Unique patterns of CD8+ T-cell-mediated organ damage in the Act-mOVA/OT-I model of acute graft-versus-host disease.

T-cell receptor (TCR)-transgenic models of acute graft-versus-host disease (aGvHD) offer a straightforward and highly controlled approach to study the mechanisms and consequences of T-cell activation following allogeneic hematopoietic stem cell transplantation (aHSCT). Here, we report that aHSCT involving OT-I mice as donors, carrying an ovalbumin-specific CD8+ TCR, and Act-mOVA mice as recipients, expressing membrane-bound ovalbumin driven by the ß-actin promoter, induces lethal aGvHD in a CD8+ T-cell-dependent, highly reproducible manner, within 4-7 days. Tracking of UBC-GFP/OT-I graft CD8+ T cells disclosed heavy infiltration of the gastrointestinal tract, liver, and lungs at the onset of the disease, and histology confirmed hallmark features of gastrointestinal aGVHD, hepatic aGvHD, and aGvHD-associated lymphocytic bronchitis in infiltrated organs. However, T-cell infiltration was virtually absent in the skin, a key target organ of human aGvHD, and histology confirmed the absence of cutaneous aGVHD, as well. We show that the model allows studying CD8+ T-cell responses in situ, as selective recovery of graft CD45.1/OT-I CD8+ T cells from target organs is simple and feasible by automated tissue dissociation and subsequent cell sorting. Assessment of interferon-gamma production by flow cytometry, granzyme-B release by ELISA, TREC assay, and whole-genome gene expression profiling confirmed that isolated graft CD8+ T cells remained intact, underwent clonal expansion, and exerted effector functions in all affected tissues. Taken together, these data demonstrate that the OT-I/Act-mOVA model is suitable to study the CD8+ T-cell-mediated effector mechanisms in a disease closely resembling fatal human gastrointestinal and hepatic aGVHD that may develop after aHSCT using HLA-matched unrelated donors.

Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide.

Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.

Gene expression profiling of experimental asthma reveals a possible role of paraoxonase-1 in the disease.

In this study, we aimed to identify novel genes involved in experimental and human asthma, importance of which has not yet been recognized. In an ovalbumin-induced murine model of asthma, we applied microarray gene expression analysis at different time points after allergen challenges. Advanced statistical methods were used to relate gene expression changes to cellular processes and to integrate our results into multiple levels of information available in public databases. At 4 h after the first allergen challenge, gene expression pattern reflected mainly an acute, but non-atopic, inflammatory response and strong chemotactic activity. At 24 h after the third allergen challenge, gene set enrichment analysis revealed significant over-representation of gene sets corresponding to T(h)2-type inflammation models. Among the top down-regulated transcripts, an anti-oxidant enzyme, paraoxonase-1 (PON1), was identified. In human asthmatic patients, we found that serum PON1 activity was reduced at exacerbation, but increased parallel with improving asthma symptoms. PON1 gene polymorphisms did not influence the susceptibility to the disease. Our observations suggest that an altered PON1 activity might be involved in the pathogenesis of asthma, and serum PON1 level might be used for following up the effect of therapy.

IL-18 induces a marked gene expression profile change and increased Ccl1 (I-309) production in mouse mucosal mast cell homologs.

Helminthic infections, which are particularly common in the developing world, are associated with the accumulation of mucosal mast cells (MMCs) in the epithelial layer of the gut. Although intestinal parasite infection models argue that IL-18 plays a role in MMC differentiation and function, the direct effect of IL-18 on MMCs is still not well understood. To clarify the role of IL-18 in mast cell biology, we analyzed gene expression changes in MMCs in vitro. DNA microarray technology uncovered a group of chemokines regulated by IL-18, among which Ccl1 (I-309, TCA-3) showed the highest up-regulation. Ccl1 induction was only transient in mast cells and was characteristic for both immature and mature MMCs, but not for connective tissue-type mast cells. IL-18 exerts its Ccl1-inducing effect in MMCs primarily via the activation of NFkappaB. Moreover, IL-18 was effective both in the absence and the presence of IgE-antigen complex. The Ccl1 receptor (CCR8) is known to be expressed by T(h)2 cells and is involved in their recruitment. Our present findings suggest that IL-18 may contribute to mast cell-influenced Th2 responses by inducing Ccl1 production.

Decreased expression of histamine H1 and H4 receptors suggests disturbance of local regulation in human colorectal tumours by histamine.

