Sami Junnikkala

COMPLEMENT ACTIVATION ASSOCIATES WITH SACCULARCEREBRAL ARTERY ANEURYSM WALL DEGENERATION AND RUPTURE

OBJECTIVE Saccular cerebral artery aneurysm (SCAA) wall degeneration and inflammatory cell infiltrations associate with aneurysm rupture and subarachnoid hemorrhage, resulting in a devastating form of stroke. The complement system is the key mediator of inflammation and household processing of injured tissue. We studied how complement activation associates with SCAA wall degeneration and rupture to better understand the pathobiology of SCAA wall rupture. METHODS Unruptured (n = 26) and ruptured (n = 32) SCAA fundi resected after microsurgical clipping were studied by immunostaining for complement activation (membrane attack complex [MAC]) and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction for related cell death. Complement activation was correlated with clinical and other histological parameters. Electromicroscopy and immunoelectron microscopy were used for locating MAC depositions at the ultrastructural level. RESULTS MAC localized consistently in a decellularized layer in the outer SCAA wall, and was found in all SCAA samples. The percentage of MAC-positive area relative to the total SCAA wall surface area (range, 5-77%) was greater in ruptured (n = 25; median, 39%) than in unruptured SCAAs (n = 18; median, 20%; P = 0.005). It also associated significantly with SCAA wall degeneration (P < 0.001), de-endothelialization(P < 0.001), and CD163+ macrophage (P = 0.023) and T-lymphocyte (P = 0.030) infiltrations. Apoptotic terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive nuclei and MAC were located at the same wall areas in four out of 14 double-stained samples, but no double-positive cells were found. Electromicroscopy and immunoelectron microscopy of an unruptured SCAA showed cell death in the MAC-positive layers in the outer SCAA wall. CONCLUSION These data suggests that complement activation and MAC formation are involved in SCAA wall degeneration and rupture.

Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets.

Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of this strain on the growth and immune function of recently weaned piglets during a feeding trial. For this, 20 piglets were divided evenly into a treatment and a control group. Piglets in the treatment group were fed L. brevis cells (1×10(10)) daily for three weeks, whereas those in the control group were provided an equivalent amount of probiotic-free placebo. For assessing the impact of L. brevis supplementation during the feeding trial, health status and weight gain of the piglets were monitored, pre- and post-trial samples of serum and feces were obtained, and specimens of the small and large intestinal mucosa and digesta were collected at slaughter. The results we obtained indicated that L. brevis-supplemented feeding induced a non-significant increase in piglet body weight and caused no change in the morphology of the intestinal mucosa. L. brevis cells were found to localize mainly in the large intestine, but they could not be isolated from feces. To a lesser extent, L. brevis was detected in the small intestine, although there was no specific attachment to the Peyers patches. Changes in total serum IgG and IgA concentrations were not caused by supplemented L. brevis and no measurable rise in L. brevis-specific IgG was observed. However, analysis of cytokine gene expression in intestinal mucosa revealed downregulation of TGF-β1 in the ileum and upregulation of IL-6 in the cecum in the L. brevis-supplemented group. Based on the results from this study, we conclude that whereas L. brevis appears to have some intestinal immunomodulatory effects, the ability of this strain to survive and colonize within the porcine gut appears to be limited.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest‐derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12‐myristate 13‐acetate was analyzed by differential display reverse transcription PCR. A strongly up‐regulated expression of decay‐accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement‐mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up‐regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

A functional complement system is required for normal T helper cell differentiation.

Complement is a fundamental part of the innate immune system, and also modulates B cell responses. Its effects on T cells, however, are less well studied. Here we have studied antigen-specific T cell responses in C3-knockout (C3-KO) C57BL/6 mice. The animals were immunized with ovalbumin (OVA) in complete Freunds adjuvant, which favors T helper 1 (Th1)-type responses. Splenic lymphocytes from C3-KO mice proliferated less in response to OVA stimulation than splenocytes from control wild type (WT) mice. The response in the C3-KO mice was also qualitatively different. The expression of Th1 lineage determining transcription factor T-bet was decreased in OVA-stimulated splenocytes, and the induction of Th1-associated IgG subclasses impaired. In WT mice T cell proliferation in response to OVA was positively correlated with antigen-specific IgG2a and IgG3 levels. In C3-KO mice the proliferative response correlated with antigen-specific IgE levels, consistent with Th2 deviation. The expression of Th1-inducing cytokines IL-12 and IFN-γ was also decreased in the collecting lymph nodes in the C3-KO mice after immunization. Our results show that the complement system and its component C3 participate in the regulation of T cell responses, and that complement function is required for normal T helper cell differentiation.

