Saša Simčič

Chitotriosidase activity in sarcoidosis and some other pulmonary diseases

Abstract Background: Patients with sarcoidosis have elevated levels of several markers of inflammation. Particularly high levels have been reported for chitotriosidase. In this study, we evaluate whether determining chitotriosidase in serum would be useful in the diagnosis and clinical management of patients with sarcoidosis. Methods: Patients with newly diagnosed sarcoidosis and patients with asthma, fibrosis, asbestosis, lung cancer or chronic obstructive pulmonary disease (n=190) were recruited from an outpatient department. Individuals with no disease (n=26) served as controls. An X-ray was taken, diffusion capacity was measured and blood samples were taken for analysis of chitotriosidase, soluble receptor for interleukin-2, tumour necrosis factor alpha and angiotensin converting enzyme. In most patients with sarcoidosis, the analyses were done before and after regular treatment with corticosteroids over 6 months. Results: Some patients with sarcoidosis had markedly high activities of chitotriosidase, but activities above controls were also found among patients with asbestos, fibrosis and lung cancer. There were significant relationships between chitotriosidase and interleukin-2 receptor and angiotensin-converting enzyme. After treatment, chitotriosidase activity decreased in 52 of 69 patients. Conclusions: The results confirm that chitotriosidase activity is markedly increased in some cases of sarcoidosis. As increased activities are also found in other diseases, chitotriosidase cannot be considered a specific marker of sarcoidosis. In cases of sarcoidosis where high CTO activities are found, this enzyme could serve as a useful marker supporting the diagnosis of sarcoidosis when following the effects of treatment and in surveillance for recurrence of the disease.

In vitro and in vivo reactivity to fungal cell wall agents in sarcoidosis.

Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n = 22) and controls (n = 20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate β‐glucan (S‐glucan, P‐glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, IL‐10 and IL‐12 were measured. The severity of sarcoidosis was determined using a chest X‐ray‐based score. Serum cytokines (IL‐2R, IL‐6, IL‐10 and IL‐12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters. N‐acetylhexosaminidase (NAHA) on the filters was measured as a marker of fungal cell biomass. The induced secretion of cytokines was higher from peripheral blood mononuclear cells (PBMC) from subjects with sarcoidosis. P‐glucan was more potent than S‐glucan inducing a secretion. Chitin had a small effect. Among subjects with sarcoidosis there was a significant relation between the spontaneous PBMC production of IL‐6, IL‐10 and IL‐12 and the NAHA levels at home. The P‐glucan induced secretion of IL‐12 was related to the duration of symptoms at the time of diagnosis. Their X‐ray scores were related to an increased secretion of cytokines after stimulation with LPS or P‐glucan. Subjects with sarcoidosis have a higher reactivity to FCWA in vitro and to home exposure. The influence of FCWA on inflammatory cells and their interference with the inflammatory defense mechanisms in terms of cytokine secretion could be important factors for the development of sarcoidosis.

Solid-state cultivation of Grifola frondosa (Dicks: Fr) S.F. Gray biomass and immunostimulatory effects of fungal intra- and extracellular β-polysaccharides

Grifola frondosa strain GF3, was cultivated on solid-state substrate consisting of milled whole corn plant (Zea mays) and olive press cake supplemented with mineral additives and olive oil. Maintenance of the moisture content in the solid substrate is of crucial importance. Moistures higher than 70% promote growth of G. frondosa mycelium and polysaccharide production. Four fractions of pure extracellular beta-D-glucans with total mass 127.2mg and four fractions of intracellular polysaccharides with total mass 47.2mg were isolated. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. Isolated polysaccharide fractions from fungal mycelium proved to induce moderate amounts of TNF-alpha in PBMC cells in vitro. The extent of TNF-alpha induction was up to 322pgmL(-1) at a polysaccharide concentration of 200microgmL(-1) for the intracellular fraction. The TNF-alpha inducing activity is comparable to romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.

Fungal cell wall agents suppress the innate inflammatory cytokine responses of human peripheral blood mononuclear cells challenged with lipopolysaccharide in vitro

Exposure to high levels of fungi might lead to diseases, such as airway inflammation, hypersensitivity pneumonitis and allergy. To comprehend the mechanisms behind the exposure to fungi and a disease, we examined the in vitro innate inflammatory cytokine response of human peripheral blood mononuclear cells (PBMC) challenged by fungal cell wall agents (FCWAs), i.e., soluble and particulate (1→3)-β-D-glucan-curdlan (BGS and BGP), zymosan (ZYM) and chitosan (CHT) in the absence or presence of lipopolysaccharide (LPS). We also studied FCWA effects on the mRNA expression of dectin-1, TLR2, TLR4 and mannose receptor (MR) by real-time RT-PCR. Our results demonstrated that BGP strongly induced the secretion of TNF-α, IL-6, IL-10 and IL-12; BGS, ZYM and CHT were weaker, but still significant cytokine inducers. We showed that BGS significantly augmented the LPS-induced in vitro secretion of TNF-α. On the other hand, BGP, ZYM and CHT suppressed the LPS-induced production of all cytokines. At the mRNA level, the dectin-1, TLR2 and TLR4 expressions were significantly reduced by all FCWAs in the absence of LPS and even more in the presence of LPS. While we demonstrated that the innate inflammatory cytokine response of PBMC induced by CHT was mediated by MR, the MR mRNA expression was significantly reduced by CHT. On the contrary, BGS significantly enhanced the MR mRNA expression. In conclusion, a long-term and massive exposure to LPS and FCWA (e.g., organic dust) may cause an important disruption of normal immune response and allow development and/or persistence of various immunopathological events.

Apyrogenic synthetic desmuramyldipeptide, LK-409, with immunomodulatory properties

The synthesis and some immunological characteristics of a new desmuramyl dipeptide 7-oxooctanoyl-l-alanyl-d-isoglutamine (LK-409) are presented. The effects of this compound were compared with those ofN-acteylmuramyl-l-alanyl-d-isoglutamine (MDP). The influence of LK-409 on the number of B and T cells in spleen and the number of peritoneal macrophages was studied; Jernes plaque forming cells assay was performed to monitor the effect of B cell differentiation. The blast transformation of T cells stimulated with concanavalin A was used to detect the influences on T lymphocytes. The activation of macrophages was studied as well. In contrast to MDP, LK-409 was apyrogenic in the doses applied but had similar immunomodulatory properties. Tested immunological properties and the absence of pyrogenicity and low toxicity make LK-409 a candidate for an immunomodulatory drug and a model molecule suitable for studying and understanding the dual activity of the MDP and its analogues.

Identification of rare genetic variation of NLRP1 gene in familial multiple sclerosis

The genetic etiology and the contribution of rare genetic variation in multiple sclerosis (MS) has not yet been elucidated. Although familial forms of MS have been described, no convincing rare and penetrant variants have been reported to date. We aimed to characterize the contribution of rare genetic variation in familial and sporadic MS and have identified a family with two sibs affected by concomitant MS and malignant melanoma (MM). We performed whole exome sequencing in this primary family and 38 multiplex MS families and 44 sporadic MS cases and performed transcriptional and immunologic assessment of the identified variants. We identified a potentially causative homozygous missense variant in NLRP1 gene (Gly587Ser) in the primary family. Further possibly pathogenic NLRP1 variants were identified in the expanded cohort of patients. Stimulation of peripheral blood mononuclear cells from MS patients with putatively pathogenic NLRP1 variants showed an increase in IL-1B gene expression and active cytokine IL-1β production, as well as global activation of NLRP1-driven immunologic pathways. We report a novel familial association of MS and MM, and propose a possible underlying genetic basis in NLRP1 gene. Furthermore, we provide initial evidence of the broader implications of NLRP1-related pathway dysfunction in MS.

Modulation of tumour necrosis factor production with desmuramyldipeptide analogues

Abstract Some synthetic analogues of the immuno-modulatory agent muramyl dipeptide (MDP), i.e. phthalimido- (LK-511, LK-413, LK-512, LK-423, LK-508), adamantyl- (LK-415, LK-517), 7-oxoaIkyl-(LK-409) desmuramylpeptides were assessed for the tumour necrosis factor (TNF) inducing activity and the ability to modulate TNF production in in vitro phorbol 12-myristate 13-acetate (PMA) & ionomycin stimulated cultures of human peripheral blood mononuclear cells. A kinetic study over a 40-hour period indicated that desmuramyldipeptides were weak TNF inducers compared to romurtide, PMA & ionomycin or lipopolysaccharide. By contrast, they showed the potential to up- or down-regulate the production of TNF evoked by PMA & ionomycin, which was strongly dependent on the time of the stimulation. After 4h of stimulation, the TNF secretion was augmented by LK-508, LK-409 and LK-511, after 18 h by LK-409 and LK-423, and after 40 h by LK-423, LK-511, LK-415 and LK-512. However, LK-517 and LK-512 inhibited the secretion of TNF after the 18-h period.

