The potential etiopathogenetic role and diagnostic utility of CD3+ CD56+ regulatory T lymphocytes in Myelodysplastic Syndromes

Serio et al. show a significant reduction of CD3+CD56+ regulatory T cells (TR3-56) in bone marrow (BM) of low-risk myelodysplastic subjects, as compared with the high-risk and the AML group; in addition, the BM frequency of mature granulocytes, a recognised marker of residual effective haematopoiesis, was observed to inversely correlate with TR3-56 in the MDS cohort. Such data are of great interest and confirm and extend, in an independent MDS cohort, the trend-increase of BM TR3-56 from very low/low risk to high/very high risk MDS and the inverse correlation with the cytotoxic T-cell (CTL) activity, likely fostering the escape of leukaemic blasts to immune-surveillance, by us recently described

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori

BACKGROUND: Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection. RESULTS: In the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD(50) lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes. CONCLUSIONS: G. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status

T cell activation induces CuZn Superoxide Dismutase (SOD)-1) intracellular re-localization, production and secretion

Reactive Oxygen Species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. CuZn Superoxide dismutase (SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T Cell Receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors

Mechanical phenotyping of K562 cells by the Micropipette Aspiration Technique allows identifying mechanical changes induced by drugs

Mechanical properties of living cells can be used as reliable markers of their state, such as the presence of a pathological state or their differentiation phase. The mechanical behavior of cells depends on the organization of their cytoskeletal network and the main contribution typically comes from the actomyosin contractile system, in both suspended and adherent cells. In the present study, we investigated the effect of a pharmaceutical formulation (OTC - Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the inflammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells. Here we show that by measuring the mechanical properties of cells exposed to OTC for different incubation times, it is possible to infer modifications induced by the formulation to the actomyosin contractile system. We emphasize that this system is involved in the first stages of the apoptotic process where an increase of the cortical tension leads to the formation of blebs. We discuss the possible relation between the observed mechanical behavior of cells aspirated inside a micropipette and apoptosis

Bone marrow CD3+ CD56+ regulatory T lymphocytes (TR3 -56 cells) are inversely associated with activation and expansion of bone marrow cytotoxic T cells in IPSS-R very-low/low risk MDS patients

Background Emergence of dysplastic haematopoietic precursor/s, cytopenia and variable leukaemia risk characterise myelodysplastic syndromes (MDS). Impaired immune-regulation, preferentially affecting cytotoxic T cells (CTL), has been largely observed in MDS. Recently, we described the TR3-56 T cell subset, characterised by the co-expression of CD3 and CD56, as a novel immune-regulatory population, able to modulate cytotoxic functions. Here, we address the involvement of TR3-56 cells in MDS pathogenesis/progression. Objectives To analyse the relationship between TR3-56 and CTL activation/expansion in bone marrow (BM) of very-low/low-risk MDS subjects. Methods Peripheral blood and BM specimens, obtained at disease onset in a cohort of 58 subjects, were analysed by immune-fluorescence and flow cytometry, to preserve the complexity of the biological sample. Results We observed that a trend-increase of BM TR3-56 in high/very-high MDS stage, as compared with very-low/low group, associates with a decreased activation of BM resident CTL; significant correlation of TR3-56 with BM blasts has been also revealed. In addition, in very-low/low-risk subjects the TR3-56 amount in BM inversely correlates with the presence of activated BM CTL showing a skewed V beta T-cell repertoire. Conclusions These data add TR3-56 to the immune-regulatory network involved in MDS pathogenesis/progression. Better knowledge of the immune-mediated processes associated with the disease might improve MDS clinical management

Effects of crp deletion in Salmonella enterica serotype Gallinarum

Background. Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested. Methods. Mutants were constructed in S. Gallinarum by P22 transduction from Salmonella Typhimurium (S. Typhimurium) with deletion of the crp gene. The effect was characterized by measuring biochemical properties and by testing of invasion in a chicken loop model and by challenge of six-day-old chickens. Further, birds were immunized with the deleted strain and challenged with the wild type isolate. Results. The crp deletions caused complete attenuation of S. Gallinarum. This was shown by ileal loop experiments not to be due to significantly reduced invasion. Strains with such deletions may have vaccine potential, since oral inoculatoin with S. Gallinarum Δcrp completely protected against challenge with the same dose of wild type S. Gallinarum ten days post immunization. Interestingly, the mutations did not cause the same biochemical and growth changes to the two biotypes of S. Gallinarum. All biochemical effects but not virulence could be complemented by providing an intact crp-gene from S. Typhimurium on the plasmid pSD110. Conclusion. Transduction of a Tn10 disrupted crp gene from S. Typhimurium caused attenuation in S. Gallinarum and mutated strains are possible candidates for live vaccines against fowl typhoid

An Open Question in the COVID-19 Pandemic: Can Humans Transmit the Disease to Pets and Vice Versa?

