Victor Prima

Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats

Adiponectin (Acrp30) is a physiologically active polypeptide hormone secreted by adipose tissue that shows insulin-sensitizing, antiinflammatory, and antiatherogenic properties. In humans, Acrp30 levels are inversely related to the degree of adiposity. In the current study, we tested the long-term weight-reducing and insulin-enhancing effects of Acrp30 cDNA delivered peripherally by a viral vector. To this end, we have generated a series of recombinant adeno-associated virus vectors of serotypes 1 and 5 encoding mouse Acrp30 cDNAs. The long-term expression of recombinant adeno-associated virus-Acrp30 vectors was tested after intramuscular or intraportal injection in female Sprague–Dawley rats with diet-induced obesity. We show that a single peripheral injection of 1012 physical particles of Acrp30-encoding vectors resulted in sustained (up to 280 days) significant reduction in body weight, concomitant with the reduction in daily food intake. Acrp30 treatment resulted in higher peripheral insulin sensitivity measured by the i.p. glucose tolerance test in fasted animals. Ectopic expression of the Acrp30 transgene resulted in modulation of hepatic gluconeogenesis and lipogenesis, as demonstrated by the reduction of the expression of two key genes: PEPCK (phosphoenolpyruvate carboxykinase) and SREBP-1c (sterol regulatory element-binding protein 1c) in the liver. These data show successful peripheral therapy in a clinically relevant model for human obesity and insulin resistance.

Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats

The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged-obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance.

Leptin-Induced Leptin Resistance Reveals Separate Roles for the Anorexic and Thermogenic Responses in Weight Maintenance

The purpose of this study was to determine whether leptin induces leptin resistance by examining the temporal attenuation of the anorexic and energy expenditure responses to leptin. We administered recombinant adeno-associated virus encoding rat leptin cDNA or control viral vector into mildly obese rats for 138 d and compared these results with those from pair-fed rats. We measured food consumption, body weight, oxygen consumption, leptin signal transduction, and brown adipose tissue uncoupling protein 1. The anorexic response attenuated by d 25, whereas the increase in energy expenditure persisted for 83 d before attenuating. Despite attenuation of physiological responses, phosphorylated signal transducer and activator of transcription-3 remained elevated for the duration of the study. The temporal differential attenuation of the anorexic and thermogenic responses allowed us to determine the relative contributions of each response to weight maintenance. The anorexic response predominantly mediated the initial loss of body weight, but only the energy expenditure response was necessary to maintain the reduced weight. This study provides evidence that leptin induces leptin resistance. The leptin resistance was associated with persistent elevation in hypothalamic phosphorylated signal transducer and activator of transcription-3 and was characterized by a rapid attenuation of the anorexic response and slower onset for the attenuation of the energy expenditure response. We propose that both elevated leptin and obesity may be necessary for the development of leptin resistance.

Neuro-Glial and Systemic Mechanisms of Pathological Responses in Rat Models of Primary Blast Overpressure Compared to “Composite” Blast

A number of experimental models of blast brain injury have been implemented in rodents and larger animals. However, the variety of blast sources and the complexity of blast wave biophysics have made data on injury mechanisms and biomarkers difficult to analyze and compare. Recently, we showed the importance of rat position toward blast generated by an external shock tube. In this study, we further characterized blast producing moderate traumatic brain injury and defined “composite” blast and primary blast exposure set-ups. Schlieren optics visualized interaction between the head and a shock wave generated by external shock tube, revealing strong head acceleration upon positioning the rat on-axis with the shock tube (composite blast), but negligible skull movement upon peak overpressure exposure off-axis (primary blast). Brain injury signatures of a primary blast hitting the frontal head were assessed and compared to damage produced by composite blast. Low to negligible levels of neurodegeneration were found following primary blast compared to composite blast by silver staining. However, persistent gliosis in hippocampus and accumulation of GFAP/CNPase in circulation was detected after both primary and composite blast. Also, markers of vascular/endothelial inflammation integrin alpha/beta, soluble intercellular adhesion molecule-1, and L-selectin along with neurotrophic factor nerve growth factor-beta were increased in serum within 6 h post-blasts and persisted for 7 days thereafter. In contrast, systemic IL-1, IL-10, fractalkine, neuroendocrine peptide Orexin A, and VEGF receptor Neuropilin-2 (NRP-2) were raised predominantly after primary blast exposure. In conclusion, biomarkers of major pathological pathways were elevated at all blast set-ups. The most significant and persistent changes in neuro-glial markers were found after composite blast, while primary blast instigated prominent systemic cytokine/chemokine, Orexin A, and Neuropilin-2 release, particularly when primary blast impacted rats with unprotected body.

Assessing neuro-systemic & behavioral components in the pathophysiology of blast-related brain injury

Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the “distinct” but shared and partially identified biochemical pathways and neuro-histopathological changes that might be linked to behavioral deficits observed. Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.

Cloning and functional characterization of MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins created by a variant t(1;19)(q23;p13.3) in acute lymphoblastic leukemia.

