Mast cell chymase degrades fibrinogen and fibrin.

BACKGROUND: The accumulation of immunoreactants and fibrinoid necrosis of postcapillary vessel walls are common pathological features of cutaneous immune complex vasculitis. In more advanced lesions, these immunoreactants are subject to proteolysis. Mast cell chymase is a powerful enzyme that can degrade several substrates including the extracellular matrix. Heparin can influence the catalytic properties of chymase. OBJECTIVES: To study the effects of recombinant human (rh) chymase on fibrinogen, coagulation and fibrinolysis, and to relate these effects to the pathogenesis of vasculitis. METHODS: The colocalization of chymase and fibrin in vasculitis specimens was analysed by immunohistochemical double staining. Fibrinogen and fibrin were treated with rh-chymase and the effects were studied in vitro by sodium dodecylsulfate polyacrylamide gel electrophoresis and a variety of clotting and fibrin gel experiments. The effects of rh-chymase on vasculitis cryosections were analysed by direct immunofluorescence. RESULTS: Chymase-positive mast cells were associated with fibrin-positive vessels in vasculitis cryosections. Rh-chymase degraded the alpha-, beta- and gamma-chains of fibrinogen, while heparin enhanced the degradation of the beta-chain. Rh-chymase pretreatment of fibrinogen prolonged thrombin-induced clotting time. Fibrinogen degradation products induced by rh-chymase increased the clotting time of human plasma. Rh-chymase degraded fibrin gel prepared from fibrinogen or human plasma. Immunofluorescence staining positivity of fibrin in vasculitis cryosections decreased after pretreatment with rh-chymase for 24 h, and heparin enhanced this effect. CONCLUSIONS: Mast cell chymase may constitute a previously unrecognized endogenous anticoagulant and fibrinolytic enzyme, and may be involved in the clearance of fibrin from vessel walls in aged vasculitis lesions.

Role of mast cells and sensory nerves in skin inflammation.

Mast cells are powerful inflammatory cells which are in close functional and anatomical association with sensory nerves in the skin. During psychological stress the neuroendocrine system and peripheral sensory nerves are activated leading to release of mediators, such as neuropeptides, neurotrophins, corticotropin-releasing hormone and a-melanocyte-stimulating hormone, which are capable of activating mast cells. On the other hand, mast cell mediators released, e.g. histamine, tryptase and nerve growth factor, can in turn excite and stimulate surrounding neuropeptide-containing C-fibers possibly resulting in feedforward loop and potentiation of neurogenic inflammation. In these mechanisms, proinflammatory cytokines and chemokines are released from mast cells. In chronic skin diseases, psoriasis, atopic dermatitis and palmoplantar pustulosis, the contacts between tryptase-positive mast cells and sensory nerves are increased in number, which provides the morphological basis for increased mast cell - sensory nerve interaction in chronically inflamed skin. Hence, in this review the current understanding of the role of cutaneous mast cells and sensory nerves and their activation in psychic stress is discussed.

Adventitial mast cells connect with sensory nerve fibers in atherosclerotic coronary arteries.

BACKGROUND: The number of activated mast cells is increased in the adventitia of coronary segments with plaque rupture and in spastic atherosclerotic coronary segments. Neurogenic activation of mast cells has been demonstrated previously in other tissues. Here we identified and quantified contacts between mast cells and nerves in the adventitia of normal and atherosclerotic coronary segments. METHODS AND RESULTS: Normal (types 0 or I) and atherosclerotic (lesion types II, III, and IV) coronary segments from 22 unselected autopsy cases were stained for mast cells and sensory nerves by a histochemical double-labeling method. Contacts between mast cells and sensory nerves were quantified morphometrically and also identified by confocal microscopy. Coronary arteries obtained during heart transplantation were stained for the neuropeptides capable of stimulating mast cells, ie, substance P and calcitonin gene-related peptide. In the adventitia of atherosclerotic coronary segments with type IV lesions, the numbers of mast cells and mast cell-nerve contacts (104+/-15 mast cells/mm(2) and 30+/-5 nerve contacts/mm(2); mean+/-SEM) were significantly greater than in segments with type III lesions (79+/-12 [P<0.001] and 24+/-6 [P<0.001]), those with type II lesions (54+/-4 [P<0.001] and 12+/-2 [P<0.001]), or those with normal intima (31+/-3 [P<0.001] and 4+/-1 [P<0.001]). The nerve fibers connected with mast cells contained both substance P and calcitonin gene-related peptide, which identified them as sensory nerves. CONCLUSIONS: Neurogenic stimulation of mast cells in the adventitia of coronary arteries may release vasoactive compounds, such as histamine and leukotrienes, which can contribute to the complex neurohormonal response that leads to abnormal coronary vasoconstriction.

