Cells In Pathogenesis Research Articles

PReS-FINAL-2354: Laboratory investigation of the role of toll-like receptros on kidney cells in pathogenesis of lupus nephritis

Lupus nephritis (LN) is a serious complication of juvenile-onset systemic lupus erythematosus (JSLE). Current treatments include long-term immunosuppressants with significant side effects. There is a need to identify targets for more effective therapies. Toll-like receptors (TLRs) perform an important role in the innate immune response by recognising conserved molecules associated with pathogens. Previous studies have suggested a role for TLR7 and TLR9 in lymphocytes in the pathogenesis of SLE. Podocytes are specialised cells forming an important part of the glomerular filtration barrier. Biopsies from LN patients have demonstrated higher TLR7 and TLR9 expression in glomeruli compared with controls and suggest that these TLRs are localised to podocytes. We hypothesise that stimulation of TLR7 and/or TLR9 in podocytes, acting via nuclear factor kappa B (NFkB), leads to cellular damage and resultant kidney disease.

PReS-FINAL-2358: T helper cells in henoch-schönlein purpura/iga vasculitis

Henoch-Schonlein purpura (HSP)/IgA vasculitis is the most common childhood vasculitis in Turkey. During the course of the disease, renal involvement may be observed in 30-60% of patients. In recent years, the role of T helper (Th) cells in pathogenesis of HSP/IgA vasculitis has become a focus for research.

Involvement Of Autoreactive T Lymphocytes In Pathogenesis Of Chronic Lymphocytic Leukemia (CLL): Specific T-Cell Immune Responses Against Autoantigens Recognized By CLL Cells

The etiology of chronic lymphocytic leukemia (CLL) has remained unknown but recently it has been speculated that antigens stimulation could be involved in the pathogenesis of CLL. Cytoskeletal proteins exposed on the surface of apoptotic cells could represent autoantigens involved in the development of CLL since monoclonal antibodies derived from CLL-specific B-cell receptors (BCR) recognized MYHIIA (non-muscle myosin heavy chain IIA), vimentin, and cofilin-1. Since the humoral immune response against these autoantigens could play a crucial role in pathogenesis of CLL we suspect that it might be accompanied by T-cell immune responses. We defined HLA-A2-restricted epitopes in silico using two independent computer prediction algorithms. Next 36 most immunogenic peptides derived from MYHIIA, cofilin-1 and vimentin were selected and synthesized. To determine whether peptides could bind to HLA-A2 also in vivo we performed cellular-peptide binding affinity assay utilizing T2 cell line. The expression of HLA-A2 was measured using flow cytometry with FITC-conjugated anti-HLA-A2 monoclonal antibodies. Fluorescence index (FI) for particular peptides was calculated as mean fluorescence intensity of HLA-A2 on T2 cells with peptide versus without peptide. We selected peptides with the highest FI M6 (KLKDVLLQV, FI=0.43), M1 (GLLRVISGV, FI=0.20), M2 (YLLEKSRAI, FI=0.16), M3 (VLMIKALEL, FI=0.15) derived from MYHIIA, V1 (SLQEEIAFL, FI=4.93) and V4 (ILLAELEQL, FI=1.49) derived from vimentin and C1 (ALYDATYET, FI=0.60) C7 (LQANCYEEV, FI=0.68), C11 (ILEEGKEIL FI=0.88) derived from cofilin-1. Mixed lymphocyte/peptide cultures (MLPC) were performed to define immunogenicity of synthesized peptides in vitro. CD8+ cells from HLA-A2 positive 15 CLL patients and 8 healthy volunteers were selected by magnetic-activated cell sorting. Afterwards, CD8- antigen presenting cells were irradiated and pulsed for 2 hours with respective peptides derived from MYHIIA, vimentin and cofilin-1 or control peptide at a concentration of 10 μg/ml and without peptide for negative control. Cells were supplemented with IL-2 and IL-7 on day +1 and restimulated every week. At day +21 of MLPC, cells were harvested and evaluated for their specific cytotoxicity in ELISpot assays with peptides, with the unspecific positive control Pokeweed Mitogen (PWM), or without peptide, as negative control. The ELISpot assay was performed under the conditions recommended by the Cancer Immunotherapy Monitoring Panel. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 71%, vimentin in 83% and cofilin-1 in 50% CLL patients. Additionally, we found positive responses in 37.5% healthy volunteers. Finally, we assessed the frequency of the specific T cells in 14 CLL patients along with detailed phenotypical characteristics using dextrameric peptide-specific complexes in 7-colour panel using FACSCantoII Flow Cytometer ex vivo. To characterize naïve/effector/effector-memory and memory cells were stained for CD8, CD3, CCR7, CD45RA with exclusion of CD19+, CD56+ and CD4+ cells and 7AAD+ dead cells. We detected low frequency of peptide-specific T cells, with the highest level of 3.6% CD8+ cells specific for M3 peptide. Most cells (83%) represented effector-memory phenotype. Subsequently, we isolated RNA from 100 CLL patients to assess MYHIIA, VIM, and CFL-1 expression using qRT-PCR. Quantity mean values for gene expression were calculated according to the Standard Curve method. GAPDH was used as a constitutive gene. All three genes were overexpressed with no difference between males and females, Binet stages A, B and C nor different cytogenetic and molecular groups of CLL patients. These data suggest that MYHIIA, VIM and CFL-1 might be considered as a novel constitutive genes, highly expressed in CLL. In summary, our findings suggest presence of cytotoxic immune responses against three autoantigens recognized by BCR of CLL cells. The existence of autoreactive CD8+ T cells against MYHIIA, vimentin, cofilin-1 in CLL patients points to the involvement of antigen-specific autoreactive T cells in pathogenesis of CLL. Disclosures:No relevant conflicts of interest to declare.

