LX4211, a dual SGLT1/SGLT2 inhibitor, improved glycemic control in patients with type 2 diabetes in a randomized, placebo-controlled trial.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA(1c); and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

Swine as models in biomedical research and toxicology testing.

Swine are considered to be one of the major animal species used in translational research, surgical models, and procedural training and are increasingly being used as an alternative to the dog or monkey as the choice of nonrodent species in preclinical toxicologic testing of pharmaceuticals. There are unique advantages to the use of swine in this setting given that they share with humans similar anatomic and physiologic characteristics involving the cardiovascular, urinary, integumentary, and digestive systems. However, the investigator needs to be familiar with important anatomic, histopathologic, and clinicopathologic features of the laboratory pig and minipig in order to put background lesions or xenobiotically induced toxicologic changes in their proper perspective and also needs to consider specific anatomic differences when using the pig as a surgical model. Ethical considerations, as well as the existence of significant amounts of background data, from a regulatory perspective, provide further support for the use of this species in experimental or pharmaceutical research studies. It is likely that pigs and minipigs will become an increasingly important animal model for research and pharmaceutical development applications.

P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.

In vitro culture decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF in avian tendon explants.

Weight-bearing tendons in many species, including humans, chickens and horses, are prone to failure, in many cases without a discernible cause. The normal function of the tendon depends on the proper assembly of fibrils of type I collagen, the main structural component of the tendon. We studied the effect of in vitro culture, temperature (37 degrees C vs. 43 degrees C) and wounding on the expression of mRNAs for several collagen regulators, transforming growth factor beta (TGF(beta)), heat shock protein 47 (Hsp47) and connective tissue growth factor (CTGF), in chicken embryonic gastrocnemius tendon explants. The expression of mRNAs for TGF(beta) and Hsp47, a chaperone of collagen assembly, remained strong during the first day of in vitro culture, but then it decreased, slightly more at higher temperature. Additional injury in selected tendons had no significant effect on the levels of TGF(beta) and Hsp47 mRNAs. Likewise, the level of immunostained type I procollagen also decreased with the length of culture. The expression of CTGF gradually increased from 0 at the time of tendon removal with the duration of culture to strong after three days of culture when the expression of TGF(beta) and Hsp47 was low. We conclude that in vitro culture over the period of several days rather than an increase in temperature or additional wounding decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF.

Angiolipomatous tumors in dogs and a cat.

Variants of lipoma are uncommon, although fibrolipoma and infiltrative lipoma have been well documented. This report describes two cases of rare angiolipoma in dogs and the first documentation of angiofibrolipoma and infiltrating angiolipoma in a cat and a dog, respectively. Tumors were solitary, and most were located on the thorax of middle-aged patients. Angiolipomas were composed of mature adipose tissue mixed with variable numbers of blood vessels. In addition to the adipose and vascular components, the angiofibrolipoma contained bundles of collagenous connective tissue. The infiltrative angiolipoma had a primary mass external to the muscle and was histologically similar to a mixed intramuscular hemangioma that was confined to the muscle. Both disrupted bundles of striated muscle and were associated with segmental degeneration and loss of myofibers.

The potential toxicity of Ilex myrtifolia in beef cattle.

Spatial and temporal patterns of death loss in a beef cowherd indicated a possible relationship between the loss of 11 cows and the consumption of Ilex myrtifolia (myrtle leaf holly). To investigate this relationship, plant material from Ilex myrtifolia was harvested and 2 feeding trials were performed. The 1st trial involved intermittent feeding of plant material to 4-mo-o calves for 2 w, and the 2nd trial was continuous plant feeding to 2-mo-o calves for 35 d. No significant clinical pathology, histological or gross lesions resulted and no clinical signs consistent with the original herd problem were observed, suggesting that berries, leaves and stems from Ilex myrtifolia were not sufficiently toxic to induce clinical effects under these experimental conditions.

