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Diagnostic Microbiology and Infectious... Mar 2023The study aims to investigate the potential of convolutional neural network (CNN) based on spot image of T-SPOT assay for distinguishing active tuberculosis (ATB) from...
OBJECTIVES
The study aims to investigate the potential of convolutional neural network (CNN) based on spot image of T-SPOT assay for distinguishing active tuberculosis (ATB) from latent tuberculosis infection (LTBI).
METHODS
CNN was applied to recognize and classify T-SPOT spot image. Logistic regression was used to establish prediction model based on CNN.
RESULTS
Areas under the receiver operating characteristic curve (AUCs) of early secreted antigenic target 6 (ESAT-6) CNN, culture filtrate protein 10 (CFP-10) CNN, and phytohemagglutinin (PHA) CNN were more than 0.7 in differentiating ATB from LTBI, while the performance of these indicators was significantly better than that of spot number. Furthermore, prediction model based on the combination of CNNs yielded an AUC of 0.898. The model presented a sensitivity of 85.76% and a specificity of 90.23%.
CONCLUSIONS
The current study identified CNN based on T-SPOT spot image with the potential to serve as a tool for TB diagnostics.
Topics: Humans; Latent Tuberculosis; Mycobacterium tuberculosis; Antigens, Bacterial; Sensitivity and Specificity; Tuberculosis
PubMed: 36702072
DOI: 10.1016/j.diagmicrobio.2023.115892 -
International Journal of Molecular... Jan 2023Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side...
Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side effects, especially an increased risk of infections, have been reported in recent years. The underlying mechanism has still not yet been fully uncovered. Hence, in this study, we analyzed the T cell response after treatment with pantoprazole in vitro. Pantoprazole preincubation reduced the production and secretion of interferon (IFN)-γ and interleukin (IL)-2 after the T cells were activated with phytohemagglutinin (PHA)-L or toxic shock syndrome toxin-1 (TSST-1). Moreover, a lower zinc concentration in the cytoplasm and a higher concentration in the lysosomes were observed in the pantoprazole-treated group compared to the untreated group. We also tested the expression of the zinc transporter Zrt- and Irt-like protein (Zip)8, which is located in the lysosomal membrane and plays a key role in regulating intracellular zinc distribution after T cell activation. Pantoprazole reduced the expression of Zip8. Furthermore, we measured the expression of cAMP-responsive element modulator (CREM) α, which directly suppresses the expression of IL-2, and the expression of the phosphorylated cAMP response element-binding protein (pCREB), which can promote the expression of IFN-γ. The expression of CREMα was dramatically increased, and different isoforms appeared, whereas the expression of pCREB was downregulated after the T cells were treated with pantoprazole. In conclusion, pantoprazole downregulates IFN-γ and IL-2 expression by regulating the expression of Zip8 and pCREB or CREMα, respectively.
Topics: Proton Pump Inhibitors; Pantoprazole; Interleukin-2; 2-Pyridinylmethylsulfinylbenzimidazoles; Zinc; T-Lymphocytes; Acids
PubMed: 36674704
DOI: 10.3390/ijms24021191 -
Cell Death Discovery Jan 2023Intrahepatic stem/progenitor cells and cytotoxic CD8 T cells (CD8 T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer....
Intrahepatic stem/progenitor cells and cytotoxic CD8 T cells (CD8 T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8 T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8 T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA) cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8 T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8 T cells (pn-msCD8 T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8 T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8 T cells and healthy human peripheral blood CD8 T cells (hp-CD8 T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.
PubMed: 36658107
DOI: 10.1038/s41420-023-01313-4 -
Infection Aug 2023Human tuberculosis is characterized by immunopathology that affects T-cell phenotype and functions. Previous studies found impaired T-cell response to phytohemagglutinin...
Impaired T-cell response to phytohemagglutinin (PHA) in tuberculosis patients is associated with high IL-6 plasma levels and normalizes early during anti-mycobacterial treatment.
PURPOSE
Human tuberculosis is characterized by immunopathology that affects T-cell phenotype and functions. Previous studies found impaired T-cell response to phytohemagglutinin (PHA) in patients with acute tuberculosis. However, the influence of disease severity, affected T-cell subsets, and underlying mechanisms remain elusive.
