Understanding the role of haptoglobin in psoriasis: effects of ultraviolet B.

BACKGROUND: Haptoglobin (Hp) is one of the acute phase proteins, whose main function is to bind free haemoglobin (Hb) and transport it to the liver for degradation and iron recycling. In addition to its role as an Hb scavenger, Hp has been shown to behave as an anti-inflammatory, antioxidant and angiogenic factor. We previously investigated the role of Hp in the pathogenesis of psoriasis, and found that it displays some structural modifications that might be associated with protein function in the disease. Phototherapy is an efficacious treatment for psoriasis, although the biological mechanisms by which phototherapy improves psoriasis are still unclear. AIM: To investigate the effects of ultraviolet (UV)B on Hp to clarify the role of Hp in psoriasis. METHODS: Expression of the genes encoding Hp, interleukin (IL)-6 and IL-10 was assessed in UVB-irradiated and unirradiated HaCaT cells. The biological significance of Hp modulation of UVB treatment was confirmed by ELISA and Western blotting. The Hp gene and protein expression in the skin of patients with psoriasis was also investigated. RESULTS: In vitro results showed that UVB modulated IL-6 and IL-10 gene expression and Hp gene and protein expression in HaCaT cells. The in vivo data also showed that Hp levels were increased in the skin of patients with psoriasis compared with healthy controls. CONCLUSIONS: UVB irradiation was able to modulate Hp production in immortalized keratinocytes. The higher levels of Hp in vivo in both lesional and nonlesional skin suggest that it might have a role in the pathogenesis of the disease.

Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects.

Preservation of the chemical architecture of a cell or of an organism under changing and perhaps stressful conditions is termed homeostasis. An integral feature of homeostasis is the rapid expression of genes whose products are specifically dedicated to protect cellular functions against stress. One of the best known mechanisms protecting cells from various stresses is the heat-shock response which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). A large body of information supports that stress proteins--many of them molecular chaperones--are crucial for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions, and thus can be considered "homeostatic proteins." Recently emphasis is being placed on the potential use of these proteins in preventing and/or treating diseases. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce the accumulation of HSPs in patients with chronic disorders such as diabetes mellitus, heart disease or kidney failure. Here we show that a novel cytoprotective hydroxylamine derivative, [2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoil-chloride maleate, Bimoclomol, facilitates the formation of chaperone molecules in eukaryotic cells by inducing or amplifying expression of heat-shock genes. The cytoprotective effects observed under several experimental conditions, including a murine model of ischemia and wound healing in the diabetic rat, are likely mediated by the coordinate expression of all major HSPs. This nontoxic drug, which is under Phase II clinical trials, has enormous potential therapeutic applications.

Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum.

p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens.

Does the membrane's physical state control the expression of heat shock and other genes?

Membranes provide the structural framework that divides cells from their environment and that, in eukaryotic cells, permits compartmentation. They are not simply passive barriers that are liable to be damaged during environmental challenge or pathological states, but are involved in cellular responses and in modulating intracellular signalling. Recent data show that the expression of several genes, particularly those that respond to changes in temperature, ageing or disease, is influenced and/or controlled by the membrane's physical state.

Haptoglobin from psoriatic patients exhibits decreased activity in binding haemoglobin and inhibiting lecithin-cholesterol acyltransferase activity.

OBJECTIVE: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of haptoglobin (Hpt). BACKGROUND: Hpt is a plasma acute-phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free haemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT). STUDY DESIGN: Hpt was isolated from patients with psoriasis vulgaris, and its activity in haemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. METHODS: Two affinity chromatography steps, the first using resin-coupled haemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by Enzyme-linked immunosorbent assay with stationary haemoglobin or ApoA-I, Hpt in solution and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. RESULTS: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by haemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both haemoglobin binding and LCAT inhibition. CONCLUSIONS: In psoriasis, Hpt displays some structure modification(s), which might be associated with the protein function in the disease.

Correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum.