Survey of selected tick-borne diseases in dogs in Finland

BackgroundDue to climate changes during the last decades, ticks have progressively spread into higher latitudes in northern Europe. Although some tick borne diseases are known to be endemic in Finland, to date there is limited information with regard to the prevalence of these infections in companion animals. We determined the antibody and DNA prevalence of the following organisms in randomly selected client-owned and clinically healthy hunting dogs living in Finland: Ehrlichia canis (Ec), Anaplasma phagocytophilum (Ap), Borrelia burgdorferi (Bb) and Bartonella.MethodsAnti-Ap, −Bb and –Ec antibodies were determined in 340 Finnish pet dogs and 50 healthy hunting dogs using the 4DX Snap®Test (IDEXX Laboratories). In addition, PCRs for the detection of Ap and Bartonella DNA were performed. Univariate and multivariate logistic regression analyses were used to identify risk factors associated with seropositivity to a vector borne agent.ResultsThe overall seroprevalence was highest for Ap (5.3%), followed by Bb (2.9%), and Ec (0.3%). Seropositivities to Ap and Bb were significantly higher in the Åland Islands (p <0.001), with prevalence of Ap and Bb antibodies of 45 and 20%, respectively. In healthy hunting dogs, seropositivity rates of 4% (2/50) and 2% (1/50) were recorded for Ap and Bb, respectively. One client-owned dog and one hunting dog, both healthy, were infected with Ap as determined by PCR, while being seronegative. For Bartonella spp., none of the dogs tested was positive by PCR.ConclusionsThis study represents the first data of seroprevalence to tick borne diseases in the Finnish dog population. Our results indicate that dogs in Finland are exposed to vector borne diseases, with Ap being the most seroprevalent of the diseases tested, followed by Bb. Almost 50% of dogs living in Åland Islands were Ap seropositive. This finding suggests the possibility of a high incidence of Ap infection in humans in this region. Knowing the distribution of seroprevalence in dogs may help predict the pattern of a tick borne disease and may aid in diagnostic and prevention efforts.

Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (Fcytosol/Fnucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells.

Activation of complement system in kidney after ketoprofen-induced kidney injury in sheep

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammatory pain in humans and animals. An overdose of an NSAID is nephrotoxic and can lead to acute kidney injury (AKI). Complement activation occurs in several types of renal disorders with proteinuria. The aim of this study was to investigate whether complement system becomes activated in kidneys after a high dose of NSAID. Kidney tissue and urine samples were collected from six sheep with ketoprofen-induced AKI and from six healthy control sheep. The localization of complement proteins in kidney tissue was carried out using immunohistochemical stainings, and excretion of C3 was tested by immunoblotting.ResultsThe complement system was found to become activated in the kidney tissue as demonstrated by positive immunostaining for C1q, C3c, C4c, C5, C9 and factor H and by Western blotting analysis of C3 activation products in urine samples in sheep with AKI.ConclusionsOur results thus suggest that the alternative complement pathway is activated, and it may contribute to the acute tubular injury seen in the kidneys of NSAID-induced AKI sheep. Inhibition of complement activation may serve as potential therapeutic target for intervention in drug-induced AKI.

Dietary supplementation with yeast hydrolysate in pregnancy influences colostrum yield and gut microbiota of sows and piglets after birth

Dietary supplementation with yeast derivatives (YD) contributes to the health and physiology of sows and piglets, but few studies have focused on how it influences gut health and performance of sows and piglets. The goal was therefore to examine whether YD, based on brewer’s yeast hydrolysate added to pregnancy diet, would affect colostrum composition, yield (CY) and gut microbiota of sows and piglets. Sows were allocated to either a control diet (n = 19) or a control diet supplemented with 2g YD/kg (n = 18) during the pregnancy. Piglets suckling belonging to the control sows (n = 114) and supplemented sows (n = 108) were also included in the study. Gut microbiota populations of sows at farrowing and piglets at one and four weeks of age were assessed using 16S rRNA gene sequencing. Colostrum samples were examined for nutritional composition and immunoglobulin (Ig) content. All piglets were individually weighed at birth and 24 hours later in order to calculate CY, and later at four weeks to calculate average daily gain (ADG). Protein, lactose and dry matter content of colostrum did not significantly differ between the two groups, while sows fed YD had higher levels of fat in their colostrum (P < 0.05). Immunoglobulin A, IgM and IgG levels in colostrum did not differ between the two groups (P >0.05). Colostrum yield was lower in the control than that in YD group (3701g vs. 4581 g; P <0.05). Although the YD supplementation did not change fecal bacteria diversity in sow, more beneficial and fermentative bacteria (Roseburia, Paraprevotella, Eubacterium) were found in the YD fed group (P <0.01) while, some opportunistic pathogens, including Proteobacteria, especially the genera Desulfovibrio, Escherichia/Shigella and Helicobacter, were suppressed. Piglets at one week of age from sows fed YD had more beneficial microbial populations with significant diversity and fewer opportunistic pathogens. Additionally, we established a Pearson’s correlations between CY, colostrum components, piglet birth weight and fecal microbiota. Therefore, YD added to the sow diet during pregnancy increases colostrum availability and its energy content for neonate piglets, also promoting beneficial maternal microbial sources for neonate.