cytokines, including IL-6, IL-15, IFN-γ, fractalkine and MCP-1. Fifteen patients (75%) achieved CR and 2 (10%), PR. One-year OS and PFS estimates were 83% and 73%. Prophylactic tocilizumab was associated with low CRS incidence and severity. There were no adverse events associated with tocilizumab, no increase in frequency or severity of ICANS and excellent disease control and overall survival., Competing Interests: BD is a previous employee of Lentigen, a Miltenyi Biotec Company, and has Patents and Royalties related to CAR-T immunotherapy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Caimi, Pacheco Sanchez, Sharma, Otegbeye, Ahmed, Rojas, Patel, Kleinsorge Block, Schiavone, Zamborsky, Boughan, Hillian, Reese-Koc, Maschan, Dropulic, Sekaly and de Lima.)

Background: COVID-19 is affecting the entire population of India. Understanding district level correlates of the COVID-19's infection ratio (IR) is essential for formulating policies and interventions., Objective: The present study aims to investigate the district level variation in COVID-19 during March-October 2020. The present study also examines the association between India's socioeconomic and demographic characteristics and the COVID-19 infection ratio at the district level., Data and Methods: We used publicly available crowdsourced district-level data on COVID-19 from March 14, 2020, to October 31, 2020. We identified hotspot and cold spot districts for COVID-19 cases and infection ratio. We have also carried out two sets of regression analysis to highlight the district level demographic, socioeconomic, household infrastructure facilities, and health-related correlates of the COVID-19 infection ratio., Results: The results showed on October 31, 2020, the IR in India was 42.85 per hundred thousand population, with the highest in Kerala (259.63) and the lowest in Bihar (6.58). About 80 percent infected cases and 61 percent deaths were observed in nine states (Delhi, Gujarat, West Bengal, Uttar Pradesh, Andhra Pradesh, Maharashtra, Karnataka, Tamil Nadu, and Telangana). Moran's- I showed a positive yet poor spatial clustering in the COVID-19 IR over neighboring districts. Our regression analysis demonstrated that percent of 15-59 aged population, district population density, percent of the urban population, district-level testing ratio, and percent of stunted children were significantly and positively associated with the COVID-19 infection ratio. We also found that, with an increasing percentage of literacy, there is a lower infection ratio in Indian districts., Conclusion: The COVID-19 infection ratio was found to be more rampant in districts with a higher working-age population, higher population density, a higher urban population, a higher testing ratio, and a higher level of stunted children. The study findings provide crucial information for policy discourse, emphasizing the vulnerability of the highly urbanized and densely populated areas., Competing Interests: The authors have declared that no competing interests exist.

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion., Competing Interests: Declaration of interests D.S., B.D., and B.W. have submitted a patent application regarding treating cancer with self-driving chimeric antigen receptors (PCT/US2020/021320) on the basis of this work. This study was funded by Lentigen Technology, a Miltenyi Biotec Company, and Miltenyi Biotec. Y.X., P.H., D.W., L.A., D.S., and B.D. are employees of Lentigen, a Miltenyi Biotec company, and B.W. is an employee of Miltenyi Biotec., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

This study aimed to examine the impact of ewe's age at first lambing (AFL) on days in milk (DIM), average daily milk yield (DMY), and total milk yield (TMY). Symmetrical bimodal distribution of AFL enabled classification of maidens in those mated in the first (47%) or second year of life (53%). After accounting for all available sources of phenotypic variability with the linear mixed model for repeated records, it was estimated that AFL had a statistically significant effect only on DIM (p < 0.001). The litter size had a significant effect only on TMY ( p < 0.001), while the effect of the parity was significant for all the examined traits ( p < 0.001). The results of the study suggest that prolongation of age at first mating to the second year of life is not justified in dairy-orientated sheep farms. However, more evidence on this issue is needed for generalization, especially considering some other traits that can impact profitability of dual-purpose sheep farms (reproduction traits, growth rate of lambs, etc.).

