High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

IL-31-dependent neurogenic inflammation restrains cutaneous type 2 immune response in allergic dermatitis.

Despite robust literature associating IL-31 with pruritic inflammatory skin diseases, its influence on cutaneous inflammation and the interplay between inflammatory and neurosensory pathways remain unmapped. Here, we examined the consequences of disrupting Il31 and its receptor Il31ra in a mouse model of house dust mite (HDM)-induced allergic dermatitis. Il31-deficient mice displayed a deficit in HDM dermatitis-associated scratching, consistent with its well-established role as a pruritogen. In contrast, Il31 deficiency increased the number and proportion of cutaneous type 2 cytokine-producing CD4+ T cells and serum IgE in response to HDM. Furthermore, Il4ra+ monocytes and macrophages capable of fueling a feedforward type 2 inflammatory loop were selectively enriched in Il31ra-deficient HDM dermatitis skin. Thus, IL-31 is not strictly a proinflammatory cytokine but rather an immunoregulatory factor that limits the magnitude of type 2 inflammatory responses in skin. Our data support a model wherein IL-31 activation of IL31RA+ pruritoceptors triggers release of calcitonin gene-related protein (CGRP), which can mediate neurogenic inflammation, inhibit CD4+ T cell proliferation, and reduce T cell production of the type 2 cytokine IL-13. Together, these results illustrate a previously unrecognized neuroimmune pathway that constrains type 2 tissue inflammation in the setting of chronic cutaneous allergen exposure and may explain paradoxical dermatitis flares in atopic patients treated with anti-IL31RA therapy.

Conservative treatment of Head and Neck hemangiomas using Intralesional Bleomycin.

Background: Hemangiomas are vascular tumours and commonly occur in head and neck region. Complete treatment of these vascular swellings are often challenging. As surgical excision carries high risk of complications and poor cosmetic outcome, treatment is shifted more towards non-surgical modalities like steroid therapy, radiotherapy, chemotherapy and sclerotherapy. Objective: To study the efficacy of intralesional Bleomycin sclerotherapy for head and neck hemangiomas. Method: Seventeen patients ( 9 males and 8 females) with head and neck hemangiomas were treated with sclerotherapy using intralesional bleomycin injection ( 0.1-0.5 mg/kg/ dose) in a medical college of India. Details of the patients were recorded along with serial photographs of lesion. Post injection outcomes in form of reduction of lesion size, patient satisfaction and complications were also recorded. Results: 11 patients were completely cured, 3 had more than 50% reduction in size, one patient showed minimal reduction and two had no response. One patient had superficial ulceration, one case of slough and fever was reported. None of the patients developed severe toxic side effects and pulmonary fibrosis with follow up of 18 months. Conclusions: Bleomycin injection is safe, easy and well tolerated method for treatment of head and neck hemangiomas. We recommend surgery only if there is no response to other conservative treatment modalities.

A Comparative Evaluation of Soft and Hard Tissue Changes Around Dental Implants Placed With and Without Platelet-Rich Fibrin.

BACKGROUND: A patient's ability to maintain a healthy bone-implant interface seems to be a major predictor of implant longevity over the long term. The implant surface is protected from the oral environment, the bone, and the implant itself by the peri-implant tissues. Platelet-rich fibrin (PRF) has been shown to help in the regeneration of bone and other connective tissues. Since there has been inadequate information on the role of PRF in maintaining soft tissue integrity and crestal bone changes, the present study aimed to evaluate these challenges clinically and radiographically in human patients who had dental implants placed with PRF. MATERIALS AND METHODS: There were a total of 15 patients who were recalled for the analysis, and they were split into two groups. PRF was used to complete the implant procedure in the experimental group, but PRF was not used in the control group. Cone beam computed tomography (CBCT) was used to evaluate the amount of alveolar bone prior to dental implant placement and intra-oral periapical radiograph (IOPAR) for postoperative assessment. Gingival index, plaque index, probing depths, papilla bleeding index, and crestal bone changes were used to document clinical limits. IOPAR using a similar approach was used to evaluate the crestal bone level alterations. Patients were evaluated clinically and radiographically for changes in the peri-implant soft tissue and crestal bone during implant placement, six and nine months postoperatively. RESULTS: From baseline (p=0.02) to six months (p=0.04) and nine months (p=0.04), both groups showed changes in crestal bone loss and soft tissue although the changes in the test group were smaller. Soft tissue changes showed significant differences for probing depth and papilla index score at baseline and at the end of the six and nine months (p<0.05), whereas no significant difference was noted with bleeding index and plaque index score during the follow-up (p>0.05). CONCLUSION:  To conclude, the provided data demonstrated that the local injection of PRF during implant placement has the potential to favorably stimulate bone formation, and may be used as a therapeutic adjuvant in the clinical setting of implant placement.