Involution of the chicken bursa of Fabricius: a light microscopic study with special reference to transport of colloidal carbon in the involuting bursa.

The involution of the bursa of Fabricius in White Leghorn chickens and the transport of colloidal carbon in the involuting bursa were studied on light microscopy. In chickens, from newly hatched to 23.5 weeks of age, the bursa was weighed, its histology was described, and the mitoses in the cortical and medullary compartments of the lymphoid follicles were counted. Decrease in bursal weight and in the number of mitoses were observed between 10 and 16 weeks of age. During week 17.5, the follicle-associated epithelium began to lose its endocytic capability, and mucin droplets appeared in the follicular medulla initiating the large mucoid cysts that were seen in the later phases of involution. The involutionary process was almost completed by week 23.5, when the bursa appeared as a fibrotic residue without intact lymphoepithelial structures. The particulate tracer used, colloidal carbon, was not observed to be transported to other organs from the bursa. The present study gives further support to the idea that after completing its central immune function in 6 weeks [21] the bursa, as estimated histologically, still possesses capacity to function like a peripheral lymphoid organ until 16 weeks of age.

Quantification of cutaneous sensory nerves and their substance P content in psoriasis.

The aim of the present study was to extend our previous hypothesis that the inflammatory reaction in psoriasis is neurogenic, and that substance P mediates the inflammation. For this purpose, the pattern of neurofilament-positive sensory nerve fibers was studied and the lengths and substance P content of these fibers measured morphometrically in dermal and epidermal compartments of the psoriatic lesion, psoriatic but lesion-free skin, and control skin. The epidermis and dermis of the psoriatic lesions were significantly more densely innervated with neurofilament-positive fibers than either lesion-free psoriatic or control skin. Although substance P is known to be rapidly degraded in tissues, and its actual concentrations in the sections were unknown, there was an increase in substance P containing nerves in the psoriatic lesion, the increase being significant in the epidermal nerve fibers. No significant differences in the measured parameters were obtained between lesion-free psoriatic and control skin. These results indicate that there is an altered pattern of sensory nerves in a psoriatic plaque and that substance P may be an important mediator in the inflammatory processes that contribute either to the initiation or maintenance of a psoriatic lesion.

Focal dermal-epidermal separation and fibronectin cleavage in basement membrane by human mast cell tryptase.

Mast cell proteases are believed to participate in the basement membrane destruction in blistering diseases. Thus, normal human skin specimens were incubated with purified human skin tryptase or compound 48/80 (a mast cell degranulator) for up to 24 h. Thereafter, the specimens were studied immunohistochemically. Tryptase caused, in the presence and absence of 1,10-phenanthroline, focal dermal-epidermal separation above laminin and almost complete disappearance of the staining of the extra domain A region of cellular fibronectin in and beneath the basement membrane. The immunopositivity of the cell-binding region of fibronectin, laminin, and collagens IV and VII, however, was unaltered. Compound 48/80 induced almost complete dermal-epidermal separation above intact laminin and only focal reduction in the extra domain A region of cellular fibronectin staining. These alterations by compound 48/80 were prevented partially by Nalpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors were present suggesting the involvement of tryptic serine proteinases, probably also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phenylalanine chloromethyl ketone was weak suggesting minor function of chymotryptic serine proteinases. When tryptase was incubated with heparin and pure plasma fibronectin, an abrupt decrease in the adherence of cultured keratinocytes on to plastic surface coated with these substances and a gradual plasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, tryptase can cause focal dermal-epidermal separation above laminin in skin specimens but it is not known to what extent the decreased keratinocyte adherence in vitro and fibronectin cleavage are related to this dermal-epidermal separation.

Expression of HER2 and its association with AP-2 in breast cancer.

