Prenatal blood metals, per- and polyfluoroalkyl substances and antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion.

BACKGROUND: Evidence suggests that prenatal per- and polyfluoroalkyl substances (PFAS) and metals, two classes of chemicals found ubiquitously in human populations, influence immune system development and response. OBJECTIVE: We evaluated whether first trimester blood PFAS and metals were associated with antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion. METHODS: We measured six PFAS, as well as six nonessential and four essential metals, in first trimester blood from participants in the longitudinal pre-birth Project Viva cohort, recruited between 1999 and 2000 in eastern Massachusetts. We measured antigen- or mitogen-stimulated cord blood mononuclear cell proliferation responses (n = 269-314) and cytokine secretion (n = 217-302). We used covariate-adjusted least absolute shrinkage and selection operator (LASSO) for variable selection and multivariable regression to estimate associations with the immune markers. RESULTS: Each ng/mL of MeFOSAA was associated with a 3.6% (1.4, 5.8) higher lymphocyte proliferation response after stimulation with egg antigen, as well as 0.8 (0.7, 1.0) reduced odds of having IFN-γ detected in response to dust mite. Each ng/g increment of cesium was associated with 27.8% (-45.1, -4.9) lower IL-10 levels in response to dust mite. Each ng/g increment of mercury was associated with 12.0% (1.3, 23.8) higher IL-13 levels in response to mitogen PHA. Each ng/g increment of selenium and zinc was associated with 0.2% (0.01, 0.4) and 0.01% (0.002, 0.02) higher TNF-α in response to mitogen PHA, respectively. CONCLUSIONS: Prenatal metals and PFAS influence cord blood lymphocyte proliferation and cytokine secretion in ways that may increase risk for atopic disease in childhood.

Strategies to determine positive anti-SARS-CoV-2 memory T lymphocyte response during the evolution of an epidemic.

During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit "negative" patients.

Annual rhythm in immune functions of blood leucocytes in an ophidian, Natrix piscator.

Annual variations in animal's physiological functions are an essential strategy to deal with seasonal challenges which also vary according to the time of year. Information regarding annual adaptations in the immune-competence to cope with seasonal stressors in reptiles is scarce. The present research plan was designed to analyze the presence of circannual immune rhythms in defense responses of the leucocytes in an ophidian, Natrix piscator. Peripheral blood leucocytes were obtained, counted, and superoxide anion production, neutrophil phagocytosis, and nitrite release were tested to assess the innate immune functions. Peripheral blood lymphocytes were separated by centrifugation (utilizing density gradient) and the cell proliferation was measured. The Cosinor rhythmometry disclosed the presence of significant annual rhythms in the number of leucocytes, superoxide anion production, nitric oxide production, and proliferation of stimulated lymphocytes. The authors found that respiratory burst activity and proliferative responses of lymphocytes were crucial immune responses that showed the annual rhythm. It was summarized that the immune function of the N. piscator is a labile attribute that makes the animal competent to cope with the seasonal stressor by adjustment in the potency of response.

Lymphocyte Function at Baseline Could Be a New Predictor of Tumor Burden following Six Cycles of Radium-223 Therapy in Patients with Metastasized, Castration-Resistant Prostate Cancer.

Previous data indicate that one cycle of treatment with radium-223 (223Ra) did not significantly impair lymphocyte function in patients with metastasized, castration-resistant prostate cancer. The aim of the current study was to assess in 21 patients whether six cycles of this therapy had an effect on lymphocyte proliferation and interferon-γ and interleukin (IL)-10 ELISpot results. Lymphocyte proliferation after stimulation with microbial antigens and the production of interferon-γ continuously decreased after six cycles of radionuclide therapy, reaching statistical significance (p < 0.05) at months 1, 2, 4, and/or 6 after therapy. One month after the last cycle of therapy, 67% of patients showed a decrease in tumor burden. The tumor burden correlated negatively with IL-10 secretion at baseline, e.g., after stimulation with tetanus antigen (p < 0.0001, r = -0.82). As determined by receiver operating characteristic (ROC) curve analysis, tetanus-specific IL-10 spots at baseline had the highest predictive value (p = 0.005) for tumor burden at month 6, with an area under the curve (AUC) of 0.90 (sensitivity 100%, specificity 78%). In conclusion, we observed an additive effect of treatment with 223Ra on immune function and found that IL-10 secretion at baseline predicted tumor burden at month 6 after treatment.

