Sebaceous glands in acne patients express high levels of neutral endopeptidase.

Acne is a complex, chronic and common skin disorder of pilosebaceous units. Although it is known that exacerbation of acne results from emotional stress, the nature of the association between stress and acne remains unclear. This is due in part to the lack of substantial evidence regarding the participation of cutaneous neurogenic factors in the pathogenesis of acne. To examine the possible involvement of neurogenic factors in the etiology of acne, we used immunohistochemistry to compare the distribution of SP-containing nerve fibers around sebaceous glands and the expression of neutral endopeptidase in sebaceous acini of the facial skin of acne patients and of healthy subjects. More numerous substance P immunoreactive nerve fibers in close apposition to the sebaceous glands and an increase in expression of neutral endopeptidase in sebaceous acini were observed in acne patients compared with the controls. Immunoelectron microscopy revealed that the subcellular localization of neutral endopeptidase was restricted to the Golgi apparatus and the endoplasmic reticulum within sebaceous germinative cells. In addition, in vitro experiments using an organ culture system demonstrated that substance P induced expression of neutral endopeptidase in sebaceous glands in a dose dependent manner. This study reveals that substance P and its degrading enzymes are involved in the pathogenesis of acne, which in turn might partially explain the pathologic significance of neurogenic and psychogenic aspects in the disease process.

Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis.

BACKGROUND: Neurogenic components, such as neurotrophic factors and neuropeptides, are probably involved in the pathogenesis of atopic dermatitis (AD) via the neuroimmunocutaneous system. Numerous in vitro and in vivo studies have shown that nerve growth factor (NGF), the best-characterized member of the neurotrophin family, modulates the synthesis of the neuropeptide substance P (SP), both of which may be associated with the pathogenesis of human allergic diseases. OBJECTIVES: To evaluate the levels of NGF and SP in the plasma of patients with AD and to examine their possible correlation with disease activity. METHODS: We measured plasma levels of NGF by an immunoenzymatic assay and of SP by aradioimmunoassay in 52 patients with AD, and compared them with 35 normal non-atopic controls. The severity of the disease in AD patients was evaluated using validated clinical scoring systems. RESULTS: Patients with AD had significant increases in plasma levels of NGF and SP compared with controls (P < 0.0005 and P < 0.0001, respectively). A positive correlation between the plasma levels of NGF and SP was found in AD patients (correlation coefficient, Cc = 0.920, P < 0.0001). There was a significant correlation of plasma NGF and SP levels with disease activity evaluated using three different scoring systems: the grading system of Rajka and Langeland (P < 0.001 and P < 0.01, respectively), the objective Severity Scoring of AD (Cc = 0.656, P < 0.005 and Cc = 0.752, P < 0.0005, respectively) and the Eczema Area and Severity Index (Cc = 0.740, P < 0.001 and Cc = 0.765, P < 0.005, respectively). CONCLUSIONS: These data represent the first reported evidence of increased plasma levels of NGF and SP in an allergic human skin disease. They suggest that these neurogenic factors systemically modulate the allergic response in AD, probably through interactions with cells of the immune-inflammatory component. In addition, NGF and SP may be useful markers of disease activity in patients with AD.

Ultrastructural characteristics of trichilemmal cysts: report of two cases.

Two trichilemmal cysts (TC) on the scalps of two Japanese women were examined at the electron microscopic level. The ultrastructural characteristics of the TC included an abrupt transition from nucleate to anucleate cells, a few oval, small, keratohyaline granules mixed with increasing numbers of tonofibrils from the peripheral layer towards the center, spherical particles with lipid droplets, desmosomal structures in keratinized cells, and the interdigitation of keratin in the uppermost keratinizing cells. The keratinization pattern of the TC was unique in that typical soft or hard keratinization processes did not occur. Trichilemmal keratinization is observed in the trichilemmal sacs that surround the lower ends of catagen or telogen hairs, as well as in the outer root sheaths in the follicular isthmus of anagen hairs. Comparison of the ultrastructure of these two known types of trichilemmal keratinization with the keratinization pattern of the TC in our patients indicates that TC may differentiate into, or originate from, the proliferation of the outer root sheath in the follicular isthmus of anagen hairs.

