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Background: The transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is complex and multifactorial. We aimed to identify the risk factors for infection among the household contacts of index patients and to determine the incubation period, serial interval, and estimates of secondary infection rate. Methods: We conducted a study in three districts of Kerala among the inhabitants of households of reverse transcriptase polymerase chain reaction (RT-PCR)-positive coronavirus disease (COVID-19) patients between January and July 2021. COVID-19-positive patients and corresponding contacts were enrolled and followed up for 28 days to determine RT-PCR positivity and the presence of total antibodies against SARS-CoV-2 on days 1, 7, 14, and 28 from the date of enrolment. Results: The mean incubation period, serial interval, and generation time were 1.6, 3, and 3.9 days, respectively. The secondary infection rate was 43.0%. Individuals who worked outside the home were protected, whereas those who had kissed the COVID-19-positive patients during illness were more than twice at risk of infection than those who had not kissed the COVID-19-positive patients. Similarly, the contacts who had shared a toilet with the COVID-19-positive patients were more at risk than those who had not shared a toilet. However, the contacts who reported using masks were at a higher risk of infection in household settings. Conclusions Assessment of SARS-CoV-2 transmission in household settings is important, considering its high secondary infection rate. Close physical contact and toilet sharing increase the risk of infection. This study demonstrates shorter incubation period and serial interval.

S1. Combination of lenalidomide (1uM) and bortezomib (different concentrations) induces apoptosis in myeloma cell lines (MM.1S and U266) compared to bortezomib alone treated cells (n=3) at the end of 48 hours. S2. Representative Immunoblot showing induction of apoptosis in MM.1S cell line upon treatment with lenalidomide (1uM) and bortezomib (5nM), lysates were collected the end of 24 and 48 hours post treatment with drugs (n=3). The proteins analysed were mentioned in the image. S3. a) Immunoblot showing that the combination of lenalidomide and bortezomib degraded IKZF1 in U266 cell line (n=3). b) Immunofluorescence assays showing degradation of IKZF1 in U266 cell line upon treatment with Lenalidomide and bortezomib (n=3). S4. Immunoprecipitation of Ubiquitin after treatment with lenalidomide (1uM) and bortezomib (5nM) revealed an interaction with IKZF1 at 8 hours. S5. Inhibition of autophagy by hydroxycholoroquine (HCQ) and bafilomycin A1 (BAFA1) in MM1.S cell line was confirmed using cyto-ID stain, where accumulation of autophagosome was observed (n=3) which was further confirmed by western blot (n=3). S6. Inhibition of autophagy by hydroxycholoroquine (HCQ) and bafilomycin A1 (BAFA1) did not inhibit the degradation of IKZF1 in U266 cell line (n=3). S7. Inhibition of calpain and caspase by PD150606 and zVAD.fmk inhibited the degradation of IKZF1 in U266 cell line (n=3). S8. Inhibition of calpain and chelation of calcium resulted in inhibition of PARP cleavage and IKZF3 degradation in MM1.S cell line (n=3) at the end of 24 hours of drug treatments. S9. Immunoblot representing modulation of calcium flux with ionomycin in MM.1S cell line enhanced IKZF1 degradation only in Ionomycin alone or in combination with LEN treated cells for 12 hours (n=3). S10. Viability of MM1.S cell line at post LEN (1uM) and BTZ (5nM) treatment after 48 hours (n=3). Viability was assessed by Annexin V- and 7 AAD - viability assay.

Key Points Metabolic rewiring promotes ATO resistance in APL, independent of PML mutation status.Inhibition of mitochondrial respiration combined with ATO is a potential therapeutic option for relapsed APL and non-M3 AML., Visual Abstract, Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are less sensitive to all-trans retinoic acid (ATRA) and the combination of ATO and ATRA compared with the sensitive cell line. Characterization of these resistant cell lines that were generated in-house showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO-resistant APL cell lines. Glycolytic inhibition by 2-deoxyglucose (2-DG) was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO-resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that combining mitocans with ATO can overcome ATO resistance. We also show that this combination has potential for treating non-M3 acute myeloid leukemia (AML) and relapsed APL. The translation of this approach in the clinic needs to be explored further.

The standard of care for patients with acute promyelocytic leukaemia (APL) relapsing after front-line treatment with arsenic trioxide (ATO)-based regimens remains to be defined. A total of 67 patients who relapsed after receiving ATO-based up-front therapy and were also salvaged using an ATO-based regimen were evaluated. The median (range) age of patients was 28 (4-54) years. While 63/67 (94%) achieved a second molecular remission (MR) after salvage therapy, three (4·5%) died during salvage therapy. An autologous stem cell transplant (auto-SCT) was offered to all patients who achieved MR, 35/63 (55·6%) opted for auto-SCT the rest were administered an ATO + all-trans retinoic acid maintenance regimen. The mean (SD) 5-year Kaplan-Meier estimate of overall survival and event-free survival of those who received auto-SCT versus those who did not was 90·3 (5·3)% versus 58·6 (10·4)% (P = 0·004), and 87·1 (6·0)% versus 47·7 (10·3)% (P = 0·001) respectively. On multivariate analysis, failure to consolidate MR with an auto-SCT was associated with a significantly increased risk of relapse [hazard ratio (HR) 4·91, 95% confidence interval (CI) 1·56-15·41; P = 0·006]. MR induction with ATO-based regimens followed by an auto-SCT in children and young adults with relapsed APL who were treated with front-line ATO-based regimens was associated with excellent long-term survival.

