Alterations of Blood Immune Cells in Elderly Patients with COVID-19 and with or without Pre-Existing Type 2 Diabetes

Elderly individuals, especially those with pre-existing conditions like type 2 diabetes mellitus (T2DM), have a high risk for developing severe cases of COVID-19. The aim of this work was to characterize the alterations of blood immune cells (BIC) in patients with symptomatic COVID-19 and confirmed SARS-CoV-2 infection, ≥60 years and who needed hospitalization in the Centro de Salud Hospital of Tucuman, Argentina, during the second peak of the pandemic in Argentina. Ten patients were enrolled from December 2020 to May 2021. Blood samples were taken at the time of admission (day 0) and five days after (day 5) for routine laboratory tests and the characterization of BIC by flow cytometry. Most of the patients were men (70%) aged between 60 and 78 years. The 70% of patients had T2DM while 50% had arterial hypertension. At day 0, all the patients had increased neutrophils and inflammatory markers (C reactive protein and D-dimers) and reduced numbers of lymphocytes, HLA-DRhi monocytes, CD16+CD56+ NK cells, CD3+HLA−DR+CD25+ cells, CD4+ and CD8+ T cells in blood. Patients received a standard treatment for COVID-19 care (O2, corticosteroids and antibiotics). The hospital treatment normalized the levels of BIC (day 5) in 30% of patients who were those with no comorbidities. In patients with T2DM, BIC recovery was variable. In T2DM patients who required administration of plasma (30%), prolonged O2 therapy (40%) or referral to the intensive care unit (10%) significant reductions of CD16+CD56+, CD3+HLA−DR+CD25+, CD4+ and CD8+ cells were observed between days 0 and 5. In line with previous studies, our results show that absolute counts of major lymphocyte subsets in blood are significantly and substantially decreased during the course of severe COVID-19 disease in elderly patients. These BIC alterations may persist despite clinical care in elderly patients with T2DM. Further studies are needed to investigate the utility of early lymphocyte subset measurements as prognostic biomarkers of disease severity, mortality, and response to treatment in COVID-19 elderly patients with T2DM.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.

Evaluation of the Immunoregulatory Capacities of Feed Microbial Materials in Porcine Intestinal Immune and Epithelial Cells

The establishment of drug-free feeding systems has been required for secure and healthy livestock production. Although functional feed materials containing microorganisms as alternatives to enhance intestinal immunity are expected to be beneficial for reducing diarrhoea caused by pathogens in weaned piglets, the effects of such materials on porcine intestinal cells have not been investigated in detail. Therefore, this work evaluated the immunoregulatory functions of microbial feed materials in porcine intestinal immune and epithelial cells. Porcine immune cells isolated from Peyer’s patches and mesenteric lymph nodes were stimulated with six different feed materials containing microorganisms, and evaluated for lymphocyte mitogenicity and cytokine inductions. In addition, porcine intestinal epithelial cells were stimulated with the materials before treatment with heat-killed enterotoxigenic Escherichia coli (ETEC), and analyzed for the proinflammatory cytokine expressions. The material containing Bifidobacterium thermophilum significantly augmented lymphocytes’ mitogenicity and also induced a high expression of IL-2, IL-6 and IFN-γ in immune cells, and inhibited ETEC-induced overexpression of IL-6 and IL-8 via regulation of Toll-like receptor signaling. These results suggest that this feed material stimulates intestinal epithelial and immune cells to exert immunoregulation, suggesting that this feed is expected to contribute to promoting the health of piglets without using antimicrobial feed materials.

Bifidobacteria Upregulate Expression of Toll-Like Receptor Negative Regulators Counteracting Enterotoxigenic <i>Escherichia coli</i>Mediated Inflammation in Bovine Intestinal Epitheliocytes

