Ursula Forster

Activation of nuclear factor-κB in dogs with chronic enteropathies.

Homeostasis in the intestinal microenvironment between the immune system and luminal antigens appears disturbed in chronic enteropathies. Pro-inflammatory cytokines likely play a role in the pathogenesis of intestinal inflammation. Several inflammatory and immunoregulatory genes have associated nuclear factor-kappaB (NF-kappaB) binding sites, which allow NF-kappaB to regulate gene transcription. The purpose of this study was to investigate (1) the occurrence of NF-kappaB activation during mucosal inflammation in situ, (2) the mucosal distribution pattern of cells expressing activated NF-kappaB within treatment groups, and (3) the effect of specific therapy on NF-kappaB activation. Dogs with chronic enteropathy were studied (n=26) and compared with 13 healthy dogs. Ten dogs had food responsive disease (FRD) and 16 had inflammatory bowel disease (IBD). NF-kappaB activation was detected in duodenal mucosal biopsies using a mouse monoclonal antibody (MAB 3026) that selectively binds the nuclear localization sequence of activated NF-kappaB. To identify macrophages, biopsies were stained using the MAC 387 antibody. Macrophages in the lamina propria double-stained for MAC 387 and NF-kappaB were quantitated; epithelial cell expression of activated NF-kappaB was determined semi-quantitatively. Results showed that more macrophages positive for activated NF-kappaB were present in lamina propria of dogs with chronic enteropathy compared to control dogs (p<0.01). More NF-kappaB positive epithelial cells were observed in FRD dogs compared to IBD dogs (p<0.05). After therapy, the number of macrophages and epithelial cells staining positive for activated NF-kappaB decreased (p<0.01) in chronic enteropathy dogs. In conclusion, activation of NF-kappaB is closely associated with the pathophysiology of canine chronic enteropathy. Down-regulation follows successful therapy.

Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PCR.

Improved polymerase chain reaction-based method to detect early- stage epitheliotropic T-cell lymphoma (mycosis fungoides) in formalin-fixed, paraffin-embedded skin biopsy specimens of the dog

In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRγ) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRγ gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRγ gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRγ gene, localized approximately 2.3 kb upstream of J5, was identified.

Comparative spatiotemporal analysis of the intrathecal immune response in natural listeric rhombencephalitis of cattle and small ruminants

This study examined the spatiotemporal immune response in listeric rhombencephalitis of ruminants in situ. Our data support the view that astrocytes facilitate the containment of infectious lesions. Results on the natural disease recapitulate observations in experimental rodent models and suggest that the mounted adaptive lymphocytic response of ruminants is effective in eliminating Listeria monocytogenes (LM). However, our data indicate earlier participation of the adaptive immune response, a stronger B lymphocyte contribution and a more protracted macrophage infiltration in the natural disease than it has been deduced from experimental models. Therefore, such models should be complemented by studies in natural host systems. Various macrophage and microglia subsets are involved in listeric rhombencephalitis and their differential contribution may account for species differences in clinical course and outcome of infection as might species differences in the B-cell response. Future functional ex vivo and in vitro studies are necessary to further investigate the findings obtained in the present study.