A case of 'smouldering' mastocytosis with high mast cell burden, monoclonal myeloid cells, and C-KIT mutation Asp-816-Val.

Mastocytosis is a term used for a group of disorders characterized by abnormal growth and accumulation of tissue mast cells (MC) in one or more organ systems. In patients with systemic mastocytosis (SM) the clinical course may be indolent or aggressive or even complicated by leukemic progression or an associated clonal hematologic non mast cell lineage disease (AHNMD). However, at first presentation (diagnosis) it may be difficult to define the category of disease and the prognosis. We report on a 48-year-old female patient with SM with urticaria pigmentosa-like skin lesions and mediator-related symptoms. She was found to have splenomegaly, a high infiltration grade (MC) in bone marrow biopsies (>30%), mild anemia, and a high serum tryptase level (>500 ng/ml). In addition, she exhibited discrete histologic signs of myeloproliferation in the 'non-affected' marrow and monoclonal blood cells established by C-KIT 2468A-->T mutation (Asp-816-Val) -analysis and HUMARA assay. Despite these findings, however, the clinical course was stable over years and no AHNMD or organ impairment developed. Because of the 'intermediate' clinical signs and absence of progression to aggressive disease, we proposed the term 'smouldering mastocytosis'.

Physical changes of amniotic membranes through glycerolization for the use as an epidermal substitute. Light and electron microscopic studies.

Our Department of Plastic and Reconstructive Surgery has routinely been using amnion preserved in glycerol for the treatment of debrided II degrees burns. This treatment is almost pain free and requires fewer changes of dressings and fewer anaesthetics. It also prevents overgrowing granulation tissue and lessens scarring.Since 1910 amnion has been used as biological wound dressing. Its advantages such as reduced loss of protein and electrolytes, fluids and energy as well as reducing the risk of infection and accelerated regeneration of the epithelium have been well documented in medical literature.In order to more closely examine the question of possible changes to the micro structure of the amnion through preservation and rehydration as well as the interaction between transplanted tissue and recipient, we have carried out several light and electron microscopic studies.Results showed that neither the treatment with glycerol, nor the pretransplantation rehydration eliminates the monolayer of surface epithelium of the amnion. Its complex architecture remains intact during the preservation process and is therefore suitable as a matrix for the growth of keratinocytes and thereby the healing process.In clinical use we found amnion to be an excellent wound dressing as it allows proper control of fluid, has sufficient permeability for gases, has good thermal properties, is impervious to micro-organisms and is free from toxic material.

Hydroxyethylstarch deposits in human skin--a model for pruritus?

Severe itching for unknown reasons has been reported after administration of hydroxyethylstarch (HES) in haemodilution therapy of humans. After HES treatment, vacuoles in cells of various organs in humans have been shown, predominantly affecting the mononuclear phagocyte system. These vacuoles present indirect evidence for phagocytosis of HES particles. Since phagocytosis is also known to occur in the skin, this organ might represent a target for HES deposition, resulting in subsequent release of mediators responsible for the observed itching. The aim of the present investigation was to study skin biopsies of patients, who had received HES and suffered subsequently from itch. Skin sections were investigated for morphological impairment by means of light and electron microscopy, immunohistochemistry and immunoelectron microscopy using a polyclonal anti-HES antiserum. Storage of HES was demonstrated in the skin of all patients, mainly in dermal macrophages, endothelial cells of blood and lymph vessels, some perineural cells and endoneural macrophages of larger nerve fascicles, some keratinocytes and Langerhans cells. Treatment with antihistaminic agents proved ineffective in these patients; this fits with the observation that morphological signs of histamine release from mast cells were absent. These findings indicate that other mediators from HES-affected cells must be responsible for the development of the itching. Thus, investigation of HES storage may be a useful contribution to the elucidation of release of itch mediators and induction of pruritus.

Human papillomavirus in cervix carcinoma and condylomata acuminata--identification of HPV-DNA by improved dot-blot-hybridization.

