Mihir Gupta

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.

Mast cell activation contributes to sickle cell pathobiology and pain in mice

Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.

Morphine for the Treatment of Pain in Sickle Cell Disease

Pain is a hallmark of sickle cell disease (SCD) and its treatment remains challenging. Opioids are the major family of analgesics that are commonly used for treating severe pain. However, these are not always effective and are associated with the liabilities of their own. The pharmacology and multiorgan side effects of opioids are rapidly emerging areas of investigation, but there remains a scarcity of clinical studies. Due to opioid-induced endothelial-, mast cell-, renal mesangial-, and epithelial-cell-specific effects and proinflammatory as well as growth influencing signaling, it is likely that when used for analgesia, opioids may have organ specific pathological effects. Experimental and clinical studies, even though extremely few, suggest that opioids may exacerbate existent organ damage and also stimulate pathologies of their own. Because of the recurrent and/or chronic use of large doses of opioids in SCD, it is critical to evaluate the role and contribution of opioids in many complications of SCD. The aim of this review is to initiate inquiry to develop strategies that may prevent the inadvertent effect of opioids on organ function in SCD, should it occur, without compromising analgesia.

Spinal glial activation and oxidative stress are alleviated by treatment with curcumin or coenzyme Q in sickle mice.

Sickle cell anemia (SCA) is accompanied by unpredictable episodes of recurrent acute pain during vaso-occlusive crises (VOC), superimposed on chronic pain.1 Pain in SCA can start in infancy and may continue throughout life, leading to sustained activation of the nociceptive mechanisms resulting in poor therapeutic outcomes. Pain is an outcome of nociceptive processing in the central nervous system (CNS), triggered by peripheral nervous system response to exogenous and endogenous stimuli. Activation of transient receptor potential vanilloid 1 (TRPV1) channels on C-fibers, neurogenic inflammation, mast cell activation, systemic inflammation, and oxidative stress in the periphery have been demonstrated in SCA.2,3 However, the extent and mechanisms of CNS involvement remain unknown in SCA. The activation of inflammatory and neuronal cells in the CNS plays a pivotal role in nociception.4 We recently observed that spinal nociceptive neurons are sensitized in sickle mice, suggestive of central sensitization.5 Bidirectional signaling occurs between neurons and immunocompetent cells present in the CNS, including microglia, astrocytes and oligodendrocytes.4 Activated microglia release reactive oxidative species (ROS), inflammatory cytokines, neurotrophic factors, and prostaglandins that excite nociceptive neurons and contribute to the persistence of chronic pain.4 It is therefore likely that the activation of central nociceptive mechanisms contributes to chronic pain in SCA. Remarkable decreases in inflammation, thiobarbituric acid reactive substances (TBARS, an indicator of oxidative stress), and VOC have been observed in SCA patients receiving coenzyme Q10 (CoQ10).6 Additionally, curcumin reduced markers of oxidative stress in thalassemia patients and also ameliorates pain hypersensitivity in rats with monoarthritis by decreasing spinal neuroinflammation.7,8 Since excess free iron due to hemolysis contributes to oxidative stress and inflammation in SCA, we examined glial activation, inflammation and oxidative stress in the spinal cords of sickle mice and tested the possibility of a synergistic effect of CoQ10 and/or curcumin to ameliorate spinal oxidative stress, glial activation and hyperalgesia. To examine our hypotheses, we used female transgenic HbSS-BERK sickle mice with murine α and β globin knockouts and expressing human sickle, or normal haemoglobin A (designated sickle or control mice, henceforth, respectively). We bred and characterized these mice by pheno- and genotyping (see Online Supplementary Appendix for details).3,9 These sickle mice have severe hematologic disease, organ damage and tonic hyperalgesia similar to that observed in human SCA.9–11 Sickle mice received either vehicle (olive oil), curcumin (15 mg/kg), CoQ10 (45 mg/kg), or both CoQ10 and curcumin (cotreatment) daily for 4 weeks by gavage. Female mice were used because BERK female mice show more hyperalgesia as compared to males.11 Pain behaviors were evaluated during the proestrous/estrous cycle before treatment and weekly. The mice were tested for mechanical hyperalgesia using paw withdrawal frequency (PWF) in response to von Frey filaments, paw withdrawal latency (PWL) in response to a heat stimulus using a Hargreave’s apparatus; and sensitivity to cold was determined by PWL and PWF per 2 min on a cold plate (see detailed procedures in the Online Supplementary Appendix).11 Spinal cords were harvested after 4 weeks of treatment. Sections were examined by laser scanning confocal microscopy (LSCM) for Iba1, a microglial marker (Wako, Richmond, VA, USA), glial fibrillary acidic protein (GFAP), an astrocyte marker (Abcam, Cambridge, MA, USA), neuropeptide substance P (SP, Abcam) and detection of ROS with dihydroethidium (DHE, Life Technologies, Grand Island, NY, USA).