Immunohistochemical study of inducible nitric oxide synthase in skin cancers.

BACKGROUND: Nitric oxide (NO) is synthesized from the amino acid L-arginine by NO synthase (NOS). Experimental evidence suggests that increased express of inducible NOS (iNOS), which is an NOS isoform and calcium independent, is related to various pathological processes, such as inflammation and cancer. METHODS: In this study, we used immunohistochemistry to investigate iNOS expression in a series of basal cell carcinomas (BCC), Bowen's disease, squamous cell carcinomas (SCC), extramammary Paget's disease (EPD) and metastatic tumors of the skin. RESULTS: Only 1 of 16 BCC cases was positive for iNOS and the intensity of staining was weak. In most of the 10 cases of Bowen's disease, iNOS was weakly expressed and there was a wide range in the percentage of positive tumor cells. Twelve of the 16 cases of SCC were positive for iNOS and the extent of positivity was greater than in Bowen's disease. Two of the 7 cases of EPD were positive for iNOS, and 12 of the 15 cases of metastatic cancer were positive. Well-differentiated adenocarcinomas were diffusely positive, whereas poorly-differentiated ones showed strong and heterogeneous staining. CONCLUSIONS: These results indicated that the expression of iNOS may reflect the proliferation of tumor cells and that a heterogeneous distribution of iNOS may correlate with a wide variety of biological behavior of tumor cells.

Immunohistochemical expression of cyclooxygenase-2 in skin cancers.

BACKGROUND: Cyclooxygenase (COX), also known as prostaglandin endoperoxide synthase, catalyses the conversion of arachidonic acid to prostanoids. There are two different isoforms of COX, referred to as COX-1 and COX-2. Overexpression of COX-2 has been demonstrated in various neoplasms, such as experimentally promoted tumors, gastrointestinal cancers and breast tumors. METHODS: In this study, we used immunohistochemistry to investigate COX-2 expression in a series of basal cell epitheliomas (BCE), Bowen's disease, squamous cell carcinomas (SCC) and metastatic tumors of the skin. RESULTS: Four of 16 BCE showed a positive reaction for COX-2 and the adenoid type of BCE was the most strongly positive. In Bowen's disease, the extent of positive staining for COX-2 was even higher than that in BCE. Eleven of 15 SCC showed a positive reaction for COX-2 and the pattern of staining was heterogeneous with more intense staining in the center of the tumor nests. In metastatic tumors, the percentage of COX-2-positive tumor cells and the intensity of their staining was low compared with Bowen's disease and SCC. CONCLUSIONS: These results indicate that the intensity of COX-2 staining and its heterogeneous distribution are related to the degree of cellular differentiation and the various phenotypes of tumor cells, but the extent of COX-2 staining did not correlate with the degree of malignancy.

Expression of neuropeptide-degrading enzymes in alopecia areata: an immunohistochemical study.