Using the benzoylated naphthoylated DEAE cellulose method (BND-method) we have designed a more efficient approach for the detection of human papillomavirus-DNA (HPV-DNA) via dot-blot and hybridization. Biopsy material from anogenital warts (40 patients), invasive carcinoma uteri (12 patients) and normal controls (20 patients) were studied for the presence of HPV-DNA. Phenol extracted DNA from representative lesions was loaded onto a pretreated nitrocellulose filter, was incubated under stringent conditions with 32-P-dCTP labelled HPV-DNA and exposed to a Kodak X-OMAT film. DNA of HPV types 11, 16, 18 were cloned into plasmid vectors. The common, time-consuming caesium-chloride density-gradient centrifugation used for purification of plasmid DNA (20-36 h), was substituted by the BND-method (15 min). Complete HPV genomes were excised using the restriction endonucleases Eco RI and Bam HI. The HPV-DNA fragments obtained were then electroeluted using the 'Bio-Trap' method and subsequently labelled with 32P-dCTP by nick translation. Without resorting to more complex and sensitive technology, such as the polymerase chain reaction, efficiency of specific analysis of large numbers of cervical samples and condylomata was achieved without loss of accuracy or increased costs. The time required for HPV identification from biopsy or sample receipt was shortened considerably (approximately 50%).

Comparative histochemistry of port-wine stains after copper vapor laser (578 nm) and argon laser treatment.

The present study compared the histologic changes occurring 15 min after copper vapor laser (CVL; operating at 578 nm) and argon laser (488/514 nm) treatment of port-wine stains (PWS) over a range of energy densities (8-32 J/cm2) with corresponding pulse widths of 50-200 ms. Frozen tissue sections were stained with nitroblue tetrazolium chloride (NBTC). This histochemical method permits an accurate color differentiation between blue-stained viable and unstained thermally damaged cells. At 8, 10, and 12 J/cm2 the argon-laser injury was confined to epidermal cell layers; none to superficial dermal effects were found. Fluences of at least 15 J/cm2 produced a diffuse NBTC-negative coagulation necrosis. Exposure of PWS skin to 8-12 J/cm2 at 578 nm did not alter the integrity of epidermal cells. In the dermis, damage was confined to blood vessels and surrounding collagen, showing a clear demarcation from adjacent viable structures. The maximum penetration depth achieved with these vessel selective energy densities was 0.44 mm. At 15 J/cm2, besides vascular injury, damage to the basal cell layer also occurred. At fluences of 17-20 J/cm2 a diffuse necrosis similar to that induced by the argon laser was found. Vessel selectivity of the 578 nm wave band was achieved with pulse widths from 50-74 ms, exceeding the estimated "ideal" exposure time (0.1-10.0 ms) for a vascular selective laser effect. The NBTC method allowed identification of subtle laser-induced tissue changes providing accurate quantitative data relating to the extent of vascular injury.

Quinolones as an alternative treatment of chlamydial, mycoplasma and gonococcal urogenital infections.

We report the results of several trials aimed at evaluating the quinolones in urogenital infections. In Chlamydia trachomatis infections, ofloxacin (200 mg b.i.d. for 10 days) gave a cure rate of 98% (n = 66), and fleroxacin (400 mg s.i.d. for 7 days) provided a cure rate of 89% (n = 19). A double-blind study comparing fleroxacin (600 mg s.i.d.) to doxycycline (100 mg b.i.d.) for 7 days showed similar high cure rates for both regimens (100%; n = 23). In Mycoplasma hominis infections, ofloxacin (200 mg b.i.d. for 10 days) yielded a cure rate of 86% (n = 50) for M. hominis and 55% (n = 43) for Ureaplasma urealyticum. Gonococcal infections (n = 122) were all cured by a single dose of 200 mg ofloxacin. Both ofloxacin and fleroxacin were well tolerated and may be recommended for patients with chlamydial or uncomplicated gonococcal infections, although 600 mg fleroxacin showed a higher incidence of adverse events compared to doxycycline.

