Phenotyping, functional characterization, and developmental changes in canine intestinal intraepithelial lymphocytes

Little is currently known about the lymphocyte populations in the normal and diseased canine gut. The aim of this study was thus the phenotypical and functional characterization of canine intestinal intraepithelial lymphocytes (IEL). IEL were isolated from full-thickness biopsies of 15 adult Swiss Beagle dogs (mean age 8.2 +/-2.8 years) and compared to mesenteric lymph node cells. The phenotypical characterization by multi-parameter flow cytometry revealed that canine IEL differ substantially from lymph node T cells, and consist of various unconventional lymphocyte subsets, unique to mucosal surfaces. These include gammasigma T cells, and CD4(-)CD8(-) and CD8alphaalpha(+) T cells. IEL populations in adult dogs were also compared to those isolated from neonatal Beagle dogs. Analysis revealed a high frequency of undifferentiated CD4(-)CD8(-) T cells in newborn dogs whereas mature CD4(+) and CD8(+) T cells predominate in adult dogs, indicating maturation of the intestinal immune system during development. As IEL in other species are thought to exhibit regulatory functions, we investigated the role of IEL on the activation-induced proliferation of lymph node T cells. While IEL alone did not show activation-induced proliferation, they significantly inhibited the proliferation of activated lymph node T cells in a cell number-dependent manner. These findings are the first to demonstrate that canine intestinal IEL have an immunoregulatory phenotype, which may contribute to the maintenance of intestinal immune homeostasis and may, therefore, be lost in canine chronic enteropathies.

Immunohistochemical localization of RANK, RANKL and OPG in healthy and arthritic canine elbow joints

OBJECTIVE: To determine if the receptor activator of nuclear factor-kappaB-receptor activator of nuclear factor-kappaB ligand-osteoprotegerin (RANK-RANKL-OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease. METHODS: Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints. RESULTS: All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few. CONCLUSIONS: The RANK-RANKL-OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption. CLINICAL RELEVANCE: Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.

Bone response to loaded implants with non-matching implant-abutment diameters in the canine mandible

BACKGROUND: One way to evaluate various implant restorations is to measure the amount of bone change that occurs at the crestal bone. The objective of this study was to histologically evaluate the alveolar bone change around a bone-level, non-matching implant-abutment diameter configuration that incorporated a horizontal offset and a Morse taper internal connection. METHODS: The study design included extraction of all mandibular premolars and first molars in five canines. After 3 months, 12 dental implants were placed at three levels in each dog: even with the alveolar crest, 1 mm above the alveolar crest, and 1 mm below the alveolar crest. The implants were submerged on one side of the mandible. On the other side, healing abutments were exposed to the oral cavity (non-submerged). Gold crowns were attached 2 months after implant placement. The dogs were sacrificed 6 months postloading, and specimens were processed for histologic and histometric analyses. RESULTS: Evaluation of the specimens indicated that the marginal bone remained near the top of the implants under submerged and non-submerged conditions. The amount of bone change for submerged implants placed even with, 1 mm below, and 1 mm above the alveolar crest was -0.34, -1.29, and 0.04 mm, respectively (negative values indicate bone loss). For non-submerged implants, the respective values were -0.38, -1.13, and 0.19 mm. For submerged and non-submerged implants, there were significant differences in the amount of bone change among the three groups (P <0.05). The percentage of bone-to-implant contact for submerged implants was 73.3%, 71.8%, and 71.5%. For non-submerged implants, the respective numbers were 73.2%, 74.5%, and 76%. No significant differences occurred with regard to the percentage of bone contact. CONCLUSIONS: Minimal histologic bone loss occurred when dental implants with non-matching implant-abutment diameters were placed at the bone crest and were loaded for 6 months in the canine. The bone loss was significantly less (five- to six-fold) than that reported for bone-level implants with matching implant-abutment diameters (butt-joint connections).

ESPEN Guidelines on Parenteral Nutrition: geriatrics

Older subjects are at increased risk of partial or complete loss of independence due to acute and/or chronic disease and often of concomitant protein caloric malnutrition. Nutritional care and support should be an indispensable part of their management. Enteral nutrition is always the first choice for nutrition support. However, when patients cannot meet their nutritional requirements adequately via the enteral route, parenteral nutrition (PN) is indicated. PN is a safe and effective therapeutic procedure and age per se is not a reason to exclude patients from this treatment. The use of PN should always be balanced against a realistic chance of improvement in the general condition of the patient. Lower glucose tolerance, electrolyte and micronutrient deficiencies and lower fluid tolerance should be assumed in older patients treated by PN. Parenteral nutrition can be administered either via peripheral or central veins. Subcutaneous administration is also a possible solution for basic hydration of moderately dehydrated subjects. In the terminal, demented or dying patient the use of PN or hydration should only be given in accordance with other palliative treatments.

