Intestinal Cytokine mRNA Expression in Canine Inflammatory Bowel Disease: A Meta-Analysis with Critical Appraisal

Data implicating mucosal cytokines in the pathogenesis of canine inflammatory bowel disease (IBD) are limited. The aims of the present study were to report new findings of intestinal cytokine expression in dogs with IBD and to compare these data with previous studies through meta-analysis. Cytokine mRNA abundance in intestinal biopsies collected prospectively was evaluated by using a semiquantitative RT-PCR technique. For meta-analysis, an electronic database search revealed 3 clinical trials, all of which were nonrandomized (type III) case series. Prospective analysis showed that the intestines of healthy dogs and those with IBD express numerous cytokines and that a proinflammatory expression profile is not a feature of small or large-intestinal IBD. The meta-analysis data included 158 dogs characterized as healthy (n = 45), diarrheic nonIBD dogs (n = 6), nonresponders (n = 2), small-intestinal IBD (n = 41), colonic IBD (n = 25), and chronic enteropathy (n = 39). German shepherd dogs were overrepresented in 3 of the 4 studies. Healthy dogs showed mRNA expression for most cytokines including IL2, IL4, IL5, IL10, IL12, IFNγ, TNFα, and TGFβ. Only IL12 mRNA expression was increased consistently in small-intestinal IBD, whereas IBD colitis lacked consistent patterns of expression. In summary, dogs with IBD fail to express a predominant Th1- or Th2 cytokine bias in inflamed mucosa. Heterogeneity of results among these studies might be explained by numerous factors including the method of mRNA quantification, stage of disease, and demographic differences in study populations.Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBD, inflammatory bowel disease; LP, lymphocytic–plasmacyticIdiopathic inflammatory bowel disease (IBD) in dogs is a chronic immune-mediated disorder empirically defined by clinical, histologic, and therapeutic features.^{18,26,27,29,45} Evidence suggests that intestinal inflammation in IBD results from altered interaction between the resident microflora and mucosa in a susceptible host.^48,53 Aggressive host immune responses directed against commensal bacteria play a central role in the pathogenesis of chronic mucosal inflammation. The concept of impaired immunoregulation in canine IBD is supported by observations of increased numbers of immunoglobulin-containing cells and T cells in inflamed tissues,^16,28,30,49 upregulated mucosal and luminal expression of nitric oxide metabolites,^20,29 and altered serum concentrations of select acute phase proteins, such as C-reactive protein, in diseased dogs.³¹ C-reactive protein is a marker of inflammation and tissue injury and is produced by the liver in response to stimulation by IL6, IL1β, and TNFα.^14,51Cytokines play a key role in the modulation of the mucosal immune system of humans. To maintain gut homeostasis, the normal mucosal immune system balances a network of inflammatory mediators, including proinflammatory, antiinflammatory, and regulatory cytokines.⁴⁷ Cytokines are synthesized rapidly and secreted on stimulation and induce the production of adhesion molecules and other inflammatory mediators including reactive oxygen species, nitric oxide metabolites, and lipid products such as prostaglandins, leukotrienes, and platelet-activating factor. Cytokine-producing cells induce, amplify, prolong, and mediate intestinal mucosal injury.¹³ Disturbances in the balance of proinflammatory (Th1/Th17-derived) and immunoregulatory (Th2/Tr1-derived) cytokines occur in humans with IBD as well as numerous animal models of intestinal inflammation.^16,39,44Data evaluating the role of CD4⁺ T cells and mucosal cytokines in the pathogenesis of canine IBD are limited. Lymphocytes expressing CD4⁺ are either increased¹⁶ or decreased²⁸ in dogs having small-intestinal IBD, whereas mucosal CD4⁺ T cells are increased in dogs with IBD colitis.^30,49 A recent study¹⁵ described a balance between proinflammatory and antiinflammatory cytokine mRNA expression in dogs with small-intestinal enteropathies but included only 4 dogs diagnosed with IBD. A separate investigation⁴⁴ evaluating mucosal cytokine mRNA expression in dogs with lymphocytic–plasmacytic colitis reported upregulated expression of proinflammatory cytokines IL2 and TNFα. Yet another study⁴¹ reported no difference in cytokine expression in the duodenal mucosa of dogs with or without chronic diarrhea; however, the dogs of this report were not subdivided in terms of response to therapy as having idiopathic IBD, antibiotic-responsive diarrhea, or food-responsive enteropathy. Because of these varied observations, it is unclear which cytokines, if any, control or enhance the local immune response of canine IBD because 1) few dogs with IBD have been evaluated, 2) German shepherd dogs with enteropathies were over-represented in most studies, 3) various measures of cytokine mRNA expression were used, and (4) distinctly variable patterns of cytokine expression were present among these earlier studies.The objectives of this study were to 1) assess cytokine mRNA expression in the intestinal mucosa of dogs diagnosed with small- and large-intestinal IBD by using an RT-PCR technique and 2) compare these data with previously published data to determine the putative role of cytokine expression in the pathogenesis of canine IBD and other forms of chronic enteropathy through meta-analysis of combined findings.     