Histological and immunohistochemical investigations of hydroxyethyl-starch deposits in rat tissues.

Tissue storage of hydroxyethyl starch (HES), a widely used artificial colloid, has been reported. In order to clarify whether storage of HES can be detected in tissues by immunohistochemical methods, use was made of a polyclonal rabbit anti-HES antiserum. Thirteen days after a single intravenous injection of HES rats were sacrificed and liver, spleen, lymph node, lung, kidney and skin were removed. On paraffin sections in all organs the anti-HES antiserum stained mainly cells which could be attributed to the mononuclear phagocyte system, as confirmed by the use of the antimacrophage monoclonal antibody ED1. The use of a polyclonal anti-HES antiserum may allow analysis of long-term storage and possible side effects in various tissues of man.

Accurate histochemical definition of argon-laser-induced tissue necrosis.

We present a histochemical method using the redox dye nitro blue tetrazolium chloride (NBTC) for the evaluation of argon-laser-induced tissue damage. By this method viable cells are stained blue, devitalized cells not. Our findings suggest that the NBTC staining method is more appropriate for the accurate definition of the necrosis and cell viability after laser treatment than routine histology.

Enzyme histochemical analysis of cell viability after argon laser-induced coagulation necrosis of the skin.

Reduction of nitroblue tetrazolium chloride, a redox indicator, by nicotinamide adenine dinucleotide diaphorase produces in frozen tissue sections an intense blue cytoplasmic pigment. The activity of this enzyme has been shown to subside immediately upon cell death. Twelve patients with port-wine stains were treated with an argon laser. Frozen tissue sections from biopsy specimens obtained before and 10 minutes, 24 hours, and 48 hours after laser application were processed for nitroblue tetrazolium chloride staining. In normal skin all epidermal and dermal cells displayed dense cytoplasmic blue granular pigment that spared the nuclei. In port-wine stains the laser-induced coagulation necrosis was first seen as an arc-shaped, sharply demarcated, unstained, nitroblue tetrazolium chloride-negative area. Initiation of epidermal repair could be observed in all 48-hour sections. The nitroblue tetrazolium chloride method, when compared with hematoxylin and eosin staining, allowed an easier and more accurate definition of laser injury because of the color difference between damaged and normal tissue.

Sjögren syndrome: comparative studies in local ocular and serum immunoglobulin concentrations with special reference to secretory IgA.

The objective of the present study was to examine Secretory IgA in tears and serum of Sjögren patients (34 patients and 23 controls). The test was performed in a parallel study using a polyclonal and a monoclonal method (Inter-Assay variation 9.1/Intra-Assay variation 5.5). No differences in the levels of tear secretory IgA were found between patients and controls. In serum however, secretory IgA, total IgA, IgM and IgG showed a significant increase in the Sjögren patients. The fact that secretory IgA levels remain normal in the ocular mucosa of these patients may explain that, despite the autoimmune destruction of the lacrimal gland, humoral defense factors are normally present on the surface of the eye to combat infections.

Immunoglobulins coat microorganisms of skin surface: a comparative immunohistochemical and ultrastructural study of cutaneous and oral microbial symbionts.

