Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in human T98G glioblastoma multiforme xenografts.

Although rare, the morbidity and mortality from brain tumors are significant. Chemotherapy has made only a small impact on these tumors. The human T98G glioblastoma multiforme cell line was used as a brain tumor model. The protein kinase Cbeta inhibitor 317615 x 2HCl was not highly cytotoxic toward T98G cells in culture and was additive in cytotoxicity with carmustine (BCNU). When nude mice bearing s.c. T98G tumors were treated with 317615 x 2HCl p.o. twice daily on days 14-30 after tumor cell implantation, the number of intratumoral vessels stained by CD31 was decreased to 37% of control, and the number of intratumoral vessels stained by CD105 was decreased to 50% of control. The compound 317615 x 2HCl was an active antitumor agent against s.c. growing T98G xenografts. A treatment regimen administering 317615 x 2HCl before, during, and after BCNU was compared with a treatment regimen administering 317615 x 2HCl sequentially after BCNU. In the tumor growth delay determination of the s.c. tumor, the sequential treatment regimen was more effective than the simultaneous treatment regimen. However, when the same treatments were administered to animals bearing intracranial T98G tumors, the survival of animals receiving the simultaneous treatment regimen increased from 41 days for those treated with BCNU alone to 102 days for animals treated with the combination, whereas animals receiving the sequential treatment regimen survived 74 days. Treatment with the protein kinase Cbeta inhibitor decreased T98G glioblastoma multiforme angiogenesis and improved treatment outcome with BCNU.

Overexpression of TGF-beta 1 in the central nervous system of transgenic mice results in hydrocephalus.

Transforming growth factor beta (TGF-beta) has been proposed to play a number of roles in central nervous system (CNS) development and response to injury. To test these proposals, transgenic mice were generated which overproduce TGF-beta 1 in the CNS. Surprisingly, these mice developed severe hydrocephalus and died between birth and 3 weeks of age. Ovary transplantation from an affected female founder has permitted perpetuation of one of the lines as a hydrocephalus model whose genetic defect is known. These results also demonstrate that the developing CNS is highly sensitive to TGF-beta, and suggest a role for aberrant expression of TGF-beta in the pathogenesis of developmental disease of the CNS.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in murine lewis lung carcinoma and human Calu-6 non-small-cell lung carcinoma xenografts.

In cell culture, the compound 317615.2HCl, a potent inhibitor of VEGF-stimulated HUVEC proliferation, was not very effective against Calu-6 non-small-cell lung carcinoma cells (IC50 26 microM). Exposure to combinations of paclitaxel or carboplatin and 317615.2HCl with Calu-6 cells in culture resulted in cell survival that reflected less-than-additivity to additivity of the two agents. Administration of 317615.2HCl orally twice daily to nude mice bearing subcutaneous Calu-6 tumors resulted in a decreased number of intratumoral vessels as determined by CD31 and CD105 staining to 50% of the number in control tumors. 317615.2HCl showed antitumor activity against the Lewis lung carcinoma and increased the tumor growth delay produced by paclitaxel by 5-fold, that produced by gemcitabine by 2-fold and that produced by carboplatin by 1.7-fold. There was a decrease in the number of lung metastases in the Lewis lung carcinoma that paralleled the increased response of the primary tumor with each treatment combination. Administration of 317615.2HCl also increased the tumor growth delay produced by fractionated radiation therapy in the Lewis lung tumor. Treatment with 317615.2HCl was an effective therapy in the Calu-6 non-small-cell lung carcinoma xenograft when the compound was administered early (days 4-18) or later (days 14-30). Combination treatment regimens in which 317615.2HCl was administered along with or sequentially with paclitaxel or carboplatin were much more effective than the chemotherapeutic agents administered alone. 317615.2HCl is in early clinical testing.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in human HT-29 colon carcinoma and human CaKi1 renal cell carcinoma xenografts.

