Capillary zone electrophoresis separation of recombinant human interleukin-3 and related proteins

Zone electrophoresis separations of human recombinant interleukin-3 (rh IL-3) and related proteins in untreated fused silica capillaries are presented. Results using pH 9 CHES buffer show that rh IL-3 is easily separated from a common carrier, human serum albumin, in a commercial preparation.     

Identification of structural differences between different forms of interleukin-2 (IL-2) using anti-(human recombinant) IL-2 monoclonal antibodies.

We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (Kaff approximately equal to 10(-8) M) and are of the IgG1 isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh IL-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IL-2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID50, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 micrograms/ml to 30 micrograms/ml final concentrations of the antibodies. Using different forms of IL-2 we found that the mAbs give different patterns of inhibition of the IL-2-dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2.     

IL-9, a local growth factor for synovial T cells in inflammatory arthritis

ObjectiveThe regulatory role of the T_h9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis.MethodsCD3⁺ T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3⁺ T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9.ResultsSF of PsA and RA was enriched with IL-9 producing CD3⁺ T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3⁺ T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p < .005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway.ConclusionIL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.     

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Background aimsKey obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells.MethodsWe delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3⁺Vα24⁺Vβ11⁺ iNKT cells.ResultsGene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4⁺ and CD8⁺ responder T cells.DiscussionThese data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.     

Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes

BackgroundNatural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.AimThe aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4⁺ lymphocytes.MethodsPurified human CD4⁺ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf^®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.ResultsNeither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4⁺ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4⁺ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf^®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4⁺ lymphocytes.ConclusionFor the first time, the immunomodulatory capacity of CHF5633 on CD4⁺ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4⁺ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4⁺ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.     

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of mitogen-activated protein kinases

AimWe have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.Main methodsThe levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.Key findingsPDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.SignificanceThese results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.     

Relation of pro- and anti-inflammatory cytokines and the production of nitric oxide in patients receiving high-dose immunotherapy with interleukin-2

Immunotherapy with intravenous recombinant human interleukin-2 (rh IL-2) may be accompanied by hypotension and the emergence of capillary leak syndrome. Nitric oxide (NO) is supposed to be responsible for both side effects. The aim of the current investigation was to elucidate the relationship between pro- and anti-inflammatory cytokines and the production of NO in eight tumor patients receiving intravenous rh IL-2 continuously over a time period of 120 hours. Markers of systemic inflammation, as well as nitrate plasma levels, were consecutively determined. Significant changes in the levels of pro-inflammatory cytokines IL-6 and IL-8 were observed (p < 0.05). In contrast to the anti-inflammatory cytokine IL-10, which did not increase significantly, the serum concentrations of the soluble tumor necrosis factor receptors (sTNFr) I and II rose continuously and significantly during the observation period (p < 0.05). In parallel, a significant rise in nitrate plasma levels was observed (p < 0.05). Moreover, there were highly significant correlations between nitrate and IL-6 serum levels (p < 0.05), nitrate and sTNFr-I (p < 0.05), nitrate and sTNFr-II (p < 0.05), and between IL-6 and IL-10 (p < 0.05), respectively. We conclude that immunotherapy with IL-2 promotes a pro-inflammatory state, parallelled by an increased production of nitric oxide. Although anti-inflammatory responses accompany this process, they are not able to diminish the production of nitric oxide.     

Pentraxin 3 is an anti-inflammatory protein associated with lipid-induced interleukin 10 in vitro