Only recently have human sweat glands been demonstrated to secrete immunoglobulins (Ig), paralleling Ig secretion in mucosal epithelia. It is well established that Ig protect mucosal membranes against infections by binding to surface structures of microorganisms. In view of these findings immunohistochemical studies were performed to determine if microbes on the skin surface are coated by Ig as proposed for mucosal bacteria and fungi. Smear preparations from the skin and oral cavity rich in micro-organisms were subjected to immunoperoxidase staining using anti-secretory component (SC), -IgA, -IgM, -IgG antibodies. An immunogold labeling technique of microbial suspensions of sweat and saliva was adapted to correlate the results on an ultrastructural level. Negative controls included unsuccessful staining for IgA in preparations obtained from an IgA-deficient patient as well as nonreactivity of subcultured microorganisms for all Ig classes or SC. Smear preparations from both the oral cavity and skin surface exhibited labeling of bacterial or fungal elements with anti-IgA, -IgM, -IgG, and -SC antibodies. Skin bacteria revealed a lower number of reactive microbes as compared to saliva. Staining intensity for the different Ig classes exhibited intra- and interindividual variations. Immunoelectronmicroscopically, Ig and SC could be detected either directly along the cell wall of coccal, coryneform, and fungal elements or on floccular and fimbrial material adhering to the bacterial surface. It is concluded that secretory Ig of the skin cover surface structures of microorganisms and thus modify their adhesional and/or infectious properties, resembling humoral surface immunity on mucous membranes.

Borrelia burgdorferi and different types of morphea.

Thirty patients with different clinical manifestations of morphea (circumscribed scleroderma) were investigated for serum antibodies against Borrelia burgdorferi determined by ELISA and Western blot analysis. Forty-six percent of the patients were seropositive. Western blots confirmed the ELISA results in 10 of 25 patients (40%), showing a reactivity pattern which can be seen in the course of Lyme borreliosis. In some cases the outcome after antibiotic treatment suggests a direct correlation between the further development of skin lesions and Borrelia infection. Because of these findings we suggest some morphea types to be possibly due to a B. burgdorferi infection.

Histochemical evaluation of the coagulation depth after argon laser impact on a port-wine stain.

A two-step excisional treatment of a port-wine stain (PWS) on the back of a 43-yr-old female patient was performed. Immediately before the first surgical treatment, two corresponding series of argon laser impacts were performed, each on one PWS half. Different laser parameters with irradiances ranging from 95 to 382 W/cm2 and energy fluences ranging from 19 to 114,6 J/cm2 were used. Laser spots on the first part ot be excised were biopsied 10 min after laser treatment and prepared for histochemical analysis by staining with nitro blue tetrazolium chloride (NBTC). Reduction of this redox dye by nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase) leads on frozen tissue sections to an intense blue precipitate. The activity of NADH-diaphorase subsides immediately upon cell damage. All vital epidermal and dermal cells presented a dense blue granular pigment in their cytoplasm, sparing the nuclei. Laser induced arc-shaped epidermal and dermal necrosis did not stain, showing a clear demarcation from surrounding vital tissue. The depth of the thermal injury ranged from 0.28 to 0.45 mm; it did not correlate with the chosen fluences. With these penetration depths, the vast majority of PWS vessels was affected. Assessment of the remaining part of the PWS 8 months later yielded blanching of all laser-treated areas. With the NBTC method, an accurate definition of laser-induced tissue damage is feasible. It could be shown that the exposure time is the most relevant parameter influencing the penetration depth.

Xanthoma disseminatum with progressive involvement of the central nervous and hepatobiliary systems.

A rapidly progressive disease with multiple organ involvement and symptoms of polyuria and polydipsia developed in a 32-year-old woman, with an initial presentation of xanthoma disseminatum. Within 6 months increasing visual impairment of the right eye developed; a computed tomographic scan revealed multiple cerebral and cerebellar lesions. Progress of her disease resulted in blindness of the right eye, internal hydrocephalus with involvement of both ventricles, and cerebellar ataxia. Ultrasound examination of the abdomen and a subsequent endoscopic retrograde cholangiopancreatographic examination revealed a xanthomatous gallbladder and liver involvement. The gallbladder and liver showed histologic changes compatible with xanthomatosis. The patient died 4 years after the appearance of skin lesions.

Disseminated syringomas of the upper extremities. Case history and immunohistochemical and ultrastructural study.