Glomerular abundance of complement proteins characterized by proteomic analysis of laser-captured microdissected glomeruli associates with progressive disease in IgA nephropathy

Background

The clinical course of IgA nephropathy (IgAN) is variable and complement activation may predict prognosis. The present study investigated whether glomerular abundance of complement proteins associates with progression to end-stage renal disease (ESRD) in patients for whom prognosis could not be predicted based on clinical variables.

Methods

Based on data from the Norwegian Kidney Biopsy Registry and the Norwegian Renal Registry, three groups were included: IgAN patients with (n = 9) or without (n = 16) progression to ESRD during 10 years, and controls (n = 15) with a normal kidney biopsy. IgAN patients had eGFR > 45 ml/min/1.73 m² and non-nephrotic proteinuria at time of biopsy. Using stored formalin-fixed paraffin embedded kidney biopsy tissue, about 100 glomerular cross sections were microdissected for each patient. Samples were analyzed by liquid chromatography–tandem mass spectrometry and relative abundances of complement proteins were compared between groups.

Results

Proteomic analyses quantified 2018 proteins, of which 28 proteins belong to the complement system. As compared to IgAN patients without progressive disease, glomeruli from patients with progressive IgAN had significantly higher abundance of components of the classical and the terminal complement pathways, and inhibitory factors such as Factor H and factor H related proteins. Abundance of complement proteins classified progressors from non-progressors with an area under ROC curve of 0.91 (p = 0.001). Clinical and morphological data were similar between the two patient groups and could not predict progressive IgAN.

Conclusions

In conclusion, higher glomerular abundance of complement proteins was associated with a progressive clinical course in IgAN and are candidate biomarkers to predict prognosis.

     

Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19

SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses.     

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.     

Case-control study to evaluate the link between immunosuppression and Candida spp. infection

Candidiasis is one of the fungal infections with the highest incidence in the immunosuppressed host. The evolution of infection and the increase of antifungal medical drugs resistance could both contribute to the mortality attributable to Candida infection in the immunosuppressed host. Even though the data from international studies are well known, few studies have been published in Romania on this subject. In the case-control study we demonstrated the link between the immunosuppression and the presence of Candida infection. Further studies are to be carried out in order to identify more accurately this link and to extend the study to other fungi. There is a need to increase the microbiological diagnosis use at least at the hospital laboratory level in order to better identify the real situation of fungal infections and the link between them and the concrete status of different hosts. Continued surveillance for infections caused by C. albicans and other species of Candida among hospitalized patients is recommended. Control of antimicrobial resistance among nosocomial infections caused by C. albicans and other species of Candida requires rational policies for use of both antifungal and antibacterial agents and appropriate surveillance for the emergence of resistant strains and species.     

Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using beta-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury-glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury.