Soluble Vascular Cell Adhesion Molecule-1 (VCAM-1) as a Biomarker in the Mouse Model of Experimental Autoimmune Myocarditis (EAM).

Vascular cell adhesion molecule-1 (VCAM-1) is strongly upregulated in hearts of mice with coxsackie virus-induced as well as in patients with viral infection-triggered dilated cardiomyopathy. Nevertheless, the role of its soluble form as a biomarker in inflammatory heart diseases remains unclear. Therefore, we investigated whether plasma levels of soluble VCAM-1 (sVCAM-1) directly correlated with disease activity and progression of cardiac dysfunction in the mouse model of experimental autoimmune myocarditis (EAM). EAM was induced by immunization of BALB/c mice with heart-specific myosin-alpha heavy chain peptide together with complete Freund`s adjuvant. ELISA revealed strong expression of cardiac VCAM-1 (cVCAM-1) throughout the course of EAM in immunized mice compared to control animals. Furthermore, sVCAM-1 was elevated in the plasma of immunized compared to control mice at acute and chronic stages of the disease. sVCAM-1 did not correlate with the degree of acute cardiac inflammation analyzed by histology or cardiac cytokine expression investigated by ELISA. Nevertheless, heart to body weight ratio correlated significantly with sVCAM-1 at chronic stages of EAM. Cardiac systolic dysfunction studied with positron emission tomography indicated a weak relationship with sVCAM-1 at the chronic stage of the disease. Our data provide evidence that plasma levels of sVCAM-1 are elevated throughout all stages of the disease but showed no strong correlation with the severity of EAM.

Age-related Qualitative Histological and Quantitative Stereological Changes in the Equine Pituitary.

The aim of this study was to characterize the age-related morphological changes in the equine pituitary and to identify features that allow distinction between pituitary pars intermedia dysfunction (PPID)-associated and non-functional/age-associated pars intermedia (PI) adenoma. Pituitary glands of all horses submitted for necropsy examination at the Institute of Veterinary Pathology, Ludwig Maximilians University Munich, between 2008 and 2012 were examined. The pituitary glands of 124 horses were weighed, cut into ∼2 mm slices and examined histologically. A slightly modified grading scheme (grades 1-5) was applied to evaluate histological alterations of the PI semiquantitatively. The volume fractions and total volumes of the three pituitary lobes, PI, pars distalis (PD) and pars nervosa (PN), as well as the total number and mean size of PI cells (PICs), were determined using state-of-the-art quantitative stereological methods. There were significant associations between histological grade, the appearance of PI adenomas, follicles and cysts in the PI, lipofuscin in the PN (P <0.001) and focal hyperplasia of chromophobes in the PD and age. In contrast, the appearance of follicles and cysts in the PD, invasion of basophil cells into the PN, haemorrhage and necrosis were not age dependent. PI adenomas were observed in 18% (22/124) of the animals, but only four horses were evidently suffering from PPID, therefore clinically overt/PPID-associated PI adenomas were found in 3% (4/124) of all horses. Most PI adenomas were incidental and considered non-functional. Pituitary weight, PI volume, total number and mean volume of PICs increased with age in all horses. The total PI volume and the number of PICs of horses exhibiting PPID-associated PI adenomas were significantly higher, while the mean size of PICs was smaller compared with that of horses showing non-PPID-associated adenoma, which suggests that different growth processes are responsible for adenoma formation. The present study demonstrated various age-associated lesions of the PD and PN and revealed a high frequency of incidental, non-PPID-associated PI adenomas in aged horses. Therefore, post-mortem diagnosis of PPID in horses is possible, by determination of pituitary weight and by demonstration of PIC hyperplasia, using quantitative stereological methods.

Chronic Pancreatitis with Acinar-Ductal Metaplasia and Ductal Dysplasia in a Horse.

A 16-year-old Friesian gelding with relapsing colic was humanely destroyed during diagnostic laparotomy due to suspected abdominal neoplasia. On post-mortem examination, the pancreas appeared as a firm mass (20 × 8 × 8 cm). The cut surface had a lobular structure with multiple cavities. Histological examination revealed severe chronic fibrosing pancreatitis with acinar-ductal metaplasia and duct dysplasia, which was considered to be the cause of the recurrent colic. Formation of tubular complexes within a background of acinar-ductal metaplasia is similar to the regressive lesions detected in the human pancreas in the context of inflammation, duct obstruction, cystic fibrosis and neoplasia. Pancreatic acinar-ductal metaplasia and ductal dysplasia are considered to be preneoplastic conditions in man and in the mouse.

Acaricide treatment prevents adrenocortical hyperplasia as a long-term stress reaction to psoroptic mange in cattle.

In cattle, infestation with Psoroptes ovis mites may cause severe dermatitis (psoroptic mange) which compromises the health and welfare of the animals and may lead to significant economic losses. To investigate yet undocumented effects of psoroptic mange mite infestations and how successful therapy promotes animal health, the present study examined alterations of the skin, lymph nodes and adrenal glands of P. ovis infested Fleckvieh (Simmental) bulls treated with either ivermectin long-acting injection (IVM LAI; IVOMEC(®) GOLD, Merial; 3.15% ivermectin w/v) or saline (n=16 each). Approximately 8 weeks subsequent to experimental infestation with P. ovis, the bulls had developed mange and were administered either IVM LAI or saline once at 1 mL/50 kg body weight by subcutaneous injection. Mite counts were conducted in weekly intervals for determination of efficacy of treatment, and following humane euthanasia of the animals 8 weeks after treatment, skin samples from affected (mangy or previously mangy) and unaffected areas, prescapular lymph nodes and adrenal glands were collected for gross and pathohistological examination. In addition, four age-matching, uninfested Simmental bulls were sampled as controls for comparison. No P. ovis mites were detected on any IVM LAI-treated bull after 28 days following treatment whereas saline-treated bulls maintained infestation throughout the study. At sampling (approximately 16 weeks after experimental infestation and 8 weeks following saline or IVM LAI treatment), saline-treated bulls displayed a severe, exsudative dermatitis with significantly increased skin thickness and inflammatory cell infiltration, significantly enlarged, hyperplastic prescapular lymph nodes, as well as significantly increased adrenal gland weights and volumes as compared to P. ovis-infested, IVM LAI-treated bulls and uninfested controls. Quantitative stereological analysis revealed that the adrenal gland enlargement in P. ovis-infested, saline-treated bulls was due to a selective increase of the volume of the zona fasciculata in the adrenal cortex. Compared to uninfested controls and P. ovis-infested, IVM LAI-treated bulls, the number of epithelial cells in the zona fasciculata was significantly increased in P. ovis-infested, saline-treated bulls, while the zona fasciculata cell volumes did not differ between the three groups of cattle. While the single point determination of serum cortisol concentrations did not reveal significant differences between the three groups of cattle at tissue sampling, the hyperplastic growth of the adrenal cortex in the P. ovis-infested, saline-treated bulls provides morphologic evidence that a chronic stress reaction is one consequence of mange mite infestations that can be prevented by efficacious acaricidal treatment.

Genetic dissection of IGF1-dependent and -independent effects of permanent GH excess on postnatal growth and organ pathology of mice.

To study insulin-like growth factor 1 (IGF1)-independent effects of permanent growth hormone (GH) excess on body and organ growth and pathology in vivo, hemizygous bovine GH transgenic mice with homozygous disruption of the Igf1 gene (Igf1(-/-)/GH) were generated, and examined in comparison to Igf1(-/-), Igf1(+/-), wild-type (WT), Igf1(+/-)/GH, and GH mice. GH mice and Igf1(+/-)/GH mice showed increased serum IGF1 levels and the well-known giant-phenotype of GH transgenic mice. In contrast, the typical dwarf-phenotype of Igf1(-/-) mice was only slightly ameliorated in Igf1(-/-)/GH mice. Similar to GH mice, Igf1(-/-)/GH mice displayed hepatocellular hypertrophy, glomerulosclerosis, and reduced volumes of acidophilic cells in the pituitary gland. However, GH excess associated skin lesions of male GH mice were not observed in Igf1(-/-)/GH mice. Therefore, development of GH excess induced liver-, kidney-, and pituitary gland-alterations in GH transgenic mice is independent of IGF1 whereas GH stimulated body growth depends on IGF1.

Multiple glucagon-producing pancreatic neuroendocrine tumors in a horse (Equus caballus).