Multiple myeloma had been successfully treated by combining lenalidomide and bortezomib with reports suggesting benefits of such a combination even in relapsed/refractory cases. Recently, it was demonstrated that Ikaros degradation by lenalidomide happens via proteasome-dependent pathway and this process is critical for the eradication of myeloma cells. On the basis of this, an antagonistic effect should be observed if a combination of both these agents were used, which however is not the observation seen in the clinical setting. Our study demonstrates that when these agents are combined they exhibit a synergistic activity against myeloma cells and degradation of Ikaros happens by a proteasome-independent calcium-induced calpain pathway. Our study identifies the crucial role of calcium-induced calpain pathway in inducing apoptosis of myeloma cells when this combination or lenalidomide and bortezomib is used. We also report that this combination enhanced the expression of CD38 compared with lenalidomide alone. Thus, data from our study would establish the rationale for the addition of daratumumab along with this combination to further enhance therapeutic activity against multiple myeloma. Implications: Lenalidomide and bortezomib combination degrades IKZF1 in multiple myeloma through a calcium-dependent calpain and caspase pathway. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/4/529/F1.large.jpg.

The standard‐of‐care for patients with acute promyelocytic leukemia (APL) relapsing after upfront arsenic trioxide (ATO) therapy is not defined. The present study was undertaken to evaluate the safety of addition of bortezomib to ATO in the treatment of relapsed APL based on our previously reported preclinical data demonstrating synergy between these agents. This was an open label, nonrandomized, phase II, single‐center study. We enrolled 22 consecutive patients with relapsed APL. The median age was 26.5 years (interquartile range 17.5 to 41.5). The median time from initial diagnosis to relapse was 23.1 months (interquartile range 15.6 to 43.8). All patients achieved hematological remission at a median time of 45 days (range 40‐63). Nineteen patients were in molecular remission at the end of induction. Grade 3 adverse events occurred in eight instances with one patient requiring discontinuation of therapy for grade 3 neuropathy. Twelve (54.5%) patients underwent autologous transplantation (auto‐SCT) in molecular remission while the rest opted for maintenance therapy. The median follow‐up was 48 months (range 28‐56.3). Of the patients undergoing auto‐SCT, all except one was alive and relapse free at last follow‐up. Of the patients who opted for maintenance therapy, three developed a second relapse. For treatment of APL relapsing after upfront ATO therapy, addition of bortezomib to a standard ATO‐based salvage regimen is safe and effective. This trial was registered at http://www.clinicaltrials.gov as NCT01950611., The standard‐of‐care for patients with acute promyelocytic leukemia (APL) relapsing after upfront arsenic trioxide (ATO) therapy is not defined. In this phase II study we demonstrate the safety of adding a proteasome inhibitor with ATO in the management of relapsed APL treated with upfront ATO. We also demonstrate the efficacy of this combination and the potential for its utilization in this setting.

Not available.

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further.Key pointsMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation.Combination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients.

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

Introduction: Factor replacement therapy in treatment of haemophilia A is complicated by the production of neutralising antibodies known as inhibitors. The formation of inhibitors is multifactorial being associated with both genetic and environmental factors. Aim: To document the prevalence of inhibitors in severe haemophilia in the community where most patients receive only infrequent episodic replacement therapy and evaluate the factors which could be contributing to it. Methods: Community based camps were conducted in different parts of the country. Patients were assessed through a structured questionnaire and blood samples were obtained for laboratory evaluation of inhibitors and defined immunological parameters. Results: Inhibitors were present in 87/447 (19.5%) of the evaluated patients. High-titre inhibitor (>5 Bethesda Units [BU]) was identified in 31 (35.6%) patients. HLA DRB1-13-positive cases (RR = 2.04; 95% CI 1.06-3.911; P = 0.033) had an increased risk of inhibitor formation which was retained in the high-titre subset. A decreased risk of inhibitor formation was noted with heterozygous IL4-590 C/T allele (RR = 0.22; 95% CI 0.108-0.442: P = 0.000). There were no significant correlations between any of the evaluated environmental factors and the development of inhibitors in this study. Conclusion: The overall prevalence of inhibitors in patients with severe haemophilia A is similar to that reported among patients receiving regular replacement therapy. The data from this study, limited by its retrospective and cross-sectional study design, would suggest that genetic rather than environmental are more likely to impact the development of inhibitors.

Acute promyelocytic leukemia (APL) is a subtype of leukemia which is associated with unique and distinctive coagulopathy. In the absence of treatment it is rapidly fatal and even after initiation of therapy the major cause of early mortality is related to hemorrhagic complications. The coagulopathy can be exacerbated with the start of treatment. In the absence of early hemorrhage related deaths the probability of cure exceeds 90% in low and intermediate risk patients and 80% even in high risk patients, highlighting the importance of understanding the pathophysiology of this complication and instituting prompt and appropriate management strategies. The coagulopathy in APL is complex and results from a combination of thrombocytopenia, disseminated intravascular coagulation and hyperfibronlysis. Recently the effect of all-trans retinioc acid (ATRA) induced ETosis on exacerbating coagulopathy in the first few days after starting therapy with this agent raises the potential for potentially novel strategies to reduce the risk of hemorrhage. Currently management is mainly related to rapid initiation of therapy with ATRA along with appropriate and adequate replacement of blood products to correct the coagulopathy. There is limited role for the use of low dose anti-coagulants and anti-fibrinolytic agents in the initial management of this disease. There is limited data on the use of rFVIIa or the use of global tests of hemostasis in the management of this condition.

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bβ- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bβ: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bβ: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia–retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL.

The nose being highly vascular with communications with intracranial venous sinuses, it is highly prone for emergencies due to trauma, infections and allergy.

Pituitary failure occurs due to multiple reasons including genetic abnormalities, trauma, infections, empty sella syndrome, Sheehan’s syndrome, etc.

