Thiopurine S-methyltransferase genotypic analysis in autoimmune bullous diseases.

Thiopurine S-methyltransferase (TPMT) activity is inversely related to the risk of developing severe hematopoietic toxicity in patients treated with azathioprine. The aim of this study was to evaluate the usefulness of TPMT genotyping in severe cases of autoimmune bullous diseases treated with azathioprine. A retrospective study of TPMT genotyping was performed in patients with autoimmune bullous diseases hospitalized in a single centre between 1999 and 2006 and susceptible of being treated by azathioprine. Among 75 patients tested, 70 (93%) had a high TPMT activity and 5 (7%) an intermediate activity. TPMT genotyping was performed in 33/34 patients currently treated with azathioprine. Haematopoietic side-effects (usually moderate) were observed in 12/34 patients treated with a mean dosage of 2.7 mg/kg/day and occurred, despite a high predicted TPMT activity. No myelotoxicity was observed in the two patients with intermediate predicted TPMT activity (mean dosage: 1.7 mg/kg/day), who obtained a clinically complete remission. Although strongly recommended before azathioprine treatment, predicting TPMT activity appears only marginally helpful in patients with autoimmune bullous diseases, mainly for adjusting the azathioprine dosage. In addition, a normal TPMT genotyping is not a guarantee against the occurrence of haematological side-effects.

p38 MAPK signaling in oral-related diseases.

Multiple dental diseases are characterized by chronic inflammation, due to the production of cytokines, chemokines, and prostanoids by immune and non-immune cells. Membrane-bound receptors provide a link between the extracellular environment and the initiation of intracellular signaling events that activate common signaling components, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor (NF)-kappaB. Although ERK pathways regulate cell survival and are responsive to extracellular mitogens, p38 MAPK, JNK, and NF-kappaB are involved in environmental stress responses, including inflammatory stimuli. Over the past decade, significant advances have been made relative to our understanding of the fundamental intracellular signaling mechanisms that govern inflammatory cytokine expression. The p38 MAPK pathway has been shown to play a pivotal role in inflammatory cytokine and chemokine gene regulation at both the transcriptional and the post-transcriptional levels. In this review, we present evidence for the significance of p38 MAPK signaling in diverse dental diseases, including chronic pain, desquamative disorders, and periodontal diseases. Additional information is presented on the molecular mechanisms whereby p38 signaling controls post-transcriptional gene expression in inflammatory states.

Mast cells in developing subepidermal bullous diseases: emphasis on tryptase, chymase and protease inhibitors.

The possible involvement of mast cell tryptase and chymase in subepidermal bullous diseases was studied enzyme-histochemically in specimens from erythematous and vesicular skin and from non-involved skin of patients with dermatitis herpetiformis, bullous pemphigoid, erythema multiforme, infective bullous eruption and linear IgA dermatosis. Patients with pemphigus were biopsied for comparison. The immunoreactivity of chymase inhibitors, alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC), in mast cells was demonstrated using the sequential double staining method. Tryptase-positive mast cells were unchanged or only slightly increased in number in erythematous lesions and slightly decreased in blistering skin compared with healthy-looking skin. Only occasionally were mast cells seen in apparent contact with the basement membrane zone. Chymase-positive mast cells and the chymase/tryptase ratio steadily decreased during the development of the lesions in each subepidermal bullous disease. The percentage of alpha1-PI+ and/or alpha1-AC+ mast cells increased simultaneously, which could explain the disappearance of chymase activity. Similar results were obtained regardless of the bullous disease. The results were also similar in pemphigus, which is an intraepidermal bullous disease. In conclusion, these results show significant alterations in mast cell chymase and protease inhibitors in a range of different bullous diseases, suggesting mast cell involvement. The apparent inactivation of chymase could be due to the action of chymase inhibitors detected in numerous mast cells. However, these alterations probably reflect general inflammation rather than a specific reaction in a certain bullous disease.

Suction blister formation in skin after acute and repeated mast cell degranulation.

