Knowledge and attitude regarding human immunodeficiency virus/acquired immunodeficiency syndrome in dermatological outpatients.

BACKGROUND: Dermatologists are often the first-line specialists who recognize and diagnose human immunodeficiency virus (HIV) infection because of pathognomic skin signs. It is therefore important to investigate attitudes and knowledge regarding HIV/acquired immunodeficiency syndrome (AIDS) amongst dermatological patients in order to provide information for dermatologists and to draw their attention to the issues. OBJECTIVES: Awareness of HIV/AIDS, its prevention, and hypothetical behaviour were surveyed in dermatological outpatients. PATIENTS/METHOD: The anonymous cross-sectional survey was conducted with consecutive German-speaking outpatients aged 18-65 years, who registered at the dermatological outpatient's clinic (excluding venereology, genitourinary or HIV medicine) of the University of Munich (Germany). RESULTS: Three hundred forty-seven (77.5%) questionnaires were accepted for analysis. Most of the patients knew about HIV incurability (89.4%), HIV transmissibility during needle sharing (95.3%), or vaginal (87.4%) and anal intercourse (79.5%), as well as about HIV prevention by condom use (97.8%), and use of single needles (76.2%). However, knowledge gaps and misconceptions were detected regarding the risk of HIV transmission during oral sex, and the efficacy of sexual fidelity and avoidance of blood transfusions in HIV prevention. The lowest knowledge level (< 50% correct answers) was detected in patients aged 50-59 years, in unemployed, divorced/widowed, and in those without or with incomplete school education. CONCLUSIONS: Patient education about HIV/AIDS in dermatological ambulant settings should be performed differentially with regard to socio-demographic factors, and focused on the topic of oral sexual HIV transmission and on some other specific misconceptions.

Alterations in the rap1 signaling pathway are common in human gliomas.

Several inherited predisposition to cancer syndromes are associated with the development of nervous system tumors. Tuberous sclerosis complex (TSC) is an autosomal dominant disorder in which affected individuals are at risk for developing astrocytomas. One of the genes responsible for this disorder is TSC2, located on chromosome 16p, and encoding a 180 kDa protein (tuberin) that functions in part as a negative regulator of rap1. Previous studies from our laboratory demonstrated that 30% of sporadic astrocytomas have reduced or absent tuberin expression. In addition to loss of tuberin in sporadic astrocytomas, aberrant rap1 mediated signaling may also result from overexpression of rap1. In this study, we test the hypothesis that alterations in the rap1 signaling pathway are frequently observed in certain subsets of gliomas compared to other tumors of the nervous system. Analysis of sporadic astrocytomas and ependymomas demonstrated either increased rap1 or reduced/absent tuberin protein expression in 50-60% of different cohorts of these gliomas, compared to 30-33% of sporadic schwannomas and meningiomas and none of eight oligodendrocyte tumors. These results suggest that alterations in the rap1 signaling pathway are important in the development of certain sporadic human gliomas.

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

No evidence for Borrelia burgdorferi-specific DNA in lesions of localized scleroderma.

A possible association of Borrelia burgdorferi with localized scleroderma is currently the focus of intense research and discussion. Skin biopsies from 30 patients with localized scleroderma (28 of the plaque type/morphea; two linear scleroderma) were analyzed for the presence of Borrelia burgdorferi using three different polymerase chain reaction systems for amplification of segments of borrelial genes. Formalin-fixed, paraffin-embedded biopsies of 14 patients and fresh-frozen, cryo-conserved biopsies of 16 patients with localized scleroderma were obtained. Lesions of all patients showed clear signs of scleroderma and disease progression at the time of biopsy. Fresh-frozen as well as formalin-fixed biopsies from patients with erythema migrans or acrodermatitis chronica atrophicans were used as positive controls. With all three polymerase chain reaction systems, borrelial DNA was detected in none of the 30 specimens of localized scleroderma. In contrast, with one polymerase chain reaction system, Borrelia burgdorferi-specific DNA was found in 24 of 27 frozen biopsies from patients with erythema migrans and in all 5 analyzed frozen biopsies of patients with acrodermatitis chronica atrophicans. In approximately half of the paraffin-embedded biopsies from patients with erythema migrans (nine of 23) and acrodermatitis chronica atrophicans (13 of 27), Borrelia burgdorferi-specific DNA was identified. These results question the association of localized scleroderma with known subtypes of Borrelia burgdorferi.

Conformational polymorphisms of cRNA of T-cell-receptor genes as a clone-specific molecular marker for cutaneous lymphoma.