The third trimester is the period of mainly “big 4” – conditions which result in maternal deaths – PIH, eclampsia, placenta praevia and abruptio placenta.

Sore throat is a common complaint in the emergency department and is often associated with benign conditions, such as pharyngitis. The neck and throat are regions rich in vascular and lymphatic activity. They also house the food passage and airway; hence, any pathology – allergic, infective, neoplastic as well as foreign bodies – causes significant discomfort. A high index of suspicion should be maintained to diagnose less common but serious pathology, such as epiglottitis and retropharyngeal abscess. This chapter briefly highlights the salient emergencies concerning the throat.

The emergency department is often the first port of call for a variety of ear, nose and throat (ENT) disorders. Although most of these conditions are benign, there are important critical ENT disorders that must be immediately recognised and managed. This chapter, divided as three subsections, attempts to discuss salient disorders from the perspective of the emergency physician.

Purpose: The use of dried blood spots (DBS) for HIV-1 viral load determination could greatly enhance the management of HIV infected individuals in resource-limited countries. Objective: To compare the HIV-1 viral load values obtained between parallel collected plasma and DBS. Materials and Methods: DBS and plasma samples were collected from 62 HIV-1 infected individuals and were used for determination of HIV-1 RNA concentrations using the Abbot real-time HIV-1 PCR. Result: Mean of the log difference of viral load values between plasma and DBS was -0.41 log. DBS viral load values significantly correlated with plasma viral load (r = 0.9818, P < 0.0001). Conclusion: These results suggest that DBS samples can be used as an alternative to plasma for the estimation of HIV-1 viral load if samples are appropriately stored.

Human immunodeficiency virus type-2 (HIV-2) belongs to the family retroviridae which is phylogenetically clusters with SIV SM from sooty mangabeys. This virus is morphologically similar to human immunodeficiency virus type-1 (HIV-1) but has got only a 40% homology at the nucleotide level. There is a distinct geographical distribution of HIV-2 unlike HIV-1. There are currently eight subtypes/groups identified with subtype/group A responsible for the majority of infections. HIV-2 shows a considerable difference in the course of the disease. Clinical, haematological and immunological evaluation of individuals infected with HIV-2 has shown the virus to be less pathogenic than HIV-1 although the exact mechanism underlying this difference is not well defined. Similar to HIV-1, the HIV-2 isolates also showed distinct replicative and cytopathic characteristics. The transmission rate for HIV-2 compared to HIV-1 is very low both by heterosexual route and mother to child transmission. The clinical signs and symptoms of immunodeficiency associated with HIV-2 are similar to the ones seen among the HIV-1-infected individuals and they can also progress to AIDS. It is naturally resistant to NNRTI and hence the diagnosis become important as it affects the treatment strategy. Similar to HIV-1, HIV-2 strains of infected individuals also show mutations that can cause drug resistance. The current evidence suggests that there is no protective effective for HIV-2 against HIV-1.

Traditional treatment of organophosphate poisoning (OP) with oximes has had limited success. Fresh frozen plasma (FFP) or albumin, acting as bioscavengers to mop up free organophosphate, has been recently proposed as a treatment modality. In this pilot open-label, three-arm, randomized controlled study exploring proof of concept, we evaluated if bioscavenger therapy had a role in OP.Sixty patients with significant poisoning presenting within 12 hours, with suppression of pseudocholinesterase activity to1,000 U/L, were randomized to receive FFP (8 bags, 250 mL each over 3 days), 20% human albumin (4 × 100 mL over 3 days), or saline (2,000 mL over 3 days) in addition to atropine and supportive care. Pseudocholinesterase and organophosphate levels were measured pretreatment, post-infusion (Day 2, Day 3), and predischarge and expressed as mean ± standard error. The incidence of intermediate syndrome, need for mechanical ventilation, atropine requirement, and mortality were assessed.Twenty patients received albumin and 19 patients each FFP or saline. FFP increased pseudocholinesterase levels (250 ± 44-1,241 ± 364 U/L) significantly (p = 0.007). Small, nonsignificant increases were observed with saline (160 ± 30-259 ± 78) and albumin (146 ± 18-220 ± 61). Organophosphate levels reduced in all 3 arms; no clear-cut trends were observed. We observed more cases of intermediate syndrome with FFP [10/19 (53%) vs. 5/20 (25%) vs. 5/19 (26%), FFP, albumin, and saline arms (p = 0.15)]. The interventions did not affect ventilatory requirements (14/19 vs. 15/20 vs. 14/19) or prevent delayed intubation. There were no differences in mean (±standard error) atropine requirement (in milligrams) in the first 3 days (536 ± 132 vs. 361 ± 125 vs. 789 ± 334) and duration (in days) of ventilation (10.0 ± 2.1 vs. 7.1 ± 1.5 vs. 7.5 ± 1.5) or hospital stay (12.4 ± 2.2 vs. 9.8 ± 1.4 vs. 9.8 ± 1.6). Two patients developed adverse effects with FFP. Mortality was similar (4/19 vs. 5/20 vs. 2/19, p = 0.6).Despite significant increase in pseudocholinesterase levels with FFP, this pilot study did not demonstrate favorable trends in clinical outcomes with FFP or albumin.