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

The variation of CD4+ memory T cells in the development of asthma in mice (P6233)

Abstract Objective To understand the role of CD4+ memory T cells in pathogenesis of asthma in mice Method Female BALB/c mice were sensitized and challenged with OVA for continuous 7 days. After one month, another challenge was given for one day. We detected the ratio changes of CD4+ memory T cells in CD4+ T cells during the treatment course, the membrane surface markers of CD3+, CD4+, CD44High, CD62L- were examined by using a flow cytometry. We further compared the different challenge time durations effect on CD4+ memory T cell numbers. Results In control mice, the ratios of CD4+ memory T cells in lung and spleen were 11.32% and 20.48%, respectively; After challenge, the ratio of CD4+ memory T cells increased gradually and reached the peak value ( 53% in lung and 45% in spleen) at one week after the last challenge. After one month these ratios returned to the levels of before the challenge. However, these memory T cells raised up rapidly on the next day during the second challenge, especially in the lung and kept relatively stable levels (about 35% in lung and 30% in spleen) within one week. We observed that the different challenge time durations had similar effect on the ratio of CD4+ memory T cells in lung, but it differ in spleen the peak value of CD4+ memory T cells at 30min challenge was 10% lower than that with 1 hour challenge and dropped earlier. Conclusion The CD4+ memory T cells play important roles in asthma pathogenesis, the detailed mechanisms warrant further exploration.

Implication of vascular endothelial cell-reactive invariant NKT cells in pathogenesis of small vessel vasculitis in rats

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.

Mediators of Neuroinflammation

Mediators of Neuroinflammation

Role of Regulatory T Cells in Pathogenesis and Biological Therapy of Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory disease in which the myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. It is caused by an autoimmune response to self-antigens in a genetically susceptible individual induced by unknown environmental factors. Principal cells of the immune system that drive the immunopathological processes are T cells, especially of TH1 and TH17 subsets. However, in recent years, it was disclosed that regulatory T cells took part in, too. Subsequently, there was endeavour to develop ways how to re-establish their physiological functions. In this review, we describe known mechanisms of action, efficacy, and side-effects of contemporary and emerging MS immunotherapeutical agents on Treg cells and other cells of the immune system involved in the immunopathogenesis of the disease. Furthermore, we discuss how laboratory immunology can offer physicians its help in the diagnosis process and decisions what kind of biological therapy should be used.

Roles of aberrant endothelial progenitor cells in pathogenesis of systemic sclerosis

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by excessive fibrosis and microvasculopathy, along with poor vascular formation and repair. The maintenance of the postnatal vascular system requires constant remodeling through vasculogenesis, which is mediated by the de novo differentiation of mature endothelial cells from endothelial progenitor cells (EPCs). However, a great deal of controversy about EPCs and their roles in postnatal vascular formation has arisen because of discrepancies in how EPCs are defined. The current consensus is that EPCs are heterogeneous cell population containing an extremely small count of "true EPCs", and pro-angiogenic hematopoietic cells (PHCs) that promotes vascular formation and repair through secretion of pro-angiogenic factors, and differentiation into endothelial cells and mural cells. In 2004, we reported a reduced number and impaired function of circulating CD34(+)CD133(+)CD309(+)CD45(dim)CD14(-) EPCs, which are now regarded as an immature subset of PHCs, in patients with SSc, and proposed a theory that defective vascular repair machinery as one of important mechanisms contributing to SSc vasculopathy. In addition, we showed that in SSc patients, circulating monocytic PHCs were increased and have enhanced angiogenic potency and differentiation potential to fibroblast-like cells. In summary, EPCs are involved in the pathogenesis of SSc by participating in two major pathological features, microvasculopathy and excessive fibrosis. Understanding the roles of EPCs in disease process of SSc may be key to dissecting its pathogenesis and to developing novel therapeutic strategies for this intractable condition.

Role of retinal glial cells in pathogenesis of macular oedema

AbstractPurpose Macular edema is a complex nonspecific reaction that results in intraretinal fluid accumulation and leads to a decrease of retinal transparency and visual impairment. Virtually all retinal disorders associated with ischemia, inflammation and/or mechanical traction may be accompanied by the development of macular edema. The pathogenic mechanisms underlying the development of macular edema are largely unresolved. Most studies favor vasogenic edema as key mechanism in the development of macular edema. However there are indications that a swelling of retinal Müller cells may also contribute to the development of macular edema.Methods Immunohistochemical staining as well as recording of Müller cells behavior was investigated under normal conditions, under osmotic stress and under different pathological processes, e.g., retinal ischemia, ocular inflammation, and diabetes.Results The Müller cells are responsible for the maintenance of the retinal homeostasis including intraretinal osmolyte and water homeostasis through coupling of transglial water fluxes (via aquaporins) to potassium currents clearance. Functional alterations of Müller cells in course of ischemia and/or inflammation contribute to the downregulation of potassium conductance and intracellular overload of potassium ions. This results in osmotically driven water movements from the blood and vitreous into the glial cells and causes neuronal hyperexcitability and glial cellConclusion An impairment of ion and water movements through Müller cells (in addition to vascular leakage) may contribute to the development of macular edema. A better understanding of the mechanisms of cellular ion and water homeostasis will facilitate the development of efficient edema‐resolving drugs.

Status and Development of The Tumor Microenvironment in Hepatocellular Carcinoma*

A large quantity of literature highlights the interaction between tumor cells and the surrounding microenvironment which is a very important modulator of the processes in hepatocarcinogenesis.The microenvironment of hepatocellular carcinoma(HCC) can be obviously classified into cellular and produced non-cellular components,including hepatic stellate cells,tumor associated fibroblasts,immune cells and sinusoidal endothelial cells,extracellular matrix(ECM) proteins,growth factors and inflammatory cytokines which play an important role in the development and metastasis of HCC.In this review,we discuss the current cross-talk about the tumor microenvironment and tumor cells in pathogenesis of HCC.

Intratumoral mast cell number is negatively correlated with tumor size and mitosis in dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous tumor with intermediate malignancy. The purpose of this study was to evaluate the utility of p53, smooth muscle actin (SMA) and c-kit as immunohistochemical markers and toluidine blue staining for mast cell that can correlate with the clinical outcome and clarify role of mast cells in pathogenesis of tumor. We analysed data for 32 lesions from 31 patients. Fibrosarcomatous type DFSP showed high immunoreactivity for SMA compared with other subtypes (P = 0.026). No differences in p53 immunoreactivity were observed between subtypes. None of tumor cells were immunoreactive with c-kit. The mast cell counts showed a negative correlation with mitosis and tumor size (P < 0.05), implying that mast cells do not have a causative primary role in tumorigenesis but rather play a secondary role.