Anticryptococcal cytotoxicity of murine nonadherent cells is perforin and nonperforin mediated

The encapsulated fungal pathogen Cryptococcus neoformans is a significant agent of life-threatening infections, particularly in people with suppressed cell-mediated immunity. The cellular cytotoxicity against C. neoformans infection is mainly mediated by NK and T cells, but effector mechanisms are not well understood. The objective of this study was (i) to determine whether prior exposure to the cryptococcal antigens enhances anticryptococcal activity of cytotoxic cells in mice and (ii) the contribution of perforin- and nonperforin-mediated cytotoxicity of NK and T cells in growth inhibition of C. neoformans. Our data showed that in vitro exposure of nonadherent (NA) spleen mononuclear cells from nonimmunized mice to heat-killed C. neoformans strain Cap67 unencapsulated mutant of B3501 (Ag1) or its supernatant (Ag2) demonstrated higher anticryptococcal activity. This effector mechanism can be enhanced further after immunization with either Ag1 or Ag2. There is a synergistic effect of immunization and in vitro incubation of the NA cells with the same antigens. Concanamycin A (CMA) and strontium chloride (SrCl2) inhibition assays were performed to clarify the contribution of perforin- and nonperforin-mediated anticryptococcal cytotoxicity of NA cells in these events. Treatment with these inhibitors demonstrated that anticryptococcal cytotoxicity of non-primed NA cells was primarily perforin mediated. Anticryptococcal activity of the NA cells obtained from immunized mice after in vitro incubation with cryptococcal antigens was both perforin and non-perforin mediated. Taken together these data demonstrate that in mice a nonperforin-mediated pathway of anticryptococcal cytotoxicity can be induced by immunization. Further research is needed to examine their potential role for human vaccines strategies and/or therapies.

Fungal exposure and low levels of IL-10 in patients with sarcoidosis.

Background and Objectives. Sarcoidosis is an inflammatory disease with increased levels of inflammatory cytokines. Previous studies have shown a relation between the degree of granuloma infiltration and serum cytokine levels, except for interleukin- (IL-) 10. The aim of the study was to further investigate the serum levels of IL-10 in patients with sarcoidosis and relate them to fungal exposure in terms of the amount of fungi in the air of their homes and β-glucan in bronchoalveolar lavage (BAL) fluid. Methods. Patients with sarcoidosis (n = 71) and healthy controls (n = 27) were enrolled. IL-10 was determined in serum. BAL was performed and the amount of β-glucan was measured. Domestic exposure to fungi was determined by measuring airborne β-N-acetylhexosaminidase (NAHA) in the bedrooms. Results. At high levels of fungal exposure (domestic fungal exposure and β-glucan in BAL), serum IL-10 values were lower than at low and intermediate exposure levels. Conclusion. The low serum IL-10 values at high fungal exposure suggest that fungal cell wall agents play a role in granuloma formation in sarcoidosis by inhibiting the secretion of the anti-inflammatory cytokine IL-10.

Fungal cell wall agents and bacterial lipopolysaccharide in organic dust as possible risk factors for pulmonary sarcoidosis

BackgroundComposition of organic dust is very complex, involving particles of microbial, animal and plant origin. Several environmental exposure studies associate microbial cell wall agents in organic dust with various respiratory symptoms and diseases. The aim of the present study was to investigate the in vitro effects of the co-exposure of fungal cell wall agents (FCWAs) and bacterial lipopolysaccharide (LPS) on inflammatory immune responses of peripheral blood mononuclear cells (PBMCs) from patients with pulmonary sarcoidosis.MethodsPBMCs from 22 patients with pulmonary sarcoidosis and 20 healthy subjects were isolated and stimulated in vitro with FCWAs (soluble and particulate (1 → 3)-β-D-glucan, zymosan and chitosan) and/or LPS. Subsequently, cytokines were measured by ELISA and the mRNA expression of dectin-1, toll-like receptor 2 (TLR2), TLR4 and mannose receptor (MR) was analysed by real-time RT-PCR.ResultsPatients with sarcoidosis had a significantly higher secretion of inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-10 and IL-12 (1.7-fold, 2.0-fold, 2.2-fold, and 2.8-fold, respectively; all p < 0.05) after in vitro co-stimulation of PBMCs with FCWAs and LPS. We showed that PBMCs from patients with sarcoidosis had a higher baseline mRNA expression of dectin-1, TLR2, TLR4 and MR (6-fold, 11-fold, 18-fold, and 4-fold, respectively). Furthermore, we found a reduced expression of dectin-1, TLR2 and TLR4 after stimulation with FCWAs and/or LPS, although the reduction was significantly weaker in patients than in healthy subjects.ConclusionsIn conclusion, co-stimulation with FCWAs and LPS of PBMC from patients with sarcoidosis caused a weaker reduction of dectin-1, TLR2, TLR4 receptors expression, which could increase the sensitivity of PBMCs, leading to excessive inflammatory cytokine responses and result in the development or progression of pulmonary sarcoidosis.