Sequential Increase in Complement Factor I, iC3b, and Cells Expressing CD11b or CD14 in Cutaneous Vasculitis.

Mast cells contribute to the pathogenesis of cutaneous vasculitis through complement C3 that is cleaved to C3b and then to iC3b by complement factor I. The receptor of iC3b, CD11b, is expressed on neutrophils and monocytes and CD14 on monocytes. Their role in vasculitis is obscure. In this study, frozen skin biopsies from the nonlesional skin, initial petechial lesion, and palpable purpura lesion from 10 patients with immunocomplex-mediated small vessel vasculitis were studied immunohistochemically for complement factor I, iC3b, CD11b, and CD14. Peripheral blood mononuclear cells from 5 healthy subjects were used to study cell migration and cytokine secretion. Already, the nonlesional skin revealed marked immunostaining of complement factor I, iC3b, CD11b, and CD14, and their expression increased sequentially in initial petechial and palpable purpura lesions. Mast cell C3c correlated to iC3b, and both of them correlated to CD11b+ and CD14+ cells, in the nonlesional skin. The stimulation of mononuclear cells with 0.01-0.1 µg/ml iC3b induced cell migration in the transwell assay. C3a stimulated slightly interleukin-8 secretion, whereas 1 µg/ml iC3b inhibited it slightly, in 4/5 subjects. In conclusion, the C3-C3b-iC3b axis is activated already in the early vasculitis lesion leading to progressive accumulation of CD11b+ and CD14+ cells.

Cardiomyopathy associated with the Ala143Thr variant of the α-galactosidase A gene.

OBJECTIVE: To investigate whether the Ala143Thr variant of the α-galactosidase A gene (A143T/GLA), with conflicting interpretations of pathogenicity, is associated with Fabry cardiomyopathy. METHODS: The index patient, a woman in her 60s with cardiomyopathy, was screened for variants in 59 cardiomyopathy-related genes. A143T/GLA, the only rare variant found, was screened in 10 relatives. GLA activity and lyso-Gb3 levels were measured and echocardiography was performed in 8 of 9 subjects carrying A143T/GLA. Cardiac magnetic resonance (CMR) imaging and 18F-fluorodeoxyglucose (FDG) positron emission tomography/CT (PET/CT) were performed in four adult A143T/GLA carriers. Endomyocardial biopsy was obtained from two adult A143T/GLA carrying sons of the index patient. RESULTS: The index patient and her elder son had a pacemaker implantation because of sick sinus syndrome and atrioventricular block. GLA activities were decreased to 25%-40% of normal in both sons and one granddaughter. Lyso-Gb3 levels were elevated in both sons. In CMR, the index patient and her two sons had left ventricular (LV) hypertrophy and/or dilatation. The elder son had late gadolinium enhancement, high CMR-derived T1 time and positive FDG signal in PET/CT in the basal inferolateral LV wall. The younger son had low T1 time and the mother had positive FDG signal in PET/CT in the basal inferolateral LV wall. Endomyocardial biopsy of both sons showed myocardial accumulation compatible with glycolipids in light and electron microscopy, staining with anti-Gb3 antibody available for the younger son. Five female relatives with A143T/GLA had no cardiomyopathy in cardiac imaging. CONCLUSIONS: A143T/GLA is likely a late-onset Fabry cardiomyopathy causing variant with incomplete penetrance.

Mast cell CD30 ligand is upregulated in cutaneous inflammation and mediates degranulation-independent chemokine secretion.

Mast cells are involved in many disorders where the triggering mechanism that leads to degranulation and/or cytokine secretion has not been defined. Several chronic inflammatory diseases are associated with increased mast cell numbers and upregulation of the TNF receptor family member CD30, but the role of elevated CD30 expression is poorly understood. Here we report what we believe to be a novel way to activate mast cells with CD30 that leads to degranulation-independent secretion of chemokines. CD30 induced a de novo synthesis and secretion of the chemokines IL-8, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, a process involving the MAPK/ERK pathway. Mast cells were found to be the predominant CD30 ligand-positive (CD30L-positive) cell in the chronic inflammatory skin diseases psoriasis and atopic dermatitis, and both CD30 and CD30L expression were upregulated in lesional skin in these conditions. Furthermore, the number of IL-8-positive mast cells was elevated both in psoriatic and atopic dermatitis lesional skin as well as in ex vivo CD30-treated healthy skin organ cultures. In summary, characterization of CD30 activation of mast cells has uncovered an IgE-independent pathway that is of importance in understanding the entirety of the role of mast cells in diseases associated with mast cells and CD30 expression. These diseases include Hodgkin lymphoma, atopic dermatitis, and psoriasis.