Comparative evaluation of two different xenogenic acellular matrices on full-thickness skin wound healing.

OBJECTIVE: The purpose of the study was to compare the healing potential of bubaline small intestinal matrix (bSIM) and fish swim bladder matrix (FSBM) on full-thickness skin wounds in rabbits. METHOD: Four full-thickness skin wounds (each 20×20mm) were created on the dorsum of 18 rabbits that were divided into three groups based on treatment: untreated sham control (I), implanted with double layers of bSIM (II) and implanted with double layers of FSBM (III). Macroscopic, immunologic and histologic observations were made to evaluate wound healing. RESULTS: Gross healing progression in the bSIM and FSBM groups showed significantly (p<0.05) less wound contraction compared with the sham group. The IgG concentration in rabbit sera was significantly (p<0.05) lower in the FSBM group compared with the bSIM group by enzyme-linked immunosorbent assay. The stimulation index of peripheral blood lymphocytes was significantly (p<0.05) lower in the FSBM group compared with the bSIM group by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Implantation of FSBM resulted in improved re-epithelialisation, neovascularisation and fibroplasia. CONCLUSION: The FSBM is a more effective dermal substitute when compared with the bSIM for full-thickness skin wound repair in rabbit.

A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1).

BACKGROUND: Proinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific pro-inflammatory milieu. METHODS: Peripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation. RESULTS: IDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation. CONCLUSION: IDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells.

Hepatic P53 upregulation and the genotoxic potential of acesulfame-K treatment in rats with a special emphasis on in vitro lymphocyte and macrophage activity testing.

Acesulfame-k (Ace-k) is a widely used artificial sweetener in various products, and long-term cumulative and multisource exposure is possible despite inadequate toxicological data confirming its safety. Ninety male rats were divided into two main groups according to their body weight into immature and mature rats. Each group was subdivided into 3 subgroups: control untreated, 30 and 90 mg/kg b. w of Ace-k via gastric intubation. The treatment was performed daily 5 days per week for 12 weeks. At the end of the experimental period, blood samples were collected for in vitro testing of lymphocyte proliferation rate, comet assay, and macrophage activity about nitric oxide (NO) production. In addition, the collection of liver specimens was performed for P53 gene expression and histopathological evaluation. The results revealed that Ace-k induced modulation in lymphocyte proliferation rate and affected the production of NO by macrophages while increasing in tail moment in a dose-dependent manner that varied among different age groups. The upregulation of P53 in the liver was correlated with increased polyploidization and necro apoptotic reaction and various histopathological hepatic alterations. The present data revealed that chronic treatment of rats with Ace-k affects lymphocyte proliferation and macrophage activity in a dose-dependent manner. In addition, the genotoxic and hepatotoxic potential of Ace-k were confirmed.

Modified cell trace violet proliferation assay preserves lymphocyte viability and allows spectral flow cytometry analysis.

In this study we describe three different methods for labeling T lymphocytes with cell trace violet (CTV), in order to track cell division in mouse and human cells, in both the in vitro and in vivo setting. We identified a modified method of CTV labeling that can be applied directly to either conventional or spectral flow cytometry, that maintained lymphocyte viability and function, yet minimized dye spill-over into other fluorochrome channels. Our optimized method for CTV labeling allowed us to identify up to eight cell divisions and the replication index for in vitro-stimulated mouse and human lymphocytes, and the co-expression of T-cell subset markers. Furthermore, the homeostatic trafficking, expansion and division of CTV-labeled congenic donor T cells could be detected using spectral cytometry, in an adoptive T-cell transfer mouse model. Our optimized CTV method can be applied to both in vitro and in vivo settings to examine the behavior and phenotype of activated T cells.

Adenosine 2A receptor antagonists promote lymphocyte proliferation in sepsis by inhibiting Treg expression of PD-L1 in spleen.