A novel molecular assay for the detection and characterization of monoclonal lymphoid populations in clinical specimens was developed. The assay is based on the principle that upon non-denaturing polyacrylamide gel electrophoresis RNA molecules separate into several metastable conformational forms. These conformational polymorphisms strictly depend on the nucleotide sequence of the individual molecule. Using DNA from formalin-fixed, paraffin-embedded tissue of patients with mycosis fungoides, highly variable junctional sequences of rearranged T-cell receptor gamma genes were amplified by polymerase chain reaction. Subsequently, the polymerase chain reactions products were transcribed into complementary RNA and analyzed by non-denaturing polyacrylamide gel electrophoresis. In clinical specimens with a monoclonal lymphoid population, a clone-specific pattern of bands was identified representing conformational polymorphisms of cRNA molecules of rearranged T-cell receptor gamma genes of the predominant lymphoid clone. Three biopsies from one patient taken from different sites of the body over 3 years yielded an identical pattern of bands. This methodology provides a novel and rapid tool for the molecular identification and characterization of clonal lymphoid populations in clinical specimens. It is likely to be of special value for studies on the clonal evolution of lymphoid disorders of the skin.

Localization of tuberous sclerosis 2 mRNA and its protein product tuberin in normal human brain and in cerebral lesions of patients with tuberous sclerosis.

Tuberous sclerosis (TSC), an autosomal dominant disorder, is characterized by malformations, hamartomas and tumors in various organs including the brain. TSC is genetically linked to two loci: TSC1 on chromosome 9q34 and TSC2 on 16p13.3. TSC2 has been cloned, sequenced and encodes a protein (tuberin) which functions as a tumor suppressor. We have analyzed the distribution of TSC2 mRNA and tuberin in the brains of TSC patients and non-affected individuals using both autopsy and biopsy material. High levels of transcript and protein expression were observed in choroid plexus epithelium, ependymal cells, most brainstem and spinal cord motor neurons, Purkinje cells and the external granule cell layer of the cerebellum in both TSC and control cases. Individual balloon cells from TSC patients showed very faint expression while other glia showed no expression of either transcript or tuberin. Neocortical and hippocampal neurons expressed high levels of TSC2 transcript, but only modest levels of tuberin. The internal granule cell layer of the cerebellum expressed abundant transcript but low levels of tuberin. These observations suggest either that tuberin expression is controlled at the level of both transcription and translation or the antibody and in-situ hybridization recognize different splice variants of the TSC2 gene. In TSC patients, dysmorphic cytomegalic neurons expressed high levels of tuberin and transcript, particularly when in an 'ectopic' location. Individual cells within subependymal giant cell astrocytomas (SEGAs) and hamartomas from TSC patients expressed moderate to high levels of TSC2 transcript and tuberin. While the TSC2 transcript is widely expressed primarily within neurons, tuberin is demonstrable primarily within dysplastic/cytomegalic cells of the cortex and subependymal hamartomas/SEGAs. CNS expression of tuberin is unique in that primarily non-dividing cells express it in this location, whereas extra-CNS expression of tuberin is mainly found in actively proliferating cell types such as epithelium.

No detection of Borrelia burgdorferi-specific DNA in erythema migrans lesions after minocycline treatment.

BACKGROUND AND DESIGN: Early treatment of erythema migrans is important to prevent late complications. Minocycline possesses several attributes, making it potentially useful in the treatment of borrelial infections. In our study, minocycline was administered to 14 patients with erythema migrans. Punch biopsy specimens were obtained from the (affected) skin of all patients before and after therapy. The formalin-fixed, paraffin-embedded specimens were analyzed by polymerase chain reaction for the presence of Borrelia burgdorferi-specific DNA. RESULTS: Polymerase chain reaction assay succeeded in amplifying B burgdorferi-specific DNA from the first biopsy specimen, obtained from the border of erythema migrans before initiating treatment, in eight (57%) of 14 patients. At the end of minocycline therapy, however, polymerase chain reaction analysis disclosed no B burgdorferi-specific DNA in any of the 14 patients. The good clinical response of our patients with erythema migrans substantiates our molecular findings. CONCLUSIONS: The presented polymerase chain reaction data, together with the clinical outcome, indicate that minocycline may be useful for treatment of early Lyme borreliosis.

Detection of monoclonal lymphoid cell populations by polymerase chain reaction technology.

Molecular studies have substantially broadened our understanding of cutaneous lymphoproliferative disorders and have been introduced in the routine assessment of lesions suggestive of a lymphoma. As compared with traditional Southern blot analyses, PCR-based assays for the detection of monoclonal lymphoid populations in clinical specimens offer several advantages. In general, they are faster to perform and allow for the detailed molecular analysis of very small tissue specimens, including sections from formalin-fixed, paraffin-embedded biopsy specimens. The PCR-based assays for the analysis of CTCL involve the amplification of highly variable junctional sequences of rearranged TCR-gamma genes, which represent a unique molecular marker for an individual lymphoid cell and a clonal disease developing from this cell. Using various strategies and gel electrophoretic systems, the PCR products can be separated to detect subsets of identical amplification products representing identical gene rearrangements in a clonal lymphoid population in the analyzed specimen. Although the molecular evidence for a monoclonal cell population as provided by PCR technology is an important factor in the diagnosis of a cutaneous lymphoma, only the synopsis of all available clinical, histomorphologic, immunohistochemical, and molecular data will lead to the adequate assessment of a cutaneous lesion.

