The molecular basis for inherited bullous diseases

In the past 5 years enormous progress have been made in our understanding of the molecular basis for a number of inherited skin diseases characterized by easy blistering of the skin and the mucous membranes after minor physical trauma. This increased fragility of the skin or its appendages is due to molecular defects in genes coding for different intra- and extracellular structural proteins which are responsible for mechanical strength at their sites of expression. These diseases encompass the group of epidermolysis bullosa and disorders of cornification such as bullous forms of ichthyosis, palmoplantar keratoderma, and pachyonychia congenita. On the basis of clinical, morphological, and ultrastructural observations the epidermolysis bullosa group has been divided into three major categories. In epidermolysis bullosa simplex blister formation appears within the basal cell layer of the epidermis, and many mutations have been found in the genes of keratin 5 and 14 which are both expressed in basal keratinocytes. Epidermolytic hyperkeratosis leads to an epidermal separation in the suprabasal cell layers. In these patients numerous point mutations have now been described in the suprabasally expressed genes of keratin 1 and 10. In ichthyosis bullosa of Siemens blisters occur in the more upper suprabasal epidermis coincidental with the expression of keratin 2e, and mutations have been detected in the corresponding gene. In epidermolytic palmoplantar hyperkeratosis the suprabasal epidermal splitting is restricted to palms and soles of the patient. In keratin 9, which reveals such an exclusive expression pattern, molecular defects have indeed been recognized. Most recently in two different clinical subtypes of pachyonychia congenita, which is characterized by defective nails and focal palmoplantar hyperkeratosis, point mutations have been found in the genes coding for keratins 6, 16, and 17. In junctional epidermolysis bullosa the separation takes place within the dermal-epidermal basement membrane at the level of the lamina lucida, and mutations have been found in three genes coding for different laminin chains, in the ₄ gene of ₆₄ integrin, and in the gene of collagen XVII. In dystrophic epidermolysis bullosa the tissue separation occurs beneath the basement membrane within the papillary dermis at the level of the anchoring fibrils, and several mutations have been identified in the collagen VII gene. The rapid unraveling of molecular defects in these disabling or even lethal inherited skin diseases makes possible a more precise and earlier prenatal diagnosis, creates new options for suitable therapeutic regimens, and even offers the hope of curing these diseases by means of somatic cell gene therapy.Abbreviations BM Basement membrane - BPAg Bullous pemphigoid antigen - DEB Dystrophic epidermolysis bullosa - EB Epidermolysis bullosa - EBS Epidermolysis bullosa simplex - EHK Epidermolytic hyperkeratosis - EPPK Epidermolytic palmoplantar keratoderma - IBS Ichthyosis bullosa of Siemens - JEB Junctional epidermolysis bullosa - KIF Keratin intermediate filaments - NC Noncollagenous domain - NEPPK Nonepidermolytic palmoplantar keratoderma - PC Pachyonychia congenita     

Resistance of mucoid Pseudomonas aeruginosa to nonopsonic phagocytosis by alveolar macrophages in vitro.

A unique, recently described rat alveolar macrophage cell line (NR8383) was used to study the interaction of the pulmonary immune system with a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa (SRM-3), its nonmucoid revertant (SRM-3R), and a non-cystic fibrosis isolate (PAO-1). Strain SRM-3 was cultivated in a chemostat system to allow maintenance of an entirely mucoid population. The alveolar macrophage response to the mucoid and nonmucoid strains of P. aeruginosa was determined by visually quantitating phagocytosis in acridine orange-stained monolayers and measuring the induction of an oxidative burst as indicated by chemiluminescence and H2O2 production. In all experiments, fewer than 2% of the NR8383 cells engulfed the mucoid SRM-3 isolate, while SRM-3R and PAO-1 were phagocytized by 15 and 41%, respectively. Opsonization by normal serum (complement) provided minimal phagocytic enhancement of these strains, whereas specific anti-P. aeruginosa antibody slightly elevated phagocytic responses to strains with nonmucoid phenotypes while providing a sevenfold increase in uptake of SRM-3. Chemiluminescent and H2O2 responses were comparable with the levels of phagocytosis observed, with very little or no response to the mucoid strain SRM-3. The data indicate that the strains with mucoid phenotypes are refractile to ingestion and that studies which describe ingestion of mucoid strains were likely measuring ingestion of revertants. Alginic acid (2 mg/ml) was found to inhibit stimulation of macrophage response to the opsonized and unopsonized nonmucoid strain PAO-1.     

Improved biotyping schemes for Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.     

Aeration selects for mucoid phenotype of Pseudomonas aeruginosa.

A mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis and its nonmucoid revertant were grown in a chemically defined alginate-promoting medium under batch and continuous culture conditions. Selection for the mucoid and nonmucoid phenotype was accomplished by varying the levels of air available to the culture. The addition of air at a rate of 0.5 liters/min to the nonmucoid revertant growing under batch or continuous culture conditions resulted in a greater than 50% decrease in viability over a 10-h incubation period. In contrast, aeration of the mucoid culture maintained a totally mucoid population and there was no decrease in viability over a 55-h incubation. Aeration of a mixed population of the mucoid and nonmucoid phenotype (1:1) resulted in selection for the mucoid phenotype within the first 20 h of cocultivation. The correlation between the mucoid phenotype and alginic acid was demonstrated by the production of 580 micrograms of uronic acid per mg (dry weight) of cells by the mucoid phenotype and less than 1 microgram of uronic acid per mg (dry weight) of cells by the nonmucoid revertant. These results suggest that nonmucoid revertants may have an unusual sensitivity to aeration, which may indicate a mechanism for natural selection of the mucoid phenotype in vivo.     