The compound 317615 x 2HCl, a selective protein kinase Cbeta inhibitor, was not very cytotoxic toward human CaKi1 renal cell carcinoma cells or human HT-29 colon carcinoma cells in monolayer culture. Isobologram analysis was used to determine additivity or synergy of the combination regimens. Exposure of CaKi1 cells to 317615 x 2HCl (10 or 100 mM) along with gemcitabine or 5-fluorouracil for 24 hours resulted in cytotoxicity that appeared to be less-than-additive to additive for the two agents. Exposure of HT-29 cells to gemcitabine along with 317615 x 2HCl (10 mM or 100 mM) resulted in a synergistic cytotoxicity while combinations with 5-fluorouracil resulted in additive to greater-than-additive cytotoxicity for the agents. After treatment of CaKi1 or HT-29 xenograft-bearing mice with 317615 x 2HCl, immunohistochemical staining for expression of endothelial specific markers, either CD31 or CD105, was used to quantify the number of intratumoral vessels in the samples. CaKi1 tumor angiogenesis was very responsive to treatment with 317615 x 2HCl such that the number of intratumoral vessels stained by CD31 or CD105 was decreased to 20% of the control. The HT-29 colon carcinoma angiogenesis was also responsive to 317615 x 2HCl, such that the number of intratumoral vessels stained by CD31 or CD105 was decreased to 40% to 50% of the controL 5-fluorouracil, cisplatin or fractionated radiation therapy was combined with treatment with 317615 x 2HCl in the simultaneous combination treatment regimen in animals bearing HT-29 colon carcinoma xenografts. The resulting tumor growth delays indicated that administration of 317615 x 2HCl increased the effects of the cytotoxic therapy. Both a simultaneous or an overlapping treatment regimen and a sequential treatment regimen were used to assess 317615 x 2HCl alone and along with fractionated radiation therapy or gemcitabine against the human CaKi1 renal cell carcinoma xenograft. The CaKi1 tumor was quite sensitive to fractionated radiation therapy and to gemcitabine and, although 317615 x 2HCl was an effective single agent in this tumor, the combination regimens did not reach additivity for the combination regimens in vivo. 317615 x 2HCl is in early clinical testing.

Evaluation of intestinal villus height in rats after ischemia and reperfusion by administration of superoxide dismutase, polyethylene glycol-conjugated superoxide dismutase, and two 21-aminosteroids.

Intestinal ischemia was induced and maintained for 60 minutes in male Sprague-Dawley rats weighing 175 to 225 g. Prior to reperfusion, the following drugs were administered via the caudal vena cava: 0.9% NaCl (0.5 ml), superoxide dismutase (SOD; 1,000 IU/kg of body weight), polyethylene glycol-conjugated SOD (PEG-SOD; 1,000 IU/kg), or the 21-aminosteroids, U74006F (3 mg/kg) or U78715G (3 mg/kg). A sham-operated control group was included. Animals from each group were euthanatized at 5 periods of reperfusion: 5 minutes, 30 minutes, 18 hours, 3 days, and 7 days after reperfusion. Fixed tissues were embedded in paraffin, sectioned at 5 microns, and stained with H&E. Villi profiled in cross section were measured from the crypt villus junction to the tip of the villus. The mean villus height for each rat was calculated and compared by two-way ANOVA to determine the effects of time and treatment. Villus height was maintained after 30 minutes of reperfusion in rats of the sham- and U74006F-treated groups; U78715G and SOD treatment attenuated the loss in villus height, and villus height was not maintained in the PEG-SOD- and 0.9% NaCl-treated rats. In all rats, villus height was comparable to, or was greater than villus height in sham-operated controls by 18 hours after reperfusion in all animals and remained constant through 7 days. Administration of the 21-aminosteroids maintained villus height after ischemia and reperfusion. Treatment with PEG-SOD did not maintain villus height to the degree observed in rats treated with SOD.

Photoreceptor cell death by apoptosis in dogs with sudden acquired retinal degeneration syndrome.