[Human papilloma virus-induced disease in HIV-positive patients]. / HPV-induzierte Erkrankungen bei HIV-positiven Patienten.

Since the introduction of highly active antiretroviral therapy (HAART), opportunistic infections by bacteria and fungi have been reduced in human immunodeficiency virus (HIV)-positive patients. However, diseases caused by human papilloma virus (HPV) have become more frequent despite HAART. There is an increased incidence of anal and cervical carcinomas, their precancerous lesions such as anal/cervical intraepithelial neoplasia, and condylomas and oral warts. In order to prevent anal carcinomas, HIV-positive patients should receive proctoscopic examinations regularly. The examination should include anoscopy and smears for cytology and HPV polymerase chain reaction.

Photodynamic therapy of genital condylomata in men.

Current treatments for genital condylomata are not completely satisfactory, as they fail to clear lesions in a proportion of patients, and relapses after successful treatment are frequently seen. Photodynamic therapy (PDT) using topical 5-aminolaevulinic acid (5-ALA) has been suggested as a novel treatment option. We performed a small open study using topical 5-ALA and red light (630 nm) in nine men with genital condylomata and a history of at least one previous unsuccessful conventional treatment. Complete cure was achieved in three patients, one of whom experienced a relapse after 3 weeks. Three patients showed partial responses, and three showed no response. Based on the currently available evidence, PDT is a viable treatment option for selected cases that fail to respond to other therapies.

Molecular detection of Borrelia burgdorferi in formalin-fixed, paraffin-embedded lesions of Lyme disease.

A system for the detection of a Borrelia burgdorferi (Bb)-specific gene segment in formalin-fixed, paraffin-embedded skin lesions is described. A nested polymerase chain reaction technique is used to selectively amplify in vitro a short segment of a Bb-specific gene recently described by Rosa et al. (J Infect Dis 1989: 160: 1018). The design of oligonucleotide primers for the amplification of a relatively short gene segment allows the successful analysis of DNA which has been altered by fixation in formalin. Using this technique, Bb-specific DNA was clearly identified in 8 of 12 specimens of erythema chronicum migrans and in 1 case of lymphadenosis benigna cutis. These skin lesions are known to represent cutaneous manifestations of Lyme disease. Negative control reactions, using DNA from borrelial strains not related to Lyme disease, were negative. The system enables the dermatopathologist to identify Bb in routinely fixed clinical specimens and allows the rapid analysis of various skin diseases for which an association with Bb so far has only been hypothesized.

Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity.

Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.

p53 protein in benign and malignant sweat gland tumors.

Mutation of the p53 gene and increased levels of p53 protein are among the most frequent alterations in human cancers. To date, very little is known about the mechanisms underlying the development of sweat gland carcinomas. In this study, we analyzed 43 benign and 39 malignant sweat gland tumors for p53 protein level using the antibody PAB1801. Nine (23%) of 39 sweat gland carcinomas were positive for p53 protein. Among these carcinomas, six of 12 cases of extramammary Paget's disease were positive using immunohistochemistry. No other correlation between tumor subtype and p53 reactivity was detected. Among 43 benign sweat gland tumors, only one case displayed staining for p53. We conclude that p53 protein plays a role in a subset of sweat gland tumors, especially in extramammary Paget's disease.

Cowpox virus infection in an 11-year-old girl.

We describe an 11-year-old girl with a cowpox virus infection, who presented with a 14-day-old crusted, ulcerated nodule on the chin/neck and a 6-day-old eroded blister on the left leg. The girl lived in a rural environment, had close contact to several cats from the neighborhood, and had an atopic predisposition. The presence of orthopox virus in the lesion on the left leg was demonstrated by electron microscopy (negative staining, transmission electron microscopy) and virus isolation. Classification as a cowpox virus was determined by polymerase chain reaction (PCR), followed by restriction enzyme digestion of the PCR product.

Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription factor E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically growing cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested fibroblasts to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.

The polymerase chain reaction. Method and applications in dermatopathology.

Since it was first reported in 1985, the polymerase chain reaction (PCR) has revolutionized the way molecular studies are performed, and has developed into one of the most powerful tools in molecular pathology. By use of a cyclic change of temperature, a specific and exponential in vitro amplification of a target DNA sequence can be achieved within hours. As a template for PCR reactions, total genomic DNA is used; this can be readily extracted from clinical specimens. Very low quantities of DNA, as well as DNA degraded by fixation, can also be used as a template for PCR reactions, allowing formalin-fixed, paraffin-embedded tissue to become amenable to detailed molecular analysis. Sequences specific for certain viruses and other microorganisms, as well as molecular marker sequences associated with various types of human cancer, can be readily detected in paraffin-embedded tissue sections. The methodology of PCR, along with various applications in dermatopathology, are reviewed.