Implications of Th1 and Th17 cells in pathogenesis of oral lichen planus

Oral lichen planus (OLP) is considered a T cell-mediated autoimmune disease with unknown aetiology. T helper cells appear to play an important role in the pathogenesis of OLP. We investigated the possible role of T helper cells, Th1 and Th17, in the lesions and circulation of patients with OLP. Forty patients with OLP and 15 healthy volunteers were recruited. Double immunofluorescence staining was used to detect Th1 and Th17 cells in the OLP lesions, and intracellular cytokine staining and flow cytometry to evaluate the proportion of Th1 and Th17 cells in peripheral blood. The levels of serum interferon (IFN)-γ and interleukin (IL)-17 were assessed by using an enzyme-linked immunosorbent assay. It was found that Th17 cells, as well as Th1 cells, were present in OLP lesions. The proportion of peripheral Th1 and Th17 cells was significantly increased in patients with OLP. The proportion of Th17 cells in atrophic-erosive OLP was elevated as compared with that in reticular OLP. Serum IL-17 levels in OLP patients were significantly higher than in controls, and those in the atrophic-erosive OLP group were increased as compared with the reticular OLP group. However, the levels of serum IFN-γ were slightly decreased in OLP patients. Our data suggested that Th1 and Th17 cells in the local lesions and peripheral blood may be associated with the pathogenesis of OLP, and that IL-17 may be an important proinflammatory cytokine in OLP. These findings enhance our understanding of OLP pathogenesis.

Hepatic steato-fibrosis and non-alcoholic liver disease in elderly

The critical role of the hepatic stellate cells in pathogenesis and evolution of hepatic fibrosis is stressed. The authors, also, illustrate the most recent acquisitions about morphological and bioumoral aspects of complex sinusoidal-Disse space-stellate cells and their importance for the risk of evolution towards non-alcoholic liver disease.

Dendritic Cells in Pathogenesis of COPD

COPD (Chronic Obstructive Pulmonary Disease) is an important lung and airway disease which affects the lives of around 200 million people worldwide with an increasing incidence particularly in developing countries. The pathogenesis of COPD is based on the innate and adaptive inflammatory immune response to the inhalation of toxic particles and gases. Although cigarette smoking is the primary cause of this inflammation, many other environmental and occupational exposures contribute to the pathology of COPD. The immune inflammatory changes associated with COPD are linked to a tissue repair and remodeling process that increases mucus production and causes emphysematous destruction of the gas-exchanging surface of the lung. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages, T lymphocytes, and dendritic cells (DCs). Little is known about the relative contribution of DCs in the pathogenesis of COPD. However the number of DCs is changed in smokers and COPD patients and cigarette smoke (CS) induces the release of chemokines from DCs that play a role in the pathogenesis of COPD. In this review paper, an overview is presented on the role of DCs and their mediators in the pathogenesis of COPD. The activation of DCs and their signaling in response to CS will also be highlighted and discussed.

Identification of HnRNP-A2/B1 as a Target Antigen of Anti-Endothelial Cell IgA Antibody in Behçet's Disease

Behçet's disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease. We detected the target protein using western blotting and immunoprecipitation and determined the amino-acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/TOF). Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by ELISA. The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino-acid sequences of heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP-A2/B1). Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), 4 of 30 systemic lupus erythematosus patients (13.3%), 8 of 30 rheumatoid arthritis patients (26.7%), 9 of 30 Takayasu's arteritis patients (30%), 6 of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 were correlated with those against the recombinant streptococcal hsp-65.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

The Role of Natural Killer T Cells in the Pathogenesis of Acute Exacerbation of Human Asthma