The aim of the study was to investigate the relationship between the expression of human epidermal growth factor receptor 2 (HER2) and activator protein 2 (AP-2), as well as the prognostic significance of HER2 in breast cancer. HER2 and AP-2 expressions were immunohistochemically (IHC) analysed in a large prospective, consecutive series of 425 breast cancer patients diagnosed and treated between 1990 and 1995 at the Kuopio University Hospital, Kuopio, Finland. In a subset of patients (n = 71), the gene amplification status was examined by using a chromogenic in situ hybridisation (CISH) analysis. Expression of HER2 was studied in relation to AP-2, clinicopathological parameters and patients' survival. Pathological membranous overexpression of the HER2 receptor was seen in 13% of the carcinomas, which was related both to gene amplification (78% of the cases) and high nuclear expression of AP-2 (67%, P = 0.007). HER2-positivity was most often seen in carcinomas having both high nuclear and high cytoplasmic AP-2 expression (P < 0.001). In the univariate survival analyses HER2-positivity predicted a shorter recurrence-free survival (RFS) (P < 0.0001) and a shorter breast cancer-related survival (BCRS) (P = 0.0063) in the whole patient group, as well as in the subgroup of node-negative patients. In the node-positive patients, HER2-positivity predicted only a shorter RFS. Combined expression of HER2 and nuclear AP-2 resulted in the separation of four groups with different prognoses, the best prognosis being for patients in the HER2-/AP-2+ group and the worse prognosis for those in the HER2+/AP-2- group. In the multivariate survival analyses, HER2-positivity independently predicted a shorter RFS in the whole patient group (P = 0.0067), as well as in the subgroup of node-positive patients (P = 0.0209). In conclusion, pathological membranous overexpression of the HER2 receptor in breast cancer is related both to gene amplification and a high AP-2 expression. Combining HER2 and AP-2 expressions may provide valuable information on the prognosis of breast cancer patients.

Development of the peripheral immune function in the chicken. A study on the bursa of Fabricius isolated from the rest of the gut-associated lymphoid tissue (GALT).

The bursa of Fabricius was surgically separated from the rest of the gut-associated lymphoid tissue (GALT). The antigens, sheep red blood cells (SRBC) and killed Brucella abortus organisms, were introduced into the bursal lumen, into the gut only, or allowed to get in contact with both (controls). The antibody titres in serum, the development of the bursal weight and the bursal histology were studied at various ages. The control birds responded vigorously to both antigens at between 2 and 8 weeks of age. At that age, the isolated bursa was capable of producing full anti-Brucella response, but not the anti-SRBC response, suggesting that SRBC need collaboration between both bursal and intestinal T and B cells. After 8 weeks, the cells in the gut alone were as potent as those in the gut and the bursa together at producing antibodies to SRBC. The bursal weight was significantly higher in the per bursam immunized chickens, indicating that continuous presence of antigens in the bursa may postpone its involution.

Effects of antigen and antigen concentration on serum antibody titres in chickens primed per bursam.

A novel method for intrabursal immunization was applied to study the priming effect in 2-day-old and 4-week-old chickens. This immunization method separates the bursa of Fabricius totally from the rest of the gut-associated lymphoid tissue (GALT), and closely mimics the natural way antigens come in contact with the bursal epithelium. Sheep red blood cells (SRBC) and Brucella abortus organisms in 4 different concentrations were used as antigens. In 4-week-old birds, a suppressive effect of per bursam priming on the secondary anti-SRBC titres was recorded, an observation not reported earlier. Priming had no effect on the secondary anti-SRBC titres in 2-day-old chickens. In both age groups, a positive priming effect on anti-Brucella titres was observed. Antigen concentrations too low to induce primary immune responses in serum were still capable of causing the priming effect. A certain minimum concentration, however, was required also for the priming effect to occur. The observed suppressive effect of priming on the secondary anti-SRBC titres suggests that bursa exerts inhibitory immuno-regulatory functions when handling certain antigens. Important factors are the age of the chickens and the antigen concentration used, which influence both the primary and the secondary immune responses.

Effects of local and systemic immunization on serum antibody titres in splenectomized chickens.

The role of spleen in local (per anum) and systemic (intravenous = i.v.) immunization was studied by splenectomizing chickens and immunizing them twice with sheep red blood cells (SRBC) and Brucella abortus. The number of germinal centers in spleen and the splenic weight were also recorded. Splenectomy had no effect on the serum titres evoked by the per anum immunization but it affected the anti-SRBC and, to a lesser extent, anti-Brucella titres in serum after i.v. immunization. The effect of splenectomy was less obvious both in the secondary response and when the chickens were younger at operation and the time between splenectomy and immunization was increased. The i.v. immunizations induced germinal centre formation in the spleen whereas immunization per anum did not significantly do so. Also the splenic weight in the i.v. immunized chickens was higher than that in the per anum immunized birds. The present study shows that the spleen is the major site of antibody production against i.v. administered antigens in chickens. Antibody production to antigens applied per anum occurs mainly in other lymphatic organs, most probably in the gut-associated lymphoid tissues.

The prevalence and regional distribution of antibodies against Chlamydia pneumoniae (strain TWAR) in Finland in 1958.

