Organ Donation after Circulatory Determination of Death in India: A Joint Position Paper.

Organ donation following circulatory determination of death (DCDD) has contributed significantly to the donor pool in several countries. In India, majority of deceased donations happen following brain death (BD). While existing legislation allows for DCDD, there have been only few reports of kidney transplantation following DCDD from India. This document, prepared by a multidisciplinary group of experts, reviews international best practices in DCDD and outlines the path for DCDD in India. Ethical, medical, legal, economic, procedural, and logistic challenges unique to India have been addressed. The practice of withdrawal of life-sustaining treatment (WLST) in India, laid down by the Supreme Court of India, is time-consuming, possible only in patients in a permanent vegetative state, and too cumbersome for day-to-day practice. In patients where continued medical care is futile, the procedure for WLST is described. In controlled DCDD (category-III), decision for WLST is independent of and delinked from the subsequent possibility of organ donation. Families that are inclined toward organ donation are explained the procedure including the timing and location of WLST, consent for antemortem measures, no-touch period, and the possibility of stand-down and return to the intensive care unit (ICU) without donation. In donation following neurologic determination of death (DNDD), if cardiac arrest occurs during the process of BD declaration, the protocol for DCDD category-IV has been described in detail. In DCDD category-V, organ donation may be possible following unsuccessful cardiopulmonary resuscitation of cardiac arrest in the ICU. An outline of organ-specific requisites for kidney, liver, heart, and lung transplantation following DCDD and techniques, such as normothermic regional perfusion (nRP) and ex vivo machine perfusion, has been provided. The outcomes of transplantation following DCDD are comparable to those following DBDD or living donor transplantation. Documents and checklists necessary for successful execution of DCDD in India are described. How to cite this article: Seth AK, Mohanka R, Navin S, Gokhale AGK, Sharma A, Kumar A, et al. Organ Donation after Circulatory Determination of Death in India: A Joint Position Paper. Indian J Crit Care Med 2022;26(4):421-438.

Critical amino acid variations in HLA-DQB1* molecules confers susceptibility to Autoimmune Thyroid Disease in south India.

The HLA-DQB1* region exhibits complex associations with autoimmune thyroid disease (AITD). AITD patients (Hashimoto's thyroiditis, HT = 180; Graves' disease, GD = 55) and age/sex matched controls (n = 235) were genotyped for DQB1* alleles by PCR-SSP. Alleles DQB1*02:02, *06:03, *06:09, *03:02, and *03:03 showed an increased risk and *02:01, *05:02, and *06:02 showed a protection toward AITD. Multiple sequence alignment was used to find out the amino acid variations within the peptide-binding pockets of susceptible and/or protective DQB1* alleles. We observed susceptible associations for amino acids 'Glu86(P < 0.0007)' and 'Leu87(P < 3.8 × 10-4)' in P1, 'Leu26(P < 4.0 × 10-12)' in P4, 'His9(P < 5.0 × 10-4)' and 'Ala57(P < 3.6 × 10-4)' in P9 toward HT; and 'Gly86(P < 0.0004)' in P1 and 'Asp57(P < 1.9 × 10-4)' in P9 towards GD. Protective associations were observed for amino acids 'Ala86(P < 8.2 × 10-6)' and 'Tyr87(P < 0.0003)' in P1, 'Gly26(P < 4.9 × 10-5)' and 'Ser74(P < 4.9 × 10-5)' in P4, 'Phe9(P < 0.0007)' and 'Ser57(P < 0.0016)' in P9 towards HT. Thus, the present study revealed that DQB1* alleles and putative amino acid residues play an important role in susceptibility toward AITD in south India.

Comparative Study of Structural, Morphological, Magneto-Optical and Photo-Catalytic Properties of Magnetically Reusable Spinel MnFe2O4 Nano-Catalysts.