Cervical squamous carcinoma cells are resistant to the combined action of tumor necrosis factor-alpha and histamine whereas normal keratinocytes undergo cytolysis.

BACKGROUND: Previous reports showed that mast cells can typically be found in the peritumoral stroma of cervix carcinomas as well as in many other cancers. Both histamine and TNF-alpha are potent preformed mast cell mediators and they can act simultaneously after release from mast cells. Thus, the effect of TNF-alpha and histamine on cervical carcinoma cell lines was studied. METHODS AND RESULTS: TNF-alpha alone induced slight growth inhibition and cell cycle arrest at G0/G1 phase in SiHa cells, but increased their migration. Histamine alone had no effect on cells. In addition, TNF-alpha and histamine in combination showed no additional effect over that by TNF-alpha alone, although SiHa cells were even pretreated with a protein synthesis inhibitor. Furthermore, TNF-alpha-sensitive ME-180 carcinoma cells were also resistant to the combination effect of TNF-alpha and histamine. In comparison, TNF-alpha or histamine alone induced growth inhibition in a non-cytolytic manner in normal keratinocytes, an effect that was further enhanced to cell cytolysis when both mediators acted in combination. Keratinocytes displayed strong TNF receptor (TNFR) I and II immunoreactivity, whereas SiHa and ME-180 cells did not. Furthermore, cervix carcinoma specimens revealed TNF-alpha immunoreactivity in peritumoral cells and carcinoma cells. However, the immunoreactivity of both TNFRs was less intense in carcinoma cells than that in epithelial cells in cervical specimens with non-specific inflammatory changes. CONCLUSION: SiHa and ME-180 cells are resistant to the cytolytic effect of TNF-alpha and histamine whereas normal keratinocytes undergo cytolysis, possibly due to the smaller amount of TNFRs in SiHa and ME-180 cells. In the cervix carcinoma, the malignant cells may resist this endogenous cytolytic action and TNF-alpha could even enhance carcinoma cell migration.

Hyaluronic acid inhibits the adherence and growth of monolayer keratinocytes but does not affect the growth of keratinocyte epithelium.

Hyaluronic acid (HA) is involved in epidermal biology but evidence for its functional significance is sparse. In this study, low-calcium monolayer and high-calcium epithelium cultures of human keratinocytes were used to study the effect of up to four different HA preparations on keratinocyte growth and on the adherence of proliferating keratinocytes onto the plastic surface coated with different matrix proteins. In suboptimally growing monolayer culture, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited keratinocyte growth. Instead, all HA preparations tested did not affect the growth and migration of keratinocyte epithelium using optimal or suboptimal growth conditions. In the cell adherence assays, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited the keratinocyte adherence onto the fibronectin- and collagen-coated substratum. In contrast to other HA preparations, HA from human umbilical cord did not affect the growth of monolayer keratinocytes and it increased markedly the cell adherence onto the collagen-coated substratum. This increase, however, can be attributed to chonroitin sulphate proteoglycan contaminant present in this HA preparation. In conclusion, HA can inhibit the growth and adherence of proliferating monolayer keratinocytes, but it has no apparent effect on the growth and migration of keratinocyte epithelium.

