Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice

Akt is an important intracellular mediator of beta-cell growth and survival in rodents. However, whether constitutive activation of Akt in human beta-cells enhances the survival and function of transplanted islets is unknown. In the current study, we examined the efficacy of constitutive activation of Akt in improving human islet transplant outcomes using a marginal mass model in diabetic severe combined immunodeficient (SCID) mice. Human islets transduced with adenoviruses encoding constitutively active Akt1 (Adv-CA-Akt) displayed increased total and phosphorylated Akt and Akt kinase activity compared with control islets. Expression of CA-Akt in human islets induced a significant increase in beta-cell replication and a significant decrease in beta-cell death induced by serum and glucose deprivation or chronic hyperglycemia. Two control groups of islets (1,500 uninfected or adenovirus LacZ [Adv-LacZ]-transduced human islet equivalents [IEQs]) transplanted under the kidney capsule of streptozotocin-induced diabetic SCID mice were insufficient to correct hyperglycemia. Importantly and in marked contrast to these controls, 1,500 Adv-CA-Akt-transduced IEQs were capable of restoring euglycemia in diabetic SCID mice. Moreover, blood glucose normalization persisted for at least 6 months. Human plasma insulin at day 54 after transplant was 10-fold higher in Adv-CA-Akt islet recipients (2.4 +/- 0.4 ng/ml) compared with those receiving Adv-LacZ islets (0.25 +/- 0.08 ng/ml) (P < 0.05). In summary, expression of CA-Akt in human islets improves islet transplant outcomes in a subcapsular renal graft model in SCID mice. Akt is an attractive target for future strategies aimed at reducing the number of islets required for successful islet transplantation in humans.     

Overexpression of parathyroid hormone-related protein inhibits pancreatic beta-cell death in vivo and in vitro

Pancreatic beta-cell survival is critical in the setting of diabetes as well as in islet transplantation. Transgenic mice overexpressing parathyroid hormone-related protein (PTHrP) targeted to beta-cells using the rat insulin II promoter (RIP) display hyperinsulinemia, hypoglycemia, and islet hyperplasia, without a concomitant increase in beta-cell proliferation rate or enlargement of individual beta-cell size. Thus, the mechanism for increased beta-cell mass is unknown. In this study, we demonstrated that beta-cells of transgenic mice are resistant to the cytotoxic effects of streptozotocin (STZ) in vivo, as documented by a sixfold reduction in the rate of STZ-induced beta-cell death in RIP-PTHrP mice relative to their normal siblings. The reduced cell death in transgenic mice is due neither to their increased islet mass nor to a decrease in their sensing of STZ, but rather results from PTHrP-induced resistance to beta-cell death. This is also demonstrated in vitro by markedly reduced cell death rates observed in beta-cells of transgenic mice compared with normal mice when cultured in the absence of serum and glucose or in the presence of STZ. Finally, we demonstrated that NH(2)-terminal PTHrP inhibits beta-cell death. These findings support the concept that PTHrP overexpression increases islet mass in transgenic mice through inhibition of beta-cell death.     

Decreased calcitonin gene-related peptide expression in the dorsal root ganglia of TNF-deficient mice in a monoiodoacetate-induced knee osteoarthritis model

Background: The detailed mechanisms of knee osteoarthritis (OA) pain have not been clarified, but involvement of inflammatory cytokines such as tumor necrosis factor-alpha (TNF) has been suggested. The present study aimed to investigate the more detailed neurological involvement of TNF in joint pain using a TNF-knockout mouse OA model. Methods: The right knees of twelve-week-old C57BL/6J wild and TNF-deficient knockout (TNF-ko) mice (n=15, each group) were given a single intra-articular injection of 10 µg monoiodoacetate in 10 mL sterile saline. The left knees were only punctured as the control. Evaluations were performed immediately after the injection (baseline) and at 7, 14, and 28 days after the injection with a subsequent intra-articular injection of neurotracer into both knees. The animals were evaluated for immunofluorescence of the lumbar dorsal root ganglia (DRG) innervating the knee joints. The injected knees were observed macroscopically and mouse pain-related behaviors were scored. Results: Macroscopic observation showed similar knee OA development in both wild and TNF-ko mice. Calcitonin gene-related peptide (CGRP, a neuropeptide identified as a inflammatory pain-related biomarker) was significantly increased in DRG neurons innervating OA-induced knee joints with significantly less CGRP expression in TNF-ko animals. Pain-related behavior scoring showed a significant increase in pain in OA-induced joints, but there was no significant difference in pain observed between the wild and TNF-ko mice. Conclusions: The result of the present study indicates the possible association of TNF-alpha in OA pain but not OA development.     

Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.     

Vein wrapping facilitates basic fibroblast growth factor‐induced heme oxygenase‐1 expression following chronic nerve constriction injury

The clinical efficacy of autologous vein wrapping for recurrent compressive neuropathy has been demonstrated; however, the underlying mechanisms of this technique remain unclear. Rats were divided into chronic constriction injury (CCI) and CCI + vein wrapping (CCI + VW) groups. Mechanical allodynia was evaluated using von Frey filaments. To identify the neuroprotective factors released from veins, basic fibroblast growth factor (bFGF) mRNA expression in veins was compared to that in the sciatic nerve. The response of heme oxygenase‐1 (HO‐1) expression to vein wrapping was evaluated by RT‐PCR and enzyme‐linked immunosorbent assays. The effects of exogenous bFGF on HO‐1 expression were evaluated using a sciatic nerve cell culture. Vein wrapping significantly increased the withdraw threshold levels compared to the untreated CCI group. bFGF mRNA expression in veins was higher than that in untreated sciatic nerves. HO‐1 mRNA expression was induced at higher levels in sciatic nerve cells in the presence of exogenous bFGF compared to untreated control cells. HO‐1 mRNA and protein expression in the sciatic nerve were also higher in the CCI + VW group compared with the CCI group. Our results suggest that vein‐derived bFGF contributes to the therapeutic benefit of vein wrapping through the induction of HO‐1 in the sciatic nerve. Vein wrapping is a useful technique for reducing neuropathic pain. Further understanding of the neurotrophic factors released from veins may help to optimize current procedures for treating recurrent compressive neuropathy and traumatic peripheral nerve injury, and lead to the development of new therapeutic methods using recombinant neurotrophic factors. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:898–905, 2018.

