scholarly journals Αλληλεπίδραση μεταβιβαστών φλεγμονής και κυτταρικών πληθυσμών του αναπνευστικού επιθηλίου στην παθογένεια της ιδιοπαθούς πνευμονικής ίνωσης

2018 ◽  
Author(s):  
Ευάγγελος Μπούρος
Keyword(s):  
Real Time ◽  
Tissue Factor ◽  
Real Time Pcr ◽  
Dnase I ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Η Ιδιοπαθής Πνευμονική Ίνωση (IPF/ΙΠΙ) χαρακτηρίζεται από βλάβη του κυψελιδικού επιθηλίου, ρήξη της βασικής μεμβράνης και απορρύθμιση του επουλωτικού μηχανισμού με κυρίαρχο ρόλο να έχουν οι μυοϊνοβλάστες, κύτταρα αποκλειστικά υπεύθυνα για την υπερβολική παραγωγή και εναπόθεση κολλαγόνου στο πνευμονικό παρέγχυμα, που έχει ως αποτέλεσμα την αναδιαμόρφωσή του (remodeling). Αν και τα τελευταία χρόνια υπάρχει εντατική έρευνα σε ότι αφορά την κατανόηση των μηχανισμών που οδηγούν σε αυτή, παραμένουν πολλά ανεξερεύνητα μονοπάτια της παθογένεια της.Σκοπός της παρούσας μελέτης είναι η διερεύνηση του ρόλου των υποεπιθηλιακών μυοϊνοβλαστών του πνεύμονα (ΥΜΠ) και των διαλυτών μεσολαβητικών παραγόντων που επιδρούν στην ενεργοποίησή τους και την ανάπτυξη της ίνωσης σε ασθενείς με ιδιοπαθή πνευμονική ίνωση.Για το σκοπό της μελέτης ελήφθη βρογχοκυψελιδικό έκπλυμα (ΒΚΕ) χρησιμοποιώντας το εύκαμπτο βρογχοσκόπιο με την καθιερωμένη τεχνική από ασθενείς με γνωστή ΙΠΙ βάσει των ισχυόντων διαγνωστικών κριτηρίων (ATS/ERS/JRS/ALAT 2011). Για ομάδα ελέγχου χρησιμοποιήθηκε ΒΚΕ από ασθενείς με ιδιοπαθή μη ειδική διάμεση πνευμονία (iNSIP), πάθηση η οποία ανήκει μεν στις ιδιοπαθείς διάμεσες πνευμονίες, όμως είναι διακριτή ως προς την παθογένεια, την πορεία και θεραπεία της, καθώς και από φυσιολογικά άτομα τα οποία υποβλήθηκαν σε βρογχοσκόπηση για διαγνωστικούς λόγους. Οι ΥΜΠ απομονώθηκαν από χειρουργικά δείγματα υγιούς πνευμονικού ιστού τα οποία στη συνέχεια καλλιεργήθηκαν με προ-φλεγμονώδεις παράγοντες ή με ΒΚΕ από ασθενείς με IPF ή iNSIP και εκτιμήθηκε η ινωτική τους δράση με την δοκιμασία επούλωσης τραύματος, in vitro, με διαδοχική λήψη εικόνων μέσω ανάστροφου μικροσκοπίου Olympus, καθώς και με την παραγωγή κολλαγόνου (μg/mL). H απομόνωση του ολικού RNA έγινε με την χρήση του TRIzol και ο καθαρισμός του με τη χρήση του κιτ DNase I. Η έκφραση των ινωτικών παραγόντων Tissue factor (ΤF) και (TNF-like ligand 1A) TL1A από τους ΥΜΠ έγινε με real time PCR. Παράλληλα μελετήθηκε ο ρόλος των διαλυτών μεσολαβητών IL-1α, TNF-α και IFN-γ είτε μόνων είτε σε συνδυασμό, στην προαγωγή και έκφραση των ινωτικών παραγόντων, ιστικού παράγοντα (tissue factor, TF), TGF-β και TL1A από τους ΥΜΠ. Για την ποσοτικοποίησή τους χρησιμοποιήθηκε η τεχνική της RT-PCR μετά από σύνθεση του cDNA. O χαρακτηρισμός των μυοϊνοβλαστών και η εντόπιση του TL1A έγιναν με την τεχνική του ανοσοφθορισμού. Η έκφραση των ινωτικών παραγόντων TF και TL1A, καθώς και η παραγωγή κολλαγόνου από τους ΥΜΠ έγινε με RT-PCR και Sircol assay, αντιστοίχως. Από την παρούσα μελέτη προέκυψαν τα εξής αποτελέσματα. Διέγερση των ΥΜΠ με ΒΚΕ, ασθενών και μη (n=3), ή μαζί με τη προσθήκη συνδυασμού κυτταροκινών (IL-1α+TNF-α+IFN-γ) υπήρξε στατιστικά σημαντική αύξηση επιπέδου κολλαγόνου (IPF1: 183.99 μg/ml±14.03, p<0.01, IPF2: 118.50 μg/ml±5.04, p<0.05, IPF3: 126.23 μg/ml±2.7, p<0.05 και 3C: 184.72 μg/ml±29.72, p<0.05 σε σύγκριση με τα μη διεγερμένα: 63 μg/ml±1.2). Διέγερση των ΥΜΠ με ΒΚΕ, ασθενών και μη, ή μαζί με προ-φλεγμονώδεις κυτταροκίνες (IL-1α, TNF-α) έδειξε μια στατιστικά σημαντική αύξηση στη μεταγραφική ενεργότητα του TF (IPF: 3.56-fold±0.14, IL-1α: 2.5-fold±0.08, TNF-α: 2.3-fold±0.007, 2C: 2.7-fold±0.17, p<0.05 σε σύγκριση με τα μη διεγερμένα: 1.1-fold±0.3) και TL1A (IPF: 6.5-fold±0.8, IL-1α: 156-fold±14.94, TNF-α: 240-fold±7.8, 2C: 116-fold±3.6, p<0.01 σε σύγκριση με τα μη διεγερμένα: 1.3-fold±0.6). Τα επίπεδα του TL1A επιβεβαιώθηκαν με ανοσοφθορισμό. Είναι η πρώτη φορά που ο TL1A ερευνάται στους ΥΜΠ. Διέγερση των ΥΜΠ μαζί με προ-φλεγμονώδεις κυτταροκίνες δεν επέδειξε κάποια σημαντική αύξηση στη μεταναστευτική ικανότητα σε σύγκριση με αυτή που διεγέρθηκαν με ΒΚΕ (IPF2: 43.69%±2.16, p<0.01, IPF3: 36.49%±2.37, p<0.05).Συμπερασματικά, το BALF από ασθενείς με ΙΠΙ επάγει την ινωτική δραστηριότητα σε μυοϊνοβλάστες πνεύμονα, παρόμοιο με τους μεσολαβητές που σχετίζονται με την ίνωση του πνεύμονα, υποδεικνύοντας έναν βασικό ρόλο των ΥΜΠ στην ΙΠΙ. Η κυτταροκίνη TL1A είναι μία σημαντική ινωτική κυτταροκίνη, που εκλύεται αρκετά στις ινωτικές βλάβες και επιδρά στην ινωτική δραστηριότητα των μυοϊνοβλαστών των ασθενών με ΙΠΙ.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