The occurrence of nonavian 'ornithosis' was reported in Scandinavia at the end of the 1950s. In order to find out whether Chlamydia pneumoniae had been present in Finland, we examined the IgG antibody prevalence to C pneumoniae in samples representing the whole rural population of Finland in 1958. The total number of sera studied using micro-immunofluorescence was 2000. Trend-surface analysis was used to examine the regional patterns of antibody prevalence. C pneumoniae antibodies were present throughout the country. The mean antibody prevalence was 56% among adults and 27% among children. Thus the possible role of C pneumoniae in nonavian 'ornithosis' in Finland between 1958 and 1960 cannot be excluded.

Importance of smoking for Chlamydia pneumoniae seropositivity.

BACKGROUND: Population-based studies of the association between smoking and Chlamydia pneumoniae seropositivity do not exist. The role of smoking in the association between C. pneumoniae seropositivity and coronary artery disease (CAD) suggested by several studies has been debated. The aim of this study was to determine the relationship between smoking habits and C. pneumoniae IgG antibody titres in a middle-aged population. We also wanted to find out whether the difference in smoking habits between the sexes explains the higher C. pneumoniae antibody prevalence among men compared with women. RESULTS: After controlling for the effect of smoking, the risk of C. pneumoniae seropositivity remained 1.4 times higher in men than in women. In men, the estimated risk for C. pneumoniae seropositivity (titre > or = 1:16) was significant only for smokers (adjusted odds ratio [OR] = 1.4). The adjusted OR for high seropositivity (titre > or = 1:128) was 1.5 for smokers and 1.7 for ex-smokers. The risk for women was similar to that for men. CONCLUSIONS: The results provide evidence of an association between smoking and C. pneumoniae seropositivity in the general population. The higher prevalence of smoking in men does not explain the C. pneumoniae antibody prevalence in men compared with women.

Chlamydia pneumoniae IgG antibody prevalence in south-western and eastern Finland in 1982 and 1987.

Chlamydia pneumonia IgG antibody prevalence was examined in a representative sample (2342 subjects) of the population aged 25-59 years in south-western and eastern Finland in 1982 and 1987. Microimmunofluorescence was used to measure IgG antibodies. Prevalence of C. pneumoniae was modelled using the GLIM statistical package assuming that the prevalence had a binomial distribution. The prevalence was assumed to be a function of the year, subjects' gender, age and place of residence. The effect of the year on the prevalence was significant in both regions (P < 0.05 in the south-west and P < 0.001 in the east). In each year the prevalence was higher in the south-west than in the east and it increased between 1982 and 1987 from 55% to 63% in the south-west and from 41% to 59% in the east. The prevalence increased with age, but the age-related antibody pattern was different between sexes. The highest prevalence was found in men aged 49-59 years. The different increase in prevalence with age in the south-western and the eastern parts of the country indicates that the outbreaks of infection were not related. The endemic level of C. pneumoniae infection in the south-west is consistently higher than in the east, or there was an outbreak of C. pneumoniae infection in both the years we examined. Thus it is more likely that the high prevalence in south-western Finland was connected more with the epidemics reported elsewhere in Scandinavia in the same years than with the occurrence of C. pneumoniae infection in eastern Finland.

Clinical relevance of p53 index and expression of proliferating cell nuclear antigen and Ki-67 in gastric cancer.

The prognostic value of the immunohistochemical expression of p53 protein, proliferating-cell nuclear antigen (PCNA) and Ki-67 antigen was evaluated in a series of 116 stage I-II gastric cancer patients. The staining for p53 protein (staining frequency and intensity) in malignant cells was expressed as a p53 index. Similarly, the staining frequency and intensity for PCNA and Ki-67 were evaluated. The p53 index was independent of the stage and differentiation grade, but significantly related to DNA ploidy, S-phase fraction and mitotic activity. A high p53 index was a sign of inferior survival, compared to a low or intermediate index. p53-negative tumours were also associated with poor survival. In a multivariate analysis, only the depth of tumour infiltration and the presence of nodal metastases were independent prognostic factors in stage I-II gastric cancer. PCNA expression and Ki-67 antigen expression were not related to the stage, ploidy, proliferative activity or p53 expression, and they had no impact on survival. The results indicate that p53 protein expression may be of prognostic significance in gastric cancer, while PCNA and Ki-67 antigen expression have no predictive value.

Late onset foot-drop muscular dystrophy with rimmed vacuoles.