Association of the Val66Met polymorphism of the BDNF gene and the deletional mutation of CYP2D6 gene with the prevalence and severity of depressive disorder in an Eastern Indian population.

Background: The Val66Met single nucleotide polymorphism (SNP) of the brain-derived growth factor (BDNF) and deletional mutation of the cytochrome P4502D6 (CYP2D6) have been reported to be linked to the etiology and severity of depressive disorders (DD) in a variable manner among different ethnicities and populations. Aims: The present study was aimed to find the relationship of mutational variations of these two neurotrophins with the severity of DD and their serum cortisol levels as a marker of the stress factor. Methods: In 104 drug-naïve newly diagnosed cases of DD and 106 control subjects, the severity of depression was assessed using the HAM-D score. Val66Met SNP of the BDNF was analyzed in them using restriction digestion of its polymerase chain reaction (PCR) product. CYP2D6 deletional variants were detected by the absence of their PCR products. Serum cortisol levels were measured by the enzyme-linked immunosorbent assay (ELISA) technique. Results: The Chi-square test (Χ2 = 1.42, P = 0.49) did not show any higher prevalence of Val66Met SNP of the BDNF gene in the case group. A correlation coefficient (R) of -0.14 for HAM-D score with a P value of 0.29 signified no direct link of the severity of DD with this SNP. However, a Χ2 of 12.68 with P < 0.001 indicated a significantly higher prevalence of the CYP2D6 deletional mutants in DD cases, whereas an R-value of 0.39 for HAM-D score with P < 0.001 suggested a significantly higher severity of DD having with them. Serum cortisol level showed a significant positive correlation with the deletional variants of CYP2D6 (R = 0.198, P = 0.04) and the HAM-D score (R = 0.22, P = 0.025). Conclusion: We conclude that CYP2D6 deletion significantly contributes to the severity and stress factor in the DD patients in our study population. Early identification of these mutations may provide important molecular and cellular predisposition for the disease and may lay the ground for possible more effective measures of intervention.

Impact of COVID Lockdown on the Presentation of Nasal Foreign Bodies to a Tertiary Care Center.

BACKGROUND: India announced nationwide lockdown on March 24, 2020, to control the COVID crisis. Due to lockdown, the health care system, that is, delivery and utilization of the health facilities were adversely affected. CLINICAL SETTING: Presentation and management of nasal foreign body cases over 3 months post COVID lockdown has been discussed. Factors for delayed diagnosis and management are assessed and compared with pre-COVID era. RESULT/OUTCOME: Due to lockdown and COVID phobia, more complicated cases of nasal foreign body are presented in post lockdown period. CONCLUSION: We need to formulate and standardize the management strategies to avoid such unfortunate circumstances so that even non-COVID cases are managed more appropriately and in a timely manner.

Varied Clinical Presentation and Management of Paediatric Vallecular Cyst.

The current article aims to highlight the varied presentation and management of vallecular cysts. We report three children, aged four years, 11 months and three days, diagnosed with vallecular cyst presenting to the Chacha Nehru Bal Chikitsalaya hospital, New Delhi, India in 2018 and 2019. They were reviewed retrospectively for clinical presentation, diagnostic tools and treatment options. All three cases had respiratory and feeding difficulties. The diagnosis in all of the three cases was made with laryngoscopy (flexible/direct) and imaging. All three patients were treated with the transoral approach aimed at the excision of the cyst using cold instruments. The postoperative period was uneventful. No recurrences were observed during the long-term follow-up. Vallecular cysts should be considered as one of the differentials in children with respiratory distress and dysphagia despite being a rare anomaly. A direct transoral approach is recommended for the excision of the vallecular cyst as it is a safe and reliable method with no recurrences to date.

