An Unusual Case of an Intradiploic Arachnoid Cyst of the Orbit.

This study reported a case of an arachnoid cyst of the sphenoid bone causing orbital signs and symptoms in a 58-year-old man with progressive proptosis and nonspecific discomfort in the OS. Orbital MRI showed a 3-cm homogeneous cyst within the left greater wing of the sphenoid bone. To the best of our knowledge, this is the first report of an intradiploic arachnoid cyst in the sphenoid bone with atypical radiological features, causing clinical symptoms, and managed through an eyelid approach, achieving a complete resolution with no complications.

Thulium Fiber Laser Versus Holmium:Yttrium Aluminum Garnet for Lithotripsy: A Systematic Review and Meta-analysis.

CONTEXT: Thulium fiber laser (TFL) emerged as a competitor of holmium:yttrium aluminum garnet (Ho:YAG) laser for the treatment of urinary stones. OBJECTIVE: To compare the efficacy between Ho:YAG and TFL for laser lithotripsy of renal and ureteral stones. EVIDENCE ACQUISITION: A literature search was conducted using PubMed/Medline, Embase, and Web of Science databases to identify reports published until May 2023. The Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines were followed to identify eligible studies. The primary outcome was to compare the stone-free rate (SFR) between Ho:YAG and TFL for laser lithotripsy. EVIDENCE SYNTHESIS: Eleven studies met our inclusion criteria, and data from 1286 and 880 patients who underwent, respectively, Ho:YAG and TFL laser lithotripsy were reviewed. Most studies included ureteroscopy (URS) and retrograde intrarenal surgeries as procedures, two included percutaneous nephrolithotomy, and one included URS exclusively. Only two studies reported results in pediatric patients. TFL was associated with a higher SFR (odds ratio [OR] 1.84, 95% confidence interval [CI]: 1.06-3.20; p = 0.031) when no residual fragment is considered, but not when SFR refers to the presence of fragments <3 mm (OR 2.48, 95% CI: 0.98-6.29; p = 0.055) or when only Ho:YAG with MOSES is considered (p = 0.068). According to the stones' location, TFL was associated with higher SFRs than Ho:YAG for renal (OR 3.14, 95% CI: 1.69-5.86; p < 0.001) but not for ureteral (p = 0.8) stones. TFL was associated with a lower intraoperative complication rate (OR 0.34, 95% CI: 0.19-0.63; p < 0.001). No difference was found in major (p = 0.4) or overall (p = 0.4) complication rate, operative time (p = 0.051), and laser time (p = 0.9). CONCLUSIONS: TFL is a promising laser for the treatment of urinary stones with some advantages over Ho:YAG. Further high-quality studies are needed to confirm these findings and optimize the surgical settings. PATIENT SUMMARY: The use of thulium fiber laser rather than holmium:yttrium aluminum garnet permits to reach a higher stone-free rate in stones located in the kidney rather than in the ureter.

Screening of positive dysphotopsia before multifocal intraocular lens implantation.

PURPOSE: To assess the photic phenomena (PP) and positive dysphotopsia in candidates for presbyopia or cataract surgery and to evaluate their relationship with cataract grading systems. DESIGN: Retrospective observational. METHODS: Monocular data for 82 subjects measured during the preoperative screening were retrospectively retrieved from our database. The evaluated variables consisted of two methods for PP measurement: light distortion index (LDI) and parameters obtained from a simulator, both of which were combined with subjective bother related to PP. The cutoff for LDI that better predicted patients passing from slightly to moderately bothersome was estimated. The relationships between LDI and the following objective cataract grading methods were also assessed: objective scatter index (OSI), dysfunctional lens index (DLI), and Pentacam Nucleus Staging (PNS). RESULTS: LDI was the best method for measuring PP, which showed a significant correlation with the bothersome question (rho = 0.34, P = 0.002) and also with OSI (rho = 0.67, P < 0.0005), DLI (rho = -0.29, P = 0.007), and PNS (rho = 0.48, P < 0.0005). The number/percentage of patients who found it bothersome was as follows: "Not at all" (18/22%), "Slightly" (41/50%), "Moderately" (15/18.3%), and "Very" (8/9.7%). The cutoff value that predicted the transition from slightly to moderately bothersome was ≥15.20% according to LDI, which could be estimated with the following values for grading: ≥2.8 for OSI, ≤7.6 for DLI, and ≥2 for PNS. CONCLUSIONS: Patients reporting moderately or higher bothersome levels in the preoperative period and with LDI <15.20%, <2.8 for OSI, >7.6 for DLI, and <2 for PNS might deserve special attention in the multifocal intraocular lens selection.