scholarly journals The effect of BKV reactivation on cytokines behavior in kidney transplanted patients

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Zahra Rahimi ◽  
Ramin Yaghobi ◽  
Afsoon Afshari ◽  
Jamshid Roozbeh ◽  
Mohammad Javad Mokhtari ◽  
...  
Keyword(s):  
Real Time ◽  
Roc Curve ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Expression Level ◽  
Control Group ◽  
Roc Curve Analysis ◽  
Tnf Α ◽  
Ifn Γ ◽  
Inactive Group

Abstract Background BK virus associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplanted recipients (KTRs). The current treatment for BKV nephropathy is decreasing the immunosuppressive regimen in KTRs. Interleukin-27 (IL-27) is a multifunctional cytokine that might be the front-runner of an important pathway in this regard. Therefore, in current study it is tried to evaluate the changes in the expression level of IL-27 and some related molecules, resulting from BKV reactivation in KTR patients. Methods EDTA-treated blood samples were collected from all participants. Patients were divided into two groups, 31 kidney transplant recipients with active and 32 inactive BKV infection, after being monitored by Real time PCR (Taq-Man) in plasma. Total of 30 normal individuals were considered as healthy control group. Real time PCR (SYBR Green) technique is used to determine the expression level of studied genes. Results The results of gene expression comparisons showed that the expression level of IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 genes was significantly higher in inactive group in comparison to active group. The expression level of TLR4 was lower in both active and inactive groups in comparison to control group. ROC curve analysis showed that IL-27 and IRF7 are significantly different amongst other studied genes. Finally, the analyses revealed that the expression level of most of the studied genes (except for TNF-α and TLR4) have significant correlation with viral load. Conclusions Our findings revealed that IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 expression level is higher in inactive group and TLR4 expression level is lower in patients’ groups in comparison to control group. Also, ROC curve analysis showed IL-27 and IRF7 can significantly differentiate studied groups (BKV active vs. inactive). Therefore, these results might help elucidating the pattern in charge of BKV reactivation in kidney transplanted patients.