Current perspectives in hypertrophic cardiomyopathy with the focus on patients in the Finnish population: a review.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, with the prevalence of about 1/500. During the last two decades, the knowledge of the etiology, pathogenesis, risk stratification and prevention of sudden death in HCM has substantially advanced. Most often, HCM is familial and caused by mutations in sarcomere genes, inherited in an autosomal dominant manner. In Finland, genetic background of HCM is unique, with a few founder mutations in cardiac sarcomere genes accounting for a considerable proportion of the disease. Pathogenic mechanisms induced by disease-causing mutations are still poorly understood, although alterations in intracellular calcium handling and inefficient generation of contractile force in myocytes are considered key features in triggering the hypertrophic response. Clinical features of the disease are highly variable from no symptoms to the spectrum of exertional dyspnea, angina, palpitations, syncope and sudden death. In the current patient care, implantable cardioverter defibrillators (ICDs) are successfully used to prevent sudden cardiac death in high risk subjects. Targeted genetic testing is recommended to confirm the diagnosis in patients with HCM and to identify family members with the disease. Future research is needed to elucidate key cellular mechanisms leading to HCM, which may allow specific prevention and treatment of the disease. Key messages Hypertrophic cardiomyopathy, most often caused by defects in sarcomere genes, is the most common inherited heart disease, and a common cause of sudden cardiac death (SCD) in athletes and young subjects. Cardiac imaging, ECG and genetic testing are pivotal in the diagnosis of the disease in patients and first-degree relatives. Implantable cardioverter defibrillators in patients with high risk for SCD and tailored pharmacotherapy are efficient tools in patient care, but so far, exact mechanisms leading to cardiac hypertrophy in HCM are only partially understood, and there is no curative treatment for the disease.

Complement C3 is expressed by mast cells in cutaneous vasculitis and is degraded by chymase.

The complement factor C3 and chymase released from tryptase(+), chymase(+) mast cells may be involved in the pathogenesis of cutaneous leukocytoclastic vasculitis. To study whether mast cells contain C3 in vasculitis and whether chymase interacts with C3, cryosections from vasculitis biopsies were double-stained histochemically for C3c in tryptase(+) mast cells, as well as for chymase and vessel wall C3c, or they were treated with 5 µg/ml rh-chymase for 24 h followed by immunofluorescence (IF) analysis of C3c, IgG, IgM and IgA. The effect of rh-chymase on purified human C3, C3a and IgG was studied using SDS-PAGE electrophoresis and LAD2 mast cell cultures. The results show that 34.2 ± 17.9, 37.4 ± 15.5 and 43.4 ± 18.6 % (mean ± SD) of the mast cells express C3c immunoreactivity in the healthy skin, initial petechial (IP) and palpable purpura (PP) lesions, respectively. About 9.4-12.1 % of the chymase(+) mast cells were in apparent contact with C3c(+) vessels in IP and PP. The treatment of cryosections with rh-chymase decreased the IF staining of C3c, but not that of immunoglobulins. In SDS-PAGE, 1-10 µg/ml rh-chymase degraded the alpha- and beta-chains of C3, but did not degrade IgG. Unexpectedly, the rh-chymase treatment of C3 produced fragments that resulted in the release of tryptase and histamine from LAD2 cells. However, rh-chymase degraded C3a and consequently inhibited C3a activity on LAD2. In conclusion, mast cells can be one source for C3 in the early and late phases of vasculitis pathogenesis. However, rh-chymase degraded native C3, vessel wall C3c, and biologically active C3a. Therefore, chymase may control C3-related pathology.

Mast cell tryptase and chymase in the progress of cutaneous vasculitis.

In animal models of vasculitis, mast cells are essential in the pathogenesis, but their involvement in human skin vasculitis is obscure. Because tryptase and chymase are potent serine proteinases in the secretory granules of mast cells, the purpose was to examine the number of mast cells expressing tryptase and chymase during the progress of cutaneous vasculitis. These numbers were correlated with the appearance of immunoreactants (C3c, fibrin, IgM, IgA and IgG) in vessel walls. For this, skin biopsies were taken from the healthy-looking skin, initial petechial lesion (IP), and palpable purpura (PP) of the leg of patients with leukocytoclastic vasculitis (n = 10). The frozen biopsies were analysed using enzyme- and immunohistochemistry and direct immunofluorescence (IF) staining. The results show that there are no marked changes in the numbers of mast cells expressing chymase or tryptase proteins. Instead, chymase enzyme activity decreased, but the score of α1-antichymotrypsin staining increased, during the progress of vasculitis. The IF positivity of fibrin correlated positively with chymase activity (p = 0.01) and the ratio of chymase activity to tryptase protein in IP (p = 0.03), as well as with mast cells showing tryptase (p = 0.03) and chymase (p = 0.01) proteins in PP. The IF positivity of C3c correlated with the ratio of chymase activity to tryptase protein in IP (p = 0.01). In conclusion, chymase is partially inactivated in vasculitis possibly due to α1-antichymotrypsin. Several positive correlations between chymase and fibrin and/or C3c in IP or PP suggest that this enzyme is involved in the deposition of immunoreactants in the vessel wall.