Spinel MnFe2O4 nanostructures were synthesized by simple, economical and eco-friendly microwave combustion (MCM) and conventional combustion (CCM) methods using metal nitrates and glycine used as the fuel, instead of toxic inorganic/organic catalyst, template and surfactant. Powder XRD and FT-IR, EDX and SAED results were confirmed the products have a cubic phase spinel structure. EDX and SAED results confirmed purity and high crystallinity without any other secondary phase impurities. HR-SEM and HR-TEM analysis indicate that the MCM and CCM products consist of nano- and microstructures, respectively. The optical band gap (Eg) was measured using Kubelka-Munk model and it shows higher value (2.37 eV) for MnFe2O4-MCM than MnFe2O4-CCM (2.15 eV), due to the smaller particle size of MnFe2O4-MCM. VSM results showed a superparamagnetic behavior and the magnetization (Ms) value of MnFe2O4-MCM is higher i.e., 39.68 emu/g than MnFe2O4-CCM (33.59 emu/g). It was found that the sample MnFe2O4-MCM have higher surface area than MnFe2O4-CCM, which in turn leads to the improved performance towards the photocatalytic degradation (PCD) of methylene blue (MB) and it was found that the sample MnFe2O4-MCM show higher PCD efficiency (96.48%) than MnFe2O4-CCM (84.95%). Also, MnFe2O4 show higher activity with good reusability, and eco-friendly materials for industrial and technological applications.

Susceptible and protective associations of HLA DRB1*/DQB1* alleles and haplotypes with ischaemic stroke.

Stroke has emerged as the second commonest cause of mortality worldwide and is a major public health problem. For the first time, we present here the association of human leucocyte antigen (HLA)-DRB1*/DQB1* alleles and haplotypes with ischaemic stroke in South Indian patients. Ischaemic stroke (IS) cases and controls were genotyped for HLA-DRB1*/DQB1* alleles by polymerase chain reaction sequence-specific primers (PCR-SSP) method. The frequencies of HLA class II alleles such as DRB1*04, DRB1*07, DRB1*11, DRB1*12, DRB1*13, DQB1*02 and DQB1*07 were high in IS patients than in the age- and gender-matched controls, suggesting that the individuals with these alleles are susceptible to ischaemic stroke in South India. The frequencies of alleles such as DRB1*03, DRB1*10, DRB1*14, DQB1*04 and DQB1*05 were less in IS cases than in the controls, suggesting a protective association. Haplotypes DRB1*04-DQB1*0301, DRB1*07-DQB1*02, DRB1*07-DQB1*0301, DRB1*11-DQB1*0301 and DRB1*13-DQB1*06 were found to be high in IS patients conferring susceptibility. The frequency of haplotype DRB1*10-DQB1*05 was high in controls conferring protection. IS-LVD and gender-stratified analysis too confirmed these susceptible and protective associations. Thus, HLA-DRB1*/DQB1* alleles and haplotypes strongly predispose South Indian population to ischaemic stroke. Further studies in different populations with large sample size or the meta-analysis are needed to explain the exact mechanism of associations of HLA gene(s) with IS.

Wilms' tumour 1 gene mutations in south Indian children with steroid-resistant nephrotic syndrome.

BACKGROUND & OBJECTIVES: Clinically, nephrotic syndrome (NS) is a diverse group of symptoms; about 20 per cent of NS cases are resistant to steroid treatment, and within ten years they progress to end-stage renal disease. The present study was undertaken to identify the mutations of Wilms' tumour 1 (WT1) gene in steroid-resistant NS (SRNS) children. METHODS: A total of 173 children with SRNS and 100 children in the control group were enrolled in the study. DNA extraction was done, screened for WT1 (exons 8 and 9) gene amplified by polymerase chain reaction and direct sequencing. Karyotype analyses were done for WT1 mutation cases. RESULTS: WT1 mutations were found in three of 173 SRNS cases (2 girls, 1 boy). All of them had intron 9 (IVS 9 + 4 C>T, 2; IVS + 5 G>A, 1) mutation. Of these three cases, one had familial and another two had sporadic history. Renal histology analysis showed two cases with focal segmental glomerulosclerosis (FSGS) and they had external female genitalia but 46,XY karyotype. Both of them had streak gonads. Of the three cases, one expired. INTERPRETATION & CONCLUSIONS: The findings of the present study indicate that all females with SRNS-FSGS should be screened for WT1 gene mutation to diagnose whether they have FS for possible gonadectomy.

RAPSTROM™ first-in-man study long-term results of a biodegradable polymer sustained-release sirolimus-eluting stent in de novo coronary stenoses.