The spleen is essential for lymphocyte proliferation, which is associated to sepsis prognosis. Adenosine 2A receptor (A2AR) blocking promotes lymphocyte proliferation in sepsis, however the mechanism is uncertain. Our sepsis cecum ligation perforation model showed that blocking A2AR increased survival and CD4+ cell numbers in a spleen-dependent mechanism. The sequencing of the transcriptome of the spleen indicated alterations in the expression of genes involved in the control of lymphocyte proliferation by inhibiting A2AR, including a reduction in the expression of PD-L1. Flow cytometry analysis of PD-L1 expression intensity in splenic cell subpopulations revealed that the Treg cell subpopulation was the strongest PD-L1-expressing cell population, and Treg PD-L1 expression decreased after blocking A2AR. In vitro activation of A2AR was able to upregulate PD-L1 expression of Treg and boost Treg capacity to limit lymphocyte proliferation, while blockage of PD-L1 partly reduced A2AR-activated Treg's ability to inhibit lymphocyte proliferation. In addition, blocking CREB phosphorylation significantly inhibited A2AR-induced PD-L1 expression. According to the findings of our research, inhibiting A2AR improves the prognosis of sepsis by lowering the level of PD-L1 expression by Treg in the spleen and reducing the inhibition of lymphocyte proliferation.

Alterations in Immune Responses Are Associated with Dysfunctional Intracellular Signaling in Peripheral Blood Mononuclear Cells of Men and Women with Mild Cognitive Impairment and Alzheimer's disease.

Deficits in the neuroendocrine-immune network in the periphery associated with the onset and progression of mild cognitive impairment (MCI) and Alzheimer's disease (AD) have not been extensively studied. The present study correlatively examines the association between cell-mediated immune responses, stress hormones, amyloid precursor protein (APP) expression, peripheral blood mononuclear cells (PBMC), and intracellular signaling molecules in the pathophysiology of MCI and AD compared to adults. Serum APP, lymphocyte proliferation, total cholinesterase (TChE), butyrylcholinesterase (BChE) activities, cytokines (IL-2, IFN-γ, IL-6, and TNF-α), and intracellular signaling molecules (p-ERK, p-CREB, and p-Akt) were measured in the PBMCs of adult, old, MCI, and AD men and women initially and after 3 years in the same population. An age- and disease-associated decline in mini-mental state examination (MMSE) scores and lymphocyte proliferation of MCI and AD men and women were observed. An age- and disease-related increase in serum APP, cortisol levels, and TChE activity were observed in men and women. Enhanced production of Th1 cytokine, IL-2, pro-inflammatory cytokines, and suppressed intracellular transcription factors may promote the inflammatory environment in MCI and AD patients. The expression of CREB and Akt was lower in MCI and AD men, while the expression of p-ERK was higher, and p-CREB was lower in MCI and AD women after 3 years. These results suggest that changes in specific intracellular signaling pathways may influence alterations in cell-mediated immunity to promote disease progression in MCI and AD patients.

Comparing [3H] thymidine LPT and CFSE assay to assess lymphocyte proliferation in beryllium-exposed sarcoidosis patients.

The detection of antigen specific lymphocyte responses plays a vital role in the diagnosis of various diseases. Beryllium-specific [3H] thymidine lymphocyte proliferation test (LPT) is regarded as a gold standard in identifying chronic beryllium disease (CBD) cases. Alternatively, flow cytometric based carboxyfluorescein succinimidyl ester (CFSE) assay, has several benefits as opposed to LPT, since it further permits both phenotypical characterization and functional analysis of proliferating lymphocyte subsets. The suitability of both LPT and CFSE assay to therefore detect beryllium sensitivity in a group of Be-exposed sarcoidosis patients with suspected beryllium exposure, was evaluated in this study. The clinical relevance of the test responses, expressed as stimulation indices (SI), were additionally compared on a group and individual level. Agreement in clinical interpretation of the test responses between both methods was observed in 4 out of 5 recruited patients, when considering total lymphocyte population i.e., CD3+ and CD19+-cells combined, on day 7 and with CFSE-SI >1.5, when compared with LPT-SI >2.5. Variability in responses to beryllium was additionally evaluated in Be-exposed sarcoidosis patients and compared with healthy controls. To conclude, both LPT and CFSE assay are suitable assays to detect Be sensitivity in Be-exposed sarcoidosis patients. At the same time, flow cytometric based CFSE assay has the edge over LPT in identifying the relevant proliferating lymphocyte populations. As such, when comparing two or more methods, factors that contribute to assay variability such as timepoints, lymphocyte subsets and number of replicates should always be accounted for.