Mast cells, nerves and neuropeptides in atopic dermatitis and nummular eczema.

The association between mast cells and sensory nerves and the distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) were studied immunohistochemically in lesional and nonlesional skin of 26 atopic dermatitis (AD) and 23 nonatopic nummular eczema (NE) patients. Mast cell-nerve contacts were counted morphometrically and confirmed by confocal laser scanning microscopy. Neuropeptide positivity was assessed semiquantitatively. Dermal contacts between mast cells and nerves were increased in number in both lesional and nonlesional samples of AD and NE when compared to those in normal controls, although only the values in lesional AD reached statistical significance ( P<0.05). Nerve-mast cell contacts in the basement membrane zone were seen practically only in lesional NE. SP and CGRP fibres were prominently increased in lesional samples when compared to their nonlesional controls both in AD and NE in the epidermis and in the papillary dermis. In both AD and NE, only small differences were found regarding VIP positivity in lesional and nonlesional biopsies. The epidermis was devoid of VIP positivity. In conclusion, SP and CGRP but not VIP fibres were more frequent in lesional than in nonlesional papillary dermis of both AD and NE. Since mast cells are also increased in number in lesions of AD and NE, they are able to maintain neurogenic inflammation through activation by SP and CGRP. The increased SP/CGRP nerves in the epidermis of AD and NE lesions may stimulate keratinocytes to release cytokines which affect various cell types enhancing inflammation.

Transient production of stem cell factor in dermal cells but increasing expression of Kit receptor in mast cells during normal wound healing.

Mast cells are involved in inflammatory skin disorders and wound healing processes, but the mechanism behind mast cell activation is obscure. In this study, we stained the stem cell factor (SCF) and the Kit receptor in tryptase-positive mast cells, since these molecules are essential for mast cell survival, growth, migration and activation. For this purpose, biopsies were taken from the edge of normally healing wounds of 12 patients undergoing skin transplantation on days 0, 1, 3, 7 and 14, and from chronic leg ulcers and psoriatic skin for comparison. In healing wounds, SCF-positive cells rapidly increased in number in the dermis peaking on day 1, but declined thereafter to their baseline values. The percentage of Kit-positive mast cells increased slowly but steadily reaching a maximum (73+/-22%, P=0.02) on day 14. In chronic ulcers, most of the mast cells were Kit-positive both in the wound bed and in the perilesional skin (87+/-9% and 86+/-13%, respectively). The number of SCF-positive cells was higher in the wound bed than in the dermis of perilesional skin. In the psoriatic skin of ten patients, lesional specimens showed significantly higher numbers of SCF-positive dermal cells as well as a higher percentage (88+/-12% vs 46+/-26%, P=0.004) of Kit-positive mast cells than nonlesional skin. In conclusion, our findings show that the expression of SCF increases rapidly in the early stages of wound healing but declines thereafter, whereas the expression of Kit in mast cells is induced slowly in healing wounds. In chronic wounds as well as in psoriatic lesions, both SCF and Kit are intensely expressed. Thus, it seems possible that SCF and Kit receptor interact, and this could lead to persistent mast cell activation and growth in chronic wounds and psoriasis, whereas only temporary mast cell activation is apparently needed in healing wounds.

Low-grade inflammation and the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy.