Expression of inflammatory cytokine mRNA in lymphoid tissue from swine experimentally infected with Mycobacterium avium serovar 2.

OBJECTIVE: To evaluate in situ expression of inflammatory cytokine mRNA in lymphoid tissue of swine experimentally infected with Mycobacterium avium serovar 2. ANIMALS: 7 noninfected pigs and 7 pigs infected with M. avium serovar 2. PROCEDURE: Expression of mRNA of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta IL-6, and IL-8 in formalin-fixed paraffin-embedded blocks of lymphoid tissue (lymph nodes and tonsil) of swine experimentally infected with M. avium serovar 2 was compared with that of noninfected pigs. Tissues were evaluated by use of morphologic localization of cytokine mRNA, using in situ hybridization at 160 days after inoculation. RESULTS: A noticeable increase in mRNA expression for TNFalpha and mild increases in mRNA expression of IL-8 and IL-1beta were detected in mandibular lymph nodes from infected swine, compared with noninfected swine. Mild increase in mRNA expression for 1L-6 also was observed in tonsils from infected swine. Cytokine mRNA was detected in macrophages and lymphocytes, primarily within cortical follicles and adjacent mantle zones. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of mRNA for inflammatory cytokines was increased in lymphoid tissue of infected swine, possibly resulting from local factors on, or secreted by, M. avium. These results suggest that alterations in cytokine mRNA expression are important in the pathogenesis and clinical course of mycobacteriosis in swine. Modulation of the immune response by vaccines that selectively target cytokine expression and secretion in response to mycobacterial challenge may be effective in prevention of mycobacteriosis in swine.

Connective tissue growth factor expression in the rat remnant kidney model and association with tubular epithelial cells undergoing transdifferentiation.

Connective tissue growth factor (CTGF) has been shown to mediate many actions of transforming growth factor-beta (TGF-beta) in the fibrotic response in several diseases. We compared expression of CTGF, TGF-beta, platelet-derived growth factor (PDGF), TNF-alpha, and interleukin-1 (IL-1) by in situ hybridization in Sprague-Dawley rats euthanized at 0, 2, 4, and 8 weeks after 5/6 nephrectomy using the rat remnant kidney model of renal failure. Collagen was evaluated by trichrome stains, immunohistochemistry, and electron microscopy. We compared expression patterns to cells undergoing metaplasia. Tubular epithelial regeneration and transdifferentiation to myofibroblasts were assessed morphologically and by proliferating cell nuclear antigen, smooth muscle actin, desmin, and vimentin immunohistochemistry. CTGF expression was minimal in controls, mild at 2 weeks and marked by 4 to 8 weeks in interstitial fibroblasts, coinciding with damage, regeneration, and fibrosis. TGF-beta expression was increased in many cell types at 2 weeks, increased further by 4 weeks, then remained constant. PDGF-B messenger RNA was found in many stromal cells at 2-4 weeks, but expression decreased at 8 weeks. No significant IL-1 or TNF-alpha staining was detected. We conclude that CTGF and interacting factors are associated with development or progression of chronic interstitial fibrosis. Proximity of CTGF, TGF-beta, and PDGF mRNA expression to regenerative epithelial cells and those transdifferentiating to myofibroblasts suggests that growth factors may modulate renal tubular epithelial differentiation.

Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP.

Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.

Efficacy of vaccination for Mycobacterium avium with whole cell and subunit vaccines in experimentally infected swine.