Access technique and its problems in parenteral nutrition - Guidelines on Parenteral Nutrition, Chapter 9

Catheter type, access technique, and the catheter position should be selected considering to the anticipated duration of PN aiming at the lowest complication risks (infectious and non-infectious). Long-term (>7-10 days) parenteral nutrition (PN) requires central venous access whereas for PN <3 weeks percutaneously inserted catheters and for PN >3 weeks subcutaneous tunnelled catheters or port systems are appropriate. CVC (central venous catheter) should be flushed with isotonic NaCl solution before and after PN application and during CVC occlusions. Strict indications are required for central venous access placement and the catheter should be removed as soon as possible if not required any more. Blood samples should not to be taken from the CVC. If catheter infection is suspected, peripheral blood-culture samples and culture samples from each catheter lumen should be taken simultaneously. Removal of the CVC should be carried out immediately if there are pronounced signs of local infection at the insertion site and/or clinical suspicion of catheter-induced sepsis. In case PN is indicated for a short period (max. 7-10 days), a peripheral venous access can be used if no hyperosmolar solutions (>800 mosm/L) or solutions with a high titration acidity or alkalinity are used. A peripheral venous catheter (PVC) can remain in situ for as long as it is clinically required unless there are signs of inflammation at the insertion site.

Practical handling of AIO admixtures - Guidelines on Parenteral Nutrition, Chapter 10

All-in-one admixtures (AIO-admixtures) provide safe, effective and low-risk PN (parenteral nutrition) for practically all indications and applications. Water, energy (carbohydrates and lipids), amino acids, vitamins and trace elements are infused together with PN either as industrially-manufactured AIO admixtures provided as two- or three-chamber bags (shelf life usually more than 12 months) completed with electrolytes and micronutrients where appropriate or as individually compounded ready-to-use AIO admixtures (compounding, usually prepared by a pharmacy on either a daily or weekly basis and stored at 2-8 degrees C). Physico-chemical and microbial stability of an AIO admixture is essential for the safety and effectiveness of patient-specific PN, and its assurance requires specialist pharmaceutical knowledge. The stability should be documented for an application period of 24 (-48) hours. It is advisable to offer a limited selection of different PN regimes in each hospital. For reasons of drug and medication safety, PN admixtures prepared for individual patients must be correctly labelled and specifications for storage conditions must also be followed during transport. Monitoring is required where applicable. Micronutrients are usually administered separately to AIO admixtures. In case compatibility and stability have been well documented trace elements and/or combination preparations including water-soluble or water-soluble/fat soluble vitamin supplements can be added to PN admixtures under strict aseptic conditions. AIO admixtures are usually not used as vehicles for drugs (incompatibilities).

Selective iNOS-inhibition does not influence apoptosis in ruptured canine cranial cruciate ligaments

Abnormal patterns of cell death, including increased apoptosis, can influence homeostasis of ligaments and could be involved in the pathogenesis of cranial cruciate ligament (CCL) rupture. Increased nitric oxide (NO) production has been implicated as a stimulus to increased apoptosis in articular cartilage. This study investigated apoptotic cell death in ruptured canine CCL (CCL group, n = 15), in ruptured CCL of dogs treated with oral L-N6-(1-iminoethyl)-lysine (L-NIL), a selective NO-synthetase(NOS)-inhibitor, (L-NIL group, n = 15) and compared the results with normal canine CCL (control group, n = 10). Orally administered L-NIL at a dosage of 25mg/m2 of body surface area was effective in inhibiting NO production in the articular cartilage of dogs in the L-NIL group, but it did not significantly influence the increased quantity of apoptotic cells found in ruptured CCL specimens. The results of this study suggest that apoptosis of ligamentocytes in the canine CCL is not primarily influenced by increased NO production within the stifle joint.

Impact of beta-galactosidase mutations on the expression of the canine lysosomal multienzyme complex

beta-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1(+/+), GLB1(+/-) and GLB1(-/-) fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total beta-galactosidase, beta-galactosidase variants and protective protein for beta-galactosidase gene (PPGB) in GLB1(+/-) and GLB1(-/-) compared to GLB1(+/+) fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant beta-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1(+/+), GLB1(+/-) and GLB1(-/-) genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.