     

Level, distribution and correlates of platelet-derived microparticles in healthy individuals with special reference to the metabolic syndrome

Platelet-derived microparticles (PDMPs), a procoagulant factor, are reportedly elevated in type 2 diabetes mellitus and acute coronary syndrome. The metabolic syndrome (MS) is strongly associated with cardio- and cerebrovascular disease-related atherothrombotic events. To clarify the level, distribution and correlates of PDMPs with special reference to MS, we conducted a cross-sectional study of 467 healthy Japanese volunteers without signs, symptoms, or a history of cardio- or cerebrovascular disease. They were 211 men and 256 women (median age 39 and 35 years, respectively). Using an ELISA kit and monoclonal antibodies against CD42b and CD42a (glycoprotein Ib and IX) we assayed the PDMP levels. Total cholesterol, low-density and high-density lipoprotein cholesterol, remnant cholesterol, triglycerides, C-reactive protein, and traditional cardiovascular risk factors were also recorded. There was a significant difference in the level of PDMPs between men and women. The median value and the interquartile range of PDMPs was 8.3 IU/ml and 6.2-10.5 IU/ml and 6.8 IU/ml and 5.2-8.6 IU/ml, respectively, in men and women. PDMPs were significantly associated with MS criteria in men (p < 0.001) and women (p = 0.040). Logistic regression analysis revealed a significant odds ratio of 3.9 [95% confidence interval (CI): 1.4-10.5] in men and of 4.2 [95% CI: 1.6-10.7] in the entire study population. Our results suggest that PDMPs identified by glycoprotein CD42b and CD42a are positively associated with MS.     

Possible enhancing effects of atrazine on growth of 7,12-dimethylbenz(a) anthracene-induced mammary tumors in ovariectomized Sprague-Dawley rats

Atrazine, one of the most commonly used herbicides in the world, has been reported to have endocrine disrupting effects in vivo. In the present experiment, influence of dietary atrazine on the late promotion/progression stage of mammary carcinogenesis in ovariectomized female Sprague-Dawley rats was examined after a single intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA). When the incidence of palpable mammary tumors reached about 50%, the animals were subjected to ovariectomy and divided into tumor bearing [DMBA-Tumor(+)] and non-tumor bearing [DMBA-Tumor(-)] groups, with subgroups of each fed a soybean-free diet containing 0, 5, 50, or 500 p.p.m. atrazine for 34 weeks. At the completion of the study, the tumor volume in the 50 and 500 p.p.m. treatment Tumor(+) subgroups was greater than in the 0 p.p.m. control case. In the DMBA-Tumor(-) group, higher incidences and volumes of the mammary tumors, with or without statistical significance (P <0.05), were observed in the 50 and 500 p.p.m. subgroups. Atrazine treatment tended to increase proportion of estrogen receptor alpha-positive tumors and stimulated cell proliferation in the DMBA-Tumor(+) group, but with no clear effects on serum hormone levels. The present study indicates that atrazine has a potential for enhancing the growth of mammary tumors, partly through increasing cell proliferation in the promotion/progression stage in female rats under ovarian hormone-free conditions.     

Sequence variability and candidate gene analysis in two cancer patients with complex clinical outcomes during morphine therapy.

In this case report, we present genetic differences in two morphine-related gene sequences, UDP-glucuronosyltransferase 2B7 (UGT2B7) and mu opioid receptors (MOR1), in two cancer patients whose clinical responses to morphine were very different [i.e., sensitive (patient 1) and low responder (patient 2)]. In addition, allelic variants in the UGT2B7 gene were analyzed in 46 Japanese individuals. Amplified DNA fragments for the two genes of interest were screened using single strand conformation polymorphism and then sequenced. In the UGT2B7 gene, 12 single nucleotide polymorphisms (SNPs) were newly identified with an allelic frequency ranging from 0.022 to 0.978. Six SNPs in the promoter region (A-1302G, T-1295C, T-1111C, G-899A, A-327G, and T-125C) and two coding SNPs (UGT2B7*2 in exon 2 and C1059G in exon 4) appeared to be consistently linked. Remarkable differences in the nucleotide sequence of UGT2B7 were observed between the two patients; in contrast to patient 1 who had "reference" alleles at almost SNP positions, but a rare ATTGAT*2(AT)C haplotype as homozygosity, patient 2 was a homozygous carrier for the predominant GCCAGC*1(TC)G sequence. Serum morphine and two glucuronide concentrations in patient 2 suggest that the predominant GCCAGC*1G sequence was not associated with a "poor metabolizer" phenotype. In the MOR1 gene, patient 1 had no SNPs, whereas patient 2 was a heterozygous carrier for both the G-1784A and A118G alleles. The present study describes substantial differences in genotype patterns of two genes of interest between the two patients. The results necessitate larger trials to confirm these observations in larger case control studies.