In Patients Treated by Selective Internal Radiotherapy, Cellular In Vitro Immune Function Is Predictive of Survival.

In patients with liver malignancies, the cellular immune function was impaired in vitro after selective internal radiotherapy (SIRT). Because immunosuppression varied substantially, in the current study, we investigated in 25 SIRT patients followed up for ten years whether the lymphocyte function was correlated with survival. Peripheral blood mononuclear cells were stimulated with four microbial antigens (tuberculin, tetanus toxoid, Candida albicans and CMV) before therapy and at four time points thereafter, and lymphocyte proliferation was determined by H3-thymidine uptake. The median sum of the responses to these four antigens decreased from 39,464 counts per minute (CPM) increment (range 1080-204,512) before therapy to a minimum of 700 CPM increment on day 7 after therapy (0-93,187, p < 0.0001). At all five time points, the median survival in patients with weaker responses was 2- to 3.5-fold shorter (p < 0.05). On day 7, the median survival in patients with responses below and above the cutoff of a 2 CPM increment was 185 and 523 days, respectively (χ2 = 9.4, p = 0.002). In conclusion, lymphocyte function could be a new predictor of treatment outcome after SIRT.

Seasonal Immune Rhythms of head kidney and spleen cells in the freshwater Teleost, Channa punctatus.

Annual rhythms in immune function are the reflection of a crucial physiological strategy to deal with environmental stressors. The fish are pivotal animal models to study the annual rhythm and to understand the evolution of the vertebrate biological system. The current research was planned to assess the annual changes in the innate immune functions of immune cells in a teleost, Channa punctatus. Head kidney and splenic macrophage phagocytosis, superoxide generation, and nitrite release were evaluated to assess innate immunity. Cell-mediated immunity was measured through head kidney and splenic lymphocyte proliferation in presence of mitogens. The superoxide anion generation by the cells of head kidney and spleen was maximum in October. A bimodal pattern in nitrite production was observed with the first peak in November and the second in March. Cosinor analysis revealed a statistically significant annual rhythm in nitrite production. Similarly, phagocytosis and lymphocyte proliferation also showed statistically significant annual rhythms. It was concluded that animals maintain an optimum immune response in seasonally changing environments. Elevated immunity during certain times of the year might assist animals deal with seasonal environmental stressors. Further research may be focused upon measuring survival rate and reproductive success after season induced elevated immunity.

Missing link between tissue specific expressing pattern of ERß and the clinical manifestations in LGBLEL.

Purpose: Lacrimal gland benign lymphoepithelial lesion (LGBLEL) is an IgG4-related disease of unknown etiology with a risk for malignant transformation. Estrogen is considered to be related to LGBLEL onset. Methods: Seventy-eight LGBLEL and 13 control clinical samples were collected and studied to determine the relationship between estrogen and its receptors and LGBLEL development. Results: The serological analysis revealed no significant differences in the levels of three estrogens be-tween the LGBLEL and control groups. However, immunohistochemical analyses indicated that the expression levels of ERß and its downstream receptor RERG were relatively lower in LGBLEL samples than in control samples, with higher expression in the lacrimal gland and lower expression in the lymphocyte infiltration region. However, low expression of ERα was detected. The transcriptome sequence analysis revealed upregulated genes associated with LGBLEL enriched in lymphocyte proliferation and activation function; downregulated genes were enriched in epithelial and vascular proliferation functions. The key genes and gene networks were further analyzed. Interactions between B cells and epithelial cells were analyzed due to the identified involvement of leukocyte subsets and epithelial cells. B cell proliferation was found to potentially contribute to lacrimal gland apoptosis. Conclusion: Therefore, the tissue-heterogeneous expression pattern of ERß is potentially related to the clinical manifestations and progression of LGBLEL, although further investigations are required to confirm this finding.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Biological Importance and Therapeutic Potential of Calycopterin From Dracocephalum kotschyi: An Overview of Current Scientific Research Work.