BACKGROUND: Much clinical evidence suggests that the nervous system, including psychological factors, can influence the course of alopecia areata (AA). However, there has been little substantial evidence of specific participation of cutaneous neurogenic factors in the disease process. OBJECTIVES: As previous studies have demonstrated that stress elicits the release of the neuropeptide substance P (SP) from peripheral nerves and that some patients with AA show prominent SP expression in nerves surrounding their hair follicles, we aimed to evaluate the role of SP in AA. METHODS: We used immunohistochemistry to examine the expression of SP and SP-degrading enzymes in scalp biopsies from patients with AA and from healthy controls. RESULTS: Affected hair follicles in the centre of the areas of hair loss of patients with AA were richly innervated by SP-staining nerve fibres. Strong expression of the SP-degrading enzyme, neutral endopeptidase (NEP), was observed in hair follicles not only in the acute progressive phase of AA but also in the chronic stable phase. Expression of NEP in hair follicles from the margins of areas of hair loss was stronger than in normal controls, but was weaker than in the centre of the areas of hair loss. In addition, endothelial immunoreactivity for angiotensin-converting enzyme (also capable of degrading SP) was not observed in the centre of the areas of hair loss, which was in significant contrast to normal controls as well as to the margins of areas of hair loss where it was expressed. Further, intense expression of endothelial leucocyte adhesion molecule-1 on vessels and many degranulating mast cells was observed adjacent to affected hair follicles in AA, in admixture with dense lymphocytic inflammation. CONCLUSIONS: These findings suggest that SP is endogenously released by dermal nerve fibres around hair follicles and that it may play an important part in epithelial-mesenchymal-neuroectodermal interactions in AA. This study reveals that SP and its degrading enzymes are involved in the pathogenesis of AA, which in turn might explain the pathological significance of neurogenic and psychogenic aspects in the disease process.

An ultrastructural study of lipodystrophia centrifugalis abdominalis infantilis, with special reference to fibrous long-spacing collagen.

Lipodystrophia centrifugalis abdominalis infantilis (LCAI) in a 2.5-year-old Japanese girl is reported. Clinically she had a curved cutaneous depression with a slightly elevated erythematous border on the left abdomen beyond the left groin. The regional lymph node was palpable. Histologic examination showed a decrease of the fat, an inflammatory infiltrate mainly composed of lymphocytes, and septal fibrosis in the subcutaneous tissue. Immunohistochemical analysis found that infiltrating lymphocytes were mainly positive against CD4. Ultrastructurally nuclei in the adipocytes were crescent shaped and located toward the periphery of the cell. Some banded structures with a periodicity of 150 nm, and with intraperiodic bands, referred to as fibrous long-spacing collagen (FLSC), were observed in the septal area of the fatty tissue. These results indicated that FLSC was correlated with the breakdown of fibrillar collagens in LCAI.

Characteristics of Pseudomonas aeruginosa isolated from skin infections.

From January 1995 to June 2000, 19 Pseudomonas aeruginosa were detected in 19 cases of infectious skin diseases. Coagulase-negative staphylococci coexisted with P. aeruginosa in 10 cases and P. aeruginosa was also often associated with Enterococcus sp., Staphylococcus aureus, Streptococcus sp. or other species. Twelve cases carried P. aeruginosa more predominantly than the other species. Some coagulase-negative staphylococci, Enterococcus sp., S. aureus and Streptococcus sp. were also predominant. The frequency of predominant P. aeruginosa was significantly higher in secondary infections due to ulcers and decubitus than in secondary infections due to other skin diseases. P. aeruginosa was also often detected in the legs and feet, but not in the trunk. This organism showed high sensitivities to antibiotics, except minocycline. No significant differences in sensitivity or resistance to antibiotics were found between P. aeruginosa when detected as the predominant or single strain and this microorganism when detected as the non-predominant strain. Thus, the detection of P. aeruginosa important in choosing the appropriate antibiotics to be used and this is sometimes influenced by the patient's condition.

Relationship between Propionibacterium acnes biotypes and Jumi-haidoku-to.

We examined the relationship between Propionibacterium acnes biotypes and Jumi-haidoku-to (JHT). In all the P. acnes strains tested, the production of propionic acid (PA) and butyric acid (BA) was suppressed in a medium containing 1 mg/ml JHT compared with the control medium without JHT. There were no significant differences in the rates of decreased PA and BA production between P. acnes biotype 3 (B3) and the other biotypes or between isolates from mild skin rash and more severe skin rash. P. acnes B3 was the most commonly identified biotype. The clinical effects on acne due to the anti-P. acnes lipase activity of JHT did not seem to be influenced by the degree of acne rash or the P. acnes biotype.

