Comparison Between K3EDTA and Lithium Heparin as Anticoagulant to Isolate Bovine Granulocytes From Blood

Granulocytes play a key role in the defense against invading pathogens. To study granulocyte functions, the isolation of a pure and active cell population from fresh blood is required. Anticoagulants and red blood cells (RBCs) lysis used in the isolation procedure may influence cell harvest, cell marker expression, and pre-activation of cells. In this study, the influence of the anticoagulants K3EDTA or lithium heparin and the effect of different RBCs lysis methods on bovine granulocyte population from fresh blood of healthy cows after density gradient centrifugation were investigated. Venous blood from healthy cows was collected in K3EDTA and lithium heparin tubes. Density gradient centrifugation to separate granulocytes from other cells was conducted using Biocoll. Then, RBCs were lysed with hypotonic water or 0.2% sodium chloride (NaCl). Immediately after isolation, harvest, viability, size, granularity, purity, and CD11b expression as a marker for granulocytes was analyzed by flow cytometry. In addition, as a marker for activation and reactivity of the granulocytes, we stimulated cells with phorbol-myristate-acetate to evaluate the release of reactive oxygen species. Furthermore, extracellular trap (ET) formation was investigated by confocal immunofluorescence microscopy in untreated control cells and cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin. We did not find a significant difference in percentage of dead cells when comparing the two anticoagulants or the different RBCs lysis methods. However, the percentage of granulocytes in the harvested population was significantly less using lithium heparin blood as anticoagulant compared to K3EDTA. The granulocytes harvested from lithium heparin blood and water lysis exhibited higher clumping and pre-activation of unstimulated control cells as indicated by isolation of doublet cells, increased CD11b expression, and increased oxidative burst and higher amount of ET-releasing cells. Furthermore, the combination of K3EDTA as anticoagulant and NaCl as RBCs lysis method revealed the lowest variability and highest difference between untreated and methyl-β-cyclodextrin-treated cells when quantifying ET formation. In conclusion, density gradient centrifugation of K3EDTA blood resulted in higher purity of bovine granulocytes compared to lithium heparin blood. In contrast to water lysis, NaCl lysis method is recommended to avoid pre-activation of cells which may occur during hypotonic water lysis

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps

IntroductionNeutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence.MethodsHere, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy.ResultsFor the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation.DiscussionThese findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens

Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria

IntroductionInfluenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases.MethodsIn this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared.ResultsHere, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth.DiscussionIn summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils

Comparing Cathelicidin Susceptibility of the Meningitis Pathogens Streptococcus suis and Escherichia coli in Culture Medium in Contrast to Porcine or Human Cerebrospinal Fluid

Host defense peptides or antimicrobial peptides (AMPs), e.g., cathelicidins, have recently been discussed as a potential new treatment option against bacterial infections. To test the efficacy of AMPs, standardized methods that closely mimic the physiological conditions at the site of infection are still needed. The aim of our study was to test the meningitis-causing bacteria Streptococcus suis and Escherichia coli for their susceptibility to cathelicidins in culture medium versus cerebrospinal fluid (CSF). Susceptibility testing was performed in analogy to the broth microdilution method described by the Clinical and Laboratory Standard Institute (CLSI) to determine minimum inhibitory concentrations (MICs) of antimicrobial agents. MICs were determined using cation-adjusted Mueller–Hinton broth (CA-MHB), lysogeny broth (LB), Roswell Park Memorial Institute medium (RPMI) or Dulbecco’s Modified Eagle’s Medium (DMEM) (the latter two supplemented with 5% CA-MHB or blood) and compared with MICs obtained in porcine or human CSF. Our data showed that MICs obtained in CA-MHB as recommended by CLSI do not reflect the MICs obtained in the physiological body fluid CSF. However, the MICs of clinical isolates of S. suis tested in RPMI medium supplemented with CA-MHB, were similar to those of the same strains tested in CSF. In contrast, the MICs in the human CSF for the tested E. coli K1 strain were higher compared to the RPMI medium and showed even higher values than in CA-MHB. This highlights the need for susceptibility testing of AMPs in a medium that closely mimics the clinically relevant conditions

Case study on the pathophysiology of Fabry disease: abnormalities of cellular membranes can be reversed by substrate reduction in vitro

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with N-butyldeoxynojirimycin (NB-DNJ) in vitro was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

INTRODUCTION: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. METHODS: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. RESULTS: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. DISCUSSION: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions

Companion Animals in Zoonoses Research – Ethical Considerations

Non-human animals are commonly classified according to their "role", such as "livestock", "wild" or "companion" animals. But what if those classifications overlap? This article presents a report of the retreat week "ZooCan – Zoonoses of companion animals as case study for animal ethics" at the University of Veterinary Medicine Hannover, Germany, in November 2022. The workshop included participants from different European countries with interdisciplinary backgrounds (animal law, bioethics, epidemiology, philosophy, biology and veterinary medicine). We address ethically relevant issues that emerge when companion animals are used as research animals, particularly in zoonoses research. The outcomes of the multi-disciplinary approach are used to i) define criteria to classify "companion" and "research" animals, ii) provide guidance to overcome the challenges with classificational overlaps, iii) give insights into cutting-edge zoonoses research with an example of SARS-CoV-2 in cats, and iv) discuss animal ethics approaches with regard to classifications

M Protein and Hyaluronic Acid Capsule Are Essential for In Vivo Selection of covRS Mutations Characteristic of Invasive Serotype M1T1 Group A Streptococcus

The initiation of hyperinvasive disease in group A Streptococcus (GAS) serotype M1T1 occurs by mutation within the covRS two-component regulon (named covRS for control of virulence regulatory sensor kinase), which promotes resistance to neutrophil-mediated killing through the upregulation of bacteriophage-encoded Sda1 DNase. To determine whether other virulence factors contribute to this phase-switching phenomenon, we studied a panel of 10 isogenic GAS serotype M1T1 virulence gene knockout mutants. While loss of several individual virulence factors did not prevent GAS covRS switching in vivo, we found that M1 protein and hyaluronic acid capsule are indispensable for the switching phenotype, a phenomenon previously attributed uniquely to the Sda1 DNase. We demonstrate that like M1 protein and Sda1, capsule expression enhances survival of GAS serotype M1T1 within neutrophil extracellular traps. Furthermore, capsule shares with M1 protein a role in GAS resistance to human cathelicidin antimicrobial peptide LL-37. We conclude that a quorum of GAS serotype M1T1 virulence genes with cooperative roles in resistance to neutrophil extracellular killing is essential for the switch to a hyperinvasive phenotype in vivo