Background: Rectal signet ring cell carcinoma is a rare type of colorectal adenocarcinoma characterized by an aggressive biological behavior and poor prognosis. The co-occurrence of colorectal carcinoma and renal cell carcinoma (RCC) has found in many hundreds of patients, many of whom also have additional malignancies. Cancer to cancer metastasis is rare and an uncommon phenomenon in malignancy, especially at the time of initial diagnosis, suggesting a genetic susceptibility., Case Presentation: We present the case of a 66-year-old Macedonian man with synchronous rectal signet ring cell carcinoma and RCC with tumor to tumor metastasis feature. He underwent a left nephrectomy and anterior rectal resection after complaining of constipation for 3-4 months and the appearance of synchronous tumors on the imaging studies. Morphology and immunohistochemical analysis of specimens from the RCC revealed signet ring cells identical to the rectal signet ring cell carcinoma. The next-generation sequencing study revealed mutations in TP53 and ERBB2, and microsatellite stable signet ring cell carcinoma was determined by deoxyribonucleic acid (DNA) sequencing., Conclusions: Cancer to cancer metastasis, although rare, needs to be considered in synchronous tumors. RCC, when diagnosed in multiple synchronous tumors, should be examined carefully. The paucity of reported cases indicates the need for advanced research in imaging methods for metastasis and new therapeutic approaches.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

There is an increased production and demand for ewes' milk in the Republic of Croatia, as well as globally. There is also a growing concern about its quality, since milk from farm animals may become contaminated with mercury and other toxic elements. Thus, the aim of this paper is to determine the influence of lactation stage on the ewes' milk quality in western Croatia by considering concentrations of mercury and other elements in ewes' milk. The research was conducted on 36 Travnik pramenka sheep during different lactation stages. The digested milk samples were analysed with continuous flow hydride generation technique by using inductively coupled plasma mass spectrometry. Samples were taken during 40th, 80th and 120th d of lactation. Yield and quality of ewes' milk was within lactation curve. As lactation progressed, significantly lower concentrations of Hg (on 80th d compared to 40th d) and of Cd (120th d compared to 80th d) were noted, and Hg on 120th d was below the detection limit. Concentrations of Ca and Cu were lower on the 120th d compared to 40th d, while P, Mg, Fe, Zn, Mn, and Se were lower on the 80th and 120th d compared to the 40th d. Concentrations of K, Mo, and Cr differed among all stages of lactation. Regarding toxic elements, the observed low concentrations of Hg, Co, Cd and As suggest that ewes' milk in western Croatia is safe for human or animal consumption., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: We aim to explore the barriers to accessing modern healthcare services in two tribal populations in Assam., Methods: In March 2018, we conducted qualitative research through 60 in-depth interviews with men and women aged 15 to 50 from Bodo and Rabha tribes in Udalguri and Baksa districts of Assam. We interviewed a group of health-service providers from public health facilities to understand the demand-supply balance in those facilities., Findings: On the demand side, direct and indirect financial obstacles, distance to health facilities, poor public transportation, perceived negative behavior of hospital staff, and lack of infrastructure were the main barriers to utilizing healthcare facilities. On the supply side, doctors and nurses in government health facilities were overburdened by demand due to a lack of human resources., Conclusions: Our study highlights the barriers to utilizing health facilities; these are not always driven by factors linked to the patient's socio-economic status but also depend significantly on the quality of the health services and other contextual factors. Although the government has made efforts to improve the rural healthcare system through national-level programs, our qualitative study shows that these programs have not been successful in enhancing the rural healthcare system in the study area., Competing Interests: The authors have declared that there is no competing interest.