BACKGROUND: Durable polymers used for first-generation drug-eluting stents (DES) potentially contribute to persistent inflammation and late DES thrombosis. We report the first real-life human experience with the rapamycin-eluting biodegradable polymer-coated Rapstrom stent. METHODS: All consecutive patients with single de novo native coronary stenosis (<30 mm and between 2.5 and 4.0 mm) were enrolled. Major adverse cardiac events (MACE) at 1 year (cardiac death, myocardial infarction [Q and non-Q], or ischemia-driven target lesion revascularization) were the primary end-point. RESULTS: A total of 123 patients were enrolled. The stent was implanted without complications in all patients, and no MACE were recorded at 30 days. At 12-month follow-up 9 patients (7.3%) experienced a MACE and 4 (3.2%) required a target lesion revascularization, while 1 (1%) stent thrombosis was recorded. A planned angiographic follow-up (FU) was performed in 73 patients (59%) at 9.4 ± 2.6 months following the index procedure. In-stent late loss was 0.16 ± 0.09 mm, and in-segment late loss was 0.18 ± 0.8 mm. CONCLUSION: The Rapstrom biodegradable polymer rapamycin-eluting stent appeared safe and efficacious in this first real-life human experience, due to a low late lumen loss. Larger randomized studies are required to confirm these preliminary results.

A major susceptibility locus for leprosy in India maps to chromosome 10p13.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is prevalent in India, where about half of the world's estimated 800,000 cases occur. A role for the genetics of the host in variable susceptibility to leprosy has been indicated by familial clustering, twin studies, complex segregation analyses and human leukocyte antigen (HLA) association studies. We report here a genetic linkage scan of the genomes of 224 families from South India, containing 245 independent affected sibpairs with leprosy, mainly of the paucibacillary type. In a two-stage genome screen using 396 microsatellite markers, we found significant linkage (maximum lod score (MLS) = 4.09, P < 2x10-5) on chromosome 10p13 for a series of neighboring microsatellite markers, providing evidence for a major locus for this prevalent infectious disease. Thus, despite the polygenic nature of infectious disease susceptibility, some major, non-HLA-linked loci exist that may be mapped through obtainable numbers of affected sibling pairs.

Distribution of virulence genes and biofilm characterization of human isolates of Streptococcus agalactiae: A pilot study.

This study included 21 newly isolated clinical samples of Streptococcus agalactiae (Group B Streptococcus) screened in patients (six male, fifteen female) from various states of India with different infections (urinary tract infections, blood, pus and eye infections). All isolates were identified as Group B Streptococcus (GBS) using hemolytic properties, serogrouping and MALDI-TOF-MS analysis. Six virulence genes, cfb (100%), cylE (90.4%), lmp (85.7%), bca (71.4%), rib (38%) and bac (4.7%) were detected via polymerase chain reaction (PCR). Distribution studies of these six genes revealed five isolates containing five virulence genes (23.8%), followed by ten isolates containing four virulence genes (47.6%). The twenty GBS isolates selected on the glass surface included non-biofilm producers (n = 6, 30%), weak (n = 11, 55%) and moderate biofilm producers (n = 3, 15%). On the polystyrene surface, weak (n = 4, 20%), moderate (n = 2, 10%) and strong (n = 14, 70%) biofilm producers were detected. Live-dead cell staining revealed that more viable cells accumulated in the S. ag 7420 isolate than in the AH1 isolate. Scanning electron microscope (SEM) biofilm analysis showed S. ag AH1 cells appeared as chain-like structures, whereas the S. ag 7420 isolate biofilm cells appeared as fork-like structures on the glass surface. Biofilm elements were analyzed using Energy Dispersive X-Ray Analysis (EDAX) for both isolates and 13 elements with different orders of composition were found. Thus, virulence gene detection, distribution and biofilm formation by these new clinical isolates suggested the virulent nature of these pathogens, which might cause different levels of disease severity in humans.

Discrimination experienced by sexual minority males in Australia: Associations with suicidal ideation and depressive symptoms.

INTRODUCTION: Discrimination has been under-examined as a social determinant of the higher rates of poor mental health experienced by sexual minorities. The objectives of our study were to: 1) assess whether discrimination was independently associated with poor mental health among sexual minority males, and 2) assess the potential mediation role of discrimination in the associations between sexual minority status and poor mental health. METHODS: We used cross-sectional data on 13,230 males aged 18-55 years from the Australian Longitudinal Study on Male Health; bisexual and homosexual males comprised 1.5% and 1.6% of the sample, respectively. We fit Poisson regression and zero-inflated negative binomial regression models to examine suicidality, depressive symptoms and perceived discrimination in the past two years as correlates of suicidality and depressive symptoms. RESULTS: Statistically significant differences were observed in the prevalence of perceived discrimination by sexual orientation (p < 0.001), with the highest prevalence among bisexual (29.3%) and homosexual (40.4%) males, and the lowest prevalence among heterosexual males (18.6%). After adjusting for confounding, bisexual/homosexual males had higher rates of perceived discrimination (IRR = 1.88, p < 0.001), recent suicidal ideation (IRR = 1.51, p = 0.008), lifetime suicide attempt (IRR = 2.09, p < 0.001) and recent depressive symptoms (IRR = 1.34, p < 0.001) than heterosexual males. Analysis of ß-coefficients suggested that discrimination may mediate a small to moderate proportion of the association between sexual minority status and poor mental health. LIMITATIONS: Use of cross-sectional data. CONCLUSION: Poor mental health is more common among sexual minority males, and discrimination may be a contributor to these mental health disparities. Reducing discrimination should be considered as part of a strategy to improve the mental wellbeing of sexual minority males.