Vitamin D Regulates Fatty Acid Composition in Subcutaneous Adipose Tissue Through Elovl3

Fatty acids (FAs) are a major energy source in the body. White adipose tissue (WAT) is a primary site where FAs are stored as triacylglycerols. Brown adipose tissue also stores and recruits FAs as a carbon source for uncoupled β-oxidation during thermogenesis. The deletion of the vitamin D nuclear hormone receptor (VDR) gene in mice (VDRKO) results in a lean WAT phenotype with increased levels of expression of the brown adipose tissue marker Ucp1 in the WAT. However, the impact of vitamin D/VDR on FA composition in WAT has not been explored in detail. To address this question, we examined the FA composition of sc and visceral white adipose depots of VDRKO mice. We found that the levels of a subset of saturated and monounsaturated FAs of C18-C24 are specifically increased in the sc adipose depot in VDRKO mice. We revealed that a specific elongase enzyme (Elovl3), which has an important role in brown fat biology, is directly regulated by VDR and likely contributes to the altered FA composition in VDRKO mice. We also demonstrate that Elovl3 is regulated by vitamin D in vivo and tissue specifically in the sc WAT depot. We discovered that regulation of Elovl3 expression is mediated by ligand-dependent VDR occupancy of a negative-response element in the promoter proximal region of the Elovl3 gene. These data suggest that vitamin D/VDR tissue specifically modulates FA composition in sc WAT through direct regulation of Elovl3 expression.

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth‐promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration.

Transient Global Amnesia Associated with a Unilateral Infarction of the Fornix: Case Report and Review of the Literature

Stroke is an extremely uncommon cause of transient global amnesia (TGA). Unilateral lesions of the fornix rarely cause amnesia and have not previously been reported to be associated with the distinctive amnesic picture of TGA. We describe the case of a 60-year-old woman who presented with acute onset, recent retrograde, and anterograde amnesia characteristic of TGA. Serial magnetic resonance imaging showed a persistent focal infarction of the body and left column of the fornix, without acute lesions in the hippocampus or other structures. Amnesia resolved in 6 h. Infarction of the fornix should thus be included in the differential diagnosis of TGA, as it changes the management of this otherwise self-limited syndrome.

Electroacupuncture in conscious free-moving mice reduces pain by ameliorating peripheral and central nociceptive mechanisms.