Production of histamine in colon tumours has been described earlier. Histamine-mediated signals have been shown to be implicated in tumour growth, and the effects of histamine are largely determined locally by the histamine receptor expression pattern. We analysed histamine receptor expression in human colorectal cancer, adenoma and normal mucosa by quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunostaining. Real-time RT-PCR results revealed significantly decreased (p<0.001) H1R and H4R mRNA levels in tumours compared to normal colonic mucosa, without any significant change in H2R mRNA expression. H3R was absent in most samples; it was detected at low levels in 7.9% of the cases. Protein analysis showed a similar decrease in histamine receptor expression in carcinoma and adenoma compared to normal mucosa controls. Based on these results, we performed further Western blot analysis on Dukes-classified and -selected tumour samples. We found significantly decreased H4R levels in neoplastic samples compared to normal colonic tissue, but there was no significant correlation between histamine receptor expression profile and the Dukes stage of tumours. Immunohistochemical staining revealed expression patterns of H1R, H2R and H4R similar to those suggested by the mRNA and Western blot results. In the present study, we demonstrate that H1R, H2R and H4R are expressed in colon carcinoma and the adjacent normal mucosa. The results suggest a dramatic alteration in the distribution of histamine receptors in colon cancer. These findings raise the perspective of targeted pharmacological studies with selective histamine receptor antagonists or agonists in the therapy of colorectal tumours.

Locally generated VGVAPG and VAPG elastin-derived peptides amplify melanoma invasion via the galectin-3 receptor.

Melanomas containing more elastin are associated with higher stages of the disease. The interaction between elastin-derived peptides and melanoma cells appears to play an important role in the progression of melanomas. The effects of the elastin-derived peptides VGVAPG and VAPG have been investigated on the migration, invasion, adhesion and angiogenesis of human melanoma cells of different invasive potential. Elastin, tropoelastin and VGVAPG peptide were demonstrated at the invasion site of melanoma using histochemistry and immunohistochemistry. Not only the VGVAPG elastin-derived peptide, which exhibits the XGXXPG consensus sequence in its primary structure, but also the shorter VAPG bind directly to 3 cell surface receptors: galectin-3, integrin alpha v beta 3 and elastin-binding protein. Our results suggest that the increased levels of elastin-derived peptides facilitate the invasion of melanoma cells: (i) VGVAPG and VAPG elastin-derived peptides are chemotactic for melanoma cells; (ii) they can increase the migration of melanoma cells and the expression of CXCR-4 and CXCL-12 chemokines; (iii) they enhance the expression of the elastin-degrading MMP-2 and MMP-3; (iv) they increase the attachment of melanoma cells and the expression of different adhesion molecules; (v) they increase the expression of the lymphangiogenic VEGF-C and (vi) the galectin-3 receptor can mediate all these effects. Clinical and therapeutic aspects are also discussed.

HtrA1 is a novel mast cell serine protease of mice and men.

Mast cells are rich sources of proteases, such as tryptases and chymases that control many physiological and pathological processes, for example vascular permeability, smooth muscle cell proliferation or extracellular matrix remodeling. Murine mucosal mast cells mature under the influence of TGF-beta and play a role in asthmatic and anti-helminthic immune responses. In an attempt to identify novel genes that are highly upregulated during mucosal mast cell differentiation, we detected HtrA1 protease as a novel protein in mast cells by microarray experiments. HtrA1 level was much higher in murine mucosal than in connective tissue-type mast cells. Furthermore, HtrA1 is not localized in the secretory granules and is constitutively secreted by human mast cells. Although HtrA1 has been attributed a TGF-beta-inhibitory activity, it did not show any influence on TGF-beta-induced mucosal mast cell differentiation. As many extracellular target proteins have been suggested for HtrA1, this protease may participate in the mast cell-induced extracellular remodeling.

[Plasmacytic skin infiltration in multiple myeloma]. / Myeloma multiplex kórlefolyása során megjelenô plazmasejtes bôrinfiltráció.

UNLABELLED: Authors present a case of a therapy-resistant multiple myeloma who developed plasmacytic skin infiltration in the course of the disease. AIM: To define characteristics of skin infiltrating plasma cells, which differentiate them from those cells residing in the bone marrow in order to contribute to a better understanding of the epidermoinvasion process. METHODS: Histidine decarboxylase is the only enzyme capable for histamine synthesis having significance in cell proliferation. Histidine decarboxylase was determined in skin samples and bone marrow slides by immunohistochemical procedures and in bone marrow cells using flow cytometry analysis. RESULTS: The histidine decarboxylase expression of plasma cells participating in skin invasion disappeared, while that of bone marrow plasma cells remained. CONCLUSIONS: Authors conclude that the histidine decarboxylase loss would serve as an evidence for the dedifferentiation of epidermoinvasive cells as being the result of fundamental changes in histamine metabolism. As extramedullary myeloma cells differ from those residing in the bone marrow, their therapeutical response might also be different.