Mast cells and their proteases are thought to participate in the development of skin blisters in various pathological conditions. In this study, suction blistering was used as an experimental model to evaluate the significance of mast cells in blister formation after pre-treatment of normal skin with intradermal injections of 100 microg/ml compound 48/80 (a mast cell degranulator) or with 0.1% capsaicin cream. Tryptic and chymotryptic enzyme activities in blister fluids were measured with sensitive p-nitroanilide substrates. Repeated injections of compound 48/80 once a day on 3 or 5 consecutive days or capsaicin applications 3 times a day for 7 or 10 days were used to induce mast cell degranulation and inflammation in normal skin. Both treatments ultimately led to decreased wheal and erythema reactions before suction blistering, but neither treatment affected the size or formation rate of suction blisters. No suction blister fluids had detectable levels of chymotryptic activity, but blister fluids from bullous pemphigoid, herpes zoster and insect bullous eruption, used as the control, revealed clear chymotryptic activity. In addition, tryptic activity in suction blister fluids was not significantly altered after compound 48/80 and capsaicin pre-treatments. However, if the wheal reaction was induced immediately before suction blistering, a significantly increased rate in blister formation together with increased tryptic activity was found, but, unexpectedly, no chymotryptic activity could be detected in blister fluids. The results show that repeated mast cell degranulation in normal skin has no effect on the formation rate of suction blisters, which developed more rapidly on acutely whealing skin. This is probably due to skin oedema rather than mast cell proteases, since no chymotryptic activity was detected in suction blisters where tryptic activity exhibited high individual variation.

Interstitial collagenase is expressed by keratinocytes that are actively involved in reepithelialization in blistering skin disease.

Migrating keratinocytes actively involved in reepithelialization in dermal wounds acquire a collagenolytic phenotype upon contact with the dermal matrix. To determine whether this phenotype is associated with repair in other forms of wounds, we assessed collagenase expression in 50 specimens representing a variety of blistering skin diseases, including subtypes of epidermolysis bullosa, porphyria cutanea tarda, bullous pemphigoid, pemphigus, transient acantholytic dermatosis, and suction blisters. Distinct from that seen in chronic ulcers or in normal healing by second intention, reepithelialization in these blistering conditions was not necessarily associated with a complete loss of basement membrane, as determined by immunostaining for type IV collagen. Collagenase mRNA was detected in the basal keratinocytes of several specimens of epidermolysis bullosa simplex (six of 10) and of pemphigus (three of seven), as well as in one quarter of transient acantholytic dermatosis samples in the presence of an intact basement membrane. In contrast, three of nine porphyria cutanea tarda, one third of epidermolysis bullosa acquisita, and one of 10 bullous pemphigoid samples had collagenase-positive basal keratinocytes with the basement membrane disrupted. The collagenase-positive lesions generally represented older blisters with evidence of epithelial regeneration. Collagenase was also expressed in suction blisters at 2 and 5 d after induction of the blister, but was shut off when the epidermis had healed. Other metalloproteinases were expressed occasionally, if at all. Our results suggest that keratinocyte migration is associated with collagenase expression and that contact of keratinocytes with the dermal matrix is not necessarily needed for collagenase induction.

Increased serum and blister fluid levels of creatine kinase in patients with toxic epidermal necrolysis.