Integrative approaches such as electroacupuncture, devoid of drug effects are gaining prominence for treating pain. Understanding the mechanisms of electroacupuncture induced analgesia would benefit chronic pain conditions such as sickle cell disease (SCD), for which patients may require opioid analgesics throughout life. Mouse models are instructive in developing a mechanistic understanding of pain, but the anesthesia/restraint required to administer electroacupuncture may alter the underlying mechanisms. To overcome these limitations, we developed a method to perform electroacupuncture in conscious, freely moving, unrestrained mice. Using this technique we demonstrate a significant analgesic effect in transgenic mouse models of SCD and cancer as well as complete Freund’s adjuvant-induced pain. We demonstrate a comprehensive antinociceptive effect on mechanical, cold and deep tissue hyperalagesia in both genders. Interestingly, individual mice showed a variable response to electroacupuncture, categorized into high-, moderate-, and non-responders. Mechanistically, electroacupuncture significantly ameliorated inflammatory and nociceptive mediators both peripherally and centrally in sickle mice correlative to the antinociceptive response. Application of sub-optimal doses of morphine in electroacupuncture-treated moderate-responders produced equivalent antinociception as obtained in high-responders. Electroacupuncture in conscious freely moving mice offers an effective approach to develop a mechanism-based understanding of analgesia devoid of the influence of anesthetics or restraints.

Quantification of pain in sickle mice using facial expressions and body measurements

Pain is a hallmark feature of sickle cell disease (SCD). Subjects typically quantify pain by themselves, which can be biased by other factors leading to overtreatment or under-treatment. Reliable and accurate quantification of pain, in real time, might enable to provide appropriate levels of analgesic treatment. The mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability has been used to quantify varied types of pain. We hypothesized that addition of the objective parameters of body length and back curvature will strengthen the reproducibility of MGS. We examined MGS scores and body length and back curvature of transgenic BERK sickle and control mice following cold treatment or following treatment with analgesic cannabinoid CP55,940. We observed that sickle mice demonstrated decreased length and increased back curvature in response to cold. These observations correlate with changes in facial expression for the MGS score. CP55,940 treatment of sickle mice showed an increase in body length and a decrease in back curvature concordant with MGS scores indicative of an analgesic effect. Thus, body parameters combined with facial expressions may provide a quantifiable unbiased method for objective measure of pain in SCD.

Biomarkers and Electrocardiographic Evidence of Myocardial Ischemia in Patients With Human Immunodeficiency Virus Infection

We assessed the relation of inflammatory and coagulation biomarkers with electrocardiographic (ECG) evidence of myocardial ischemia. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer levels were measured at study entry for 3,085 human immunodeficiency virus-infected participants (mean age 44 years; 26.4% women; 24.6% black) in the Strategies for Management of Antiretroviral Therapy trial. Logistic regression models were used to examine the associations of these biomarkers with prevalent and incident myocardial ischemia. The latter analyses were performed for 1,411 participants who were randomly assigned to receive continuous antiretroviral therapy during follow-up to suppress the human immunodeficiency virus viral load and had ≥1 ECG reading during the follow-up period. The median hsCRP, IL-6, and D-dimer level was 1.65 μg/ml (interquartile range 0.69 to 4.11), 1.60 pg/ml (interquartile range 1.00 to 2.75), and 0.18 μg/ml (interquartile range 0.11 to 0.32), respectively. At baseline, the prevalence of major or minor Q-QS or ST-T ECG abnormalities was 18.6%. The biomarker levels were associated with prevalent major or minor ischemic abnormalities on the univariate analyses; however, adjustment for traditional risk factors attenuated these associations. The adjusted odds ratio for major or minor ischemic abnormalities and 95% confidence intervals for the greatest versus lowest quartiles was 1.3 (95% confidence interval 0.9 to 1.7) for hsCRP, 1.0 (95% confidence interval 0.7 to 1.3) for IL-6, and 1.1 (95% confidence interval 0.9 to 1.5) for D-dimer. During a median follow-up of 2.3 years, new definite or probable ischemic ECG abnormalities developed in 11.7% of participants receiving continuous antiretroviral therapy. Biomarker levels were not associated with incident abnormalities on unadjusted or adjusted analyses. In conclusion, higher levels of hsCRP, IL-6, and D-dimer were not associated with ischemic ECG abnormalities. Elevated biomarker levels and ECG abnormalities indicating myocardial ischemia might reflect different risk pathways for cardiovascular disease.