MicroRNA-directed pathway discovery elucidates an miR-221/222-mediated regulatory circuit in class switch recombination.

MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222-deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.

The Presentation and Management of Facial Artery Pseudoaneurysm: A Review of the Literature.

Pseudoaneurysm is a rare vascular complication of trauma, causing an incomplete tear of the vessel wall. We present a clinical case report arising from the distal branch of facial artery in an infant. Facial artery pseudoaneurysm is a rare complication of facial trauma and can easily be misdiagnosed especially in the paediatric age group. Prompt investigation and diagnosis with timely and apt intervention is the key to the successful management of facial artery pseudoaneurysm.

Seaweed diversity within intertidal zone of Olaikuda and Vadakkadu, Rameshwaram, southeast coast of India.

Seasonal variation of seaweed diversity was studied in Olaikuda and Vadakkadu, the two unexplored coastal areas in Rameshwaram Island in south coast of India. An extensive survey on seasonal changes of seaweed diversity was made study and the data were analyzed for different diversity indices. In each location, 3 transects at 100 m gap were taken and in each transect 10 quadrates, a total of 30 quadrates were taken for seaweeds diversity. The diversity data were analyzed with statistical methods such as graphical and statistical analysis K-dominance curve with software PRIMER package, cluster analysis, multivariate methods, anosim and diversity indices such as Shannon Weaver diversity index, Simpson species richness index, Fisher alpha index, Pielou's evenness, and taxonomic species diversity (delta) index were analyzed by using the software R statistic version 3.1.4. Package (vegan). Totally 74 species including 28 species of Chlorophyceae, 18 species of Phaeophyceae and 28 species of Rhodophyceae were recorded. The study areas are flourishing with 49-59 seaweed species. It is inferred that the seaweeds are well diversified seasonally except summer. The baseline data generated in this work will help in conservation of the seaweeds in the areas.

The Effects of Altered Membrane Cholesterol Levels on Sodium Pump Activity in Subclinical Hypothyroidism.

BACKGROUND: Metabolic dysfunctions characteristic of overt hypothyroidism (OH) start at the early stage of subclinical hypothyroidism (SCH). Na⁺/K⁺-ATPase (the sodium pump) is a transmembrane enzyme that plays a vital role in cellular activities in combination with membrane lipids. We evaluated the effects of early changes in thyroid hormone and membrane cholesterol on sodium pump activity in SCH and OH patients. METHODS: In 32 SCH patients, 35 OH patients, and 34 euthyroid patients, sodium pump activity and cholesterol levels in red blood cell membranes were measured. Serum thyroxine (T4) and thyroid stimulating hormone (TSH) levels were measured using enzyme-linked immunosorbent assays. Differences in their mean values were analysed using post hoc analysis of variance. We assessed the dependence of the sodium pump on other metabolites by multiple regression analysis. RESULTS: Sodium pump activity and membrane cholesterol were lower in both hypothyroid groups than in control group, OH group exhibiting lower values than SCH group. In SCH group, sodium pump activity showed a significant direct dependence on membrane cholesterol with an inverse relationship with serum TSH levels. In OH group, sodium pump activity depended directly on membrane cholesterol and serum T4 levels. No dependence on serum cholesterol was observed in either case. CONCLUSION: Despite the presence of elevated serum cholesterol in hypothyroidism, membrane cholesterol contributed significantly to maintain sodium pump activity in the cells. A critical reduction in membrane cholesterol levels heralds compromised enzyme activity, even in the early stage of hypothyroidism, and this can be predicted by elevated TSH levels alone, without any evident clinical manifestations.

Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation.