METHODS
Here we investigated PHA-induced and antigen-specific T-cell effector cytokines in tuberculosis patients (n = 55) as well as in healthy asymptomatic contacts (n = 32) from Ghana. Effects of Mycobacterium (M.) tuberculosis sputum burden and treatment response were analyzed and compared during follow-up. Finally, cytokine characteristics of the aberrant plasma milieu in tuberculosis were analyzed as a potential cause for impaired PHA response.
RESULTS
PHA-induced IFN-γ expression was significantly lower in sputum-positive tuberculosis patients as compared to both, contacts and paucibacillary cases, and efficiently discriminated the study groups. T-cell responses to PHA increased significantly early during treatment and this was more pronounced in tuberculosis patients with rapid treatment response. Analysis of alternative cytokines revealed distinct patterns and IL-22, as well as IL-10, showed comparable expression to IFN-γ in response to PHA. Finally, we found that high IL-6 plasma levels were strongly associated with impaired IFN-γ and IL-22 response to PHA.
CONCLUSION
We conclude that impaired T-cell response to PHA stimulation in acute tuberculosis patients (i) was potentially caused by the aberrant plasma milieu, (ii) affected differentially polarized T-cell subsets, (iii) normalized early during treatment. This study shed light on the mechanisms of impaired T-cell functions in tuberculosis and yielded promising biomarker candidates for diagnosis and monitoring of treatment response.
Topics: Humans; Cytokines; Interleukin-6; Mycobacterium tuberculosis; Phytohemagglutinins; T-Lymphocytes; Tuberculosis; Interferon-gamma; Interleukin-22
PubMed: 36650358
DOI: 10.1007/s15010-023-01977-1 -
Diagnostics (Basel, Switzerland) Dec 2022The present study aimed to clinically evaluate the effect of T-cell dysfunction in hemodialysis (HD) patients with latent tuberculosis (TB) infection (LTBI) who were...
The present study aimed to clinically evaluate the effect of T-cell dysfunction in hemodialysis (HD) patients with latent tuberculosis (TB) infection (LTBI) who were false-negatives in the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Whole blood samples from a total of 20 active TB patients, 83 HD patients, and 52 healthy individuals were collected, and the QFT-GIT test was used for measuring (MTB)-specific interferon gamma (IFN-γ) level. The positive rate of the IFN-γ release assays (IGRAs) in HD patients was lower than the negative rate. The mean value of MTB-specific IFN-γ level, which determines the positive rate of the IGRA test, was highest in active TB, followed by HD patients and healthy individuals. Among HD patients, phytohemagglutinin A (PHA)-stimulated IFN-γ levels of approximately 40% were 10.00 IU/mL or less. However, there was no low level of PHA-stimulated IFN-γ in the healthy individuals. This reveals that T-cell function in HD patients was reduced compared to healthy individuals, which leads to the possibility that QFT-GIT results in HD patients are false-negative. The clinical manifestations of TB in patients on HD are quite non-specific, making timely diagnosis difficult and delaying the initiation of curative treatment, delay being a major determinant of outcome.
PubMed: 36611380
DOI: 10.3390/diagnostics13010088 -
Parasites & Vectors Jan 2023The saliva of sand flies, vectors of Leishmania parasites, contains several components that exert pharmacological activity facilitating the acquisition of blood by the...
BACKGROUND
The saliva of sand flies, vectors of Leishmania parasites, contains several components that exert pharmacological activity facilitating the acquisition of blood by the insect and contributing to the establishment of infection. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and validated its usefulness as a predictive biomarker of disease. PpSP32, whose functions are little known to date, is an intriguing protein due to its involvement in the etiopathogenesis of pemphigus, an auto-immune disease. Herein, we aimed to better decipher its role through the screening of several immunomodulatory activity either on lymphocytes or on monocytes/macrophages.