Mycobacterium avium infections are a common problem in large swine producing states and cause substantial financial losses at slaughter inspection due to carcass condemnation. Once the infection is established in a swine herd it is difficult to effectively prevent or eliminate the disease. Previous mouse studies in our laboratory suggested that Macrophage Inhibitory Factor-A3 (MIF-A3) is a virulence factor of M. avium and potential antigen for vaccine development. In this study we evaluated the efficacy of a killed 'whole cell' M. avium serovar 2 bacterin and conjugated MIF-A3 subunit vaccine in preventing infection and disease in swine challenged with virulent M. avium serovar 2. Gross and microscopic pathology, acid-fast staining, culture and polymerase chain reaction (PCR) for the M. avium specific insertion sequence IS902 were utilized in evaluation. Results indicated that neither vaccine prevented infection in challenged animals; however, a 47% reduction in severity of disease was found in swine vaccinated with the 'whole cell' M. avium serovar 2 bacterin. Reduction in severity of disease was not detected in animals vaccinated with the subunit MIF-A3 vaccine.

Multiple persistent vitelline duct cysts in a dog.

Persistent vitelline duct remnants, with the exception of Meckel's diverticulum in pigs and horses, are rare in animals. During an ovariohysterectomy of an 8-month-old Labrador Retriever, multiple fibrous nodules with cystic centers were found attached to the ileal serosa and in a mesodiverticular band attached to the abdominal wall. Histologic and ultrastructural evaluation revealed that the cysts were composed of well-differentiated intestine with mucosa, submucosa, and muscularis layers surrounded by a thick layer of fibrous connective tissue. The morphology and arrangement of lesions were consistent with multiple persistent vitelline duct cysts, a distinct condition related to Meckel's diverticulum. This case in a dog represents a unique presentation of this congenital anomaly in domestic animals.

Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury.

Osteogenic protein-1 (OP-1) is a morphogenetic factor highly expressed in the kidney and involved in tissue repair and development. Homozygous OP-1-deficient mice die shortly after birth due mainly to arrest of renal growth and differentiation. Because postischemic injury involves several repair mechanisms, this study examined whether kidney OP-1 mRNA expression is modulated after ischemia. Acute ischemic renal injury was achieved in rats by unilateral clamping of the renal pedicle followed by reperfusion. Rats were killed at 3, 6, 12, 24, and 48 h and 7 d after reperfusion, and kidneys were microdissected and analyzed by histology and Northern and Western blots. Changes in OP-1 mRNA were determined by measuring the ratio of OP-1/glyceraldehyde 3-phosphate dehydrogenase signals for each OP-1 transcript (4.0 and 2.4 kb) from ischemic, opposite, and sham-operated rats. The OP-1 mRNA content for transcript 4.0 kb was fivefold lower in the whole ischemic kidney compared with that in sham animals 24 h after reperfusion. In the ischemic medulla, OP-1 mRNA was strikingly downregulated 20-fold when compared with the ischemic cortex. Results for transcript 2.4 kb and for the other time points were comparable. OP-1 mRNA expression was also affected in the opposite medulla compared with the sham medulla. However, only in the ischemic medulla was the relative OP-1 content significantly lower at all time points. Similar results were obtained when analyzing OP-1 protein by Western blot at 24 h after reperfusion. Seven days after reperfusion, the levels of OP-1 mRNA returned to baseline. In conclusion, kidney OP-1 mRNA and protein are selectively downregulated in the medulla after acute ischemic renal injury. OP-1 modulation may be a key element for kidney repair.

Expression of connective tissue growth factor mRNA in the fibrous stroma of mammary tumors.

Desmoplasia, the formation of highly cellular, excessive connective tissue stroma associated with some cancers, shares many features with the wound healing response. Since connective tissue growth factor (CTGF) has previously been demonstrated to play a role in wound repair, we wanted to determine if it might be involved in the pathogenesis of stromal demoplasia in mammary cancer. We assayed 11 human invasive mammary ductal carcinomas by Northern blot and 7 out of 11 were positive for both CTGF expression and transforming growth factor-beta 1 (TGF-beta 1, a principal CTGF inducer). One specimen was positive only for TGF-beta 1. The remaining 3 tumors lacked significant stromal involvement and were negative for either factor. In every case we assayed, in which there was marked connective tissue involvement, both CTGF and TGF-beta 1 messages were found. We also assayed 3 murine mammary tumor models. The GI-101 xenograft model had marked stroma and was positive for both factors in-vivo, but positive for only TGF-beta 1 mRNA expression in culture where fibroblasts were absent. The DMBA murine tumor lacked significant stroma and was negative for CTGF and TGF-beta 1 expression by Northern blot, while the stromal rich DMBA-MMTV tumor contained multifocal desmoplasia and was positive for both factors. We performed in-situ hybridization for CTGF and TGF-beta 1 on the GI-101 and DMBA-MMTV tumors. CTGF message was observed only in the fibroblasts of the stroma, while TGF-beta 1 mRNA hybridization was present in tumor epithelial cells and leukocytes. These results suggest that cancer stroma formation involves induction of similar fibroproliferative growth factors (TGF-beta 1 and CTGF) as wound repair.