Applicability of the Barcelona scale to assess the quality of cleanliness of mucosa at esophagogastroduodenoscopy.

BACKGROUND AND OBJECTIVES: There are few scales with prospective validation for the assessment of the upper gastrointestinal mucosal cleanliness during an esophagogastroduodenoscopy (EGD). The aim of this study was to develop a valid and reproducible cleanliness scale for use during an EGD. METHODS: We developed a cleanliness scale (Barcelona scale) with a score (0-2 points) of five segments of the upper gastrointestinal tract with thorough cleaning techniques (esophagus, fundus, body, antrum, and duodenum). First, 125 photos (25 of each area) were assessed, and a score was assigned to each image by consensus among 7 experts endoscopists. Subsequently, 100 of the 125 images were selected and the inter- and intra-observer variability of 15 previously trained endoscopists was evaluated using the same images at two different times. RESULTS: In total, 1500 assessments were performed. In 1336/1500 observations (89%) there was agreement with the consensus score, with a mean kappa value of 0.83 (0.45-0.96). In the second evaluation, in 1330/1500 observations (89%) there was agreement with the consensus score, with a mean kappa value of 0.82 (0.45-0.93). The intra-observer variability was 0.89 (0.76-0.99). CONCLUSIONS: The Barcelona cleanliness scale is a valid measure and reproducible with minimal training. Its application in clinical practice is a significant step to standardize the quality of the EGD.

Combination treatment with monoclonal antibodies: Dupilumab and ustekinumab for the treatment of severe atopic dermatitis and Crohn disease.

Evidence of effectiveness and safety in combined therapies is scarce and based on case reports and small case series. We report a case of dual biologic therapy with ustekinumab and dupilumab in a patient with severe Crohn disease and atopic dermatitis. There was no interference between these drugs after a 7-month follow-up.

Total recovery of visual acuity in a pediatric patient with compressive optic neuropathy secondary to sphenoid sinus mucocele / Recuperação total da acuidade visual na neuropatia óptica compressiva secundária à mucocele do seio esfenoidal em paciente pediátrico

ABSTRACT We present an unusual case of a 13-year-old male pediatric patient with a diagnosis of sphenoid sinus mucocele. The patient suffered a progressive loss of visual acuity over three months followed by a total recovery of his visual acuity after surgery. The patient presented at the emergency room complaining of progressive loss of visual acuity in his left eye which decreased to hand motion over the preceding months. Imaging studies revealed a cystic mass, suggestive of sphenoid sinus mucocele, which was causing compressive optic neuropathy and proptosis. The patient was scheduled for a sphenoidectomy and resection of the mass. Three days after surgery, the patient's visual acuity in the left eye was 20/20, indicating complete recovery from his symptoms. We suggest that the excellent outcome in this patient may be attributable to his age. His ongoing physical development might have been the decisive factor in the recovery of his visual acuity following compressive optic neuropathy secondary to sphenoid sinus mucocele. Further research is needed to verify this proposed explanation.

RESUMO Apresentamos um caso incomum de paciente pediátrico com diagnóstico de mucocele de seio esfenoidal, que apresentou perda progressiva da acuidade visual ao longo de três meses, resultando em recuperação total da acuidade visual após a cirurgia. Paciente do sexo masculino, 13 anos, procurou o pronto-socorro, queixando-se de perda progressiva da acuidade visual do olho esquerdo nos últimos três meses. Exames de imagem revelaram uma massa cística sugestiva de mucocele de seio esfenoidal, causando neuropatia óptica compressiva e proptose. O paciente foi agendado para esfenoidectomia e ressecção da massa. Três dias após a cirurgia, a acuidade visual do paciente no olho esquerdo era de 20/20, apresentando recuperação completa dos sintomas. Diante dos resultados de nosso paciente, sugerimos que a idade do paciente pode ser decisiva na recuperação da acuidade visual de uma neuropatia óptica compressiva secundária à mucocele de seio esfenoidal. Mais pesquisas são necessárias para verificação desses dados.

STRUGGLE FOR. Italian-Polish-Spanish-Uzbek-Vietnamese Expert Forum Position Paper 2023 for better control of classical modifiable risk factors in clinical practice.

The progress in pharmacotherapy that has been made in recent years, including the introduction of very effective and safe lipid-lowering and antihypertensive drugs, has not yet translated into the expected universal control of blood pressure, lipid disorders and diabetes. In the STRUGGLE FOR Italian- -Polish-Spanish-Uzbek-Vietnamese Expert Forum Position Paper 2023, experts from five countries recounted several points about the paradigms of cardiological and cardiometabolic care for better control of classical modifiable risk factors in the year 2023. It is believed herein, that the need to intensify treatment, actively search for patients with cardiovascular risk factors, especially with arterial hypertension, hypercholesterolemia and diabetes, should go hand in hand with the implementation of the latest therapy, based on single pill combinations including proven, effective antihypertensive, lipid-lowering and antidiabetic molecules, many of which are listed in the present document. There is a need to use both new technological concepts, completely new drugs, as well as novel treatment concepts such as metabolic treatment in coronary artery disease, try to intensify the fight against smoking in every way, including the available range of drugs and procedures reducing the harm. This approach will provide substantially better control of the underlying cardiovascular risk factors in countries as varied as Italy, Poland, Spain, Uzbekistan and Vietnam.

Reductive carboxylation epigenetically instructs T cell differentiation.

Protective immunity against pathogens or cancer is mediated by the activation and clonal expansion of antigen-specific naive T cells into effector T cells. To sustain their rapid proliferation and effector functions, naive T cells switch their quiescent metabolism to an anabolic metabolism through increased levels of aerobic glycolysis, but also through mitochondrial metabolism and oxidative phosphorylation, generating energy and signalling molecules1-3. However, how that metabolic rewiring drives and defines the differentiation of T cells remains unclear. Here we show that proliferating effector CD8+ T cells reductively carboxylate glutamine through the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2). Notably, deletion of the gene encoding IDH2 does not impair the proliferation of T cells nor their effector function, but promotes the differentiation of memory CD8+ T cells. Accordingly, inhibiting IDH2 during ex vivo manufacturing of chimeric antigen receptor (CAR) T cells induces features of memory T cells and enhances antitumour activity in melanoma, leukaemia and multiple myeloma. Mechanistically, inhibition of IDH2 activates compensating metabolic pathways that cause a disequilibrium in metabolites regulating histone-modifying enzymes, and this maintains chromatin accessibility at genes that are required for the differentiation of memory T cells. These findings show that reductive carboxylation in CD8+ T cells is dispensable for their effector response and proliferation, but that it mainly produces a pattern of metabolites that epigenetically locks CD8+ T cells into a terminal effector differentiation program. Blocking this metabolic route allows the increased formation of memory T cells, which could be exploited to optimize the therapeutic efficacy of CAR T cells.

LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson's disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 µM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, -58%) and PTEN induced putative kinase 1 (PINK1, -49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C.

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbß, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C.

An HIV-1/HIV-2 Chimeric Envelope Glycoprotein Generates Binding and Neutralising Antibodies against HIV-1 and HIV-2 Isolates.

The development of immunogens that elicit broadly reactive neutralising antibodies (bNAbs) is the highest priority for an HIV vaccine. We have shown that a prime-boost vaccination strategy with vaccinia virus expressing the envelope glycoprotein gp120 of HIV-2 and a polypeptide comprising the envelope regions C2, V3 and C3 elicits bNAbs against HIV-2. We hypothesised that a chimeric envelope gp120 containing the C2, V3 and C3 regions of HIV-2 and the remaining parts of HIV-1 would elicit a neutralising response against HIV-1 and HIV-2. This chimeric envelope was synthesised and expressed in vaccinia virus. Balb/c mice primed with the recombinant vaccinia virus and boosted with an HIV-2 C2V3C3 polypeptide or monomeric gp120 from a CRF01_AG HIV-1 isolate produced antibodies that neutralised >60% (serum dilution 1:40) of a primary HIV-2 isolate. Four out of nine mice also produced antibodies that neutralised at least one HIV-1 isolate. Neutralising epitope specificity was assessed using a panel of HIV-1 TRO.11 pseudoviruses with key neutralising epitopes disrupted by alanine substitution (N160A in V2; N278A in the CD4 binding site region; N332A in the high mannose patch). The neutralisation of the mutant pseudoviruses was reduced or abolished in one mouse, suggesting that neutralising antibodies target the three major neutralising epitopes in the HIV-1 envelope gp120. These results provide proof of concept for chimeric HIV-1/HIV-2 envelope glycoproteins as vaccine immunogens that can direct the antibody response against neutralising epitopes in the HIV-1 and HIV-2 surface glycoproteins.

Use of Moses 2.0 with extended frequency and optimized Moses vs. high-power laser in MiniPCNL: a randomized controlled trial.

The aim of the study was to compare standard high-power laser (with < 80 Hz) to extended frequency (> 100 Hz) lithotripsy during miniPCNL. 40 patients were randomized in to two groups undergoing MiniPCNL. For both groups, the Holmium Pulse laser Moses 2.0 (Lumenis) was used. For group A, standard high-power laser with < 80 Hz, with Moses distance was set using up to 3 J. For Group B, extended frequency (100-120 Hz) was used allowing up to 0.6 J. All patients underwent MiniPCNL using an 18 Fr balloon access. Demographics were comparable between groups. Mean stone diameter was 19 mm (14-23) with no differences between groups (p = 0.14). Mean operative time was 91 and 87 min for group A and B (p = 0.71), mean laser time was similar in both groups, 6.5 min and 7.5 min, respectively (p = 0.52) as well as the number of laser activations during the surgery (p = 0.43). Mean Watts used was 18 and 16 respectively being similar in both groups (p = 0.54) as well as the total KJoules (p = 0.29). Endoscopic vision was good in all surgeries. The endoscopic and radiologic stone free rate was achieved in all patients expect for two in both groups (p = 0.72). Two Clavien I complications were seen, a small bleeding for group A and a small pelvic perforation in group B. The use of high-power holmium laser with extended frequency and optimized Moses was effective and safe being comparable to standard high-power laser for MiniPCNL allowing more versatility with the setting range.

Bowel Perforation after Extracorporeal Wave Lithotripsy: A Review of the Literature.

INTRODUCTION: Extracorporeal wave lithotripsy (ESWL) is considered a first-line treatment for renal and ureteral stones up to 10-20 mm in diameter. Complications are uncommon, with a reported rate of 0-6% in the literature. Bowel perforation has only been described in a few case reports but requires rapid diagnosis and treatment. METHODS: A review of the literature from PubMed/Medline, Embase, Cochrane, and Web of Science databases was performed including studies reporting bowel perforation secondary to ESWL between January 1990 and June 2022. RESULTS: We found 16 case reports of intestinal perforation in the literature. Although some patients had previously undergone abdominal surgery or had inflammatory intestinal disease, others were without comorbidities that could lead to complications. Abdominal pain was the main symptom and imaging was required to confirm the diagnosis, which usually necessitated a surgical intervention. As regards the ESWL technique, it appears that the combination of a high energy level and the prone position constitutes a risk factor for these rare complications. At the authors' centre, only one case has been reported among 24,000 ESWL procedures over 20 years: A 59-year-old female who underwent ESWL for a distal right ureteral stone presented acute abdominal pain and free intraperitoneal pelvic fluid on ultrasound. A CT scan revealed a small bowel perforation requiring open laparotomy with primary closure. CONCLUSIONS: In conclusion, although bowel perforation after ESWL is rare, progressive abdominal pain with tenderness at physical examination requires proper imaging evaluation to exclude bowel perforation and prompt intervention if required.

First Trimester Evaluation of Maternal Visceral Fat and Its Relationship with Adverse Pregnancy Outcomes.