Predominant Staphylococcus aureus isolated from various skin diseases.

We examined predominant Staphylococcus aureus isolated from lesions of various skin diseases, and during the past 3 years, 113 methicillin-sensitive and 31 methicillin-resistant S. aureus have been isolated. The predominant species isolated from almost all of the primary bacteriological cultures was S. aureus. The skin diseases from which cultures were most frequently prepared were atopic dermatitis, followed by ulcers. S. aureus was the predominant species in two-thirds or more of cases of all of the different skin diseases examined. The predominant species identified, other than S. aureus, included Streptococcus species and Pseudomonas aeruginosa. Evaluation of the predominance of S. aureus is important to the determination of the severity of skin lesions and, if needed, appropriate antimicrobial therapy.

Correlation between Propionibacterium acnes biotypes, lipase activity and rash degree in acne patients.

We examined the possible correlation between biotypes of Propionibacterium acnes, lipase activity, and rash degree in acne patients. Among 5 P. acnes biotypes, P. acnes biotype 3 (B3) was the most common, followed by P. acnes biotypes 1, 2 and 4; P. acnes biotype 5 was not found. P. acnes B3 was isolated from more severe skin rashes than those of the other biotypes. Production of propionic acid (PA) and butyric acid (BA) by P. acnes B3 was higher than those by the other P. acnes biotypes. As the rash degree in acne patients was more severe, the production of PA and BA elevated. Although only a few P. acnes strains were examined in the present study, P. acnes B3 had the highest lipase activity and might have the greatest influence on skin rash in acne patients.

Characterization of Peptostreptococcus species in skin infections.

We examined the characteristics of Peptostreptococcus species in infectious skin diseases. P. magnus was the species identified most frequently, followed by P. asaccharolyticus. Peptostreptococcus species were mainly isolated from infected atheroma and secondary infections due to ulcers; their resistance to five antimicrobial agents was generally low. The resistance of the three predominant Peptostreptococcus species to the antimicrobials was similar to that of all of the Peptostreptococcus species. The predominant Peptostreptococcus species isolated from infected atheroma might be pathogenic. It was considered that the presence of anaerobes as well as aerobes was important to induce the infectious condition.

Immunohistochemical localization of ornithine decarboxylase in skin tumors.

BACKGROUND: Ornithine decarboxylase (ODC) plays an important role in the biosynthesis of polyamines. Induction of ODC and polyamine synthesis has been demonstrated in neoplastic tumors and is thought to be related to the degree of malignancy. METHODS: In this study, we investigated a series of basal cell epitheliomas (BCE), Bowen's disease, squamous cell carcinomas (SCC), and metastatic tumors of the skin using an antibody against ODC for immunohistochemistry. RESULTS: Eight of 12 cases of BCE failed to show a positive reaction for ODC. In Bowen's disease, 5 of 13 cases diffusely showed positive reaction for ODC. Fourteen of 15 cases of SCC showed ODC expression, the intensity of which was decreased in the peripheral layer. At higher magnification, the distribution of ODC in the positive SCC cases showed granular and heterogenous patterns. Ten of 14 cases of metastatic skin tumors exhibited positive reactions, and well-differentiated adenocarcinomas tended to show more strongly positive than poorly-differentiated ones. CONCLUSIONS: These results support the conclusion that the intensity and the incidence of positive immunohistochemical staining for ODC correlate with the degree of cellular differentiation, and furthermore, that heterogenous distribution of ODC staining may be associated with heterogeneity of tumor cells.

Alopecia mice derived from albino ddY mice: breeding, histological and ultrastructural studies.