scholarly journals The Anti-Pseudomonal Peptide D-BMAP18 Is Active in Cystic Fibrosis Sputum and Displays Anti-Inflammatory In Vitro Activity

2020 ◽  
Vol 8 (9) ◽  
pp. 1407
Author(s):  
Margherita Degasperi ◽  
Chiara Agostinis ◽  
Mario Mardirossian ◽  
Massimo Maschio ◽  
Andrea Taddio ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Dnase I ◽  
Starting Point ◽  
Tnf Α ◽  
Specific Cytotoxicity ◽  
Ifn Γ ◽  
Lung Infections ◽  
Anti Inflammatory

Most Cystic Fibrosis (CF) patients succumb to airway inflammation and pulmonary infections due to Pseudomonas aeruginosa. D-BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, was evaluated as a possible antibacterial aimed to address this issue. The antipseudomonal activity of D-BMAP18 was tested in a pathophysiological context. The peptide displayed activity against CF isolates of Pseudomonas aeruginosa in the presence of CF sputum when combined with sodium chloride and DNase I. In combination with DNase I, D-BMAP18 discouraged the deposition of new biofilm and eradicated preformed biofilms of some P. aeruginosa strains. In addition, D-BMAP18 down regulated the production of TNF-α, IL1-β, and TGF-β in LPS-stimulated or IFN-γ macrophages derived from THP-1 cells indicating an anti-inflammatory activity. The biocompatibility of D-BMAP18 was assessed using four different cell lines, showing that residual cell-specific cytotoxicity at bactericidal concentrations could be abolished by the presence of CF sputum. Overall, this study suggests that D-BMAP18 may be an interesting molecule as a starting point to develop a novel therapeutic agent to simultaneously contrast lung infections and inflammation in CF patients.

scholarly journals A specific real-time PCR assay for the detection of Bordetella pertussis

2007 ◽  
Vol 56 (7) ◽  
pp. 918-920 ◽  
Author(s):  
Benoit Vincart ◽  
Ricardo De Mendonça ◽  
Sylvianne Rottiers ◽  
Françoise Vermeulen ◽  
Marc J. Struelens ◽  
...  
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Assays ◽  
Reference Strains ◽  
Respiratory Specimens

A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

scholarly journals Decitabine Promotes Modulation in Phenotype and Function of Monocytes and Macrophages That Drive Immune Response Regulation

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 868
Author(s):  
Fabiana Albani Zambuzi ◽  
Priscilla Mariane Cardoso-Silva ◽  
Ricardo Cardoso Castro ◽  
Caroline Fontanari ◽  
Flavio da Silva Emery ◽  
...  
Keyword(s):  
Immune Response ◽  
Hematological Malignancies ◽  
M2 Macrophage ◽  
M2 Polarization ◽  
Tnf Α ◽  
Monocytes And Macrophages ◽  
Ifn Γ ◽  
And Function ◽  
The Impact

Decitabine is an approved hypomethylating agent used for treating hematological malignancies. Although decitabine targets altered cells, epidrugs can trigger immunomodulatory effects, reinforcing the hypothesis of immunoregulation in treated patients. We therefore aimed to evaluate the impact of decitabine treatment on the phenotype and functions of monocytes and macrophages, which are pivotal cells of the innate immunity system. In vitro decitabine administration increased bacterial phagocytosis and IL-8 release, but impaired microbicidal activity of monocytes. In addition, during monocyte-to-macrophage differentiation, treatment promoted the M2-like profile, with increased expression of CD206 and ALOX15. Macrophages also demonstrated reduced infection control when exposed to Mycobacterium tuberculosis in vitro. However, cytokine production remained unchanged, indicating an atypical M2 macrophage. Furthermore, when macrophages were cocultured with lymphocytes, decitabine induced a reduction in the release of inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ, maintaining IL-10 production, suggesting that decitabine could potentialize M2 polarization and might be considered as a therapeutic against the exacerbated immune response.

scholarly journals Water Extract of Rubus coreanus Prevents Inflammatory Skin Diseases In Vitro Models

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1230
Author(s):  
Sumin Pyeon ◽  
Ok-Kyung Kim ◽  
Ho-Geun Yoon ◽  
Shintae Kim ◽  
Kyung-Chul Choi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Skin Diseases ◽  
Water Extract ◽  
Total Phenolic ◽  
Hacat Cells ◽  
Rubus Coreanus ◽  
Tnf Α ◽  
Ifn Γ ◽  
Inflammatory Skin