Pentraxin 3 (PTX3) is an acute phase protein expressed in response to pro-inflammatory stimuli during atherosclerosis. However, recent findings suggest that PTX3 is a counter-regulatory protein which enhances the anti-inflammatory response.ObjectiveTherefore, the capacity of PTX3 to alter the inflammatory milieu following in vitro stimulation of PBMCs with the pro-inflammatory lipid, palmitate, was examined.MethodsPBMCs from 17 healthy male donors were isolated and cultured under four separate conditions; 200 μmol/L palmitate, a physiologically relevant concentration of PTX3, in combination (pal + PTX3), and an unstimulated time-course control.ResultsPalmitate-induced production of the counter-regulatory protein PTX3 was positively associated with the production of the anti-inflammatory cytokine interleukin 10 (IL-10) following in vitro stimulation of human PBMCs. Furthermore, stimulation of PBMCs in vitro with 500 pg/mL PTX3 elicited a significantly greater increase in IL-10 production compared to the palmitate stimulated conditions. However, PTX3 stimulation did not result in the production of the pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha, and when combined with palmitate, did not alter the pro-inflammatory milieu from PBMCs in this study.ConclusionThese findings provide evidence supporting the role of PTX3 as a mediator of the anti-inflammatory response in physiologically relevant conditions, and suggests that PTX3 counter regulates the development of atherosclerosis by enhancing the production of IL-10.     

Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies

Abstract

A variety of immunomodulatory effects have previously been attributed to haptoglobin (Hp). These are supposed to be partly mediated through binding of Hp to CD11b. In the present study, we assessed its effects on T-helper (Th) cytokine production following both in vitro and in vivo stimulation of T-cells. Hp exhibits a dose-dependent inhibitory effect on human T lymphocyte release of the Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) in vitro, whereas it has no clear effect on Th1 cytokine (IL-2 and IFN-γ) release. When administered an anti-CD3 monoclonal antibody, Hp knockout mice produced more IL-4 and less IFN-γ than did their wild-type litter-mates. Our findings imply that Hp may be regarded as a regulator of the Th1/Th2 balance in both human and murine immune systems.     

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Background aimsInterleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18.MethodsIn the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells.ResultsThere was a 4.8-fold significant increase in NK-cell population (CD3^?/16⁺/56⁺; P < 0.03), 21-fold significant increase in CD3^?/56⁺/158a⁺ (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3^?/56⁺/158b⁺ (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3^?/56⁺/NKB1⁺ (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively.ConclusionsWe have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.     

白介素3治疗小鼠造血功能低下的疗效及机理初探

通过整体实验观察国产重组白介素３（ＩＬ－３）对射线和环磷酰胺所致小鼠造血功能低下的疗效;以体外实验分析其疗效机理。实验结果表明：（１）ｒｈＩＬ－３腹腔或皮下连续５天注射能全面提高７Ｇｙ照射小鼠９天时股骨骨髓ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－Ｍｉｘ和ＣＦＵ－ＧＭ的产率和数量,其效果强弱与注射途径和用药剂量有关。ｒｈＩＬ－３对小鼠股骨骨髓有核细胞总数和内源性脾结节数的改善影响小。（２）ｒｈＩＬ－３对环磷酰胺所致小鼠造血功能低下亦有改善效果,并与起用时间和剂量有关。（３）ｒｈＩＬ－３对人骨髓细胞和ＣＦＵ－ＧＭ集落形成有明显的增强作用。小鼠骨髓细胞对ｒｈＩＬ－３缺乏反应;对ｒｍＩＬ－３有增殖分化加强的反应。ｒｍＩＬ－３体外共育能提高正常及照射２Ｇｙ小鼠骨髓细胞体外培养后ＣＦＵ－ＧＭ的产率和数量。文中讨论了ＩＬ－３的应用前景及合理方案问题。     

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

Introduction

Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).

Methods

The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.

Results

rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.

Conclusion

Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.     

The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes

BackgroundNew generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes.MethodsPurified neonatal CD14⁺ cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1β, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry.ResultsCHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1β. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected.ConclusionOur data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1β cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.     