We previously established a bovine intestinal epithelial cell line (BIE cells) and showed that BIE cells are useful in vitro model system for the study of interactions between pathogenic and beneficial microorganisms and bovine intestinal epithelial cells (IECs). In the present study, we aimed to select potential immunomodulatory bifidobacteria that may be used to beneficially modulate the inflammatory response in bovine IECs. We also aimed to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of bifidobacteria by evaluating the role of Toll-like receptor (TLR)-2 and TLR negative regulators in the regulation of proinflammatory cytokines production and MAPK, NF-κB and PI3K pathways activation in BIE cells. Five bifidobacteria strains were evaluated in this study and according to their capacity to modulate the inflammatory response of BIE cells. Despite the unique effect of each strain, four common points were found when comparing the effect of the high and moderate anti-inflammatory strains: 1) Upregulation of TLR negative regulators and the intensity of that upregulation was related to the different immunomodulatory capacity of each bifidobacteria strain;2) The balance between MAPK activation and MKP-1 upregulation affected the anti-inflammatory effect of bifidobacteria in BIE cells;3) The inhibition of PI3K pathway was related to the anti-inflammatory effect of bifidobacteria;4) The immunoregulatory effect of bifidobacteria in BIE cells is partially dependent on TLR2. This study shows that BIE cells can be used for the selection of immunoregulatory bifidobacteria and for studying the mechanisms involved in the protective activity of immunobiotics against TLR4-induced inflammatory damage. In addition, we have demonstrated that the anti-inflammatory effect of bifidobacteria was achieved by a complex interaction of multiple TLRs negative regulators as well as the inhibition/activation of multiple signaling pathways.

Quantitative Microbiological Evaluation of <i>Salmonella</i>Typhimurium Shed in Diarrhea, Loose, and Normal Stools of Infected Pigs

Control of within-herd transmission of Salmonella is important for reducing the prevalence of this organism on pig farms and for preventing Salmonella-contamination of pork. At the farm level, understanding the within-herd transmission of Salmonella can lead to more effective control. Salmonella infection is dependent on the inoculation dose;hence, quantitative evaluation of Salmonella shed in feces would provide useful information for developing effective measures. In this study, to reproduce and evaluate the number of Salmonella shed in diarrhea, loose stools, and normal feces, weaned pigs were inoculated with 3.2 × 109, 3.2 × 107, and 3.2 × 105 cfu of Salmonella Typhimurium, respectively. The number of S. Typhimurium shed in the feces peaked within 1 week post-inoculation in every group and the most amount of diarrhea and loose stools were observed within 2 weeks post-inoculation. Diarrhea occurred 10 times (six pigs), and loose stools were observed 25 times (11 pigs). The average concentration of S. Typhimurium shed in diarrhea, loose stools, and normal feces was 1.0 × 108, 1.6 × 104, and 7.1 × 101 cfu/g feces, respectively. These data suggest that diarrhea and loose stools are significant sources of within-herd transmission of Salmonella. Moreover, as some of the normal feces contained >1.0 × 106 cfu/g of S. Typhimurium, even normal feces could be a source of within-herd transmission of Salmonella. At Salmonella-positive farms, reduction of the amount of Salmonella shed even in normal feces would lead to better control of within-herd transmission of Salmonella. These data can contribute to the control of within-herd transmission of Salmonella, particularly during the weaning period.

Molecular Cloning and Functional Characterization of Porcine MyD88 Essential for TLR Signaling

We isolated cDNA encoding porcine MyD88 （poMyD88） from Peyer＇s patches （Pps） of GALT. The complete open reading frame （ORF） of poMyD88 contains 879 bp encoding a deduced 293 aa residues. The amino acid sequence of poMyD88 was characterized by N-terminal death, intermediate and C-terminal Toll/IL-1 receptor （TIR） domains. The putative poMyD88 protein shares a higher level of homology with its human （87.2% amino acid identity） than with its mouse （77.4% amino acid identity） counterpart. Overexpression of poMyD88 participated in the further enhanced activation of NF-w.B in human embryonic kidney （HEK） 293 cells expressing porcine TLR2 and porcine TLR4/MD-2, but not porcine RP105/MD-1 after stimulation with the corresponding ligands. The expression levels of MyD88 were highest in the spleen and mesenteric lymph nodes （MLNs）, and lower in digestive tissues of newborn swine. In adult swine, the expression levels in the digestive tissues were lower than those in MLNs and the spleen. These results suggest that an MyD88-dependent signaling pathway is present in newborn as well as in adult swine and that it is involved in the innate immune system of these animals.