Obese women are more likely to experience pregnancy complications. The distribution of fat, and more particularly the rise in visceral fat, is well established to be more closely linked to the onset of cardiovascular disease and metabolic syndrome than obesity itself. We aim to examine the relationship between maternal visceral fat assessment in the first trimester and the appearance of adverse pregnancy outcomes. A prospective cohort study including 416 pregnant women was conducted. During the first trimester scan (11-13 + 6 weeks), all individuals had their visceral fat and subcutaneous thicknesses measured by ultrasonography. Blood samples were obtained, and maternal demographics and clinical information were documented. After delivery, the obstetric outcomes were evaluated. We contrasted two groups: one with healthy pregnancies and the other with adverse pregnancy outcomes (APO), defined as the development of at least one of the following complications: gestational diabetes mellitus, hypertensive disorders of pregnancy, abnormal fetal growth, preterm delivery or preterm premature rupture of membranes. Median maternal age was 33 and 34 years old for the uncomplicated and adverse pregnancy outcomes groups, respectively. We found that women with adverse pregnancy outcomes had higher VFT (median 30 vs. 26.5 mm, p = 0.001) and SFT (median 18.9 vs. 17.1 mm, p = 0.03). However, the visceral/subcutaneous fat ratio was not statistically different between groups. Finally, we performed a subanalysis for metabolic and placental vascular dysfunction complications. After performing a multivariate logistic regression analysis adjusted for maternal age, smoking, and mean arterial pressure, both the VFT (aOR 1.03, p < 0.001) and the ratio of visceral/subcutaneous fat (aOR 1.37, p = 0.04) were significantly associated with the development of adverse pregnancy outcomes; however, the associations of VFT and the VFT-to-SFT ratio were higher for the occurrence of gestational diabetes (aOR 1.07, p < 0.001; aOR 2.09, p = 0.001; respectively) and showed no relationships with placental complications. When conducting a first-trimester ultrasound assessment, sonographers may measure VFT without additional time or cost involved. Identification of pregnant women with increased VFT (>37 mm) may benefit from a close follow-up, especially for the development of gestational diabetes, independent of BMI.

Incidence of hypertension in young transgender people after a 5-year follow-up: association with gender-affirming hormonal therapy.

In order to assess the risk of hypertension development, we performed a retrospective analysis of the clinical records of consecutive transgender patients who began gender-affirming hormonal therapy in our Outpatient Gender Identity Clinic with <30 years of age and had a follow-up >5 years. 149 transgender women treated with estradiol and 153 transgender men treated with testosterone were included; 129 of the transgender women received also androgen blockers (54 spironolactone, 49 cyproterone acetate and 26 LHRH agonists). The annual incidence of hypertension in young transgender men (1.18%) seemed comparable to that of the general population. In young transgender women, it seemed higher (2.14%); we found that the choice of androgen blocker had a remarkable effect, with a highly significant increase in patients treated with cyproterone acetate (4.90%) vs. the rest (0.80%); the adjusted hazard-ratio was 0.227 (p = 0.001). Correlation, logistic regression and mediation analyses were performed for the associations of the available clinical variables with the increase in systolic blood pressure and the onset of hypertension, but besides the use of cyproterone acetate, only the ponderal gain was found significant (Spearman's r: 0.361, p < 0.001); with a 36.7% mediation effect (31.2-42.3%). Cyproterone acetate has additional known risks, such as meningioma; although we cannot conclusively prove that it has a role in the development of hypertension, we conclude that the use of cyproterone acetate for this indication should be reconsidered.

Total recovery of visual acuity in a pediatric patient with compressive optic neuropathy secondary to sphenoid sinus mucocele.

We present an unusual case of a 13-year-old male pediatric patient with a diagnosis of sphenoid sinus mucocele. The patient suffered a progressive loss of visual acuity over three months followed by a total recovery of his visual acuity after surgery. The patient presented at the emergency room complaining of progressive loss of visual acuity in his left eye which decreased to hand motion over the preceding months. Imaging studies revealed a cystic mass, suggestive of sphenoid sinus mucocele, which was causing compressive optic neuropathy and proptosis. The patient was scheduled for a sphenoidectomy and resection of the mass. Three days after surgery, the patient's visual acuity in the left eye was 20/20, indicating complete recovery from his symptoms. We suggest that the excellent outcome in this patient may be attributable to his age. His ongoing physical development might have been the decisive factor in the recovery of his visual acuity following compressive optic neuropathy secondary to sphenoid sinus mucocele. Further research is needed to verify this proposed explanation.

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals.

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4+ T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4+ T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

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

[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].