SARS-CoV-2 infection apparently emerged in China in December 2019, causing the disease known as COVID-19,which can cause severe damage to vital organs (Ackermann et al, 2020). Spillover of SARS-CoV- 2 from bats to humans has been hypothesized (Ackermann et al, 2020). The virus spike protein is the main determinant of viral tropism because it is responsible for binding to the angiotensin converting enzyme 2 (ACE2) and subsequent entry of SARSCoV- 2 to host cells in humans and several animal species (Sun et al, 2020). Therefore, it is reasonable to hypothesize that the spike proteineACE2 receptor complex may represent evolutionary exploitation to overcome speciesbarriers to infection, thushighlighting the zoonotic origin and transmission of the virus

Pro-Inflammatory and Immunological Profile of Dogs with Myxomatous Mitral Valve Disease

Simple Summary Myxomatous mitral valve disease (MMVD) is the most commonly acquired cardiac disease in dogs and is responsible for congestive heart failure. In this research, some inflammatory, immunological, and echocardiographic parameters were evaluated in dogs affected by MMVD in order to assess the involvement of additional pathophysiological mechanisms during the disease. The main results revealed that inflammation parameters increased according to the severity of the disease and suggested that inflammatory activation may play an important role in cardiac remodeling associated with the progressive volumetric overload in MMVD. Also, a relative increase in Treg cells was detected, suggesting that they could represent a regulatory mechanism for limiting the inflammatory immune response. Myxomatous mitral valve disease (MMVD) is a very frequently acquired cardiac disease in dog breeds and is responsible for congestive heart failure (CHF). The involvement of the immune system and pro-inflammatory cytokines in dogs with CHF due to mitral valve disease has not yet been extensively investigated. Here, we investigate the role of pro-inflammatory cytokines and the dysfunction of the immune system in dogs with different stages of severity through the blood assessment of CD4(+)FoxP3(+)regulatory T cells (Treg) cells, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6 pro-inflammatory cytokines, and immunological and echocardiographic parameters. A total of 36 cardiopathic dogs, 14 females and 22 males, with MMVD were included. Mean age and body weight (BW) at the time of enrollment were 10.7 +/- 2.77 years and 10.9 +/- 6.69 kg, respectively. For the comparison of the pro-inflammatory and immunological parameters, two groups of healthy dogs were also established. Control group 1 consisted of young animals (n. 11; 6 females and 5 males), whose age and mean weight were 4.1 +/- 0.82 years and 13.8 +/- 4.30 kg, respectively. Control group 2 consisted of elderly dogs (n. 12; 6 females and 6 males), whose age and BW were 9.6 +/- 0.98 years and 14.8 +/- 6.15 kg, respectively. Of particular interest, an increase in Treg cells was observed in the cohort of MMVD dogs, as compared to the healthy dogs, as Treg cells are involved in the maintenance of peripheral tolerance, and they are involved in etiopathogenetic and pathophysiological mechanisms in the dog. On the other hand, TNF-alpha, IL-1 beta, and IL-6 significantly increased according to the severity of the disease in MMVD dogs. Furthermore, the positive correlation between IL-6 and the left ventricle diastolic volume suggests that inflammatory activation may be involved in cardiac remodeling associated with the progressive volumetric overload in MMVD

Superoxide Dismutase-1 intracellular content in T lymphocytes associates with increased Regulatory T Cell level in Multiple Sclerosis subjects undergoing immune-modulating treatment.

Reactive oxygen species (ROS) participate in the T-cell activation processes. ROS-dependent regulatory networks are usually mediated by peroxides, which are more stable and able to freely migrate inside cells. Superoxide dismutase (SOD)-1 represents the major physiological intracellular source of peroxides. We found that antigen-dependent activation represents a triggering element for SOD-1 production and secretion by human T lymphocytes. A deranged T-cell proinflammatory response characterizes the pathogenesis of multiple sclerosis (MS). We previously observed a decreased SOD-1 intracellular content in leukocytes of MS individuals at diagnosis, with increasing amounts of such enzyme after interferon (IFN)-b 1b treatment. Here, we analyzed in depth SOD-1 intracellular content in T cells in a cohort of MS individuals undergoing immune-modulating treatment. Higher amounts of the enzyme were associated with increased availability of regulatory T cells (Treg) preferentially expressing Foxp3-exon 2 (Foxp3-E2), as described for effective Treg. In vitro administration of recombinant human SOD-1 to activated T cells, significantly increased their IL-17 production, while SOD-1 molecules lacking dismutase activity were unable to interfere with cytokine production by activated T cells in vitro. Furthermore, hydrogen peroxide addition was observed to mimic, in vitro, the SOD-1 effect on IL-17 production. These data add SOD-1 to the molecules involved in the molecular pathways contributing to re-shaping the T-cell cytokine profile and Treg differentiation