Kelly J. Ledford

Susceptibility to Diet-Induced Hepatic Steatosis and Glucocorticoid Resistance in FK506-Binding Protein 52-Deficient Mice

Although FK506-binding protein 52 (FKBP52) is an established positive regulator of glucocorticoid receptor (GR) activity, an in vivo role for FKBP52 in glucocorticoid control of metabolism has not been reported. To address this question, FKBP52(+/-) mice were placed on a high-fat (HF) diet known to induce obesity, hepatic steatosis, and insulin resistance. Tissue profiling of wild-type mice showed high levels of FKBP52 in the liver but little to no expression in muscle or adipose tissue, predicting a restricted pattern of FKBP52 effects on metabolism. In response to HF, FKBP52(+/-) mice demonstrated a susceptibility to hyperglycemia and hyperinsulinemia that correlated with reduced insulin clearance and reduced expression of hepatic CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a mediator of clearance. Livers of HF-fed mutant mice had high lipid content and elevated expression of lipogenic genes (peroxisome proliferator-activated receptor gamma, fatty acid synthase, and sterol regulatory element-binding protein 1c) and inflammatory markers (TNFalpha). Interestingly, mutant mice under HF showed elevated serum corticosterone, but their steatotic livers had reduced expression of gluconeogenic genes (phosphoenolpyruvate carboxy kinase, glucose 6 phosphatase, and pyruvate dehydrogenase kinase 4), whereas muscle and adipose expressed normal to elevated levels of glucocorticoid markers. These data suggest a state of glucocorticoid resistance arising from liver-specific loss of GR activity. Consistent with this hypothesis, reduced expression of gluconeogenic genes and CEACAM1 was observed in dexamethasone-treated FKBP52-deficient mouse embryonic fibroblast cells. We propose a model in which FKBP52 loss reduces GR control of gluconeogenesis, predisposing the liver to steatosis under HF-diet conditions attributable to a shunting of metabolism from glucose production to lipogenesis.

Development of nonalcoholic steatohepatitis in insulin-resistant liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice.

BACKGROUND & AIMS Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 causes hyperinsulinemia and insulin resistance, which result from impaired insulin clearance, in liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice (L-SACC1). These mice also develop steatosis. Because hepatic fat accumulation precedes hepatitis, lipid peroxidation, and apoptosis in the pathogenesis of nonalcoholic steatohepatitis (NASH), we investigated whether a high-fat diet, by causing inflammation, is sufficient to induce hepatitis and other features of NASH in L-SACC1 mice. METHODS L-SACC1 and wild-type mice were placed on a high-fat diet for 3 months, then several biochemical and histologic analyses were performed to investigate the NASH phenotype. RESULTS A high-fat diet caused hepatic macrosteatosis and hepatitis, characterized by increased hepatic tumor necrosis factor alpha levels and activation of the NF-kappaB pathway in L-SACC1 but not in wild-type mice. The high-fat diet also induced necrosis and apoptosis in the livers of the L-SACC1 mice. Insulin resistance in L-SACC1 fed a high-fat diet increased the hepatic procollagen protein level, suggesting a role in the development of fibrosis. CONCLUSIONS A high-fat diet induces key features of human NASH in insulin-resistant L-SACC1 mice, validating this model as a tool to study the molecular mechanisms of NASH.

Ceacam1 deletion causes vascular alterations in large vessels

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulins stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Mice with null mutation of Ceacam1 develop nonalcoholic steatohepatitis

Transgenic liver-specific inactivation of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) impairs hepatic insulin clearance and causes hyperinsulinemia, insulin resistance, elevation in hepatic and serum triglyceride levels, and visceral obesity. It also predisposes to nonalchoholic steatohepatitis (NASH) in response to a high-fat diet. To discern whether this phenotype reflects a physiological function of CEACAM1 rather than the effect of the dominant-negative transgene, we investigated whether Ceacam1 (gene encoding CEACAM1 protein) null mice with impaired insulin clearance also develop a NASH-like phenotype on a prolonged high-fat diet. Three-month-old male null and wild-type mice were fed a high-fat diet for 3 months and their NASH phenotype was examined. While high-fat feeding elevated hepatic triglyceride content in both strains of mice, it exacerbated macrosteatosis and caused NASH-characteristic fibrogenic changes and inflammatory responses more intensely in the null mouse. This demonstrates that CEACAM1-dependent insulin clearance pathways are linked with NASH pathogenesis.