The largest democracy on earth, the second most populous country and one of the most progressive countries in the globe, India, has advanced tremendously in most conventional fields of Medicine. However, emergency medicine (EM) is a nascent specialty and is yet to receive an identity. Today, it is mostly practised by inadequately trained clinicians in poorly equipped emergency departments (EDs), with no networking. Multiple factors such as the size of the population, variation in standards of medical education, lack of pre-hospital medical systems and non-availability of health insurance schemes are some of the salient causes for this tardy response. The Indian medical system is governed by a central, regulatory body which is responsible for the introduction and monitoring of all specialties--the Medical Council of India (MCI). This organisation has not recognized EM as a distinct specialty, despite a decade of dogged attempts. Bright young clinicians who once demonstrated a keen interest in EM have eventually migrated to other conventional branches of medicine, due to the lack of MCI recognition and the lack of specialty status. The Government of India has launched a nationwide network of transport vehicles and first aid stations along the national highways to expedite the transfer of patients from a crash site. However, this system cannot be expected to decrease morbidity and mortality, unless there is a concurrent development of EDs. The present article intends to highlight factors that continue to challenge the handful of dedicated, full time emergency physicians who have tenaciously pursued the cause for the past decade. A three-pronged synchronous development strategy is recommended: (i) recognise the specialty of EM as a distinct and independent basic specialty; (ii) initiate postgraduate training in EM, thus enabling EDs in all hospitals to be staffed by trained Emergency physicians; and (iii) ensure that EMs are staffed by trained ambulance officers. The time is ripe for a paradigm shift, since the country is aware that emergency care is the felt need of the hour and it is the right of the citizen.

This study was performed to examine the association of 2 functional polymorphisms of the promoter region of the serotonin 5-HTR2C receptor gene: –759C/T (rs3813929) and –97G/C (rs518147) with risperidone-induced insulin resistance syndrome in an Indian population. In this case-control study, 52 adult patients of either sex, having no insulin resistance syndrome before initiating treatment, who were previously drug naive and who received risperidone monotherapy for ≥1 year, were recruited in 2 arms. Of them, 26 had risperidone-induced insulin resistance syndrome, and 26 did not have risperidone-induced insulin resistance syndrome. Polymerase chain reaction and DNA sequencing were performed. Multiple logistic regression analysis was performed, and adjusted odds ratio (AOR) was calculated. The polymorphisms did not deviate from Hardy-Weinberg equilibrium (P > .05). For both rs3813929 and rs518147, the variant (AOR 3.95, 95% CI 0.86-21.9 and AOR 4.12, 95% CI 0.88-23.23, respectively) and the heterozygous (AOR 5.21, 95% CI 0.7-61.38 and AOR 4.26, 95% CI 0.76-31.22, respectively) alleles were associated with risperidone-induced insulin resistance syndrome. The other factors associated with risperidone-induced insulin resistance syndrome were male sex, history of risk factors (ischemic heart disease, diabetes mellitus, or stroke) in family, risperidone dose, pretreatment mean arterial blood pressure, fasting blood glucose, and triglycerides. This is the first study conducted in an Indian population to demonstrate that the 2 functional polymorphisms in the 5-HTR2C gene (rs3813929 and rs518147) are associated with risperidone-induced insulin resistance syndrome.

Introduction: There is limited data on the best treatment strategy for patients who relapse after upfront arsenic trioxide (ATO) based therapy for acute promyelocytic leukemia (APL). We had recently reported that there was significant micro-environment mediated drug resistance (EM-DR) to ATO which we noted was predominantly mediated by upregulation of the NF-ⱪB pathway. We also noted that this upregulation was more prominent in relapsed APL and that EMDR to ATO could be overcome by the use of proteasome inhibitors (Leukemia 2016 - In Press). Pre-clinical mouse model and preliminary clinical data validated these observations. There has also been a recent concern of mutations in the oncogenic PML-RARA gene following treatment with ATO, with reports suggesting a third of relapsed APL patients having such mutations and that the clinical outcomes in this subset with conventional therapy is poor [NEJM. 2014;370(19): 1864-6]. Methods: In this prospective, open-label, non-randomized, phase 2, single center study, we enrolled APL patients in first hematological relapse. Inclusion criteria included presence of t(15; 17) and exposure to ATO as part of up front therapy. Enrolled patients received conventional therapy for relapse, included ATO and ATRA in induction, consolidation. Anthracycline (mitoxantrone) was used only in induction. Additionally, patients received two doses of bortezomib (1.4mg / m2 / dose) in induction and consolidation on days 2 and 5 and one dose in maintenance (10 days/month x 6 months) on day 2. Primary end point was safety (graded according to the NCI Common Terminology Criteria for Adverse Events version 4.0) and the key secondary end points were proportion in complete molecular remission at the end of induction and event free survival (EFS). The study is ongoing and continues to recruit patients. This trial is registered with ClinicalTrials.gov, number NCT01950611. Results: Between Sep 2013 and Mar 2016, 18 patients met eligibility criteria and were enrolled in this study. All 18 patients achieved CHR at a median of 45 days (range: 42-63). Seventeen patients (94.4%) were RT-PCR negative at the end of induction. The median time complete molecular remission was 35 days (range: 21-56 days). None of the patients had any major bleeding or thrombotic events in induction. With the exception of one patient who had Grade IV neurotoxicity which required treatment and recovered after temporarily discontinuing treatment during induction none of the other patients had any Grade III/IV non-hematological toxicity. Other non-hematological events were ≤ Grade II, usually transient and judged to be unrelated to the interventional agent. These included mild headache in 8 (33.3%) and one case (5.5%) each with diarrhea, mild rash, redness of eyes, oral ulcers, vertigo and paresthesia. Auto-SCT was offered to all patients in molecular remission post consolidation. Eight patients (44.4%) underwent an autologous SCT post consolidation while the rest opted to receive maintenance therapy. One patient who opted for maintenance therapy relapsed 6 months after completing maintenance. At an actuarial median follow up of 24 months (range: 4-35) the 3 year KM estimate of EFS was 92.3±7.4% and the cohort had 100% survival at last follow up. This clinical profile and outcome data was compared with a historical cohort that was treated with the same protocol with the exception of bortezomib and the comparative data is summarized in table 1 and figures 1A and 1B. Conclusion: Addition of proteasome inhibitors with ATO is well tolerated. In combination with conventional agents, proteasome inhibitors can potentially improve the clinical outcome of patients with relapsed APL treated with upfront ATO. Our preliminary data also suggests that this combination can obviate the need for an auto-SCT in this setting. Disclosures No relevant conflicts of interest to declare.

