Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis

Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions.

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

DASH Diet as a Proposal for Improvement in Cellular Immunity and Its Association with Metabolic Parameters in Persons with Overweight and Obesity

The development of obesity entails a chronic low-grade inflammatory state with increased pro-inflammatory cells, mainly in visceral adipose tissue (VAT). Additionally, dietary patterns have an influence on the regulation of chronic inflammation. Dietary Approaches to Stop Hypertension (DASH) include foods with an anti-inflammatory profile and that have positive impacts on body composition (BC), suggesting improvements in inflammatory processes. Objective: To analyze the impact of the DASH diet on cellular immunity, anthropometric, biochemical and BC parameters in patients with overweight and obesity, who could present metabolic syndrome. Methodology: Lymphocyte subpopulations, biochemical parameters, anthropometric parameters, and BC before and 8 weeks after intervention with the DASH diet in persons with overweight and obesity were measured. Results: Fifty-nine young adults participated in the study. After the intervention, no significant changes in biochemical parameters were observed, although a significant decrease in nearly all of the anthropometric and BC variables was found: waist circumference (p < 0.001), percentage and kilograms of fat (p < 0.001 and p < 0.025, respectively), VAT (p < 0.020), and weight (p < 0.001), as well as total lymphocytes and double-positive TCD4+ cells. A relation between changes in leukocyte subpopulations (monocytes, natural killer, helper and cytotoxic lymphocytes, and naive TCD4+ cells) and metabolic improvements (glucose, triglycerides, total cholesterol and LDL-c) was also found. Conclusions: The DASH diet promotes positive changes in lymphocyte subpopulations, anthropometric parameters and BC in persons with overweight and obesity. Future studies should elucidate the cellular and molecular mechanisms through which the DASH diet produces inmunometabolic improvement.

RELATIONSHIP OF LYMPHOCYTE SUBPOPULATIONS IN BREAST CANCER PATIENTS WITH TREATMENT RESULTS

Introduction. Breast cancer (BC) is an immunogenic tumor. Immune cells infiltration of tumor tissue can affect the clinical course of the disease. The immunogenicity of breast cancer varies depending on the molecular subtype.The aim of this work was to study the main indicators of systemic and local immunity before patient’s treatment and to determine their relationship with the immediate neoadjuvant chemotherapy results.Materials and methods. Patients with stage II–III BC received standard neoadjuvant chemotherapy in accordance with the molecular subtypes. The percentage of the main effector and regulatory lymphocytes populations of systemic and local immunity was determined by flow cytometry.Results. A decrease in the level of effector CD8 and CD4 lymphocyte populations and an increase in the level suppressor populations in tumor tissue in comparison with peripheral blood indicate an immunosuppressive state of local immunity in BC patients. In tumor tissue, a high level of CD8+ PD-1+ and CD4+ PD-1+ cells were associated with a high level of regulatory CD4+ CD25highCD127–/low and CD8+ CD11b– CD28– lymphocytes. Differences were found in the significance of individual lymphocyte populations for the immediate results of treatment between patients with different subtypes of breast cancer.Conclusion. Determination of lymphocyte subpopulations correlating with the level of PD-1 cells, and the results of treatment in patients with different molecular BC subtypes, will help a clearer understanding of the antitumor immune response in this pathology, and will also serve as a basis for identifying immune biomarkers that can be used as additional predictive factors in various treatment options for BC patients.

Correlation between leukocyte phenotypes and prognosis of amyotrophic lateral sclerosis: a longitudinal cohort study