Transforming growth factor beta induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway.

Connective tissue growth factor (CTGF) is a M(r)38,000 cysteine-rich peptide, the synthesis and secretion of which are selectively induced by transforming growth factor beta (TGF-beta). The relationship of CTGF to TGF-beta action on fibroblastic cells is not well understood. TGF-beta has the unique ability to stimulate the growth of normal fibroblasts in soft agar, a property of transformed cells. We have investigated whether CTGF can substitute for TGF-beta or whether CTGF action is essential for TGF-beta to stimulate anchorage-independent growth (AIG) of NRK fibroblasts. Our studies demonstrate that CTGF cannot induce AIG of NRK fibroblasts. However, CTGF synthesis and action are essential for the TGF-beta-induced AIG of NRK fibroblasts. Anti-CTGF antibodies specifically block TGF-beta-induced AIG but have no effect on platelet-derived growth factor or epidermal growth factor-induced growth in monolayer cultures and do not cross-react with platelet-derived growth factor or TGF-beta. Clones of NRK fibroblasts that express an antisense CTGF gene (NRK-ASCTGF), which blocks the expression of the endogenous CTGF gene, do not respond to TGF-beta in the AIG assay. The growth and morphology of the cells (NRK-ASCTGF) in monolayer culture are unaltered from the parent NRK cell line. The addition of recombinant CTGF to the NRK-ASCTGF clones in the presence of TGF-beta restores the AIG response of the cells. These studies demonstrate that the TGF-beta stimulation of NRK fibroblast AIG is dependent on events induced via the synergistic action of CTGF-dependent and CTGF-independent signaling pathways.

Analysis of DNA aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry.

To derive a method for determining malignant potential of plasma cell tumors, a retrospective analysis of the DNA ploidy and relative p62c-myc oncoprotein content using bivariate flow cytometry was performed on 23 formalin-fixed, paraffin-embedded tissues from 23 dogs. The samples included one tissue each from 17 males and six females 2 to 16 years of age (mean = 7.5 years). Twelve breeds were represented, including three Cocker Spaniels, three Golden Retrievers, and five of mixed breed. Ten of the samples were histologically classified as malignant-plasma cell tumors, and ten specimens were classified as benign. Three samples of plasmacytic inflammation, from two Cocker Spaniels and one Shih Tsu, were included as controls. The ploidy and relative c-myc content data obtained were compared with the histologic grade. A significant difference in ploidy was found between benign and malignant tumors (P < or = 0.05). Five of nine malignant plasma cell tumors were aneuploid; the remainder were diploid (4/9) or tetraploid (1/9). Only one of the benign plasmacytomas was aneuploid (1/10), whereas six were diploid (6/10), and three were tetraploid (3/10). All of the controls were diploid (3/32). When relative amounts of p62c-myc from malignant and benign tumors were compared by flow cytometry, a greater significant difference was established (P < or = 0.01) than bu using aneuploidy alone. Relative values of p62c-myc content ranged from 219 to 553 units in 8/10 malignant plasma cell tumors and from 86 to 392 units in 3/10 benign plasmacytomas.(ABSTRACT TRUNCATED AT 250 WORDS)