Chimeric antigen receptor (CAR) T cells targeting CD19 are a breakthrough treatment for relapsed, refractory B cell malignancies 1-5 . Despite impressive outcomes, relapse with CD19 - disease remains a challenge. We address this limitation through a first-in-human trial of bispecific anti-CD20, anti-CD19 (LV20.19) CAR T cells for relapsed, refractory B cell malignancies. Adult patients with B cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were treated on a phase 1 dose escalation and expansion trial (NCT03019055) to evaluate the safety of 4-1BB-CD3ζ LV20.19 CAR T cells and the feasibility of on-site manufacturing using the CliniMACS Prodigy system. CAR T cell doses ranged from 2.5 × 10 5 -2.5 × 10 6 cells per kg. Cell manufacturing was set at 14 d with the goal of infusing non-cryopreserved LV20.19 CAR T cells. The target dose of LV20.19 CAR T cells was met in all CAR-naive patients, and 22 patients received LV20.19 CAR T cells on protocol. In the absence of dose-limiting toxicity, a dose of 2.5 × 10 6 cells per kg was chosen for expansion. Grade 3-4 cytokine release syndrome occurred in one (5%) patient, and grade 3-4 neurotoxicity occurred in three (14%) patients. Eighteen (82%) patients achieved an overall response at day 28, 14 (64%) had a complete response, and 4 (18%) had a partial response. The overall response rate to the dose of 2.5 × 10 6 cells per kg with non-cryopreserved infusion (n = 12) was 100% (complete response, 92%; partial response, 8%). Notably, loss of the CD19 antigen was not seen in patients who relapsed or experienced treatment failure. In conclusion, on-site manufacturing and infusion of non-cryopreserved LV20.19 CAR T cells were feasible and therapeutically safe, showing low toxicity and high efficacy. Bispecific CARs may improve clinical responses by mitigating target antigen downregulation as a mechanism of relapse.

Drip loss, pH value, and color are among the important traits that determine meat quality. Contrary to pH and color, the method associated with drip loss is not yet standardized, and literature data are difficult to compare. Besides, to our knowledge, there is no research comparing drip loss methods and their relation with pH and color in mutton. This study aimed to assess drip loss measurements in mutton taken by different methods (EZ and bag - BM) and their relationship with pH values and color. Mutton samples ( Musculus longissimus thoracis et lumborum ) originating from 20 ewes of Istrian sheep were used to examine the effect of the method on drip loss after 24 h (EZ 24 vs. BM 24 ) and 48 h (EZ 48 vs. BM 48 ). Furthermore, correlations between drip loss, pH value, and color were analyzed. The statistical analysis was conducted in R programming environment by using different packages. Within the EZ method there was no significant difference ( p > 0.05 ) between ventral and dorsal sample cores used for the assessment of EZ drip loss. Drip loss measured with the same method at two different points of time (24 and 48 h) differed significantly ( p < 0.001 ). There was also a significant difference in drip loss determined by different methods (EZ vs. BM) at the same point of time. There were significant ( p < 0.05 ) correlations between pH 45 min and all color parameters ( L * 4 , a * , b * ). The L * , a * , and b * parameters were highly correlated ( p < 0.001 ). The strongest correlation occurred between a * and b * parameter ( r = 0.93 ). Correlations between drip loss by EZ method and other meat quality attributes were low and not significant. The b * parameter correlated with BM 24 ( r = 0.46 ) and BM 48 ( r = 0.58 ), while a * correlated only with BM 48 ( r = 0.50 ). The correlations between the EZ 24 and BM 24 as well as between the EZ 48 and BM 48 were both non-significant ( p > 0.05 ). Drip loss cannot be predicted with sufficient accuracy by using pH and color. EZ and BM method in mutton do not provide equivalent results for measuring drip loss. Comparisons of the results obtained with different methods should be avoided or at least performed with great precaution., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2020 Ana Kaić et al.)

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CAR-T cells on the automated CliniMACS Prodigy ® device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CAR-T cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CAR-T cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CAR-T cells in patient circulation up to 1-year post-infusion., (Copyright © 2020 Jackson, Roe, Sharma, Lopes, Talla, Kleinsorge-Block, Zamborsky, Schiavone, Manjappa, Schauner, Lee, Liu, Caimi, Xiong, Krueger, Worden, Kadan, Schneider, Orentas, Dropulic, Sekaly, de Lima, Wald and Reese.)

Background and Aims: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties., Methods: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgc null (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgc null (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice., Results: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice., Conclusions: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC., (Published by Elsevier Inc.)