OBJECTIVE: To investigate the role of inflammation in the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy (HCM). DESIGN: Clinical study. SETTING: Kuopio University Hospital and University of Eastern Finland, Kuopio, Finland. SUBJECTS: Twenty-four patients with a single HCM-causing mutation D175N in the α-tropomyosin gene and 17 control subjects. MAIN OUTCOME MEASURES: Endomyocardial biopsy samples taken from the patients with HCM were compared with matched myocardial autopsy specimens. Levels of high-sensitivity C-reactive protein (hsCRP) and proinflammatory cytokines were measured in patients and controls. Myocardial late gadolinium enhancement (LGE) in cardiac MRI (CMRI) was detected. RESULTS: Endomyocardial samples in patients with HCM showed variable myocyte hypertrophy and size heterogeneity, myofibre disarray, fibrosis, inflammatory cell infiltration and nuclear factor kappa B (NF-κB) activation. Levels of hsCRP and interleukins (IL-1ß, IL-1RA, IL-6, IL-10) were significantly higher in patients with HCM than in control subjects. In patients with HCM, there was a significant association between the degree of myocardial inflammatory cell infiltration, fibrosis in histopathological samples and myocardial LGE in CMRI. Levels of hsCRP were significantly associated with histopathological myocardial fibrosis. hsCRP, tumour necrosis factor α and IL-1RA levels had significant correlations with LGE in CMRI. CONCLUSIONS: A variable myocardial and systemic inflammatory response was demonstrated in patients with HCM attributable to an identified sarcometric mutation. Inflammatory response was associated with myocardial fibrosis, suggesting that myocardial fibrosis in HCM is an active process modified by an inflammatory response.

Mast cell chymase is present in uterine cervical carcinoma and it detaches viable and growing cervical squamous carcinoma cells from substratum in vitro.

Increased numbers of mast cells is a typical feature of a variety of human cancers. The major mediators in the secretory granules of the MC(TC) type of mast cells, serine proteinases tryptase and chymase, may be involved in squamous cell carcinoma (SCC) lesions by inducing matrix remodeling and epithelial cell detachment. The objective of this study was to analyze immunohistochemically whether MC(TC) mast cells as well as protease inhibitors, squamous cell carcinoma antigens (SCCAs), are present in the uterine cervical SCC. In addition, the effect of tryptase and chymase on uterine cervical SCC cell lines was studied in vitro. Here we report that tryptase- and chymase-positive mast cells are present in significant numbers in the peritumoral stroma of SCC lesions. Also, weak SCCA-2 immunoreactivity is observed in the SCC lesions, but only SCCA-1 in uterine cervical specimens with nonspecific inflammation. In cell cultures, especially chymase, but not tryptase, was shown to induce effective detachment of viable, growing and non-apoptotic SiHa SCC cells from substratum. Chymase also detached viable ME-180 SCC cells from substratum as well as degraded fibronectin. In contrast, normal keratinocytes underwent apoptotic cell death after similar prolonged chymase treatment. No inhibition of chymase was detected by SiHa cell sonicates nor did these cells express marked SCCA immunopositivity. MC(TC) mast cells containing tryptase and chymase are present in the peritumoral stroma of uterine cervical SCC and the malignant cells are only weakly immunoreactive for the chymase inhibitor SCCA-2. It is chymase that appears to be capable of inducing effective detachment of viable and growing SCC cells and therefore, it may release SCC cells from a tumor leading to spreading of malignant cells.

Is there a role for mast cells in psoriasis?

Mast cells have traditionally been considered as effector cells in allergy but during the last decade it has been realized that mast cells are essentially involved in the mechanisms of innate and acquired immunity. Upon activation by anaphylactic, piecemeal degranulation or degranulation-independent mechanisms mast cells can secrete rapidly or slowly a number of soluble mediators, such as serine proteinases, histamine, lipid-derived mediators, cytokines, chemokines and growth factors. Mast cells can express cell surface co-stimulatory receptors and ligands, and they can express MHC class II molecules and thereby present antigens. These soluble factors and cell surface molecules can interact with other cells, such as endothelial cells, keratinocytes, sensory nerves, neutrophils, T cell subsets and antigen presenting cells which are essential effectors in the development of skin inflammation. Besides promoting inflammation, mast cells may attempt in some circumstances to suppress the inflammation and epidermal growth but the regulation between suppressive and proinflammatory mechanisms is unclear. Psoriasis is characterized by epidermal hyperplasia and chronic inflammation where tryptase- and chymase-positive MC(TC) mast cells are activated early in the developing lesion and later the cells increase in number in the upper dermis with concomitant expression of cytokines and TNF superfamily ligands as well as increased contacts with neuropeptide-containing sensory nerves. Due to the intimate involvement of mast cells in immunity and chronic inflammation the role of mast cells in psoriasis is discussed in this review.