Background: Natural killer T (NKT) cells have been reported to play a crucial role in the pathogenesis of asthma in a mouse model of acute asthma. The present study aimed to investigate the role of NKT cells in the immune pathogenesis of acute exacerbation of human asthma. Methods: Blood and sputum were obtained at baseline and 8 h after a challenge in 20 asthmatics who underwent allergen bronchial provocation testing and during exacerbation and convalescence in 9 asthmatics who were admitted to hospital with an acute exacerbation after an upper respiratory tract infection. 6B11+ or Vα24+ NKT cells were measured with flow cytometry. Inflammatory cells, cytokines and chemokines were determined in sputum. Results: The number of blood NKT cells did not change after a positive allergen challenge compared to the baseline. However, blood CD4+Vα24+ NKT cells decreased during infection-associated asthma exacerbations compared to the convalescence measurements of the same patients (p < 0.05) or the baseline measurements of asthmatics who underwent allergen challenges (p < 0.01). The number of sputum NKT cells did not change after a positive allergen challenge or during infection-associated asthma exacerbations. Eosinophils and various cytokines and chemokines increased in sputum during infection-associated asthma exacerbations. Blood CD4+Vα24+ NKT cells were inversely related to sputum eosinophils (Spearman’s correlation coefficient = –0.62; p < 0.01). Conclusion: Blood NKT cells decreased during infection-associated asthma exacerbation and were inversely associated with eosinophilic airway inflammation, suggesting that blood NKT cells might be mobilized to the airways and lungs during asthma exacerbation in humans.

Mast cells distribution and variations in epithelium thickness and basement membrane in oral lichen planus lesion and oral lichenoid reaction

Oral lichen planus (OLP) is a chronic mucocutaneous lesion with unknown etiology. Oral lichenoid lesions (OLL) comprise a family of lesions with different etiologies. Both lesions have similar clinical and histopathologic characteristics although their management is different. Differential diagnosis between OLP and OLL has always been a major challenge. In this prospective analytical study, the role of mast cells in pathogenesis of these lesions was investigated by evaluation of 52 patients with clinical and histopathological diagnosis of OLP (26 cases) and OLL (26 cases) based on WHO criteria, and by applying a more accessible staining methods, Hematoxylin and Eosin, toluidine blue (histochemistry) and Periodic Acid Schiff staining. In order to distinguish these two lesions, number of mast cells and thickness of epithelium and basement membrane were measured using light microscopy. Data were analyzed by SPSS software using t-test method (P<0.001). No significant difference was observed between the total numbers of mast cells of two groups (P=0.148), but a statistically significant difference was detected between degranulated mast cells in two groups (P<0.001). A significant difference was also observed between the thickness of epithelium in two groups (P<0.001), although no difference was seen between basement membrane thickness in these lesions. Number of degranulated mast cells in reticular layer of corium in lichenoid lesions was more than that of OLP. This implies that despite the increase in number of these cells, in both groups of diseases, the role of these cells has not been the same in pathogenesis of the diseases. Moreover, the epithelium thickness was lower in lesions of OLP compared to lesions of oral lichenoid, so this parameter may be a useful criterion together with other histopathological and clinical finding to discriminate these lesions. However, discrepancy of basement membrane thickness can not be a reliable criterion. Finally we suggest more accessible staining methods which are reliable for differentiation of these two lesions.

Role of Bronchial Epithelial Cells in Pathogenesis of COPD

Role of Bronchial Epithelial Cells in Pathogenesis of COPD

OVER EXPRESSION OF CYCLOOXYGENASE 2 DETECTED IN MCF-7 BREAST CANCER CELL AND COMPARED WITH LUNG CARCINOMA CELLLINE (A549)

In advanced studies Cyclooxygenase-2 (COX-2) mRNA was determined mostly by in situ hybridization or Northern Blot analysis-methods not suitable for absolute quantification of mRNA copy numbers. Here we reported that, over expression of COX-2 mRNA was observed and calibrated through highly sensitive externally standardized real-time RT-PCR with Light Cycler instrument. The total RNA was isolated from two breast cancer cell lines (MCF-7, ZR-75-1), one lung cancer cell line (A549) and one normal breast cell line (HBL-100). The presence of COX-2 mRNA copy numbers was determined in all cell lines thourgh Reverse Transcription PCR by cDNA conversion method. In this study, we observed breast cancer cell lines and lung cancer cell lines were showing high expression levels of COX-2 mRNA at the same time there was the lowest expression was detected in normal breast cells. This low levels of COX 2 expression due to the tumorogenic action of a normal cells. Thus we evaluated COX-2 expression at different levels in breast and lung carcinoma cell lines. Results of our study provide insight view to the involvement of different carcinoma cells in pathogenesis with respect to COX-2 mRNA expression. This Light Cycler technology is currently considered to be the most precise method for nucleic acid quantification and which showed to be a powerful tool for further expression studies on cancer gene pathogenesis.