METHODS
Peripheral mononuclear cells from healthy volunteers were stimulated with anti-CD3/anti-CD28 antibodies, phytohemagglutinin, phorbol 12-myristate 13-acetate/ionomycin, or lipopolysaccharide in the presence of increasing doses of PpSP32. Cell proliferation was measured after the addition of tritiated thymidine. Monocyte activation was tested by analyzing the expression of CD86 and HLA-DR molecules by flow cytometry. Cytokine production was analyzed in culture supernatants by ELISA. THP-1-derived macrophages were stimulated with LPS in the presence of increasing doses of PpSP32, and cytokine production was analyzed in culture supernatants by ELISA and multiplex technique. The effect of PpSP32 on NF-kB signaling was tested by Western blot. The anti-inflammatory activity of PpSP32 was assessed in vivo in an experimental inflammatory model of carrageenan-induced paw edema in rats.
RESULTS
Our data showed that PpSP32 down-modulated the expression of activation markers in LPS-stimulated monocytes and THP1-derived macrophages. This protein negatively modulated the secretion of Th1 and Th2 cytokines by human lymphocytes as well as pro-inflammatory cytokines by monocytes, and THP1-derived macrophages. PpSP32 treatment led to a dose-dependent reduction of IκB phosphorylation. When PpSP32 was injected into the paw of carrageenan-injected rats, edema was significantly reduced.
CONCLUSIONS
Our data indicates that PpSP32 induces a potent immunomodulatory effect on monocytes and THP-1-derived macrophages. This inhibition could be mediated, among others, by the modulation of the NF-kB signaling pathway. The anti-inflammatory activity of PpSP32 was confirmed in vivo in the carrageenan-induced paw edema model in rats.
Topics: Humans; Rats; Animals; Phlebotomus; Monocytes; NF-kappa B; Carrageenan; Lipopolysaccharides; Lymphocytes; Macrophages; Cytokines; Salivary Proteins and Peptides
PubMed: 36593519
DOI: 10.1186/s13071-022-05627-7 -
Frontiers in Immunology 2022spp. has been associated with promising immunomodulatory results in preclinical trials. The aim of this study was to investigate the pharmacodynamic (PD) effects of... (Randomized Controlled Trial)
Randomized Controlled Trial
Impact of oral administration of single strain spp. on immune responses to keyhole limpet hemocyanin immunization and gut microbiota: A randomized placebo-controlled trial in healthy volunteers.
INTRODUCTION
spp. has been associated with promising immunomodulatory results in preclinical trials. The aim of this study was to investigate the pharmacodynamic (PD) effects of three monoclonal microbial formulations of spp. (EDP1066) on the immune response to keyhole limpet hemocyanin (KLH). Potential effects on the gut microbiota were also investigated.
METHODS
The trial was registered on Netherlands Trial Register (trial ID NL7519, https://trialsearch.who.int). Eighty-one healthy subjects (median 28, range 18-59 years) were randomized to 28 days of enteric-coated capsules at five doses (n = 13) (1.5 * 10 total cells daily), freeze-dried powder at one dose (n = 12) (3.0 * 10 total cells daily) or five doses (n = 12), minitablets at one dose (n = 12) or five doses (n = 12), or placebo (n = 20) prior to KLH immunization. Antibody responses and circulating regulatory T cells (Tregs) were measured after KLH immunization, and skin responses were evaluated after a KLH rechallenge by laser speckle contrast imaging and multispectral imaging. lymphocyte (phytohemagglutinin) and monocyte (lipopolysaccharide (LPS)) cytokine release assays were explored in the minitablet-treated groups only. The prevalence of spp. in the gastrointestinal tract and the impact on the fecal microbiota were assessed by qPCR and 16S rRNA sequencing, respectively.
RESULTS
Repeated-measures analysis of covariances revealed no significant treatment effects on the antibody responses to KLH, number of Tregs, or KLH skin rechallenge outcomes. LPS-driven cytokine responses in whole blood were lower in the low dose minitablet group compared to placebo: tumor necrosis factor (estimated difference (ED) from placebo: -44.2%, 95% confidence interval (CI) -65.3% to -10.3%), interleukin (IL)-1β (ED -41.4%, 95% CI -63.5% to -5.8%), and IL-6 (ED -39.2%, 95% CI -56.8% to -14.5%). The fecal presence of spp. increased during treatment by all EDP1066 formulations and normalized 5 days after the last dose. Microbiome α-diversity did not change by the treatments compared to placebo.