Preparation of an Intelligent pH Film Based on Biodegradable Polymers for Monitoring the Food Quality and Reducing the Microbial Contaminants.

Hydrogel refers to a three-dimensional cross-linked polymeric network made of synthetic or natural polymers that can hold water in its porous structure. The inclusion of hydrophilic groups in the polymer chains, such as amino, carboxyl, and hydroxyl groups, contributes to the hydrogel's water-holding ability. At physiological temperature and pH, these polymeric materials do not dissolve in water, but they do swell significantly in aqueous media. Hydrogel can be manufactured out of almost any water-soluble polymer, and it comes in a variety of chemical compositions and bulk physical properties. Hydrogel can also be made in a variety of ways. Hydrogel comes in a variety of physical shapes, including slabs, microparticles, nanoparticles, coatings, and films. Due to its ease of manufacture and self-application in clinical and fundamental applications, hydrogel has been widely exploited as a drug carrier. Contact lenses, artificial corneas, wound dressing, suture coating, catheters, and electrode sensors are some of the biomedical applications of hydrogels. The pigment color changes were observed from colorless to pale pink followed by dark reddish-pink. Anthocyanin was produced in large quantities and tested using a UV-visible spectrophotometer. At 450-550 nm, the largest peak (absorbance) was detected, indicating the presence of anthocyanin. The FTIR analysis of this study shows the different stretches of bonds at different peaks: 2918.309 (-C-H alkane stretch), 2812.12 (-C-H aldehyde weak intensity), 192320.37/cm (C-O bend), 21915.50, 2029.08/cm (-C=C arene group), 1906.94/cm (=C-H aromatics), 1797.78/cm (=C-H), 1707.94 (-C=O ketene), 1579.70, 1382.96 (C-H alkane strong bend), 889.18/cm (C-H aromatics plane bend), and 412.77/cm (-C-CI strong bond). The spectra of the PVA/chitosan film depict the peak's formation: 1571.88, 1529.55, 1500.62/cm (C-H alkene strong bend), 1492.90, 1483.26, 1467.83/cm (C-H alkene strong bond), 670.48, 443.63, 412.77/cm (-O-H carboxylic acids with great intensity), 1708.93 (-C=O ketone), and 1656.0/cm (alkenyl C=C stretch strong bond).

Household air pollution and COPD: cause and effect or confounding by other aspects of poverty?

SETTING: Household air pollution (HAP) and chronic obstructive pulmonary disease (COPD) are both major public health problems, reported to cause around 4 million and 3 million deaths every year, respectively. The great majority of these deaths, as well as the burden of disease during life is felt by people in low- and middle-income countries (LMICs).OBJECTIVE and DESIGN: The extent to which HAP causes COPD is controversial; we therefore undertook this review to offer a viewpoint on this from the Global Initiative for COPD (GOLD).RESULTS: We find that while COPD is well-defined in many studies on COPD and HAP, there are major limitations to the definition and measurement of HAP. It is thus difficult to disentangle HAP from other features of poverty that are themselves associated with COPD. We identify other limitations to primary research studies, including the use of cross-sectional designs that limit causal inference.CONCLUSION: There is substantial preventable morbidity and mortality associated with HAP, COPD and poverty, separately and together. Although it may not be possible to define clear causal links between HAP and COPD, there is a clear urgency to reduce the avoidable burden of disease these inflict on the world´s poor.

Bilateral ovarian teratoma complicated with carcinosarcoma in a 68 year old woman: a case report.