Drug penetration model of vinblastine-treated Caco-2 cultures.

The penetrability of new chemical entities (NCE) is routinely screened in preclinical drug research. Although Caco-2 is a well-established model for human absorption, the identification of P-glycoprotein (P-gp) substrates and therefore the predictive accuracy of this model is not always satisfactory. Vinblastine has been reported to affect P-gp expression in Caco-2 cells. Therefore, this study was intended to assess the effect of sustained vinblastine treatment on the expression of P-gp, using RT-PCR and Western blot techniques. The P-gp functionality was monitored in transport assay, and metabolic enzyme activities were studied using probe substrates. Completion of culture medium with vinblastine (10nM during both the growing and the differentiation period) increased the P-gp mRNA and the expression at protein level. These changes were associated with the sensitive and steady identification of P-gp substrates in the bidirectional transport assay. While the vinblastine-treated Caco-2 (VB-Caco-2) based model reliably identified the P-gp substrates, the native Caco-2 model failed to recognize 7 out of the 11 reference substrates. The penetrability of passively permeating compounds correlated strongly (r(2)=0.9830) in the two models as expected. Omitting vinblastine from established VB-Caco-2 cultures did not affect either the protein level or the functionality of P-gp. Vinblastine did not alter the CYP mediated activities of the cells either. The higher sensitivity of VB-Caco-2 culture is also supported by the test results of NCEs, where 37% of NCEs were found to be P-gp substrate in VB-Caco-2 verified by verapamil, but only 9% by native Caco-2.

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts.

INTRODUCTION: Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints. METHODS: Expressions of beta-D-hexosaminidase, beta-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. beta-D-Glucuronidase, beta-D-glucosaminidase and beta-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity. RESULTS: According to our data, beta-D-hexosaminidase, beta-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the beta-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-beta1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFalpha, IL-1beta and IL-17. CONCLUSIONS: According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.

Differences in the expression of histamine-related genes and proteins in normal human adrenal cortex and adrenocortical tumors.

Histamine is involved in the pathogenesis of several tumors; however, there are no data on its possible involvement in human adrenocortical tumorigenesis. The expression of genes and proteins involved in the biosynthesis (histidine decarboxylase, HDC), action (histamine receptors: HRH1-HRH4), and metabolism of histamine is largely unknown both in the normal human adrenal cortex and in adrenocortical tumors. In this study, we examined the expression of histamine-related genes and proteins and histamine content in normal adrenal cortex, benign adrenocortical adenomas, and malignant adrenocortical cancer (ACC). Fifteen normal adrenals and 43 tumors were studied. mRNA expression was examined by real time RT-PCR. Western-blotting and immunohistochemistry were used for the study of proteins. Tissue histamine content was determined by enzyme-linked immunosorbent assay. We found that all proteins involved in histamine biosynthesis and action are present both in the normal adrenal cortex and in the tumors studied. HDC expression and histamine content was highest in the normal tissues and lower in benign tumors, whereas it was significantly less in ACCs. HRH3 expression was significantly higher in ACC samples than in the other groups. Adrenocortical tumorigenesis might, thus, be characterized by reduced histamine biosynthesis; furthermore, different adrenocortical tumor subtypes may show unique histamine receptor expression profiles.

Histamine suppresses fibulin-5 and insulin-like growth factor-II receptor expression in melanoma.

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis.

INTRODUCTION: Although natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage. METHODS: To measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers. RESULTS: While anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis. CONCLUSION: The highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.

TIM-3 is expressed in melanoma cells and is upregulated in TGF-beta stimulated mast cells.

Many studies detect elevated numbers of mast cells in tumors, but it is still controversial whether they are beneficial or detrimental for tumor cells. Furthermore, many tumors, such as melanomas, produce large quantities of transforming growth factor (TGF)-beta and during tumorigenesis the apoptotic and growth-inhibitory effects of TGF-betas are lost. Based on these data we investigated the gene expression changes in TGF-betaI-treated human mast cells with DNA microarray and detected 45 differentially regulated genes, among them T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). As the major sources of TIM-3 ligand galectin-9 are not tumor cells, but rather mast cells, this raises the possibility of an autocrine mechanism resulting in local immunosuppression through the elevated TIM-3 expression by TGF-betaI. Interestingly, not only melanoma tissue sections contained TIM-3-positive mast cells, but we detected this protein also in melanoma cells. Furthermore, TIM-3 was expressed in both WM35 and HT168-M1 melanoma cell lines at a higher level than in isolated epidermal melanocytes, which can contribute to the lower adhering capacity of tumor cells. In conclusion, the immunoregulatory molecule TIM-3 in TGF-beta-stimulated mast cells and melanoma cells may support the survival of this tumor type.