Congenital Langerhans cell histiocytosis mimicking a "blueberry muffin baby".

Congenital Langerhans cell histiocytosis (LCH) is a rare condition with great diversity. A case of congenital skin-only LCH presenting as a "blueberry muffin baby" with a spontaneous regression by the age of 8 months is reported here. New insights into clinical manifestations and prognosis, which is not uniformly positive, are discussed. A thorough examination and a careful follow-up should be provided to these patients. Systemic therapy is warranted in multi-system disease; no consensus on treatment exists in case of LCH isolated to skin. The diagnosis of congenital self-healing LCH should be made only retrospectively.

Increase in CD30 ligand/CD153 and TNF-alpha expressing mast cells in basal cell carcinoma.

Mast cells are a significant source of tumor necrosis factor (TNF) superfamily members, such as TNF-alpha, CD30 ligand/CD153 (CD30L) and CD40L/CD154. Furthermore, the expression of some of these proteins in mast cells has been associated with tumorigenesis, and mast cells have been found to be increased in number in the basal cell carcinoma (BCC) lesion. In this study, we have examined the expression of TNF-alpha, CD30L and CD40L immunoreactivity in mast cells in the healthy-looking skin and lesional skin of ten patients with superficial spreading BCC. Also, the counterparts of these molecules, TNF receptor (TNFR) I and II as well as CD30 and CD40, were analysed immunohistochemically. We found that numbers of mast cells and Kit-positive cells were significantly increased in the dermal BCC lesion. The percentage of CD30L-positive mast cells and the number of CD30-positive cells were significantly increased in the upper dermis of the BCC lesion as well. In addition, the numbers of TNF-alpha-positive mast cells and cells with TNFRI and TNFRII were markedly increased in the upper lesional dermis. In contrast, no mast cells positive for CD40L could be detected, even though the lesional dermis contained increased numbers of CD40 positive cells. The BCC epithelium was positive for TNFRI, TNFRII and CD40, but not for CD30, though the larger basal buds appeared to be less intensely stained for TNFRI and CD40. In conclusion, mast cells positive for CD30L and TNF-alpha, but not CD40L, are increased in number in the lesional dermis in BCC. These data suggest plausible pathways whereby mast cells can be activated and to interact with other cells and thereby contribute to the tumorigenesis in BCC.

Gene dose effect of the DQB1*0201 allele contributes to severity of coeliac disease.

OBJECTIVE: Coeliac disease (CD) susceptibility has been shown to be associated with the HLA alleles DQA1*0501 and DQB1*0201. This HLA-associated risk has been estimated to account for 29-40% of the genetic component of CD. Conflicting data have been published on the gene dose effect of these HLA alleles on the risk and severity of CD. In this study the aim was to investigate the association between the number of HLA risk alleles and the severity of CD. MATERIAL AND METHODS: Fifty-four Finnish CD families, including 144 CD patients mainly diagnosed in adulthood (94.4%), were enrolled in the study. The association between the number of DQA1*0501 and DQB1*0201 alleles and villous atrophy, symptoms and laboratory parameters at the time of diagnosis, and the association with villous atrophy after one year of treatment on a gluten-free diet were studied. RESULTS: The homozygosity for the DQB1*0201 allele was associated with a more severe form of CD assessed by more severe villous atrophy (p=0.011), younger age (p=0.036), more severe diarrhoea (p=0.048) and a lower level of blood haemoglobin at the time of diagnosis (p=0.010). Furthermore, the homozygosity for the DQB1*0201 allele was associated with a slower recovery of villous atrophy after a gluten-free diet (p=0.041). In contrast, the DQA1*0501 allele did not have a significant association with the severity of CD. CONCLUSIONS: Our results demonstrate a gene dose effect of the DQB1*0201 allele on the clinical heterogeneity of CD and on the rate of recovery from villous atrophy in patients on a gluten-free diet.

HLA genotyping is useful in the evaluation of the risk for coeliac disease in the 1st-degree relatives of patients with coeliac disease.