OBJECTIVE: To examine the role of apoptosis in retinal photoreceptor degeneration in dogs with sudden acquired retinal degeneration syndrome (SARDS). SAMPLE POPULATION: Retinas from 3 dogs with SARDS and from 2 clinically normal adult dogs. PROCEDURE: Apoptosis was identified by in situ end-labeling and observation of characteristic morphologic changes by light microscopy. RESULTS: The degree of photoreceptor degeneration varied with duration of vision loss in SARDS-affected eyes. The retina of all 3 SARDS-affected eyes had numerous (34, 61, and 70) apoptotic nuclei per section that were overwhelmingly located in the outer nuclear layer. Apoptotic nuclei were not detected, or were rare in similarly sized retinal sections from normal dogs. Inflammation was not an important feature of SARDS. CONCLUSIONS: Apoptosis appears to be at least 1 mechanism of photoreceptor cell death in dogs with SARDS. CLINICAL RELEVANCE: Because apoptosis appears to be a final common pathway in many retinal degeneration syndromes, future treatment strategies that control apoptosis in other diseases may be applicable to dogs with SARDS. Halting this pathway may allow some photoreceptors to survive and, perhaps, preserve vision.

Adenovirus-associated pneumonia and hepatitis in four llamas.

This report focuses on the diagnostic laboratory and necropsy findings in 4 llamas with adenovirus-associated hepatitis or pneumonia. In the 2 young llamas, clinical illness was characterized by chronic respiratory tract disease. In the 2 adult llamas, clinical illness was characterized by neurologic signs and a history of respiratory tract disease. Histologic examination, electron microscopy, virus isolation, and fluorescent antibody results indicated that adenovirus infection was associated with disease in all 4 llamas.

Twelve-week monotherapy with the DPP-4 inhibitor vildagliptin improves glycemic control in subjects with type 2 diabetes.

UNLABELLED: Inhibition of dipeptidyl peptidase-4 enhances the activity of incretin hormones, improving glycemic control in subjects with type 2 diabetes. This twelve-week randomized, double-masked, placebo-controlled study assessed the efficacy and tolerability of the specific and potent oral dipeptidyl peptidase-4 inhibitor, vildagliptin (25 mg, bid, n=70) VS. placebo (bid, n=28) in previously diet-treated subjects with type 2 diabetes. Standardized meal tests were performed at baseline and endpoint. The between-group difference in adjusted mean change in HbA1c from baseline to endpoint was - 0.6 +/- 0.2 % (p=0.0012) for the whole cohort (baseline 8.0 %) and -1.2 % for subjects with baseline HbA1c 8.0 - 9.5 %. Fasting glucose and mean prandial glucose were reduced by 1.1 +/- 0.4 (p=0.0043) and 1.9 +/- 0.5 mmol/l (p <0.0001), respectively. The between-group differences in corrected insulin response at peak glucose and mean prandial C-peptide were + 0.06 +/- 0.02 (p=0.0258) and + 0.10 +/- 0.03 nmol/l (p=0.0031), respectively. Vildagliptin had no effect on fasting lipid levels or body weight. The incidence of adverse events was similar in subjects receiving placebo (71.4 %) and vildagliptin (55.7 %). CONCLUSION: monotherapy with vildagliptin is well tolerated and improves glycemic control in diet-treated subjects with type 2 diabetes. Concomitant improvements in beta-cell function were also observed. Subjects with higher baseline HbA1c levels showed greater response.

Cerebellar medulloblastoma with multiple differentiation in a dog.