A case of disseminated syringomas with unusual distribution and high age of onset is reported. The 66-year-old male patient presented with multiple lesions confined to dorsum of both hands and flexor side of both forearms. Morphological and immunohistochemical studies using keratin, S-100, carcinoembryonic antigen, and epithelial membrane antigen antibodies were performed both at light microscopical and ultrastructural level. These investigations revealed the presence of keratin filament containing colloid bodies near syringomatous epithelia. Reactivity for carcinoembryonic antigen could be demonstrated on the membranes of intracytoplasmic vesicles as well as within luminal debris. Absence of S-100 and epithelial membrane antigen in the tumor and ultrastructural features indicate ductal origin of syringoma.

Disseminated superficial actinic porokeratosis: experimental induction and exacerbation of skin lesions.

A 55-year-old woman with disseminated superficial actinic porokeratosis (DSAP) had lesions on sun-exposed skin areas that were exacerbated during the summer months and involuted in winter. This is the third report in which induction and exacerbation of DSAP lesions were achieved by irradiation with artificial ultraviolet light sources. Our data show that UVB plus UVA is more effective in inducing new or exacerbating preexisting skin lesions than either wavelength alone. We believe that testing with the appropriate ultraviolet light sources is a practical means to differentiate between DSAP and disseminated superficial porokeratosis.

Unusual presentation of porokeratosis palmaris, plantaris et disseminata.

Porokeratosis plantaris, palmaris et disseminata is an autosomal dominant genodermatosis characterized by multiple lesions on the palms and soles, and later on other areas, both sun-exposed and non-sun-exposed. We report a 66-year-old man with porokeratosis plantaris, palmaris et disseminata whose disease had an unusual evolution. To our knowledge this is the first case of the disease in which the lesions first appeared on the trunk and extremities and later involved the palms and soles.

DNA dot-blot hybridization implicates human papillomavirus type 11-DNA in epithelioma cuniculatum.

Epithelioma cuniculatum is a rare, low-grade squamous cell carcinoma, belonging to the group of verrucous carcinomas. Evidence is presented that suggests that human papilloma virus type-11 may be involved in the pathogenesis of epithelioma cuniculatum. HPV-DNA was detected by dot-blot hybridization with 32P-labelled probe DNA. Plasmids containing HPV-DNA were isolated by the benzoylated naphthoylated DEAE cellulose method (BND-method), an improved procedure for routine detection of HPV-DNA.

Giant arteriovenous hemangioma (cirsoid aneurysm) of the nose.

We describe a 97-year-old woman who presented to our clinic with an unusually large pendulous asymptomatic soft tumor on the left side of her nose. History revealed a 40-year course, with accelerated growth in the last 9 years. Histology of this 7 x 3-cm large tumor revealed vascular structures composed of densely aggregated thick- and thin-walled vessels, separated by fibrous tissue. These features are characteristic for an arteriovenous hemangioma, also called cirsoid aneurysm. The distal part of the tumor showed nearly total fibrosis and at the proximal end of the tumor signs of incipient basal cell carcinoma were observed. In spite of the accelerated growth, unusual size, and the presence of a basal cell carcinoma at the base of this tumor, giant cirsoid aneurysms appear to be of a benign nature and thus conservative, nonradical surgical treatment can be performed. The tumor was removed by argon laser surgery with excellent results and no sign of recurrence after 1 year.

Secretory immunoglobulin A in sweat gland tumors.

The presence of immunoglobulin A (IgA) and secretory component (SC) was investigated in normal human skin and in cutaneous neoplasms including a variety of sweat gland tumors. Immunohistochemistry in normal sweat glands revealed the occurrence of secretory IgA (sIgA) as indicated by reactivity for IgA and SC in serial sections. The majority of 28 cases of sweat gland tumors could be demonstrated to retain their ability to produce IgA and SC. In normal as well as in neoplastic sweat glands heaviest staining for sIgA could be found in the lumina and at the surface of lining epithelia. This is comparable with the presence of sIgA in breast or intestinal neoplasms. In contrast other epidermal cysts or solid tumors were not labelled. In view of recent immunohistochemical studies the demonstration of IgA and SC may be of differentiating value in cutaneous glandular neoplasms.