OBJECTIVE: Coeliac disease (CD) is a common disease with a strong heredity. About 10-20% of 1st-degree relatives of probands develop CD. Relatives should be screened for CD, because if not treated, CD exposes patients to numerous complications. The heterogeneity of symptoms and the lifetime-spanning risk of CD render the timing of CD antibody and/or gastroscopy screenings difficult. As CD susceptibility has been shown to be strongly associated with the HLA alleles DQA1*0501 and DQB1*0201 (together encoding the DQ2 heterodimer) and DRB1*04 (associated with the DQ8 heterodimer), our aim was to investigate whether HLA genotyping might be useful in the identification of 1st-degree relatives of CD patients who do not need further screening for CD. MATERIAL AND METHODS: The study comprised 54 Finnish CD families including 54 CD probands and 382 living 1st-degree relatives. All subjects who were willing to participate were screened for CD (duodenal and skin biopsies; endomysial, reticulin and gliadin antibodies). The DQA1*0501, DQB1*0201 and DRB1*04 allele frequencies of CD patients and the 1st-degree relatives were determined. RESULTS: Altogether 17.6% (5.9% of the parents, 15.7% of the siblings, 25.8% of the offspring) of the investigated 1st-degree relatives (n = 245) did not carry any of the alleles studied. All of the CD patients (n = 136) with the exception of one (0.7%) carried at least one of the alleles investigated. CONCLUSIONS: By using the HLA genotyping a considerable proportion of 1st-degree relatives of CD probands could be excluded from further screening for CD.

Mast cell survival and apoptosis in organ-cultured human skin.

Mast cells accumulate and persist predominantly in the upper dermis of the skin but the mechanism for this is obscure. The skin is normally exposed to external air, which is essential for the maturation of the epidermis and probably also the dermis. In order to clarify the importance of air exposure on dermal mast cells, skin organ culture at the air-liquid interface (ALI) and submerged (SM) in medium (10% fetal calf serum and Dulbecco's modification of Eagle's medium) was used to study changes in tryptase-, chymase- and Kit-positive mast cell numbers during cultivation for up to 14 days. In addition, possible apoptosis (TACS TdT in situ apoptosis detection method) in chymase-positive mast cells was studied during the culture. In the less-physiologic SM culture, the number of Kit-positive mast cells decreased rapidly on day 1-2 and tryptase-positive cells decreased markedly on day 14. This decrease in mast cell numbers can be explained by the finding that a rapid increase in the apoptosis index of mast cells was induced on day 1-2. In contrast, in the more physiologic ALI culture, the number of Kit-positive cells was sustained over 1-2 days but then decreased on day 7. In addition, tryptase-positive cells decreased steadily in number but not to the same extent as those in the SM culture. Moreover, the increase in the apoptosis index of mast cells was delayed until day 7 in the ALI culture. Addition of exogenous stem cell factor (up to 200 ng/ml) to the SM culture could not prevent the decay in tryptase- and chymase-positive cells. However, stem cell factor reduced significantly the number of Kit-positive cells already on day 2 indicating that the cells had responded. Addition of histamine (0.25 or 1 mM) or tumor necrosis factor-alpha (500 or 2000 U/ml) caused a decrease in the number of tryptase- and Kit-positive cells in the SM culture. In conclusion, a novel finding was that air exposure in the ALI culture markedly delayed the rapid apoptosis and subsequent decrease in mast cell numbers noted to occur in the SM culture. Stem cell factor could not prevent the rapid decrease in mast cell numbers. Histamine and tumor necrosis factor-alpha are possible factors promoting the decline in mast cells.

Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes.

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.

Genomewide search and association studies in a Finnish celiac disease population: Identification of a novel locus and replication of the HLA and CTLA4 loci.

It has been reported that celiac disease (CD) is strongly associated with the HLA-DQ2 alleles DQA1*0501 and DQB1*0201. However, this association only accounts for a portion of the genetic component of CD. Several non-HLA loci and candidate genes that potentially contribute to CD susceptibility have been reported, but have not been confirmed. The aim of this study was to identify loci that contribute to disease susceptibility in a CD population from Finland. We performed a genomewide linkage scan and identified two regions of significant linkage to CD (6p and 2q23-32) and one region of suggestive linkage (10p). We also performed targeted typing and analyses that replicated the associations of the HLA and CTLA4 loci.