In the present study an attempt has been made to interrogate the bulk secondary structures of some selected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3M, 6M, 9M) and GnHCl (2M, 4M, 6M) and the corresponding steady state autofluorescence spectra were recorded at 281nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using "Scratch protein predictor" at 30% threshold). Further, the influence of rigidity and conformation of the indole ring (caused by protein secondary structures) on the intrinsic fluorescence properties of proteins were also evaluated using fluorescence of ANS-HSA complexes, CD spectroscopy as well as with trypsin digestion experiments. The outcomes obtained clearly demonstrated GnHCl preferably disrupt helix as compared to the beta ß-sheets whereas, urea found was more effective in disrupting ß-sheets as compared to the helices. The other way round the proteins which have shown detectable change in the intrinsic fluorescence at lower concentrations of GnHCl were rich in helices whereas, the proteins which showed detectable change in the intrinsic fluorescence at lower concentrations of urea were rich in ß-sheets. Since high salt concentrations like GnHCl and urea interfere in the secondary structure analysis by circular dichroism Spectrometry, the present method of analyzing secondary structures using laser induced autofluorescence will be highly advantageous over existing tools for the same.

Protective Role of Bilirubin Against Increase in hsCRP in Different Stages of Hypothyroidism.

In conjunction with thyroxine, bilirubin may play an important role for regulation of hsCRP level and a consequent pro-inflammatory condition in hypothyroidism. In present study we evaluated the dependence of hsCRP changes on total bilirubin (BT) and fT4 level in thirty overt (OH) and thirty subclinical hypothyroidism (SH). Serum BT, hsCRP, thyroxine and TSH were measured in both groups and compared with forty control subjects. Serum values of TSH, hsCRP showed raised (P < 0.001 for both) values with lower levels for fT4 and BT (P < 0.001 and 0.03 respectively) in hypothyroid patients compared to the controls. ANOVA showed significant increments in TSH and hsCRP values with decreases in fT4 among the control, SH and OH groups respectively (P < 0.001). BT values showed decrease in OH group only in comparison to controls (P = 0.04). Regression analysis revealed that hsCRP was negatively dependent on fT4 (ß = -0.35, P = 0.002) and serum bilirubin (ß = -0.40 and P < 0.001 respectively). Univariate general linear model analysis showed this dependence persisted even when carried out distinctly in SH and OH groups separately (P < 0.001). TSH did not show any significant predictive value on the hsCRP level in either of these two tests. From these analyses we suggest that serum hsCRP is closely integrated to a lowered synthesis of bilirubin and fT4 in hypothyroid patients. Furthermore, this causal relationship is not only limited to overt but also extends to the SH.

Role of membrane cholesterol and lipid peroxidation in regulating the Na+/K+-ATPase activity in schizophrenia.

BACKGROUND: Na+/K+-ATPase (NKA) activity is compromised in several neuropsychiatric disorders. Oxidative stress and membrane lipid composition play important roles in regulating NKA activity. AIMS: The present study was undertaken to evaluate the effects of oxidative stress-induced membrane lipid damage and membrane cholesterol composition on NKA pump activity in schizophrenia. SETTINGS AND DESIGN: It was a hospital-based, cross-sectional, observational study in 49 cases and 51 controls for 1 year. MATERIALS AND METHODS: NKA pump activity in red blood cell membrane, serum levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC) adducts, and cholesterol were measured by standard spectrophotometric techniques in newly diagnosed schizophrenia patients by Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision criteria. Membrane cholesterol was analyzed by chloroform and isopropanol extraction followed by measuring the cholesterol concentration by spectrophotometric technique. STATISTICAL ANALYSIS AND RESULTS: Mean values for NKA pump activity, membrane cholesterol level, and serum cholesterol levels were significantly lower in the case group (P < 0.001). The activity of NKA pump was found to be directly correlated to membrane cholesterol level rather than with the serum cholesterol values. Although the NKA pump activity showed inverse relationship with the serum values of TBARS and PC products both, on multiple linear regression analysis, it was found to be significantly positively dependent on the membrane cholesterol (ß = 0.268, P = 0.01) and negatively dependent on the serum TBARS (ß = -0.63, P < 0.001) levels only. CONCLUSION: Reduced membrane cholesterol and oxidative stress-induced damage to membrane lipids play crucial roles in decreasing the NKA activity in schizophrenia. Hence, for a better prognosis and treatment, measures are required to maintain optimum levels of cholesterol in neuronal tissues along with a proper control on oxidative stress.

Organocatalytic kinetic resolution of racemic secondary nitroallylic alcohols combined with simultaneous desymmetrization of prochiral cyclic anhydrides.