Pancreatic neuroendocrine tumors of glucagon-producing cells are extremely rare in domestic animals. In this report, we describe for the first time, to our knowledge, the incidental finding of multiple glucagon-producing neuroendocrine tumors of the pancreas of a horse. The animal was euthanized due to severe local infection after tooth extraction. On postmortem examination, multiple white nodules of up to 4 cm in diameter were observed in the pancreas. Histologically, pancreatic nodules had the appearance of neuroendocrine neoplasms with positive immunoreactivity for glucagon, synaptophysin, chromogranin A, and neuron-specific enolase. Electron microscopy revealed numerous electron-dense granules, similar to those observed in normal pancreatic alpha cells, in the neoplastic cells. In addition, the left adrenal gland showed multiple hyperplastic foci and adenomas in the medulla that were identified as pheochromocytomas. Based on the morphologic appearance and immunohistochemical staining pattern of pancreatic nodules, a diagnosis of multiple glucagon-producing neuroendocrine tumors was made.

Oxazolone and ethanol induce colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγ(null) mice engrafted with human peripheral blood mononuclear cells.

Oxazolone-induced colitis in mice has become a recognized model to study the efficacy of therapeutics targeting the immunological response underlying the development of inflammatory bowel disease. However, this model cannot be used when therapeutics designed to address human targets do not interact with the respective murine counterpart. In this study, we examined the induction of oxazolone mediated colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγ(null) (NOD-SCID IL2Rγ(null)) mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from ulcerative colitis (UC), atopic dermatitis (AD) and healthy volunteers. NOD-SCID IL2Rγ (null) mice were engrafted with hPBMC followed by challenge with oxazolone or ethanol vehicle. Mice developed the same symptoms as observed previously in immunocompetent mice. The clinical activity score increased and the colon architecture was characterized by the development of oedema, fibrosis, crypt loss and dense infiltration of predominantly T cells into the lamina propria. Fluorescence activated cell sorter (FACS) analysis of lymphocytes in the colon identified natural killer (NK) T cells as a major constituent. In contrast to studies with immunocompetent mice, we observed the same phenotype in the group challenged with ethanol vehicle. The phenotype was most pronounced in mice engrafted with PBMC derived from a patient suffering from UC, suggesting that the immunological history of the donors predisposes the engrafted mice to react to ethanol. The model described here has the potential to study the efficacy of therapeutics targeting human lymphocytes in a model which is more reflective of the human disease. In addition, it might be developed to elucidate molecular mechanisms underlying the disease.

[Pathogenesis of diabetes mellitus and diabetic complications. Studies on diabetic mouse models]. / Pathogenese des Diabetes mellitus und diabetischer Spätkomplikationen. Studien an diabetischen Mausmodellen.

Diabetic mouse models created via random mutagenesis or genetic modification are essential tools to unravel the mechanisms involved in the development of diabetes mellitus and associated diseases. Three diabetic mutant mouse lines derived from the Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project and one transgenic mouse line were analyzed with respect to diabetes-relevant clinical, pathomorphological and therapeutic aspects. An Ins2 mutation and two Gck mutations were identified as the cause of diabetes mellitus in the mutant lines. Heterozygous Ins2 and homozygous Gck mutants serve as model for permanent neonatal diabetes mellitus (PNDM) and heterozygous Gck mutants develop maturity onset diabetes of the young type 2. Dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) transgenic mice exhibit defective postnatal islet growth, develop PNDM and progressive diabetes-associated kidney lesions. The mutant and transgenic diabetic mouse models analyzed in the study were shown to represent valuable models to study the pathogenesis of monogenic diabetes and to establish novel treatment strategies.

Enhanced oxidative stress and endocrine pancreas alterations are linked to a novel glucokinase missense mutation in ENU-derived Munich Gck(D217V) mutants.