We analyzed the TS-2 acute lymphoblastic leukemia (ALL) cell line that contains a t(1;19)(q23;p13.3) but lacks E2A-PBX1 fusion typically present in leukemias with this translocation. We found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (Deleted in Azoospermia-Associated Protein 1) to the 1q23 gene MEF2D (Myocyte Enhancer Factor 2D), leading to expression of reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. MEF2D is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in control of muscle cell differentiation, and signaling pathways that mediate response to mitogenic signals and survival of neurons and T-lymphocytes. DAZAP1 is a novel RNA binding protein expressed most abundantly in the testis. We demonstrate that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D and is a substantially more potent transcriptional activator than MEF2D. We also show that DAZAP1/MEF2D is a sequence-specific RNA-binding protein. MEF2D has been identified as a candidate oncogene in murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contribute to leukemogenesis by altering signaling pathways normally regulated by wild-type MEF2D and DAZAP1.

Cooperative transformation by MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins generated by the variant t(1;19) in acute lymphoblastic leukemia

A variant t(1;19)(q23;p13.3) translocation creates reciprocal DAZAP1/MEF2D and MEF2D/DAZAP1 fusion genes that are expressed in acute lymphoblastic leukemia. We used retroviral gene transfer to ectopically express wild-type and chimeric DAZAP1 and MEF2D fusion proteins in NIH 3T3 cells. In soft agar assays, each of the fusion proteins transformed 3T3 cells with a 20-fold increase in colony formation as compared to empty vector or native MEF2D or DAZAP1 proteins. Co-expression of both DAZAP1/MEF2D and MEF2D/DAZAP1 led to a threefold increase in colony formation as compared to either fusion protein alone. Expression of wild-type DAZAP1, MEF2D or DAZAP1/MEF2D allowed 3T3 cells to proliferate under low serum (0.5%) conditions and suppressed apoptosis. In contrast, MEF2D/DAZAP1 expression did not facilitate proliferation in low serum and led to a modest increase in apoptosis. Both MEF2D/DAZAP1 and DAZAP1/MEF2D have oncogenic properties, and co-expression of both fusion proteins is synergistic.

Overpressure blast-wave induced brain injury elevates oxidative stress in the hypothalamus and catecholamine biosynthesis in the rat adrenal medulla

Explosive overpressure brain injury (OBI) impacts the lives of both military and civilian population. We hypothesize that a single exposure to OBI results in increased hypothalamic expression of oxidative stress and activation of the sympatho-adrenal medullary axis. Since a key component of blast-induced organ injury is the primary overpressure wave, we assessed selective biochemical markers of autonomic function and oxidative stress in male Sprague Dawley rats subjected to head-directed overpressure insult. Rats were subjected to single head-directed OBI with a 358kPa peak overpressure at the target. Control rats were exposed to just noise signal being placed at ~2m distance from the shock tube nozzle. Sympathetic nervous system activation of the adrenal medullae (AM) was evaluated at 6h following blast injury by assessing the expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH), dopamine-β hydroxylase (DβH), neuropeptide Y (NPY) along with plasma norepinephrine (NE). TH, DβH and NPY expression increased 20%, 25%, and 91% respectively, following OBI (P<0.05). Plasma NE was also significantly elevated by 23% (P<0.05) following OBI. OBI significantly elevated TH (49%, P<0.05) in the nucleus tractus solitarius (NTS) of the brain stem while AT1 receptor expression and NADPH oxidase activity, a marker of oxidative stress, was elevated in the hypothalamus following OBI. Collectively, the increased levels of TH, DβH and NPY expression in the rat AM, elevated TH in NTS along with increased plasma NE suggest that single OBI exposure results in increased sympathoexcitation. The mechanism may involve the elevated AT1 receptor expression and NADPH oxidase levels in the hypothalamus. Taken together, such effects may be important factors contributing to pathology of brain injury and autonomic dysfunction associated with the clinical profile of patients following OBI.

E2A - ZNF384 and NOL1 - E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)–PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3′ portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5′ regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT–PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

ASS and SULT2A1 are Novel and Sensitive Biomarkers of Acute Hepatic Injury-A Comparative Study in Animal Models

Liver and kidney damage associated with polytrauma, endotoxic shock/sepsis, and organ transplantation, are among the leading causes of the multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver and kidney injury is vital for the effective diagnostics and treatment of these life-threatening conditions. Previously, we identified several hepatic proteins, including Argininosuccinate Synthase (ASS) and sulfotransferases which were degraded in the liver and rapidly released into circulation during Ischemia/Reperfusion (I/R) injury. Here we compared sensitivity and specificity of the newly developed sandwich ELISA assays for ASS and the sulfotransferase isoform SULT2A1 with the standard clinical liver and kidney tests Alanine Aminotransferase (ALT) and Aspartate Transaminase (AST) in various pre-clinical models of acute injury. Our data suggest that ASS and SULT2A1 have superior characteristics for liver and kidney health assessment in endotoxemia, Ischemia/Reperfusion (I/R), chemical and drug-induced liver injury and may be of high potential value for clinical applications.