Targeted Deletion of Murine CEACAM 1 Activates PI3K-Akt Signaling and Contributes to the Expression of (Pro)Renin Receptor via CREB Family and NF-κB Transcription Factors

The carcinoembryonic antigen–related cell adhesion molecule 1 regulates insulin sensitivity by promoting hepatic insulin clearance. Mice bearing a null mutation of Ceacam1 gene (Cc1–/–) develop impaired insulin clearance followed by hyperinsulinemia and insulin resistance, in addition to visceral obesity and increased plasma fatty acids. Because insulin resistance is associated with increased blood pressure, we investigated whether they develop higher blood pressure with activated renal renin-angiotensin system and whether this is mediated, in part, by the upregulation of renal (pro)renin receptor (PRR) expression. Compared with age-matched wild-type littermates, Cc1–/– mice exhibited increased blood pressure with increased activation of renal renin-angiotensin systems and renal PRR expression. Cytoplasmic and nuclear immunostaining of phospho-PI3K p85&agr; and phospho-Akt was enhanced in the kidney of Cc1–/– mice. In murine renal inner medullary collecting duct epithelial cells with lentiviral-mediated small hairpin RNA knockdown of carcinoembryonic antigen–related cell adhesion molecule 1, PRR expression was upregulated and phosphorylation of PI3K (Tyr508), Akt (Ser473), NF-&kgr;B p65 (Ser276), cAMP response element–binding protein/activated transcription factor (ATF)-1 (Ser133), and ATF-2 (Thr71) was enhanced. Inhibiting PI3K with LY294002 or Akt with Akt inhibitor VIII attenuated PRR expression. In conclusion, global null deletion of Ceacam1 caused an increase in blood pressure with increased renin-angiotensin system activation together with upregulation of PRR via PI3K-Akt activation of cAMP response element–binding protein 1, ATF-1, ATF-2, and NF-&kgr;B p65 transcription factors.

Therapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy for treatment of ischemic cardiovascular diseases

IntroductionBone marrow derived cellular therapies are an emerging approach to promoting therapeutic angiogenesis in ischemic cardiovascular disease. However, the percentage of regenerative cells in bone marrow mononuclear cells (BMMNCs) is small, and large amounts of BMMNCs are required. Ixmyelocel-T, an expanded autologous multicellular therapy, is manufactured from a small sample of bone marrow aspirate. Ixmyelocel-T contains expanded populations of mesenchymal stromal cells (MSCs) and M2-like macrophages, as well as many of the CD45+ cells found in the bone marrow. It is hypothesized that this expanded multi-cellular therapy would induce angiogenesis and endothelial repair.MethodsA rat model of hind limb ischemia was used to determine the effects of ixmyelocel-T on blood flow recovery. To further determine the effects on endothelial cells, ixmyelocel-T was co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell® inserts.ResultsCo-culture of HUVECs with ixmyelocel-T resulted secretion of a variety of pro-angiogenic factors. HUVECs stimulated by ixmyelocel-T exhibited enhanced migration, proliferation, and branch formation. Ixmyelocel-T co-culture also resulted in increased endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. In tumor necrosis factor alpha (TNFα)-stimulated HUVECs, ixmyelocel-T co-culture decreased apoptosis and reactive oxygen species generation, increased super oxide dismutase activity, and decreased nuclear factor kappa B (NFκB) activation. Treatment with ixmyelocel-T in a rat model of hind limb ischemia resulted in significantly increased blood flow perfusion and capillary density, gene expression and plasma levels of the anti-inflammatory cytokine interleukin (IL)-10, plasma nitrates, plasma platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF) expression, and significantly decreased plasma thiobarbituric acid reactive substances (TBARS).ConclusionsThis work demonstrates that ixmyelocel-T interacts with endothelial cells in a paracrine manner, resulting in angiogenesis and endothelial protection. This data suggests that ixmyelocel-T could be useful for promoting of angiogenesis and tissue repair in ischemic cardiovascular diseases. In conclusion, ixmyelocel-T therapy may provide a new aspect of therapeutic angiogenesis in this patient population where expanded populations of regenerative cells might be required.

Therapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy, derived from bone marrow, for treatment of ischemic cardiovascular diseases

Innate immune system is a universal form of host defense against infections. The recognition of the innate immunity is based on a limited number of encoded receptors that have evolved to recognize microbial metabolism products. The recognition of these molecular structures allows the immune system to distinguish its own infectious components from non-communicable structures. The immune suppression is a hallmark of sepsis. The complement system is activated in the early stages of sepsis, generating large amounts of anaphylatoxin C5a. Complement and TLRs (toll-like receptors) family are two major upstream sensors and effectors systems of innate immunity. It was found that TLR4 and complement system are involved in the initiation of the inflammatory response in sepsis. Clinical studies in which TLR4 was blocked have not shown beneficial effects. TLRs, that are a subfamily of PRRs (pattern recognition receptors), have emerged as the crucial receptors for the recognition of DAMPs (Damage-associated molecular pattern molecules). Recently, a special form of non-coding genetic material called microRNA has been highlighted in the complex cascade of sepsis. The individual role of every microRNA and the exact role of microRNA network are under investigation. Currently, studies are performed in order to find micro RNA to be used as biomarkers of sepsis. Researches are performed to determine microRNA, small fragments of non-coding RNA, in order to distinguish between patients with sepsis and healthy patients, and if the plasma levels of microRNA correlate with the severity of the disease. Recent researches report that the regulation of gene expression through microRNA plays a very important role in the following cellular processes, for example: apoptosis, the differentiation process, and the cell cycle.Background: Vitamin D is a pivotal factor not only in disorders that involve calcium metabolism, such as osteoporosis and osteomalacia as well as an immunomodulatory effect as noted in several autoimmune conditions in diseases. Very low levels of vitamin D were also demonstrated in systemic sclerosis (SSC) patients. Patients with vitamin D deficiency showed longer and more severe disease. Furthermore, an inverse relationship was found between skin involvement and vitamin D serum concentrations. Associations were found Between Systemic Sclerosis pattern of disease and Scleroderma-Specific Autoantibodies. Novel research demonstrated the presence and importance of anti-vitamin D antibodies in SLE; this motivated our research team to seek for similar antibodies among scleroderma patients.T role of intracellular recognition molecules like NODs expressed by cells of the female reproductive tract (e.g. Fallopian tube epithelium) in the response to chlamydial infection, or any STI, remains poorly understood. A suboptimal innate immune response may result in a permissive environment for pathogen colonization, whereas an over-exuberant response will cause excessive inflammation and tissue damage. Modulation of the host response to infection is an attractive alternative or adjuvant approach to antibiotic therapies in treatment of genital tract infections. Genome sequence analysis has revealed that Chlamydia possesses numerous novel genes that might be involved in the manipulation of the host cells. The infected cells often display altered metabolic, immunological and cell biological characteristics, however, at the same time the microbes have to maintain the integrity and viability of host cells before completing their own intracellular replication. To achieve this goal chlamydiae have evolved the ability to both prevent the infected cells from undergoing apoptosis induced by intracellular stress and to protect these cells from recognition and attack by lymphocytes. Analysis of C. trachomatis genome had identified more than two dozens of open reading frames encoding proteins with potential proteolytic activity. Some of these proteases may be used to target host cell proteins because some proteins are cleaved and/or degraded in infected cells. The attacked host proteins include transcriptional factors, pro and anti apoptoytic proteins, DNA repairing enzymes, cyclins and cytoskeletal protein. However, survival strategies of Chlamydia at the tissue level and the relevance of these findings in disease pathogenesis have yet to be determined. Since till date a Chlamydia vaccine is unavailable, a better definition of human immune response along with Chlamydial survival strategies needs to remain an important research priority if we are to develop a vaccine against C. trachomatis infection that has protective and not deleterious effects.I of angiogenesis is currently perceived as one of the promising strategies in the treatment of cancer. The antiangiogenetic property of thalidomide has inspired a second wave of research on this teratogenic drug. The aim of the current study is to investigate the anti-proliferative and the anti-angiogenic activities of two thalidomide dithiocarbamate analogues by determining their anti-proliferative abilities towards