A 4-year-old female, spayed Border Collie Dog was brought to the Veterinary Medical Teaching Hospital for evaluation of a progressive head tilt and ataxia that were unresponsive to therapy. Neurologic examination localized a right-sided lesion. The owner refused additional diagnostic tests, and necropsy was performed after euthanasia. Gross findings included atrophy of the temporal muscles and a moderately well delineated, 2.5- x 1.5- x 1.0-cm, gray soft-tissue mass compressing the right cerebellar hemisphere and dorsal hindbrain, resulting in massive dilatation of the lateral, third, and fourth ventricles and hydrocephalus. Histologic examination revealed two distinct features: undifferentiated, primitive, polygonal to fusiform cells with typical morphologic characteristics of medulloblastoma and interspersed areas containing myelinated axons and cells with glial and neuronal differentiation. Immunohistochemical examination confirmed the presence of primitive neuroepithelium and cells with glial and neuronal differentiation.

Liver, renal and subcutaneous histopathology in PEPCK-bGH transgenic pigs.

Transgenic pigs were created that harboured a phosphoenolpyruvate carboxykinase-bovine growth hormone construct (PEPCK-bGH). Four founder animals and two transgenic offspring from one line were evaluated between 6 1/2 and 12 months of age. There was no evidence of severe hepatic or renal lesions in these pigs, which characterised transgenic PEPCK-bGH mice previously described. While glomerular and tubular lesions in kidney sections were not identified in the transgenic pigs, mesangial cell proliferation was observed in two transgenic offspring from a single line. Additionally, glomerular size was significantly increased in four of four puberal transgenic swine when compared to age- and sex-matched controls (28.30 +/- 4.1 vs. 14.2 +/- 2.7 x 10(5) microns 3; representing 3 transgenic lines, p < 0.05). Surprisingly, no mature adipocytes were observed in subcutaneous sections obtained in transgenic GH pigs. Histological evaluation of these transgenic pigs further illustrates the requirement for precise control of growth-related genes and their protein products.

Peripheral neuroblastoma in a dog.

Primitive neuroectodermal tumors are composed of primitive neuroepithelial cells and include tumors of the central and peripheral nervous system. Neuroblastoma, medulloblastoma and retinoblastoma are examples of these rare malignant tumors that usually occur in young patients. This report describes a peripheral neuroblastoma in a 2 year old Boxer that presented with signs of renal disease and a palpable abdominal mass. The purpose of this paper is to describe the clinical presentation, imaging and immunohistological studies of this abdominal tumor in a young dog and to review the literature.

Rectal and colonic injury in the llama. Anatomic considerations and surgical management in four llamas.

Transrectal palpation in llamas can result in iatrogenic rectal and colonic injury. The purpose of this report is to define the caudal extent of the peritoneal cavity in llamas and to describe the surgical management of rectal or colonic injuries in four llamas. Measurements were made of six adult llamas during necropsy. The mean distance from the peritoneal reflection to the anus was 3.9 +/- 0.1 cm (3.4-4.3 cm). Four llamas were examined for rectal or colonic perforations. One laceration was of partial thickness and three lacerations were of full thickness. Two of the defects were repaired by a transanal approach and two by celiotomy to facilitate removal of fecal debris and abdominal lavage. Successful repair of the rectal or colonic tears was achieved in all four llamas. Wound infection and incisional hernia occurred in both llamas that underwent celiotomy. Two llamas died 3 and 18 months later, and two llamas have survived 2 years. Rectal tears in llamas are accompanied by a high risk of peritoneal contamination, and primary closure is recommended. If fecal contamination of the abdomen has occurred, celiotomy is indicated to allow mechanical removal of fecal debris and peritoneal lavage.

Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice.

Increased expression of glial fibrillary acidic protein (GFAP) is a hallmark of gliosis, the astrocytic hypertrophy that occurs during a wide variety of diseases of the central nervous system. To determine whether this increase in GFAP expression per se alters astrocyte function, we generated transgenic mice that carry copies of the human GFAP gene driven by its own promoter. Astrocytes of these mice are hypertrophic, up-regulate small heat-shock proteins, and contain inclusion bodies identical histologically and antigenically to the Rosenthal fibers of Alexander's disease. Mice in the highest expressing lines die by the second postnatal week. The results support the notion that Alexander's disease is a disorder of astrocytes, and provide an animal model for studying the causes and consequences of inclusion body disease.

Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.