Keratinocytes have recently been reported to contain creatine kinase (CK) of brain-type isoenzyme. The aim of this study was to investigate whether necrosis of keratinocytes induced raised CK levels in toxic epidermal necrolysis (TEN). The serum and blister fluid levels of creatine kinase and its isoenzymes [muscular-type (MM), brain-type (BB), myocardial-type (MB)] were measured in 40 patients with TEN, 10 patients with other bullous dermatoses, and in suction blisters in five controls. The mean serum CK was significantly higher in TEN patients than in patients with other bullous dermatoses (mean +/- SD: 480 +/- 535 U/l vs. 107 +/- 44 U/l, P < 0.05). The MM-isoenzyme was predominant (94%). A positive correlation was found between the level of the serum CK and the percentage of body surface area (BSA) involved (r = 0.49, P < 0.001). The mean blister CK was significantly higher in TEN patients than in patients with other bullous dermatoses or controls (mean +/- SD: 728 +/- 437 U/l vs. 310 +/- 244 U/l and 268 +/- 194 U/l, respectively, P < 0.02). The isoenzyme distribution of blister CK in TEN patients was: 76.8% MM, 18.1% MB and 5% BB. Although a significant part of blister CK comigrating with CK-MB, after preincubation with protein A-Sepharose, appeared to be CK-BB/IgG complex, the CK-BB fraction constituted less than 25% of blister CK. Therefore, the CK present in increased amounts in serum and blister fluid in TEN was not directly produced by keratinocytes.

Demonstration of 72-kDa and 92-kDa forms of type IV collagenase in human skin: variable expression in various blistering diseases, induction during re-epithelialization, and decrease by topical glucocorticoids.

Type IV collagenases have been shown to play an important role in tumor metastasis and wound healing. In the present study, we have demonstrated the presence of 72-kDa and 92-kDa forms of type IV collagenase in human skin by biochemical and in situ hybridization techniques. In situ hybridization allowed us to localize the 72-kDa form mostly to fibroblasts and the 92-kDa form to the epidermis and endothelial cells. The presence of type IV collagenase was confirmed by Western blotting. Enzyme activity was assayed in spontaneous blisters (18 subjects) and suction-induced blisters (29 subjects) by the zymography method, and by using type IV collagen as the substrate. Thus, it was possible to detect both the 92-kDa and 72-kDa forms in spontaneous and induced blisters. An especially high level of the 92-kDa enzyme was found in a bullous pemphigoid patient. Type IV collagenases were studied during re-epithelialization of the blister, using the suction-blister model. There was a marked induction of the 92-kDa type that was confirmed to be in the regenerating, migratory, epithelium by in situ hybridization studies. These results indicate that 92-kDa type IV collagenase may play an essential role in the normal physiology and integrity of the skin and may be an important regulator of re-epithelialization. It was also shown that potent topical glucocorticoid down-regulated the 92-kDa type collagenase, suggesting that glucocorticoids may have a beneficial role in some skin diseases by decreasing type IV collagenase activity and, thus, reducing tissue destruction.

Cytotoxic proteases in blister fluid of pemphigus and pemphigoid patients.

Investigation of blister fluid (BF) from 49 pemphigus vulgaris and 27 bullous pemphigoid patients revealed direct interrelation between proteolytic and cytotoxic activities of BF. Human epidermal keratinocytes proved to be more sensitive to the cytolytic effect of BF as compared to human endotheliocytes and fibroplasts. Epidermal keratinocyte cultivation in patients' BF led to proteolytic activity enhancement in culture supernatant. Inasmuch as cytotoxic and proteolytic activity of BF sharply decreased under the effect of inhibitors of serine proteinases only, it was concluded that proteolytic enzymes of this class play the role of a "cytotoxic factor" causing the lysis of epidermal cells in pemphigus and pemphigoid.

Plasmin-like proteinase associated with high molecular weight complexes in blister fluid of bullous pemphigoid.

Blister fluid from tense bullae of 10 patients with bullous pemphigoid was investigated using a radial caseinolysis assay and zymography. Proteolytic activity, varying from 4.7 to 10 micrograms/ml, was found in 3 out of 10 patients, by using the caseinolysis assay. Zymography revealed that a major part of this caseinolytic activity co-migrated with plasmin standard. In addition, in the zymography, proteolytically active high molecular weight complexes were seen. This characteristic pattern was seen in the zymography of both positive and negative samples in the caseinolysis assay. These high molecular weight complexes were not seen in the zymography of the blister fluid of 2 patients with epidermolysis bullosa or in the suction blister fluid of 3 healthy control patients. These findings suggest that plasmin is generated at some phase of the blister formation, being possibly involved in the pathomechanism of bullous pemphigoid.

Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology.

Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair.

Characterization of elastase-like enzymes in various blistering diseases.

Blister fluid samples were collected from suction induced control blisters and from spontaneous blisters from various blistering diseases for the measurement of elastase-like enzyme activities using synthetic succinyl-L-(alanyl)3-paranitroanilide (SAPNA) and alanyl paranitroanilide (ala-PNA) as the substrates. The blister fluids derived from bullous pemphigoid, pyoderma or epidermolysis bullosa dystroficans lesions contained higher levels of elastase-like enzyme activities than burn blisters or fresh suction blisters. The gel filtration studies using Sepharose CL-4B chromatography revealed two major peaks of SAPNA hydrolysing enzyme activity in burn blister and in bullous pemphigoid blister. The first peak eluted in void volume, and the second peak had an apparent molecular weight of 2.5 X 10(5) daltons. The results indicate that various proteinases are present in blisters fluids and that they may participate in the blister formation by degrading structural components of the basement membrane zone and the dermis.

N-acetyl-beta-glucosaminidase activity in serum of patients with epidermolysis bullosa.

A spectrophotometric study for serum N-acetyl-beta-glucosaminidase activity in patients with different types of epidermolysis bullosa and other bullous diseases was carried out. The activity in recessive type of epidermolysis bullosa dystrophica was higher than in dominant type of epidermolysis bullosa dystrophica and other bullous diseases. High levels of this enzymatic activity may be related to the metabolism of glycosaminoglycans, especially hyaluronic acid in recessive type of epidermolysis bullosa dystrophica.

The activity of arylsulphatases in serum of patients with several bullous diseases.

Spectrophotometric estimations of levels of activities of arylsulphatases A and B in serum of patients with several bullous diseases were carried out. The activities of these enzymes in cases of bullous pemphigoid were at high levels in comparison with those in cases of other bullous diseases and in the control group. Especially, the activity of arylsulphatase B was remarkably increased. It seems likely that there is some relationship between these disturbances and the pathogenesis of bullous pemphigoid.

Demonstration of collagenase and elastase activities in the blister fluids from bullous skin diseases. Comparison between dermatitis herpetiformis and bullous pemphigoid.

We have investigated potential mechanisms for blister formation by assaying proteolytic enzymes in the blister fluids of patients with various bullous diseases. Blister fluids were obtained from patients with dermatitis herpetiformis (DH), bullous pemphigoid (BP), chronic bullous disease of childhood (CBDC), and pemphigus vulgaris (PV). The cells were recovered by centrifugation, and the supernatants as well as the cell pellets were assayed first for collagenase activity using [3H]proline-labeled type I collagen as substrate. Collagenase activity could be detected in most cases with DH, BP, and CBDC, while no activity was found in 2 cases of PV or in 5 control blister fluids obtained from suction blisters induced in healthy control subjects. Elastase activity was assayed in the same blister fluids by using a synthetic substrate succinyl-(L-alanyl)3-paranitroanilide or soluble [14C]valine-labeled tropoelastin. High levels of elastase activity were present in all DH patients, while lower, but clearly detectable, levels were found in BP, CBDC, and PV. The enzyme activity in BP was inhibited by Na2EDTA, but not by phenylmethylsulfonyl fluoride (PMSF), and Ca2+ stimulated the activity, suggesting that the enzyme in BP was a metalloproteinase. In cell-free supernatants of the DH blister fluids, the elastase activity was markedly decreased by PMSF, indicating that most of the enzyme activity was due to a serine protease. The cells recovered from DH blister fluids also contained high levels of elastase activity which could be inhibited by PMSF but not by Na2EDTA. Thus, in DH, the elastase activity is probably derived from polymorphonuclear leukocytes abundantly present in the lesions. The results indicate that active proteases are present in the blister fluids of skin diseases, and they may play a mechanistic role in the blister formation by degrading connective tissue components of the dermis and the dermal-epidermal junction.