Latex specific IgE: performance characteristics of the IMMULITE 2000 3gAllergy assay compared with skin testing.

BACKGROUND: In the absence of a US Food and Drug Administration (FDA)-cleared latex skin testing reagent, in vitro tests remain important for the diagnosis of latex allergy. OBJECTIVE: To evaluate the performance characteristics of IMMULITE 2000 3gAllergy (Immulite), a third-generation, FDA-cleared, continuous random-access immunoanalyzer, for the quantification of latex specific IgE. METHODS: Stored serum samples (N = 201) from patients classified as having positive or negative latex puncture skin test results were measured for latex specific IgE levels using Immulite, and these data were compared with historical results from 3 second-generation, FDA-cleared IgE antilatex assays (AlaSTAT [Ala], AutoCAP [CAP], and HY*TEC enzyme immunoassay [HT]). RESULTS: The diagnostic performances of the CAP, Ala, and Immulite assays (> or = 0.35 kU/L cutoff value) were equivalent in sensitivity and specificity (P > .05). The HT assay (> or = 0.05 kU/L cutoff value) was more sensitive and less specific (P < .05). Immulite (> or = 0.10 kU/L cutoff value) had greater sensitivity than Ala and CAP and greater specificity than HT (P < .05 for both). Diagnostic efficiency was greater for Immulite than for CAP, Ala, and HT (P < .05). CONCLUSIONS: The Immulite system is superior in diagnostic performance, especially at the 0.10 kU/L or greater cutoff level, for the diagnosis of latex allergy compared with older, second-generation assays. Immulite still misclassifies 15.5% of puncture skin test-positive individuals as negative for latex specific IgE. Compared with second-generation assays, Immulite represents a technological advance, with enhanced speed and less operator intervention.     

B cells express Ly-6C in a Th1 but not Th2 cytokine environment.

Interferon-alpha (IFN-alpha) is the primary regulator of transient Ly-6C expression on T cells. B cells, which do not express Ly-6C in the resting state, have been reported to express Ly-6C following exposure to proinflammatory stimuli. This study examined the factors controlling Ly-6C expression on B cells and the kinetics of Ly-6C expression in the presence of these factors. In vivo studies demonstrated that proinflammatory (Th1) cytokines transiently upregulate B cell Ly-6C expression. In vitro studies identified Th1 cytokines, particularly IFN-alpha and IFN-gamma, as the principal cytokines responsible for this induction. Polyclonal B cell activators (anti-IgM and recombinant CD40 ligand trimer) showed minimal ability to independently induce Ly-6C expression on B cells but did enhance the ability of IFNs to induce expression. Th2 cytokine environments did not result in B cell Ly-6C expression, and interleukin-4 (IL-4) actually antagonized the IFN-driven induction of Ly-6C. Ly6.1 strains of mice consistently demonstrated a greater ability to express Ly-6C on B cells than did Ly-6.2 strains. Together, these studies demonstrate the ability of Th1 but not Th2 cytokine environments to transiently induce the expression of Ly-6C on B cells and provide additional evidence for differences in the regulation of Ly-6C expression in Ly6.1 and Ly6.2 strains.     

Role of nitric oxide in the biology, physiology and pathophysiology of reproduction

Following its benchmark discovery, nitric oxide (NO) is nowknown to play important functional roles in a variety of physiologicalsystems. Within the vasculature, NO induces vasodilation, inhibitsplatelet aggregation, prevents neutrophil/platelet adhesionto endothelial cells, inhibits smooth muscle cell proliferationand migration, regulates programmed cell death (apoptosis) andmaintains endothelial cell barrier function. NO generated byneurons acts as a neurotransmitter, whereas NO generated bymacrophages in response to invading microbes acts as an antimicrobialagent. Because neurons, blood vessels and cells of the immunesystem are integral parts of the reproductive organs, and inview of the important functional role that NO plays in thosesystems, it is likely that NO is an important regulator of thebiology and physiology of the reproductive system. Indeed, inthe past 10 years, NO has established itself as a polyvalentmolecule which plays a decisive role in regulating multiplefunctions within the female as well as the male reproductivesystem. This review provides an overview of the role of NO invarious reproductive organs under physiological and pathologicalconditions.     

Präkanzerosen des Urothels

Zusammenfassung In der Fluoreszenzdiagnostik [Lichtanregung nach Gabe des physiologischen Hämmetaboliten 5-Aminolävulinsäure (ALA)] erscheinen Präneoplasien und Neoplasien rot fluoreszierend, was wahrscheinlich auf eine tumorzellselektive Anreicherung des intrazellulär aus ALA gebildeten Metaboliten Protoporphyrin IX zurückzuführen ist. Die mit dieser Methode mögliche frühe Detektion von Tumoren und Präneoplasien erlaubt deren genetische Analyse und damit Vergleiche von Aussagen der Fluoreszenz-in-situ-Hybridisierung und Loss-of-heterozygosity-Analyse hinsichtlich Deletionsnachweis und Gensequenzierung. Neue Daten zu Deletionen und Chromosomenaberrationen sowie eine etwa 30%ige Oligoklonalität von Tumoren werden dargestellt. Die tumorselektive Fluoreszenz scheint biochemisch durch Unterschiede im Hämmetabolismus bedingt zu sein. Durch Gen- und Proteinexpressionsanalysen können evtl. weitere, mit dem Hämmetabolismus assoziierte, tumorspezifische Moleküle identifiziert werden.Die geschilderten Arbeiten erfolgten im Rahmen von Unterstützungen durch die Deutsche Krebshilfe 10-1096-Ha und 70.2200-Ba sowie Industriekooperationen von Medac GmbH und metaGen Pharmaceuticals.     

Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.