human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 human breast cancer cell lines. Furthermore, their effects on the expression level of TNF-α and VEGF165 mRNA in MDAMB-231 had been assessed. Moreover, we evaluated their effects on angiogenesis using HUVECs through tube formation assay and wound healing assay. Results illustrated that, MDA-MB-231 and HUVECs proliferations were not significantly affected by thalidomide at 6.25-100 μM. Besides, thalidomide failed to block angiogenesis at similar concentrations. However, thalidomide diminished the expression level of TNF-α and VEGF165 by 27% and 34.4%, respectively. Conversely, thalidomide dithiocarbamate analogues 1 and 2 were significantly established anti-proliferative action in MDA-MB-231 and HUVECs without causing cytotoxicity. Two analogues showing powerful anti-angiogenicity in the tube formation assay and wound healing assay as well as analogue 1 demonstrated more potent inhibition ability to suppress TNF-α (79%) and VEGF165 (52%) than analogue 2, TNF-α (56%) and VEGF165 (41%). Analogue 1 consistently showed the highest potency and efficacy in all assays. Taken together, our results support the further development and evaluation of novel thalidomide dithiocarbamate analogues as anti-tumor and anti-angiogenic agents.Background: Acinetobacter baumannii is an important opportunistic pathogen which causes complications in hospitalized patients, especially those in ICU. The aim of this study was to determine the frequency of class 1 and 2 integrons in multi-drug resistance A. baumannii and to investigate the association between the presence of integrons and antibiotic resistance patterns. Methods: A total of 40 A. baumannii strains were isolated from 372 ICU patients from June to Oct 2012. A. baumannii was detected in 50% of tracheal cultures, 15% in blood, 15% in urine samples, and 22.5% in other locations. In accordance with CLSI 2011, 12 antibiotics were used through disc diffusion method. Existence of integron classes was investigated by PCR assay with the amplification of integrase genes. Results: The most effective antibiotic against Acinetobacter baumannii was polymyxin B with 100% susceptibility, followed by meropenem, piperacillin, cotrimoxazole, ceftazidime with 100% resistance; this was followed by ciprofloxacin 97.5%, tetracycline, 92.5%, imipenem 62.5%, and gentamicin 60% resistance. The presence of integron class 1 was 7.5%, class 2 was 67.5%, and non-integron was 20%. Conclusion: The association between multidrug resistance and class 2 integron was not statistically significant. Other factors accounting for the lack of significance of the findings may be the impact of other resistance determinants such as transposons or plasmids, not investigated in the current study. Considering the increasing trend of MDR infections among ICU patients with critical problems in follow up, the use of appropriate infection control strategy and a regular surveillance system is necessary in our hospital.C (CBBN) is a RING-domain containing protein that likely functions as a substrate receptor for the DDB1/Cul4/ CRBN E3 ubiquitin ligase (UbL) complex. Recently, CRBN was shown to be a target of the immunomodulatory (IMiD) drug thalidomide. Although known for inducing severe teratogenicity in the 1950s, this drug class is now used extensively for anticancer immunotherapy. CRBN is expressed in the hematopoietic compartment, but currently has no known function in immune regulation. Ubiquitin ligases including C-Cbl, cbl-b, GRAIL, and ITCH underlie T-cell homeostatic regulation and protect against autoimmunity. To understand the role of CRBN in T-cell function, we studied a mouse with a germline deletion of exon 3 and 4 of this gene. First, crbn-/mice are viable, fertile and normal in appearance without limb malformations. In the T-cell compartment, splenic and peripheral blood lymphocytes are increased at 3 months of age. The expanded population of splenic T-cells was also evident by immunohistochemical staining. Mature differentiated cell lineages such as CD4 and CD8 single positive (SP) thymocytes, SP peripheral T-cells and naive and memory T-cells are similar to wild-type littermates with no apparent spontaneous autoimmune features at 3 months. Similar to IMiD responses, the mature T-cells from crbn-/mice showed superior activation after T-cell receptor (TCR) stimulation. Proximal TCR signaling events including pZAP70 and pLck, cytokine production and survival are increased in knockout mice relative to wild-type littermates. In summary, our findings demonstrate a novel role for CRBN, the molecular target of thalidomide and other members of the IMiD-family, in T-cell activation.Results: A gender ratio of 3.8:1, average age onset of 27±7.3, and a mean diagnostic delay of 8 years was observed. The incidence of juvenile-onset cases was 11%. Positive family history was higher than that observed in most other countries. Both gender groups were similar in terms of ethnicity, positive family history, juvenile-onset AS, and diagnostic delay. Enthesitis was present in more than two-thirds of patients with females demonstrating a higher rate (p<0.05). Uveitis was the leading extra-articular manifestation. Overall HLA-B27 positivity was 73% and four HLA-B27 subtypes were