DISCUSSION
The EDP1066 formulations did not affect the immune response to KLH immunization in healthy individuals. However, exposure to spp. in minitablet formulation impacted whole blood LPS cytokine response. The clinical impact of these effects awaits further investigations.
NETHERLANDS TRIAL REGISTER
trialsearch.who.int, trial ID NL7519.
Topics: Humans; Administration, Oral; Cytokines; Gastrointestinal Microbiome; Healthy Volunteers; Immunity; Immunization; Lactococcus lactis; Lipopolysaccharides; RNA, Ribosomal, 16S; Adolescent; Young Adult; Adult; Middle Aged
PubMed: 36582231
DOI: 10.3389/fimmu.2022.1009304 -
Frontiers in Immunology 2022The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The...
The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.
Topics: Leukocytes, Mononuclear; Cells, Cultured; Mesenchymal Stem Cells; Immunomodulation; Stromal Cells; Mitogens
PubMed: 36578497
DOI: 10.3389/fimmu.2022.1085312 -
Iranian Journal of Microbiology Oct 2022Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. Peripheral blood monocytes cells (PBMCs) may have altered function to some...
BACKGROUND AND OBJECTIVES
Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. Peripheral blood monocytes cells (PBMCs) may have altered function to some extent in women with endometriosis. is a probiotic bacterium within the human body with the ability of alleviating many inflammatory diseases. Here, we examined the effect of on PBMCs of endometriosis patients.
MATERIALS AND METHODS
In this study, peripheral blood samples were obtained from endometriosis patients (n=11) and non-endometriosis individuals (n=11). After isolation of peripheral blood mononuclear cells with Ficoll, cells were cultured in the presence and absence of phytohemagglutinin. Also, these cells were co-cultured with 1×10 CFU/ml of IL-6 and IL-1 cytokines were measured by ELISA method and the two groups were evaluated and compared.
RESULTS
The results showed that in endometriosis patients, the production of pro-inflammatory cytokines, including IL-1 and IL-6, by PBMC was increased compared to non-endometriosis subjects, and stimuli such as PHA intensified this elevation. Also, increased the levels of pro-inflammatory cytokines including IL-1 and IL-6. However, the production of these cytokines decreased due to the modulatory properties of bacterial cells after 48 h.
CONCLUSION
According to the results of the current study, IL-1 and IL-6 production was significantly increased in PMBCs of endometriosis patients compared to that of the healthy controls. Also, was considered as an antigenic compound and induced IL-1 and IL-6 production. According to these results, probiotics can be further used for the treatment of endometriosis patients and more investigations are needed to confirm these results.
PubMed: 36531824
DOI: 10.18502/ijm.v14i5.10965 -
Advanced Biomedical Research 2022Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically...
BACKGROUND
Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically modified T cells to generate powerful clinical responses has been highlighted by recent clinical success. Techniques which are used to expand large numbers of T cells from different sources are critical in adoptive cell therapy. In this study, we evaluated the expansion, proliferation, activation of T lymphocytes, in the presence of various concentrations of interleukin-2, phytohemagglutinin (PHA), and insulin.
MATERIALS AND METHODS
The effect of different supplemented culture media on T cell expansion was evaluated using MTT assay. The expression level of the Ki-67 proliferation marker was evaluated by real-time polymerase chain reaction. In addition, flow cytometry analysis was performed to access T cell subpopulations.
RESULTS
Our results showed that supplemented culture media with an optimized concentration of PHA and interleukin-2 increased total fold expansion of T cells up to 500-fold with approximately 90% cell viability over 7 days. The quantitative assessment of Ki-67 in expanded T cells showed a significant elevation of this proliferation marker. Flow cytometry was also used to assess the proportion of CD4+ and CD8+ cells, and the main expanded population was CD3+ CD8+ cells.
CONCLUSIONS
Based on these findings, we introduced a low-cost and rapid method to support the efficient expansion of T cells for adoptive cell therapy and other experiments.
PubMed: 36518860
DOI: 10.4103/abr.abr_349_21