There are numerous reports on bone marrow micro-environment mediated drug resistance (EM-DR) in cancer. We had previously reported, that there was significant EM-DR to ATO in APL. We had also shown that the effect of cross talk between leukemic cells and stromal cells leads to increased activation of NF-kB pathway in the leukemic cells. There are very limited data available on the effect of leukemic cell interaction on the stromal cells. We undertook a study to evaluate the changes induced by leukemic cells (NB4 cell line) on the HS-5 stromal cell line. After 2 days of co-culture with NB4 cells, the RNA from HS-5 stromal cells were subjected to gene expression profiling and small RNA sequencing. In a gene expression profiling, we observed an enrichment of Wnt signaling and BMP signaling pathway. When we subjected the array to functional analysis, a significant enrichment of osteoblast differentiation genes was observed in the stromal cells co-cultured with leukemic cells in comparison to HS-5 cells alone. A stringent miRNA analysis using DESeq (miRNAs whose FDR corrected p values < 0.05 are considered significant) revealed 21 miRNA were differentially regulated in the HS-5 cells co-cultured with leukemic cells in comparison to HS-5 cells alone. Consistent with the gene expression profile, we observed that most of the miRNA differentially regulated were involved in the regulation of osteoblast differentiation (such as hsa-miR-33b-5p, hsa-miR-210-3p and hsa-miR-222-5p). As previously reported (PNAS 2013;110 (23):9469) we also observed upon co-culture with leukemic cells a down-regulation of NF-kB pathway in HS-5 cells. This was demonstrated by documenting decreased translocation of p65 subunit in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This was further validated by documenting downregulation of NF-kB pathway genes by real time PCR on day 2. Along with this we also observed an increased translocation of B-catenin in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This observation was validated by a real time PCR assay where an upregulation of its target gene was seen (Fig 1a). We observed that there is an increased expression of osteoblast differentiation transcription factor (RUNX2) in HS-5 cells upon co-culture on day 2 by real time PCR and western blot (Fig 1a, 1b). We also observed an increase in alkaline phosphatase activity in HS-5 cells upon co-culture with NB4 cells at different time points detected by BCIP-NBT staining. Further validation of differentiation of HS-5 cells into osteoblast was done using alizarin red S stain on day 7. Similar differentiation effects were seen in HS-5 cells when it is co-cultured with other malignant cells (U937, U266 and Jurkat) where an increased expression of RUNX2 on day 2 of co-culture was observed, suggesting that this effect is common with different subtypes of leukemia. We did not observe any effect of PBMNC / normal cord blood CD34+cells on HS-5 cells in inducing osteoblast differentiation (no expression of RUNX2; Fig 1b). This study demonstrates a possible role of leukemic cells in establishing leukemic niche in the bone marrow by inducing osteoblast differentiation of stromal cells. The role of this leukemic cell induced osteoblast differentiation of stroma in drug resistance against various chemotherapeutic agents needs further evaluation. Disclosures No relevant conflicts of interest to declare.

Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Disclosures No relevant conflicts of interest to declare.

The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells. Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b). Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. Disclosures No relevant conflicts of interest to declare.

To the Editor:We were intrigued by the recent article of Lee et al.1 Although difference in C-reactive protein (CRP) values between initial and 24-h follow-up in organophosphate (OP) poisoning appe...