BACKGROUND: Composing of less than 1% of all ovarian cancers, immature teratoma is a malignancy that mainly affects the young, and they present with advanced disease. The treatment of immature teratoma is conservative primary surgery usually involving unilateral salpingo-oophorectomy followed by combination chemotherapy. CASE PRESENTATION: Here we present a case of a 68 year old woman with bilateral ovarian teratoma complicated with carcinosarcoma. The patient was diagnosed as FIGO stage IIIC. She underwent neoadjuvant chemotherapy and interval cytoreduction followed by optimal cytoreduction. The post operative management strategies and gynaecological follow up studies revealed no evidence of regional or distant metastasis. CONCLUSION: Thus the choice of initial treatment should be decided in a selective fashion depending on various prognostic factors in order to increase the survival of the patients.

Hypoxia due to unusual right to left shunt after arterial switch surgery.

Hypoxia can occur after repair of transposition of great arteries. The most common cause of right to left shunt after arterial switch surgery is related to increased right ventricular pressures and persistent neonatal pulmonary arterial hypertension. We report a case of TGA repair causing right to left shunt with normal right ventricular pressures. Persistence of Eustachian valve with patent foramen ovale (PFO) is the unusual cause of hypoxia and desaturation. The patient was successfully managed by excision of Eustachian valve and closure of PFO.

Early outcomes after lung transplantation for severe COVID-19: a series of the first consecutive cases from four countries.

BACKGROUND: Lung transplantation is a life-saving treatment for patients with end-stage lung disease; however, it is infrequently considered for patients with acute respiratory distress syndrome (ARDS) attributable to infectious causes. We aimed to describe the course of disease and early post-transplantation outcomes in critically ill patients with COVID-19 who failed to show lung recovery despite optimal medical management and were deemed to be at imminent risk of dying due to pulmonary complications. METHODS: We established a multi-institutional case series that included the first consecutive transplants for severe COVID-19-associated ARDS known to us in the USA, Italy, Austria, and India. De-identified data from participating centres-including information relating to patient demographics and pre-COVID-19 characteristics, pretransplantation disease course, perioperative challenges, pathology of explanted lungs, and post-transplantation outcomes-were collected by Northwestern University (Chicago, IL, USA) and analysed. FINDINGS: Between May 1 and Sept 30, 2020, 12 patients with COVID-19-associated ARDS underwent bilateral lung transplantation at six high-volume transplant centres in the USA (eight recipients at three centres), Italy (two recipients at one centre), Austria (one recipient), and India (one recipient). The median age of recipients was 48 years (IQR 41-51); three of the 12 patients were female. Chest imaging before transplantation showed severe lung damage that did not improve despite prolonged mechanical ventilation and extracorporeal membrane oxygenation. The lung transplant procedure was technically challenging, with severe pleural adhesions, hilar lymphadenopathy, and increased intraoperative transfusion requirements. Pathology of the explanted lungs showed extensive, ongoing acute lung injury with features of lung fibrosis. There was no recurrence of SARS-CoV-2 in the allografts. All patients with COVID-19 could be weaned off extracorporeal support and showed short-term survival similar to that of transplant recipients without COVID-19. INTERPRETATION: The findings from our report show that lung transplantation is the only option for survival in some patients with severe, unresolving COVID-19-associated ARDS, and that the procedure can be done successfully, with good early post-transplantation outcomes, in carefully selected patients. FUNDING: National Institutes of Health. VIDEO ABSTRACT.

Medium term results following heart transplantation for end stage heart failure: A single center experience of 257 patients.

OBJECTIVE: End stage heart failure is a lethal disease with a dismal 5 year survival. Heart transplantation has proven to be a highly effective modality of treatment in appropriately selected group of such patients. This is a retrospective analysis of medium term outcomes of heart transplantation in the setting of a private health facility in India. The objective of this study was two fold. METHODS: The outcome of 257 heart transplants done at a single centre from October 2012 to October 2019 was analyzed. Patients with combined Heart and lung transplants and those whose complete medical records were unavailable were excluded from the study. Survival was tracked at 60 days, 90 days, one year and beyond for a maximum of 7 years. Preoperative patient risk profiles were characterized on the basis of INTERMACS category. RESULTS: There were 176 male and 81 female patients. The age range was from 8 months to 78 years with a mean of 32.9 years. Survival at 2 months was 87%, at 90 days was 83%, at one year was 81%, 2 years was 75%, at 3 years was 72% and at 5 years and beyond was 62% for the whole series. Strong predictors of 90 day mortality included INTERMACS category (odd's ratio 0.289, p = 0.000) and creatinine more than 1.5 mg/dl (odd's ratio 2.48, p = 0.056). Recipient pulmonary vascular resistance and donor organ ischemic times were not found to be statistically significant factors affecting outcome. Medium term survival was influenced by INTERMACS category (Hazard ratio > 3 for INTERMACS category 1 compared to INTERMACS 4 or 5, p < 0.0001) and creatinine > 1.5 mg/dl (Hazard ratio 2.15, p = 0.003). This effect of creatinine was related to the age of the recipient. Hazard ratio 1.4, p = 0.524 if age <30 and Hazard ratio 4.78, p = 0.006, if age was >50. CONCLUSION: Satisfactory medium term outcome is possible after heart transplantation even in resource constrained environment of a developing country.