Genetic engineering is an important tool for redirecting the function of various types of immune cells and their use for therapeutic purpose. Although NK cells have many beneficial therapeutic features, genetic engineering of immune cells for targeted therapy focuses mostly on T cells. One of the major obstacles for NK cell immunotherapy is the lack of an efficient method for gene transfer. Lentiviral vectors have been proven to be a safe tool for genetic engineering, however lentiviral transduction is inefficient for NK cells. We show in this study that lentiviral vectors pseudotyped with a modified baboon envelope glycoprotein can transduce NK cells 20-fold or higher in comparison to VSV-G pseudotyped lentiviral vector. When we investigated the mechanism of transduction, we found that activated NK cells expressed baboon envelope receptor ASCT-2. Further analysis revealed that only a subset of NK cells could be expanded and transduced with an expression profile of NK56 bright , CD16 dim , TRAIL high , and CX3CR1 neg . Using CD19-CAR, we could show that CD19 redirected NK cells efficiently and specifically kill cell lines expressing CD19. Taken together, the results from this study will be important for future genetic modification and for redirecting of NK cell function for therapeutic purpose.

The aim of the present research was to determine the haemato-biochemical profile and blood acid-base status of Croatian spotted goats in a traditional Mediterranean production system. The 60 non-gravid female Croatian spotted goats of different ages were included in the research. They were divided into four groups of 15 goats according to age: group I - ≤ 1  year old; group II - 2-3 years; group III - 3-6 years; and group IV - 7-10 years. Haematological parameters were determined in whole blood, biochemical parameters in serum and acid-base status in plasma by automatic analyser. Total leukocyte number (WBC), haemoglobin (HGB) and mean corpuscular volume (MCV) in the blood were the highest, while mean haemoglobin concentration in erythrocytes (MCHCs) was the lowest in yearlings compared to other groups. Concentrations of urea, Mg, Cl, non-esterified fatty acids (NEFAs) and lactate were the highest in yearlings. Concentrations of Ca, Na, total cholesterol, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and beta hydroxybutyrate (BHB) as well as the activity of alanine aminotransferase (ALT) were higher in older goats compared to yearlings, while the opposite was determined for the activities of creatine kinase (CK) and alkaline phosphatase (ALP). Values of pH, the strong ion difference (SID), anion gap (AG) and z  values as well as the content of HCO 3 and total pressure of carbon dioxide ( ctCO 2 ) were higher in older goats compared to yearlings. The results obtained may help in monitoring the health and nutritional status and improve the management of Croatian spotted goats. Based on the results of the present study, the effect of age needs to be included in the model when preparing the reference values for the haemato-biochemical profile and acid-base status of goats., (Copyright: © 2019 Zvonko Antunović et al.)

Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and loss of function. Adipose tissue harbors mesenchymal stem/stromal cells (MSC), or medicinal signaling cells as suggested by Caplan (Caplan, 2017), used in autologous transplantation in many clinical settings. The aim of the study was to characterize a stromal vascular fraction from microfragmented lipoaspirate (SVF-MLA) applied for cartilage treatment in OA and compare it to that of autologous lipoaspirate (SVF-LA). Samples were first stained using a DuraClone SC prototype tube for the surface detection of CD31, CD34, CD45, CD73, CD90, CD105, CD146 and LIVE/DEAD Yellow Fixable Stain for dead cell detection, followed by DRAQ7 cell nuclear dye staining, and analyzed by flow cytometry. In SVF-LA and SVF-MLA samples, the following population phenotypes were identified within the CD45 - fraction: CD31 + CD34 + CD73 ± CD90 ± CD105 ± CD146 ± endothelial progenitors (EP), CD31 + CD34 - CD73 ± CD90 ± CD105 - CD146 ± mature endothelial cells, CD31 - CD34 - CD73 ± CD90 + CD105 - CD146 + pericytes, CD31-CD34 + CD73 ± CD90 + CD105-CD146 + transitional pericytes, and CD31 - CD34 + CD73 high CD90 + CD105 - CD146 - supra-adventitial-adipose stromal cells (SA-ASC). The immunophenotyping profile of SVF-MLA was dominated by a reduction of leukocytes and SA-ASC, and an increase in EP, evidencing a marked enrichment of this cell population in the course of adipose tissue microfragmentation. The role of EP in pericyte-primed MSC-mediated tissue healing, as well as the observed hormonal implication, is yet to be investigated., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: Meningiomas are the type of central nervous system tumours, derived from the cells of the arachnoid membrane that are well constrained from surrounding tissues, mainly no infiltrating neoplasm with benign features. Meningiomas consist about 15-20% of all primary intracranial neoplasms., Aim: The evaluation of the outcome of the operatively treated meningiomas in relation with the Karnofsky performance score, survival, recurrence, type of the surgical excision, histological type, mitotic count (MC), localisation and volume of the lesion., Methods: In this article 40 operatively treated patients are reviewed for the outcome of the operation about the Karnofsky performance score, survival, recurrence, type of the surgical excision, histological type, mitotic count (MC), localisation and volume of the lesion., Results: Association/interconnection between the mitotic count grade I and the regrowth of meningioma have been verified. Association/interconnection between the mitotic count grade I and the regrowth of meningioma have been verified. Association/interconnection between the mitotic count grade I and the regrowth of meningioma have been established., Conclusion: Gender, age and Karnofsky performance score have predictive value in the treatment of different types of meningiomas. The magnitude of surgical resection is associated with the regrowth of a tumour. The mitotic count in different types of meningiomas presents significant feature in the appearance of meningioma recurrence. The surgical resection and the quality and quantity of patient's survival have a significant relation to the mitotic count of the meningiomas. There is no connection between the size and the localisation of a tumour related to different values of the mitotic count.

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4 + and CD8 + T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33 + AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33 + tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33 + cell lines, but not when CAR T were cultured alone. Studies with a CD33 - cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34 + hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing in vitro and in vivo , and showed comparable functionality to the scFv-based My96CAR.

Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

BACKGROUND The aim of this study was to assess sleep architecture and respiration during sleep in children with intractable epileptic encephalopathies using overnight video-polysomnography (V-PSG). MATERIAL AND METHODS Between 2015 to 2017 overnight V-PSG recordings were made for 31 children (22 boys and 9 girls) with intractable epileptic encephalopathy with a mean age of 6.78±3.61 years and a mean body mass index (BMI) of 15.83±3.16 kg/m3. Thirty-one healthy children were matched for sex, age, and BMI as the control group. The phases of sleep studied included rapid eye movement (REM) sleep, and non-REM (NREM) phases NREM 1, NREM 2, and NREM 3. Respiratory function during sleep was evaluated. RESULTS Children with epileptic encephalopathies receiving antiepileptic treatment had significantly decreased total sleep time (TST) (p=0.038), significantly increased percentage of NREM1 (p=0.033), and a significantly lower percentage of total REM (p<0.0001), compared with the control group. All children 31/31 (100%) with epileptic encephalopathies had interictal epileptiform discharges, and 4/31 (12.9%) had ictal events. The number of respiratory events did not differ significantly between the two groups (p=0.118), but children in the epileptic encephalopathy group had a significantly shorter average duration (p=0.008) and longest duration (p=0.048) of respiratory events. Average (p=0.006) and least (p=0.0004) oxygen saturation (SatO2) were significantly lower in children with epileptic encephalopathies compared with the control group. CONCLUSIONS Children with epileptic encephalopathies had altered sleep architecture and marked oxygen desaturation, which supports the need for referral of children with epileptic encephalopathy for overnight sleep evaluation.

Background Aims: Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients., Methods: The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days., Results: Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3 + T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 10 8 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3 + with a mix of CD4 + and CD8 + cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays., Discussion: The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4 + and CD8 + T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4 + and CD8 + T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t + cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.

The authors would like to make the following corrections to the published article.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 10 6 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19 dim or CD19 - B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22 + cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.