Sparse-haired (SH) mice arose spontaneously in a stock of sib-mated albino ddY mice. Hair loss in the skin of SH mice begins at 3 weeks of age and progresses with age. On the basis of breeding studies between SH mice and albino ddY mice, the phenotype of SH mice is identified as autosomal recessive inheritance. Histological examination shows that numerous pilary canals begin to widen and the outer root sheath becomes thin at 1 week of age. The inner root sheath and the hair were separated. Ultrastructural examination reveals that electron dense materials which formed coalesced and irregularly coiled chains are seen at the level of firm attachment of the hair to its inner root sheath. At the level of hair and inner root sheath separation, linear materials with high electron density are observed in the exocuticle of the hair cuticle. These results suggest that the appearance of electron dense materials is closely related to the mechanism of hair loss in SH mice and SH mice may be useful as an animal model in human hypotrichosis.

An ultrastructural study of pigmented purpuric dermatitis with special reference to fibrous long-spacing collagen.

A case of pigmented purpuric dermatitis (PPD) in a Japanese man aged 59 years is reported with an interesting ultrastructural finding. Clinically, the lesions, which consisted of telangiectatic puncta and pigmentation, were irregular in shape and occurred predominantly on the lower legs without pruritus. Histologically, lymphocytic perivascular infiltrates and extravasation of red blood cells were observed in the papillary dermis. Ultrastructurally, endothelial cells with ovoid nuclei showed swelling and the lumen of the capillary became narrowed. Several banded structures, so-called fibrous long-spacing collagen (FLSC), were observed in the cytoplasm. They were spindle shaped, about 5 microm in length, and showed crossbands of 300-nm-wide intervals with fine intraperiodic bands. These structures were not observed in dermal connective tissue and fibroblasts. These results suggested that FLSC was synthesized in endothelial cells rather than being phagocytosed by endothelial cells, which might be helpful in investigating the etiology of PPD.

An ultrastructural and immunohistochemical study of pigmented dermatofibrosarcoma protuberans (Bednar tumor).

A case of Bednar tumor on the right shoulder of a 47-year-old Japanese woman is reported. Histological examination showed plump, spindle cells arranged in a storiform pattern in central areas of the tumor and a diffuse infiltration of the dermal stroma, which was frequently extended into the subcutis at the periphery of the tumor. The tumor contained a fairly identified population of dendritic pigmented cells. Ultrastructurally, most cells had folded nuclei, were spindle-shaped and had long, slender cytoplasmic projections. Dendritic pigmented cells, which were dispersed among neoplastic cells, contained premelanosomes and mature melanosomes. Immunohistochemically, tumor cells exhibited positive reactions for vimentin and CD 34 and failed to show a positive reaction for neuron specific enolase, HMB-45 or S-100 protein. Factor X IIIa was only expressed on tumor cells around melanin-containing cells, which reacted positively with antibodies to S-100 protein and vimentin. These results indicate that the phenotype of tumor cells around melanin-containing cells differs from other tumor cells and that this difference may be caused by the relationship of tumor cells and melanin-containing cells.

Phytol is a novel tumor promoter on ICR mouse skin.

Phytol is a branched, long-chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation-promotion model, and compared its promotion activity with that of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 microg of 7,12-dimethylbenz(a)anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 microg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose-dependent manner and showed a weak, delayed induction (which was maximal 11-12 h after treatment) as compared with the case of TPA. The specific binding of [3H]phorbol-12,13-dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 mM. These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non-TPA-type tumor promoter in this model of mouse skin carcinogenesis.

The induction by topical minoxidil of increased fenestration in the perifollicular capillary wall.

We report the induction by topical minoxidil of increased fenestration in the perifollicular capillary wall. Male 30-day-old Wistar rats were divided into two groups, i.e. an experimental group which received 4% minoxidil solution topically on the dorsal skin, and a control group which received only vehicle solution topically. Using transmission electron microscopy, follicular and subepidermal capillaries and dermal fibres were compared between both groups. There were no obvious differences in subepidermal capillaries or dermal fibres between the two groups. However, topically applied minoxidil increased fenestration in follicular capillary walls around anagen hair bulbs.