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by immune hypersensitivity reaction. The cause of AD is unclear, but its symptoms have a negative effect on quality of life; various treatment methods to alleviate these symptoms are underway. In the present study, we aimed to evaluate in vitro antioxidant and anti-inflammatory effects of Rubus coreanus water extract (RCW) on AD. Total phenolic compounds and flavonoid content of RCW were 4242.40 ± 54.84 mg GAE/g RCE and 1010.99 ± 14.75 mg CE/g RCW, respectively. RCW reduced intracellular reactive oxygen species level and increased the action of antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-stimulated HaCaT cells. Moreover, mRNA expression of the pro-inflammatory cytokines, including TNF-α, interleukin-1β, and interleukin-6, was downregulated by RCW in the TNF-α/IFN-γ-stimulated cells. The levels of inflammatory chemokines (thymus- and activation-regulated chemokine; eotaxin; macrophage-derived chemokine; regulated on activation, normal T-cell expressed and secreted; and granulocyte-macrophage colony-stimulating factor) and intercellular adhesion molecule-1 were decreased in the TNF-α/IFN-γ-stimulated HaCaT cells after RCW treatment. Additionally, the mRNA expression levels of filaggrin and involucrin, proteins that form the skin, were increased by RCW. Furthermore, RCW inhibited the nuclear factor kappa-light-chain-enhancer of the activated B cells pathway in the TNF-α/IFN-γ-stimulated HaCaT cells. Collectively, the present investigation indicates that RCW is a potent substance that inhibits AD.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals High Matrix Metalloproteinase Production Correlates with Immune Activation and Leukocyte Migration in Leprosy Reactional Lesions

2009 ◽  
Vol 78 (3) ◽  
pp. 1012-1021 ◽  
Author(s):  
Rosane M. B. Teles ◽  
Rose B. Teles ◽  
Thais P. Amadeu ◽  
Danielle F. Moura ◽  
Leila Mendonça-Lima ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Cultured Cells ◽  
Leukocyte Migration ◽  
Therapeutic Interventions ◽  
Matrix Metalloproteinase 2 ◽  
Mrna Levels ◽  
Membrane Breakdown ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-α. It was observed that IFN-γ, TNF-α, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-α, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

scholarly journals The Effects of Oral Liposomal Glutathione and In Vitro Everolimus in Altering the Immune Responses against Mycobacterium bovis BCG Strain in Individuals with Type 2 Diabetes

2021 ◽  
Vol 12 (1) ◽  
pp. 16-26
Author(s):  
Kimberly To ◽  
Ruoqiong Cao ◽  
Aram Yegiazaryan ◽  
James Owens ◽  
Kayvan Sasaninia ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Immune Cells ◽  
Mycobacterial Infection ◽  
Drug Resistant ◽  
Tnf Α ◽  
Resistant Strains ◽  
Ifn Γ ◽  
Drug Resistant Strains

Abstract Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains a devastating infectious disease in the world. There has been a daunting increase in the incidence of Type 2 Diabetes Mellitus (T2DM) worldwide. T2DM patients are three times more vulnerable to M. tb infection compared to healthy individuals. TB-T2DM coincidence is a challenge for global health control. Despite some progress in the research, M. tb still has unexplored characteristics in successfully evading host defenses. The lengthy duration of treatment, the emergence of multi-drug-resistant strains and extensive-drug-resistant strains of M. tb have made TB treatment very challenging. Previously, we have tested the antimycobacterial effects of everolimus within in vitro granulomas generated from immune cells derived from peripheral blood of healthy subjects. However, the effectiveness of everolimus treatment against mycobacterial infection in individuals with T2DM is unknown. Furthermore, the effectiveness of the combination of in vivo glutathione (GSH) supplementation in individuals with T2DM along with in vitro treatment of isolated immune cells with everolimus against mycobacterial infection has never been tested. Therefore, we postulated that liposomal glutathione (L-GSH) and everolimus would offer great hope for developing adjunctive therapy for mycobacterial infection. L-GSH or placebo was administered to T2DM individuals orally for three months. Study subjects’ blood was drawn pre- and post-L-GSH/or placebo supplementation, where Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood to conduct in vitro studies with everolimus. We found that in vitro treatment with everolimus, an mTOR (membrane target of rapamycin) inhibitor, significantly reduced intracellular M. bovis BCG infection alone and in conjunction with L-GSH supplementation. Furthermore, we found L-GSH supplementation coupled with in vitro everolimus treatment produced a greater effect in inhibiting the growth of intracellular Mycobacterium bovis BCG, than with the everolimus treatment alone. We also demonstrated the functions of L-GSH along with in vitro everolimus treatment in modulating the levels of cytokines such as IFN-γ, TNF-α, and IL-2 and IL-6, in favor of improving control of the mycobacterial infection. In summary, in vitro everolimus-treatment alone and in combination with oral L-GSH supplementation for three months in individuals with T2DM, was able to increase the levels of T-helper type 1 (Th1) cytokines IFN-γ, TNF-α, and IL-2 as well as enhance the abilities of granulomas from individuals with T2DM to improve control of a mycobacterial infection.

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