Background: Immune response changes have been reported in amyotrophic lateral sclerosis (ALS), but their clinical relevance remains undetermined. Therefore, we aim to evaluate the relationships between blood leukocyte subpopulations and prognosis of ALS.<br />Methods: A longitudinal cohort of 288 ALS patients with up to 5 years of follow-up during 2015-2020 were recruited at the only tertiary referral center for ALS in Stockholm, Sweden. Routine differential leukocyte counts, and determination of lymphocyte subpopulations including an extended T cell panel with flow cytometry, collected at diagnosis and at regular intervals thereafter. The primary outcome was risk of death (alternatively use of invasive ventilation) after diagnosis of ALS. The secondary outcomes included repeatedly measured functional status - through Amyotrophic Lateral Sclerosis Functional Rating Scale-revised (ALSFRS-R) score and disease progression rate. Cox model was used to evaluate the associations between leukocytes and risk of death. Generalized estimating equation model (GEE) was used to assess the correlation between leukocytes and ALSFRS-R score<br />and disease progression rate.<br />Results: The counts of leukocytes, neutrophils and monocytes increased gradually over time since diagnosis and were negatively correlated with ALSFRS-R score, but not associated with risk of death or disease progression rate. Focusing on lymphocyte subpopulations, increasing counts of natural killer (NK) cells (HR=0.61, 95% CI= [0.42-0.88] per SD increase) and proportions of Th2-diffrentiated CD4+ central memory (CM) T cells (HR=0.64, 95% CI= [0.48-0.85] per SD increase) were correlated with a lower risk of death. Increasing proportions of CD4+ effector memory cells re-expressing CD45RA (EMRA) T cells (HR=1.39, 95% CI= [1.01-1.92] per SD increase) and CD8+ T cells (HR=1.38, 95% CI= [1.03-1.86] per SD increase) were associated with a higher risk of death. None of the lymphocyte subpopulations was correlated with ALSFRS-R score or disease progression rate.<br />Conclusion: Our findings suggest a dual role of immune responses in ALS prognosis, where neutrophils and monocytes primarily reflect functional status whereas NK cells and different T lymphocyte populations act as prognostic markers for survival.

Cytometric analysis of patients with COVID-19: what is changed in the second wave?

Background Coronavirus disease 2019 (COVID-19) pandemic had a 1st wave in Europe from March to May 2020 and a 2nd wave since September 2020. We previously studied 35 hospitalized COVID-19 patients of the 1st wave demonstrating a cytokine storm and the exhaustion of most lymphocyte subpopulations. Herein, we describe the results obtained from COVID-19 patients of the 2nd wave. Methods We analyzed interleukin (IL)-6 by human-specific enzyme-linked immunosorbent assay and a large set of lymphocyte subpopulations by flow cytometry in 274 COVID-19 patients hospitalized from September 2020 to May 2021. Results Patients of 2nd wave compared with those of 1st wave showed lower serum IL-6 levels and a higher number of B and most T lymphocyte subpopulations in advanced stages, in relation with the age and the gender. On the other hand, we observed in 2nd wave patients: (i) a reduction of most lymphocyte subpopulations at mild and moderate stages; (ii) a reduction of natural killer cells and T regulatory cells together with a higher number of activated T helper (TH) 17 lymphocytes in all stages, which were mainly related to steroid and azithromycin therapies before hospitalization. Conclusions COVID-19 had a less severe impact in patients of the 2nd wave in advanced stages, while the impact appeared more severe in patients of mild and moderate stages, as compared with 1st wave patients. This finding suggests that in COVID-19 patients with milder expression at diagnosis, steroid and azithromycin therapies appear to worsen the immune response against the virus. Furthermore, the cytometric profile may help to drive targeted therapies by monoclonal antibodies to modulate specific IL/lymphocyte inhibition or activation in COVID-19 patients.

Clinicopathological Manifestations and Immune Phenotypes in Adult-Onset Immunodeficiency with Anti-Interferon-γ Autoantibodies

Purpose Anti-interferon (IFN)-γ autoantibodies (anti-IFN-γ Abs) is an emerging adult-onset immunodeficiency syndrome. Immune dysfunction in this distinct disorder remains to be clarified. Methods We prospectively collected blood samples of 20 patients with anti-IFN-γ Abs and 40 healthy normal subjects. The percentages of lymphocyte subpopulations, most relevant to T, B, and NK cells, and the percentages of stimulated lymphocytes with cytokine production were assessed using eight-color flow cytometry. The results were adjusted to age and absolute lymphocyte counts. Results Most (85%) patients presented nontuberculous mycobacterial infection. Skin lesions were predominantly manifested by neutrophilic dermatoses. The involved lymph nodes had granulomatous inflammation, except 22.2% showing atypical lymphoid hyperplasia without granuloma formation. The percentages of CD4 + T cells and non-activated subpopulations (recent thymic emigrants and naïve subtypes) decreased significantly with increased expression of activation markers and polarization to differentiated cells. The percentage of NK cells increased, but that of two major NK subpopulations, CD56bright and CD56 + CD16 + subsets decreased. Furthermore, the expression of NKp30 and NKp46 decreased with increased CD57 expression. Intracellular cytokine production of the lymphocyte subpopulations were significantly low in the patients compared with the control group. Conclusion We conclude that the immune system in patients with anti–IFN-γ Abs could be exhausted, contributing to the distinct clinicopathologic features.