Pharmacodynamics and proteomic analysis of acalabrutinib therapy: similarity of on-target effects to ibrutinib and rationale for combination therapy.

Acalabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, is associated with high overall response rates and durable remission in previously treated chronic lymphocytic leukemia (CLL); however, complete remissions were limited. To elucidate on-target and pharmacodynamic effects of acalabrutinib, we evaluated several laboratory endpoints, including proteomic changes, chemokine modulation and impact on cell migration. Pharmacological profiling of samples from acalabrutinib-treated CLL patients was used to identify strategies for achieving deeper responses, and to identify additive/synergistic combination regimens. Peripheral blood samples from 21 patients with relapsed/refractory CLL in acalabrutinib phase I (100-400 mg/day) and II (100 mg BID) clinical trials were collected prior to and on days 8 and 28 after treatment initiation and evaluated for plasma chemokines, reverse phase protein array, immunoblotting and pseudoemperipolesis. The on-target pharmacodynamic profile of acalabrutinib in CLL lymphocytes was comparable to ibrutinib in measures of acalabrutinib-mediated changes in CCL3/CCL4 chemokine production, migration assays and changes in B-cell receptor signaling pathway proteins and other downstream survival proteins. Among several CLL-targeted agents, venetoclax, when combined with acalabrutinib, showed optimal complementary activity in vitro, ex vivo and in vivo in TCL-1 adoptive transfer mouse model system of CLL. These findings support selective targeting and combinatorial potential of acalabrutinib.

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

We identified an extremely rare condition, isolated complete deficiency of the fourth component of complement, in a child with systemic lupus erythematosus. The genes for C4 are located within the major histocompatibility complex (MHC) on the short arm of chromosome 6. The patient expressed only paternal phenotypes for proteins encoded by the MHC (HLA and GLO), yet was 46XX with no detectable 6p deletion. Genomic DNA from patient, parents, and sibling was digested with restriction enzymes, and blots were probed for five chromosome 6 markers. At all loci, maternal and paternal RFLPs could be distinguished, and the patient showed only paternal bands. RFLP analysis of markers from four other chromosomes showed maternal and paternal contribution. The data are consistent with uniparental isodisomy 6 (inheritance of two identical chromosome 6 haplotypes from the father and none from the mother). Direct analysis of genetic material from both parents, as well as detection of multiple protein polymorphisms encoded on chromosome 6, clearly demonstrates this novel mechanism for the expression of a recessive genetic condition.

Demonstration of blood flow by color doppler in the femoral artery distal to arterial cannula during peripheral venoarterial-extracorporeal membrane oxygenation.

In spite of distal perfusion of the limb using a cannula, the limb can have ischemic events if there is an undetected kink or clot anywhere in the line or thrombus in the artery. There are several ways to monitor and assess the limb ischemia. Monitoring for clinical signs of limb ischemia like temperature change and pallor is reliable and mandatory. We report a method where we used color Doppler to document the blood flow. Curvilinear vascular probe of an echo machine is used to identify the flow in the distal femoral artery of the lower limb. . As we have demonstrated in the video attached, once flow to the distal limb perfusion system is shut off by closing the three way stop cock, we can appreciate the immediate cessation of flow in the artery by Doppler.

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199).

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

A simple and rapid method for the preparation of plasma membranes.

A simple and rapid method for preparing plasma membranes from isolated cells or tissues is described. The membranes were characterised (a) biochemically by an analysis of specific marker enzymes, (b) by quantitation of cell surface receptors, and (c) immunologically by their ability to elicit specific allogeneic responses from cytotoxic T cells in secondary in vitro stimulations. Based on both biochemical and immunologic criteria, plasma membranes prepared by the method described here are of equal or greater 'purity' compared to those prepared by two other methods that are most widely used to date and the yields are several-fold higher.