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

In patients with acute promyelocytic leukemia (APL), in spite of recent advances, early treatment related mortality (TRM) still remains a challenge and is largely related to the associated coagulopathy. It is well recognized that conventional coagulation parameters used in the clinic do not accurately predict the risk of adverse outcomes like major bleeding or thrombosis. There is limited data on the clinical utility of whole blood viscoelastic assays and extended coagulation parameters for evaluation of coagulopathy in patients with APL. Consecutive patients with PML-RARA positive APL were enrolled. Blood samples were collected prior to infusion of blood products on day 1 and again on day 4 for thromboelastometry done on whole blood and activated with recombinant tissue factor at a final dilution of 1:50,000 (ROTEM analyzer, Tem Innovations GmbH, Basel) and an extended panel of coagulation parameters done by immunoassays (TAT complex, protein C, ATIII, PIC complex, tPAIC, d-dimer and thrombomodulin). Platelet count and conventional coagulation parameters, which were prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and thrombin time (TT), were measured daily for the first 14 days (more frequently as clinically indicated). Patients received platelet transfusions to target a platelet count of 30x109/L (in the absence of bleeding). Fresh frozen plasma and cryoprecipitate transfusions were done to target a normal PT and aPTT and maintain a fibrinogen level above 140 mg%. Major bleeding, thrombosis and death during induction therapy were the outcome variables. Of the 61 patients enrolled, 11 were excluded (3 did not continue treatment at our hospital, 2 died within 48 hours of admission, and 6 took discharge against medical advice before completing induction). Of the remaining 50 cases that were evaluated, 40 were newly diagnosed and 10 were relapsed. The median age was 34.5 years (range 9-68). 25 were males (50 %). The WBC count was above 10 x 109/L in 20 patients (40%). The median platelet count was 28 x 109/L (range 4-339). There were 13 (26%) deaths in induction. Six patients had a major bleeding event. Five patients had a thrombotic event. Of the evaluated conventional coagulation parameters, extended coagulation parameters, platelet counts and ROTEM parameters at diagnosis (ROTEM data not available at diagnosis in 2 cases) only the ROTEM parameter of maximum clot firmness (MCF) as a continuous variable was significantly associated with death (p=0.012). On univariate cox regression analysis with death as the outcome, MCF≤ 30mm was a poor prognostic marker (hazard ratio of 13.24, 95%CI 1.71-102.19; p =0.013). On multivariate cox regression analysis including high WBC count (>10 x 109/L), low platelet count (< 40 x 109/L) and history of relapse, MCF ≤30mm was an independent poor prognostic variable (HR: 11.89; 95% CI 1.43 - 98.75; p 0.022). Four of the 6 major bleeding events, four of five thrombotic events and all deaths related to coagulopathy occurred in patients with MCF ≤30mm. The descriptive statistics of patients with MCF >30mm (n=21) vs those with MCF ≤ 30mm (n=27) are shown in table 1. Figure 1 illustrates the type of discordance one can see between conventional coagulation parameters and ROTEM values. This study suggests that the MCF value from a ROTEM assay at diagnosis is probably a better measure of the hemostatic defect at diagnosis in APL. Patients with APL with low MCF on ROTEM at diagnosis are at an increased risk of death during induction therapy despite an adequate blood product replacement strategy which was based on conventional coagulation parameters. In these patients, there is a need to develop novel and possibly a more intensive replacement strategy. Table 1. Comparison of baseline characteristics and clinical outcomes. Variables MCF ≤30 (n = 27)N (%) /Median (range) / Mean±SD MCF > 30 (n=21)N (%) /Median (range) / Mean±SD p value Age (years) 40(12-68) 27(9-48) 0.006 WBC >10 x x109/L 17(62.96) 2(9.52) 0.003 Platelet count (x109/L) 17(4-89) 45(10-339) 0.001 PT in seconds 14.5(11-87.5) 11.8(10.1-14.8) 0.004 aPTT in seconds 28(23.2-77.9) 30.2(22-53.6) 0.104 Fibrinogen in mg% 134.8(24.7-393) 172.2(14.7-549.5) 0.066 Major bleeding 4(14.8) 1(4.8) 0.368 Thrombosis 4(14.8) 1(4.8) 0.369 Death 12(44.4) 1(4.8) 0.003 Disclosures No relevant conflicts of interest to declare.

To cite this article: Sumitha E, Jayandharan GR, David S, Jacob RR, Sankari Devi G, Bargavi B, Shenbagapriya S, Nair SC, Abraham A, George B, Viswabandya A, Mathews V, Chandy M, Srivastava A. Molecular basis of Bernard–Soulier syndrome in 27 patients from India. J Thromb Haemost 2011; 9: 1590–8. Summary. Background: Bernard–Soulier syndrome (BSS) is an extremely rare (1:1 million) bleeding disorder of platelet adhesion, caused by defects in the glycoprotein (GP)Ib/IX/V complex. Patients and methods: The diagnosis in 27 patients was based on low platelet count, presence of giant platelets and aggregometry studies. Flow cytometry to assess the surface GPIb/IX/V complex showed reduced (7.7–57%) expression. gDNA was screened for mutations in the GPIBA, GPIBB, GP9 genes using PCR-conformation sensitive gel electrophoresis (CSGE). Results: Thirteen different disease-causing mutations, including missense (54%), frameshifts (38%) and nonsense (8%) mutations, were identified in 27 patients. Eleven of them were novel including five novel frameshifts (GPIbα: p.Gln97_98fsX113, p.Pro402_403fsX52; GPIbβ: p.Arg17fsX14; GPIX: p.Gly24fsX43, p. Pro130fs, a nonsense mutation (GPIX, p.94, Gln>X) and five novel missense mutations (GPIbα: p.492, Tyr>His; GPIbβ: p.65, Pro>Arg, p.129, Gln>His, p.132, Leu>Pro; GPIX: p.55, Phe>Cys). Interestingly, four common mutations, Cys8Arg (n = 6) and Phe55Ser (n = 2), Phe55Cys (n = 2) in GPIX and a novel 22-bp deletion in the GPIBB gene predicting p.Arg17fsX 14 (n = 10) were seen in 20 patients. Conclusion: The molecular data presented here is the largest series of BSS patients to be reported so far, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.

Data are limited on inhibitors in people with hemophilia (PWH) in developing countries. There is a perception that the overall prevalence of inhibitors, ranging from 7 to 19% in different reports, may be lower in these countries as compared with that reported from developed countries. This is possible given the fact that most patients are treated after 2 years of age with plasma-derived clotting factor concentrates. Whether genetic or other environmental factors also contribute to this needs further evaluation. There is a need to develop laboratory infrastructure and establish quality control programs for laboratory tests for inhibitors in developing countries. Management options vary widely given the socioeconomic diversity among these countries. Significant individualization of approach to management is therefore required depending on the available resources, particularly with regard to the use of bypassing agents. The limited data on immune tolerance induction with some low-dose regimens deserve further evaluation. Even in resource-constrained environments, education and a policy of systematic screening of patients associated with judicious use of bypassing agents can significantly improve the care of PWH who develop inhibitors.

Alkaptonuria is a rare autosomal recessive metabolic disorder that may present with multi-system involvement such as ochronotic arthropathy, renal, urethral and prostatic calculi, cardiac valvular lesions and pigmentation of the skin, sclera, cartilage and other connective tissues. An association of the disease with uveitis has never been reported. We report the first case of alkaptonuria with ochronotic arthropathy presenting with recurrent acute anterior uveitis as the initial manifestation. The possible common link with the HLA-B27 gene is discussed.