The Induction of IL-33 in the Sinus Epithelium and Its Influence on T-Helper Cell Responses

BackgroundChronic rhinosinusitis (CRS) is characterized by epithelial activation and chronic T-cell infiltration in sinonasal mucosa and nasal polyps. IL-33 is a new cytokine of the IL-1 cytokine family that has a pro-inflammatory and Th2 type cytokine induction property. The role of IL-33 in the pathomechanisms of CRS and its interaction with other T cell subsets remain to be fully understood.MethodsThe main trigger for IL-33 mRNA expression in primary human sinonasal epithelial cells was determined in multiple cytokine and T-cell stimulated cultures. The effects of IL-33 on naïve, Th0 and memory T-cells was studied by PCR, ELISA and flow cytometry. Biopsies from sinus tissue were analyzed by PCR and immunofluorescence for the presence of different cytokines and receptors with a special focus on IL-33.ResultsIL-33 was mainly induced by IFN-γ in primary sinonasal epithelial cells, and induced a typical CRSwNP Th2 favoring cytokine profile upon co-culture with T-helper cell subsets. IL-33 and its receptor ST2 were highly expressed in the inflamed epithelial tissue of CRS patients. While IL-33 was significantly up-regulated in the epithelium for CRSsNP, its receptor was higher expressed in sinus tissue from CRSwNP.ConclusionsThe present study delineates the influence of IL-33 in upper airway epithelium and a potential role of IL-33 in chronic inflammation of CRSwNP by enhancing Th2 type cytokine production, which could both contribute to a further increase of an established Th2 profile in CRSwNP.     

Oxidative stress-mediated epidermal growth factor receptor activation regulates PM2.5-induced over-secretion of pro-inflammatory mediators from human bronchial epithelial cells

BackgroundExposure to PM_2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM_2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B.MethodsBEAS-2B cells were exposed to 0, 20, 50, 100 and 150 μg/ml of PM_2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1β was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting.ResultsPM_2.5 exposure increased the secretion of IL-6, IL-8, and IL-1β in a concentration-dependent fashion. Moreover, exposure to PM_2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM_2.5-induced IL-6, IL-8 and IL-1β secretion, implying that both oxidative stress and EGFR activation were involved in PM_2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM_2.5-induced EGFR activation, suggesting that oxidative stress was required for PM_2.5-induced EGFR activation.ConclusionPM_2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.     

Impaired M. tuberculosis Antigen-Specific IFN-γ Response without IL-17 Enhancement in Patients with Severe Cavitary Pulmonary Tuberculosis

BackgroundTh1 cells play an essential role in immune protection against tuberculosis. Th17 cells might be involved with immune pathology in active human tuberculosis (TB). The balance between Th1 and Th17 cells in patients with cavitary tuberculosis needs to be clarified which might help understanding the immunological basis of pathologic pathogenesis in TB.MethodInitially treated pulmonary TB (PTB) patients with or without cavities were recruited before chemotherapy. We isolated peripheral blood mononuclear cells, stimulated with phytohemagglutinin (PHA), PPD, or ESAT-6 antigens, and assayed supernatant IFN-γ and IL-17 by ELISA after 24 or 72 hours incubation, respectively. Cells were also stained with antibodies to CD3, CD4, CD8, IFN-γ or IL-17 and the proportion of stained cells was measured by flow cytometry.ResultsWe found wide variation of IFN-γ response in active PTB patients, but less subject-to-subject variation of IL-17 was observed as we previously reported. There were no significant differences in IFN-γ and IL-17 between cavitary and non-cavitary PTB; however, we found decreased IFN-γ secretion in severe cavitary PTB compared to mild lesion non-cavitary PTB (p < 0.05). We also found a decrease in the proportion of CD3+CD4+ T cells in the blood of severe cavitary PTB patients (p < 0.05).ConclusionsIL-17 seemed to have no association with the formation of cavities in active PTB from the study of PBMC. Impaired IFN-γ without IL-17 enhancement occurs in peripheral blood during severe cavitary PTB. Our results demonstrate that M. tuberculosis antigen-specific Th1 response is decreased when PTB lesions develop to severe cavities.     

Anti-inflammatory effect of proteoglycan and progesterone on human uterine cervical fibroblasts

AimsThe aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix.Main methodsAfter obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24–48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting.Key findingsLPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each.SignificanceThis study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.