We studied a family with late-onset (fifth or sixth decade) or asymptomatic hereditary myopathy of the anterior tibial muscle. The occurrence of the disease in two successive generations pointed out an autosomal dominant pattern of inheritance. The initial symptom was uni- or bilateral foot drop resembling peroneal paresis. Surprisingly many of the diagnosed patients were asymptomatic and considered themselves healthy whether there was any foot drop or not. The anterior tibial muscles were atrophic in patients with foot drop but the long toe extensors were usually and the short ones were always spared. Apparently the toe extensors could relieve the foot drop symptom. As shown by computed tomography there was often an early uni- or bilateral involvement of the semimembranosus muscle in males. The proband showed also a late involvement of the femoral biceps and the minor gluteal muscles. The muscles of the upper extremity were spared. The anterior tibial muscles had a characteristic myopathic alteration with rimmed vacuoles in histopathological study. This picture was most evident in latent cases without atrophy of the anterior tibial muscle, but with distinctly abnormal EMG of that muscle. Non-affected muscles showed only slight non-specific histopathological changes. We suggest that this disease is a new mild variety of autosomal dominant distal myopathy with rimmed vacuoles.

The development of manifest psoriatic lesions is linked with the appearance of ICAM-1 positivity on keratinocytes.

The development of psoriatic lesions was studied in 36 psoriatic patients using the Koebner reaction induced by tape stripping. Two biopsies per patient were taken from non-lesional psoriatic skin before, and 6 h, 2 days, 7 days, 14 days and 21 days after tape stripping. Alterations in HLA-DR, ICAM-1, Ki-67 and FXIIIa positivities in both the dermis and the epidermis were estimated using immunohistochemical methods. A double staining for CD4+ and CD8+ T cells was also carried out to show their possible Ki-67 positivity. Results were compared with those from lesional (mature plaque) and non-lesional psoriatic skin, and control skin. Of the 36 patients, 9 were Koebner-positive. The most important finding in Koebner-positive psoriatic skin was the appearance of ICAM-1 positivity on epidermal keratinocytes simultaneously with the clinically observed lesion on day 7. The number of FXIIIa+ dendrocytes in the dermis was quite constant, and increased in mature psoriatic lesions only. The number of active HLA-DR+ immunocompetent cells increased in developing psoriatic lesions, being highest in mature lesions, but no Ki-67 positivity was detected in epidermal or dermal T cells in the psoriatic specimens. Based on these results, it is concluded that T cells divide and are activated extracutaneously in psoriasis, and also that ICAM-1/LFA-1 interactions are important in the recruitment of inflammatory cells and in controlling the effector cell functions.

The development of manifest psoriatic lesions is linked with the invasion of CD8 + T cells and CD11c + macrophages into the epidermis.

Koebner response was studied in 35 psoriatic patients. Two punch biopsies per patient were taken from non-lesional psoriatic skin before, and 6 h, 2 days, 7 days, 14 days and 21 days after, tape stripping. Alterations in the numbers of CD1+ Langerhans cells, CD4+ and CD8+ T cells and CD11c+ macrophages were mapped morphometrically. Results were compared with lesional and non-lesional psoriatic skin, and control skin. Nine of 35 patients were Koebner-positive. No statistically significant differences were noted between non-lesional psoriatic and control skin. CD4+ T cells increased in number 2 days after trauma in both the epidermis and the dermis, whereas epidermal CD8+ T cells and CD11c+ macrophages increased only in the Koebner-positive lesional skin after 7 days. The changes in lesions induced by tape-stripping resembled those seen in lesional psoriatic skin (mature plaques). The number of CD1+ cells increased in mature psoriatic lesions only. It seems possible that trauma per se stimulates the accumulation of CD4+ T cells at the site of injury, but the development of manifest psoriatic lesions correlates with invasion of CD8+ T cells and CD11c+ macrophages into the epidermis.

Mast cell proteinases and cytokines in skin inflammation.

The role of mast cells in provoking immediate-type hypersensitivity reactions is well established, but their involvement in chronic inflammation and immune reactions is not so clear. Mast cells synthesize and secrete large amounts of active proteinases, including tryptase, chymase, carboxypeptidase and cathepsin G, which can rapidly process numerous biologically active peptides and proteins or their precursors. Furthermore, mast cells are able to produce a variety of cytokines such as interleukin-4 (IL-4), IL-5, IL-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) which are known to be intensively involved in modulating and directing inflammatory responses in the skin. In this review, the role of mast cell proteinases and cytokines in skin inflammation is discussed.

Immunohistochemical analysis of sensory nerves and neuropeptides, and their contacts with mast cells in developing and mature psoriatic lesions.

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p < 0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin.

Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzamidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-anti-trypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15, 30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefulness of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.