Degradation of PML-RARA upon treatment with ATO is predominantly mediated by the proteasome complex. Reports suggest that in relapsed APL patients who were treated upfront with ATO, mutations in the B2 domain of PML in PML-RARA gene are involved in resistance to ATO. These mutations predict a poor clinical outcome in spite of subsequent combination of ATO with chemotherapy (NEJM 2014). We had previously reported that Bortezomib (Bo) was able to synergize with ATO by inducing apoptosis through increased levels of ROS and up regulation of the UPR pathway (Blood. 2012;120, 3552) and we had also noted PML-RARA degradation and nuclear body formation with this combination. We further evaluated the mechanism of degradation of PML-RARA when ATO was combined with Bo and the effect of this combination on resistant cell lines, a mouse model and in relapsed patients. We generated in-house ATO resistant NB4 cell lines (NB4EV-ASR1, ASR2 and ASR3). In NB4EV-ASR1 we confirmed the presence of A216V mutation in the PML B2 domain (previously reported to be involved in ATO resistance) while the other 2 clones did not have this or any other mutation in PML-RARA. We noted an increase in the baseline proteasomal activity in all the resistant cell lines when compared to naïve NB4 cells (n=3; data not shown). The combination of ATO and Bo induced a significant apoptosis in all the resistant cells similar to naïve NB4 cells (Figure 1A: n=4; Combination Index = 0.02).The mechanism of inducing apoptosis in the resistant cell lines was similar to naïve NB4 cells, as previously reported by us, and involved an increased level of ROS, decreased mitochondrial membrane potential, induction of UPR and activation of caspase-3 (Figure 1B). We next evaluated PML-RARA degradation in NB4 naive cells treated with a combination of ATO+Bo. At 24 hours, there was an evidence of induction in autophagy as shown by LC3II formation using western blot technique which increased at 48 hours; this time point coincides with time at which maximum PML-RARA degradation occurred (Figure1C). Similar results were seen in the resistant cell lines (with and without mutation A216V). Blocking autophagy by 3-methyl adenine showed a partial inhibition in the degradation of PML-RARA. We have also observed that there is an accumulation of p62 (ubiquitin binding protein) at 24 hours and this was degraded by 48 hours suggests that accumulated ubiquitinated products were cleared by autophagy via p62 (Figure1D). In a co-immunoprecipitation experiment, p62 and LC3II proteins precipitated along with PML-RARA (figure 1E). Knock down of p62 transcript by siRNA followed by ATO+Bo treatment showed an accumulation of PML-RARA in the treated cells in comparison to the scrambled and control. In an APL transplanted mice model, combination of ATO and Bo prolonged the life span of the mice as illustrated in Figure1F. In this group there was a significant decrease in the leukemia burden evidenced by decreased leukemic cells in bone marrow, peripheral blood and spleen by flow cytometry, RQ-PCR and decreased spleen size on day +20. A reduction in the LIC was demonstrated by secondary transplants. We also observed that transplantation of bone marrow cells from the long term surviving mice post ATO+Bo therapy did not induce leukemia (Figure1G) and no transcripts of PML-RARA were detected in the recipients. A phase II clinical study combining Bo with ATO and chemotherapy has been initiated for patients with relapsed APL (NCT01950611). In this ongoing study 11 patients have been enrolled. The median age was 32 years. 7 were males. All patients achieved hematological remission and the median time to complete molecular remission was 42 days (one patient still on induction therapy). The addition of Bo was well tolerated. None of the cases had evidence of significant neuropathy, worsening of coagulopathy, IC bleed or a differentiation syndrome. Long term follow up is awaited to comment on the efficacy of this combination. In conclusion, the mechanism of ATO+Bo synergy is multi-factorial and appears to be predominantly due to increase in ROS activity and up regulation of UPR pathway leading to apoptosis. In spite of proteasomal inhibition by addition of Bo with ATO, PML-RARA continues to be degraded and this is mediated by up-regulation of autophagy pathway. ATO+Bo synergy was further confirmed in a pre-clinical model. This combination is also effective in ATO resistant cell lines with high levels of synergism. Figure 1 Figure 1. Disclosures Off Label Use: Bortezomib in the treatment of acute promyelocytic leukemia.