This study describes an organocatalytic kinetic resolution of racemic secondary nitroallylic alcohols (2) combined with simultaneous desymmetrization of prochiral cyclic anhydrides (1). The experimental results revealed that enantioselective alcoholysis of 3-substituted glutaric anhydrides afforded hemiesters (3) with high levels of enantioselectivities (up to 99% ee) in the presence of cinchonidine-derived thiourea catalyst (IV). The highly optical enrichment (up to 95% ee) of (S)-nitroallylic alcohols (2) was recovered.

Comparison of urinary and serum CA 19-9 as markers of early stage urothelial carcinoma.

OBJECTIVES: Although the glycoprotein group tumor marker CA 19-9 has been detected in both serum and urine of bladder cancer patients, information about their comparative role in screening of low grade transitional cell carcinoma (LGTCC) and high grade transitional cell carcinoma (HGTCC) is rare. MATERIALS AND METHODS: In this study we measured both the urinary and serum levels of CA 19-9 in 35 LGTCC and 20 HGTCC patients by ELISA and determined the cut off value of both urinary and serum CA 19-9 levels by receiver operator characteristic curve (ROC) for both patient groups. Odds ratio (OR) for CA 19-9 was analyzed with its range at 95% confidence interval to analyze the role of this tumor marker as a screening parameter for both of these cancer types. RESULTS: For urinary CA 19-9 the OR was 20.16 with an interval of 4.91-82.71 whereas for the serum CA 19-9 it was 7.5 with an interval of 2.28-24.62. CONCLUSIONS: From these data we suggest that urinary CA 19-9 is a better screening parameter with optimum sensitivity and specificity than its serum counterpart for diagnosis of low grade and early stages of transitional cell carcinoma of urinary bladder. Furthermore, it can be suggested that urinary CA 19-9 can be used as better prognostic marker for LGTCC than its serum counterpart.

Comparison of Urinary and Serum CA 19-9 as Markers of Early Stage Urothelial Carcinoma

Objectives Although the glycoprotein group tumor marker CA 19-9 has been detected in both serum and urine of bladder cancer patients, information about their comparative role in screening of low grade transitional cell carcinoma (LGTCC) and high grade transitional cell carcinoma (HGTCC) is rare. Materials and Methods In this study we measured both the urinary and serum levels of CA 19-9 in 35 LGTCC and 20 HGTCC patients by ELISA and determined the cut off value of both urinary and serum CA 19-9 levels by receiver operator characteristic curve (ROC) for both patient groups. Odds ratio (OR) for CA 19-9 was analyzed with its range at 95% confidence interval to analyze the role of this tumor marker as a screening parameter for both of these cancer types. Results For urinary CA 19-9 the OR was 20.16 with an interval of 4.91-82.71 whereas for the serum CA 19-9 it was 7.5 with an interval of 2.28-24.62. Conclusions From these data we suggest that urinary CA 19-9 is a better screening parameter with optimum sensitivity and specificity than its serum counterpart for diagnosis of low grade and early stages of transitional cell carcinoma of urinary bladder. Furthermore, it can be suggested that urinary CA 19-9 can be used as better prognostic marker for LGTCC than its serum counterpart. .

Skull base chordoma presenting as nasopharyngeal mass with lymph node metastasis.

Spheno-occipital chordomas can rarely present as nasopharyngeal mass. Metastases occur only in advanced disease. They can pose a diagnostic dilemma when information about diagnosis of the primary tumor is not available. We present cytological findings in upper cervical lymph node of a case of nasopharyngeal chordoma and discuss possible differential in such a location.

Three-component organocascade kinetic resolution of racemic nitroallylic acetates via sequential iminium/enamine asymmetric catalysis.

Nitroallylic acetates 1a-f have been kinetically resolved via an asymmetric three-component coupling that involves indoles, acrolein, and nitroolefin allylic acetates and is mediated by the chiral catalyst 2 (5 mol %). The reactions proceed via iminium/enamine cascade catalysis. Both recovered starting substrates and reaction products are typically obtained in high chemical yield and in good to excellent enantiopurity (79-95% ee for 1a-f and 83-99% ee for 3a-n). For the first time, a highly efficient three-component, organocascade kinetic resolution has been demonstrated.