found. HLA-B27 positivity was higher among males (78.3% vs. 55.2%; p<0.001). Disease activity was high and the functional status was poor as indicated by mean Bath AS disease activity, functional, and metrology indices. Female disease was at least as severe as male disease and more severe impairment was present in some aspects. Extra-articular manifestations and treatments modalities presented similar frequencies among genders. Biological medication was utilized less frequently than disease modifying anti-rheumatic drugs and corticosteroids. Quality of life was considerably impaired.N is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection. Decrease in Calcium-dependent protease activity secondary to NMDAR inhibition, has been postulated as a mechanism for this approach. However, this probable mechanism remains to be proven yet. Moreover, selective antagonisation of NR2B subtype of these receptors, an NMDAR subtype believed to play a more pivotal role in neurodegeneration, has not been studied too. In this study, the effect of inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57/Bl6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle in different experimental groups. Behavioral, histological and western blot analysis of Calpain and Caspase 3-dependent α-spectrin break down were studied comparatively among groups. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation, neuro-axonal degeneration and Calpain activity when RO25-6981was administered with higher doses. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes appears to modulate the neurological disabilities and pathological changes in EAE. Decreasing the activity of Calcium-dependent protease, Calpain, by blockade of NR2B-containing NMDAR using high dose of RO25-6981 can be seen simultaneously. More experimental studies could be performed to suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS.Background: Information on the pattern of drug resistant tuberculosis (TB) among re-treatment cases is crucial to develop appropriate control strategies. Therefore, we conducted this study to assess the drug resistance pattern of M. tuberculosis complex (MTBC) isolates and associated factors among re-treatment cases in Jimma area, Southwest Ethiopia. Methods: Health facility-based cross-sectional study was conducted between March 2012 and April 2013 in Jimma area, Southwest Ethiopia. We included 79 re-treatment cases selected conveniently. Socio demographic and clinical data were collected using structured questionnaire. Sputum sample processing, mycobacterial culture, isolation and drug susceptibility testing (DST) were done at Mycobacteriology Research Centre (MRC) of Jimma University. All data were registered and entered in to SPSS version 20. Crude odds ratio (COR) and adjusted odds ratios (AOR) were calculated. P-values less than 0.05 were considered statistically significant. Results: Seventy-nine re-treatment cases included in the study; 48 (60.8 %) were males. Forty- seven (59.5 %) study participants were from rural area with the mean age of 31.67 ± 10.02 SD. DST results were available for 70 MTBC isolates. Majority (58.6 % (41/70)) isolates were resistant to at least one of the four first line drugs. The prevalence of multidrug-resistant TB (MDR-TB) was 31.4 % (22/70). Place of residence (AOR = 3.44 (95 % CI: 1.12, 10.60), duration of illness (AOR = 3.00 (95 % CI: 1.17, 10.69) and frequency of prior TB therapy (AOR = 2.99, (95 % CI: 1.01, 8.86) were significant factors for any drug resistance. Moreover, history of treatment failure was found to be associated with MDR-TB (AOR = 3.43 (95 % CI: 1.14, 10.28). Conclusion: The overall prevalence of MDR-TB among re-treatment cases around Jimma was high. The rate of MDR-TB was higher in patients with the history of anti-TB treatment failure. Timely identification and referral of patients with the history of treatment failure for culture and DST need to be strengthened.C T cells have the competence to attack cancer cells and eradicate a complete tumor. However, this mechanism has showed challenging for two motives. First, T cells and other elements of the immune system ignore self-molecules and cells. Second, tumor microenvironment has immunosuppressive elements capable of producing a mechanism called immunological tolerance. CD4+ T cells generate signaling molecules and activate immune cells that deliver efficient CD8+ T cell response. CD70 is essential for dendritic cells-facilitated delay of T cell tolerance initiation. CD80 and CD86 cells are implicated in the refunctionalizing the tolerized T cell. Here, these cells (CD4+, CD8+, CD70, CD80 and CD86) were obtained from a tumor prostate tissue treated with androgen ablation and were applied in a mouse model of human prostate cancer. Count cell number and purity for the particular cell population was realized by flow cytometric analysis. Preliminary results report that the activity of the CD8+T cells persisted for up to 45 days after treatment with CD4, CD70, CD80 and CD86 cells, and resulted in a significant diminution of tumor size. The stimulation of T cell infiltration and other immune cells in cancer tissues could have effects for the immunotherapeutic treatment of other hormone-related malignant tumors.