There has been a recent concern of arsenic trioxide (ATO) resistance in patients with acute promyelocytic leukemia (APL) treated with ATO as upfront therapy. The focus of ATO resistance has centred on mutations in PML-RARA gene (Blood 2011, NEJM 2014). NB4 cells in our laboratory were exposed to serial increasing concentrations of ATO. We subsequently generated 3 ATO resistant clones, NB4EV-ASR1, ASR2 and ASR3. In addition we have also evaluated an established ATRA resistant APL cell line UF1 (Gift from Dr. Chomienne. C). In a viability assay, we observed that these cell lines are resistant to ATO and had an IC50 above 2µm (Table1). These resistant cell lines also had a higher IC50 to other therapeutic drugs such as Daunorubicin and Cytosine arabinoside and a reduced differentiation effect on exposure to ATRA (summarized in Table 1). To identify the differences between the naïve cells and the resistant cells, we did whole exome sequencing (NGS) by Iontorrent and found that only NB4 EV-ASR1 clone had ATO resistance causing mutation (A216V) in the PML B2 domain (VAF=91.7%) while the other two cell lines (NB4EV-ASR2 and ASR3) did not have a mutation in PML-RARA. Next, we did an expression array to find the differential regulated genes between the naïve cell line and the parent resistant cell line from where all the 3 cell lines had been derived. We found that 1490 genes were differentially regulated (> 2 fold). The pathways significantly enriched for differentially expressed genes were cell survival, ABC transporters, Glutathione synthesis, Ubiquitin- proteasome degradation system and signalling pathways like PI3-AKT and PTEN. Validating the micro-array data, we found that there is an increased expression of ABC transporters such as MRP4, AQP9 (n=3; Figure1A) which are known to efflux ATO from the cells and a decreased expression of ABCA1 (known to efflux glutathione). The up regulation of these transporters also correlated with decreased levels of intracellular ATO (IC-ATO; measured using AAS, see Figure 1B for details) in the resistant cell lines. We also noted that there is a varying reduction in the basal reactive oxygen species levels and a varying increase in the amount of basal reduced glutathione (GSH) levels in the resistant cell lines (n=3, Table 1). We have noted that adding Buthionine sulphoximine (BSO - GSH inhibitor) along with ATO was able to restore the sensitivity of ATO in the resistant cells lines, however there was significant variation in the sensitivity of ATO among the cell lines when treated with the same concentration of BSO (Figure 1C). At the transcript levels we did not find any difference in expression of PML-RARA but at the protein level we noted a significant reduction in the levels of PML-RARA in the resistant cell lines (Figure 1D). We also observed an increase in the proteasome activity in the resistant cell lines compared to naïve cells (data not shown). In an immunofluorescence assay probing for PML, we found an absence of micro-speckled pattern in the resistant cell lines and UF1 cell lines compared to naive cells (Figure 1D). In conclusion, we have observed that in addition to PML-RARA mutations, variations in the Redox system, ABC transporters, intracellular ATO concentration and anti-apoptosis pathways are likely to be altered in ATO resistance. It is likely that ATO resistance is multi-factorial and that the dominant mechanism can vary between different resistant cell lines and potentially the same variation could be seen in relapsed patients. Importantly in the presence of ATO resistance there was also a decrease in sensitivity to other conventional agents used to treat APL. Novel agents and strategies based on these observations are required to address the issue of ATO resistance in patients with relapsed APL. Abstract 3554. Table. Characteristic features NB4 naïve NB4EV-AsR1 NB4EV-AsR2 NB4EV-AsR3 UF1 Sensitivity to ATO (IC 50 -µM) 0.9 3.09 3.44 2.88 4.1 Differentiation with ATRA exposure (1uM for 72hrs) (n=4) (CD11b% expression) (mean±SD) 49.2±7.3 40.1±3.0 12.4±2.5 30.5±2.6 0.57±0.23 Sensitivity to other chemotherapy drugs (IC50) (n=3) a) Daunorubicin(µM) b) Cytosine arabinoside (µM) 0.14 8.3 0.22 16.5 0.19 4.7 0.2 13.1 0.18 NA MRP4 expression (Fold difference) 1 4.2 3.8 4.2 NA Reactive oxygen species (ROS) levels (MFI Fold difference normalized to NB4 cells) (n=3) 1 0.74 0.86 0.68 0.3 Glutathionine levels measured by flowcytometry (MFI Fold difference normalized to NB4 cells) (n=3) 1 1.37 1.45 1.39 0.5 Disclosures No relevant conflicts of interest to declare.

The shift in cytokine profile during human immunodeficiency virus (HIV) disease progression is influenced by dehydroepiandrosterone sulfate (DHEAS) level. Radioimmunoassay was used to measure plasma DHEAS for 30 treatment-naïve HIV-infected and 30 uninfected individuals. There was a significant negative correlation of viral load with DHEAS level ( P < 0.05). Further studies of the use of DHEAS levels for monitoring HIV patients economically are warranted.

An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation ( r ) of the two systems for CD4 + T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies.

Sir, The article by Sarkar et al.,[1] is indeed interesting and timely as practitioners and emergency physicians have started seeing cases of cocaine intoxication in India. However, a few aspects of this paper require contemplation. The authors have administered thrombolytic agent initially for cocaine associated myocardial infraction (CAMI) which is not congruent with current evidence.[2] Current literature questions the routine use of thrombolytic therapy for patients with CAMI and there are reports of severe complications associated with thrombolytics in cocaine users.[3] The frequent presence of contraindications to thrombolysis, including severe hypertension, seizures, intracerebral hemorrhage, and aortic dissection in cocaine abusers precludes the liberal use of thrombolytic agents. Moreover, the standard electrocardiographic criteria (ST elevation) for thrombolytic therapy is hampered by the high rate of abnormal or nondiagnostic electrocardiogram (ECG)'s in patients presenting with cocaine associated chest pain. The guidelines issued by American College of Cardiology and American Heart Association[4] advocate the use of thrombolytic therapy in CAMI only if ST segments remain elevated despite nitroglycerin and calcium antagonists and coronary angiography is not possible. Secondly, the recent cocaine use can alter the specificity of cardiac biomarkers and making them difficult to interpret in CAMI, especially the serum creatinine kinase level is not a reliable indicator of myocardial injury and is increased in almost half of cocaine users irrespective of concurrent MI possibly because of cocaine-induced hyperthermia, increased skeletal muscle activity, and rhabdomyolysis.[5] The awareness of reliability of cardiac biomarkers in cocaine associated chest pain is warranted for effective management. The American Heart Association recommends nitroglycerin and benzodiazepines as first-line agents for cocaine associated chest pain. Benzodiazepines are administrated especially in cocaine-addicted patients with associated hypertension, tachycardia, or anxiety after the cocaine use. Intuitively, dexmedetomidine, a central sympatholytic agent is highly effective in reversing the cocaine's sympathomimetic actions.[6] Substance abuse remains one of the major public health issues across the globe, despite health education on prevention; the incidence of illicit substances is escalating. Since many patients with cocaine toxicity will present with chest pain to the emergency department, it is essential that these patients be recognized early and managed appropriately to prevent complications.