BACKGROUND: Dracocephalum kotschyi Boiss is an important medicinal plant of Asia, Europe and Iran. The therapeutic potential of Dracocephalum kotschyi has been mentioned in traditional as well as in modern medicine, mainly due to its therapeutic effectiveness against different types of human disorders and associated secondary complications. Calycopterin is a flavonoid class phytochemical commonly called 5,4-dihydroxy-3,6,7,8- tetramethoxyflavone, which has been isolated from Dracocephalum kotschyi. It is widely used for the treatment of human disorders and associated secondary complications. METHODS: Medicinal importance and therapeutic potential of calycopterin was investigated in the present work through scientific data analysis of different research works. Scientific data on calycopterin published in different research articles were collected from PubMed, Google, Google Scholar, Science Direct, SpringerLink, and Scopus and analyzed in the present work. Further, scientific data was collected from books and book chapters. Analytical data on calycopterin was also collected and analyzed in the present work. RESULTS: Scientific data analysis of different research work revealed the biological importance and therapeutic potential of calycopterin. Calycopterin showed therapeutic effectiveness against different types of cancerous disorders, including human prostate cancer, breast cancer, cervical cancer, lymphocyte proliferation and hepatoblastoma cancer. Moreover, the biological effectiveness of calycopterin on neuroprotection and Pseudomonas aeruginosa has also been discussed in the present work with their pharmacokinetic parameters. Nevertheless, more scientific investigations must be conducted to explore the healthbeneficial aspects of calycopterin in medicine. CONCLUSION: Present work signified the biological importance and therapeutic potential of calycopterin for the treatment of human disorders and complications, including different types of cancerous disorders.

Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently.

We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the "commensal" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4+ and CD8+ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.

Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation.

Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.

Nutritional deficiencies that may predispose to long COVID.

Multiple nutritional deficiencies (MND) confound studies designed to assess the role of a single nutrient in contributing to the initiation and progression of disease states. Despite the perception of many healthcare practitioners, up to 25% of Americans are deficient in five-or-more essential nutrients. Stress associated with the COVID-19 pandemic further increases the prevalence of deficiency states. Viral infections compete for crucial nutrients with immune cells. Viral replication and proliferation of immunocompetent cells critical to the host response require these essential nutrients, including zinc. Clinical studies have linked levels of more than 22 different dietary components to the likelihood of COVID-19 infection and the severity of the disease. People at higher risk of infection due to MND are also more likely to have long-term sequelae, known as Long COVID.

Aryltetralin lignans from Hyptis brachiata inhibiting T lymphocyte proliferation.

Increased activation and proliferation of T lymphocytes plays an essential role in the development of chronic inflammation and autoimmune diseases. Currently used immunosuppressive drugs often do not provide long-lasting relief of symptoms and show a gradual loss of efficacy over time, and are accompanied by various side effects. Therefore, novel immunosuppressive lead substances are needed. For this purpose, an in-house library consisting of 600 extracts of plants from Panama was screened for inhibition of human T lymphocyte proliferation. As one of the hits, an ethyl acetate extract from the aerial parts of Hyptis brachiata (Lamiaceae) exhibited strong inhibitory effects. Subsequent investigation resulted in the isolation of seven aryltetralin lignans, five arylnaphthalene lignans, two flavonoids, three triterpenes, and cinnamyl cinnamate. Aryltetralin lignans inhibited T lymphocyte proliferation in a concentration-dependent manner without induction of apoptosis. No relevant inhibition was observed for the arylnaphthalene lignans, flavonoids, and triterpenes. Additional cell cycle arrest investigations revealed that isolated aryltetralin lignans potently inhibited cell division in G2/M phase similarly to podophyllotoxin. Multifluorescence panel analyses of the extract also showed weak suppressive effects on the production of IL-2 and TNF-α. Therefore, preparations made out of H. brachiata could be further explored as an interesting herbal alternative in the treatment of autoimmune diseases.