In the large-scale Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project murine models recapitulating human diseases were generated. In one strain, a novel missense mutation (D217V) in the glucokinase (Gck) gene was identified, resulting in decreased glucokinase activity. Heterozygous mutants display mild hyperglycaemia, disturbed glucose tolerance, and decreased glucose-induced insulin secretion. In contrast, homozygous mutants exhibit severe but not survival affecting hyperglycaemia, mild growth retardation, diminished oxidative capacity, and increased abundance of CHOP protein in the islets. Furthermore, the total islet and ß-cell volumes and the total volume of isolated ß-cells are significantly decreased in adult homozygous mutants, whereas in neonatal mice, ß-cell mass is not yet significantly decreased and islet neogenesis is unaltered. Therefore, reduced total islet and ß-cell volumes of adult homozygous mutants might predominantly emerge from disturbed postnatal islet neogenesis. Thus, we identified a novel Gck mutation in mice, with relevance in humans, leading to glycaemic disease.

Early insulin therapy prevents beta cell loss in a mouse model for permanent neonatal diabetes (Munich Ins2(C95S)).

AIMS: Heterozygous male Munich Ins2(C95S) mutant mice, a model for permanent neonatal diabetes mellitus, demonstrate a progressive diabetic phenotype with severe loss of functional beta cell mass. The aim of this study was to investigate the influence of early insulin treatment on glucose homeostasis and beta cell destruction in male Munich Ins2(C95S) mutants. METHODS: One group of male Ins2(C95S) mutants was treated with subcutaneous insulin pellets, as soon as blood glucose levels began to rise; placebo-treated mutants and wild-type mice served as controls. An additional group of mutant mice received a sodium-dependent glucose transporter 2 (SGLT2) inhibitor (AVE2268) via rodent chow. RESULTS: Insulin treatment normalised blood glucose concentrations, improved oral glucose tolerance, preserved insulin sensitivity and inhibited oxidative stress of Munich Ins2(C95S) mutant mice. Pancreatic C-peptide content, as well as total beta cell and isolated beta cell volumes, of insulin-treated mutant mice were higher than those of placebo-treated mutants. In addition, alpha cell dysfunction and hyperplasia of non-beta cells were completely normalised in insulin-treated mutant mice. Treatment with the SGLT2 inhibitor lowered blood glucose, improved glucose tolerance and normalised insulin sensitivity as well as oxidative stress of Ins2(C95S) mutants. The abundance of the endoplasmic reticulum (ER) stress markers binding Ig protein (BiP) and phosphorylated eukaryotic translation initiation factor 2 alpha (P-eIF2α) was significantly increased in the islets of mutants, before onset of hyperglycaemia, vs wild-type mice. CONCLUSIONS: We conclude that early insulin treatment protects Munich Ins2(C95S) mutant mice from insulin resistance, alpha cell hyperfunction, beta cell loss and hyperplasia of non-beta cells, some well-known features of human diabetes mellitus. Therefore, insulin treatment may be considered early for human patients harbouring INS mutations.

Microarray analysis of equine endometrium at days 8 and 12 of pregnancy.

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Metastatic extra-adrenal sympathetic paraganglioma in a horse.

Post-mortem examination was performed on a horse that died after exhibiting signs of colic. Gross findings included haemoperitoneum and a large round encapsulated mass located in the sublumbar area cranial to the left kidney. On sectioning the mass was solid red to brown and small nodules of similar tissue were noted at the periphery of the mass. The spleen was firm and three nodules were found in one thyroid gland. Microscopically, the abdominal mass, adjacent nodules, the spleen and one thyroid nodule consisted of clusters and cords of round to oval neoplastic cells, separated by a fine collagen and reticulin fibre network. Immunohistochemically, tumour cells expressed chromogranin A, synaptophysin and neuron-specific enolase, but did not express cytokeratin. The findings were consistent with a metastatic extra-adrenal sympathetic paraganglioma.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2).

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

Betacellulin overexpression in transgenic mice improves glucose tolerance and enhances insulin secretion by isolated islets in vitro.

Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic beta-cells and to improve glucose metabolism in experimental diabetic rodent models. We employed transgenic mice (BTC-tg) to investigate the effects of long-term BTC overabundance on islet structure and glucose metabolism. Expression of BTC is increased in transgenic islets, which show normal structure and distribution of the different endocrine cell types, without pathological alterations. BTC-tg mice exhibit lower fasted glucose levels and improved glucose tolerance associated with increased glucose-induced insulin secretion. Surprisingly, quantitative stereological analyses revealed that, in spite of increased cell proliferation, the islet and beta-cell volumes were unchanged in BTC-tg mice, suggesting enhanced cell turnover. Insulin secretion in vitro was significantly higher in transgenic islets in medium containing high glucose (11.2 or 16.7mM) as compared to control islets. Our results demonstrate that long-term BTC overabundance does not alter pancreatic islet structure and beta-cell mass, but enhances glucose-induced insulin secretion in vivo as well as in vitro.

Disseminated angiostrongylosis with fatal cerebral haemorrhages in two dogs in Germany: a clinical case study.

Canine angiostrongylosis is a nematode infection in domestic dogs and wild carnivores. Few single case reports describing the occurrence of this disease in Germany exist and until recently angiostrongylosis has not been considered endemic in this country. The present report focuses on clinical, pathological and parasitological findings in two cases of fatal disseminated canine angiostrongylosis associated with multifocal haemorrhages in the central nervous system. Both animals, which lived in Germany, presented with rapidly progressive neurological signs including depression, ataxia, unilateral central blindness and epileptic seizures. Blood work revealed grossly elevated D-dimers and mild thrombocytopenia. Both animals were subsequently euthanised due to progressive clinical aggravation. Necropsy showed cerebral and lung haemorrhages in both animals. Multiple sections of nematode larvae consistent with Angiostrongylus vasorum were identified on histopathological sections of the brain, heart, kidney and lung in both animals and a predominantly granulomatous inflammation with the occurrence of multinucleated giant cells was observed. Adult nematodes were found in the larger lung arteries of one dog and Angiostrongylus infection was subsequently confirmed by PCR-analysis and sequencing in both dogs. A. vasorum larvae were not detected by faecal Baermann examination performed in one of the dogs. It was concluded that canine angiostrongylosis should be considered as differential diagnosis in dogs in Germany, even if faecal examination is negative. There is currently still a lack of studies investigating the occurrence of angiostrongylosis in dogs and intermediate hosts in Germany which would be necessary to survey the endemic realities of this disease.

Assessing and influencing the fractional contribution of erythrocyte-bound 59Fe to individual 59Fe tissue content in murine 59Fe distribution studies.

BACKGROUND: Murine proteins of iron homoeostasis are frequently manipulated to investigate the mechanisms of iron-distribution and their toxicological consequences. Beyond subtracting erythrocyte-bound 59Fe of the residual blood content determined for each tissue (subtraction method), procedures are needed to determine 59Fe distribution in murine models of, e.g. inflammation or diabetes that cause local hyperaemia and changes in microcirculation. AIM: Two new methods were developed to correct total 59Fe tissue content individually for erythrocyte-bound 59Fe-labelled haem iron. METHODS: Iron-deficiency and iron-overload was induced in male C57BL6 mice by feeding of respective diets. Distribution of 59Fe between different tissues was determined 24h, 14, and 28 days after intravenous injection of 59Fe trace amounts. Haem-bound 59Fe was separated from non-haem 59Fe in homogenates from all tissues by dispersion in a mix of lipophilic cyclohexanone and hydrophilic H3PO4 (separation method). Moreover, the reduction of 59Fe-labelled tissue blood content was determined in all organs after in vivo saline perfusion via the left ventricle (perfusion method). RESULTS AND DISCUSSION: 59Fe-labelled non-haem iron determined by the separation method was not significantly different from values determined by the subtraction method, except for the iron-deficient spleen 14 and 28 days after 59Fe injection when the separation method yielded approximately 20% higher values. Approximately 20% of 59Fe-labelled haem iron spilled over into the hydrophilic phase. The impact of this error decreases in parallel to 59Fe radioactivity in the residual tissue blood content: thus, it is higher in iron-deficient mice which accumulate more 59Fe in their erythrocytes than iron-adequate and iron-rich mice. For the same reason this type of error is more marked after long distribution periods and in organs with high residual blood content. Saline perfusion via the left ventricle reduced total blood content in mice to less than 10%. Liver (95%) and duodenum (94%) showed the highest removal of blood while it is lowest in spleen (66%) and lungs (69%). CONCLUSIONS: The separation and the perfusion method can be used to correct the impact of erythrocyte-bound haem iron individually. A margin of error below 10% was determined for all organs except for spleen, lungs, and fat. Both methods can be applied sequentially to obtain satisfactory results in spleen, lungs, and fat.