We compared the mycelial to yeast transitions of the Downs strain of Histoplasma capsulatum (low level of virulence) with those of G184A and G222B, two more virulent strains having different levels of pathogenicity for mice. When the morphological transitions are initiated by a temperature shift from 25 degrees to 37 degrees C, all three strains undergo similar physiological changes, but these are less severe in G184A and G222B than in the Downs strain. The transitions from mycelial to yeast morphology in both of the more virulent strains are also one-third more rapid than in Downs. We also find that the differences in temperature sensitivity of the three strains can be correlated with the temperature required for complete uncoupling of oxidative phosphorylation. The differences in sensitivity to elevated temperatures extend to the growth of yeast cells of all three strains. Considered together, our results suggest that sensitivity to elevated temperatures may be a key factor accounting for differences in virulence and that uncoupling of oxidative phosphorylation may be the primary event in the morphological transition in all three strains.

The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions.

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

Morphological transition in the human fungal pathogen Histoplasma capsulatum.

Considerable information has accumulated recently about specific genes of Histoplasma capsulatum that are expressed during the process of adaptation when the organism undergoes morphological transition at the onset of infection. The study of these genes is crucial to identify targets for the development of novel antifungal agents.

Molecular cloning and expression of hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum.

We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions. During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37 degrees C in the temperature tolerant and mouse-virulent G222B strain, while 34 degrees C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.

The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition.

To understand the molecular mechanisms that control the reversible morphologic transition from mycelia to yeast in dimorphic fungi, we have isolated and characterized a cdc2 gene from Histoplasma capsulatum. This organism is a dimorphic pathogenic fungus that grows as a filamentous saprobic mold in soil and as a unicellular pathogenic yeast in human tissue. The cloned gene, whose protein product has a high degree of homology with other members of the cdc2 family, is split into four exons and three introns of 95, 52 and 85 nucleotides. Analyses of cDNA clones confirm the presence of the eukaryotic splice donor (GT) and acceptor (AG) sites. The spliced gene codes for a protein of 324 amino acids (aa) with a predicted molecular mass of 36.9 kDa. The H. capsulatum cdc2 product has 71% aa identity with Saccharomyces cerevisiae and 70% with Schizosaccharomyces pombe. The deduced protein contains the sequence, PSTAIRE, that is normally found in most p34cdc2 proteins. H. capsulatum cdc2 is transcriptionally regulated during the morphologic mycelium<==>yeast transitions and is more actively transcribed in the yeast than in the mycelial phase.

Effects of membrane fatty acids on thermal and oxidative injury in the human premonocytic line U937.

Heat shock (HS) proteins (HSP) function as molecular chaperones and protect cells from thermal and oxidative injury. The signals leading to HSP synthesis, i.e. the "cellular thermometer(s)," are still a matter of debate. In the human premonocytic line U937, we investigated the effects of specific modification of membrane fatty acid (FA) composition by incubation with various saturated and unsaturated fatty acids (UFA) on the HS response and on hydrogen peroxide (H2O2)-induced cell death. FA readily incorporated into U937 cell membranes. UFA did not modulate the HS response but potentiated H2O2-mediated damage, while pre-exposure to HS protected the UFA-treated cells from this increased H2O2 toxicity.

Changes in membrane fluidity modulate heat shock gene expression and produced attenuated strains in the dimorphic fungus Histoplasma capsulatum.

In the dimorphic fungus Histoplasma capsulatum the expression of heat shock genes is modulated by addition of fatty acids. Addition at 25 degrees C of saturated fatty acid (palmitic acid) to mycelia of H. capsulatum induced a significant increase in heat shock mRNAs transcription when cells were heat shocked. Conversely, treatments with unsaturated fatty acid (oleic acid) drastically reduced the level of heat shock gene transcription at 37 degrees C, and no detectable levels were measurable with 2 mM. Addition of saturated fatty acid induced a thermotolerant state and mitochondria retained ATPase activity coupled to electron transport under severe heat shock conditions and shortened the time required for mycelium-to-yeast phase transition. Conversely, addition of unsaturated fatty acids uncoupled mitochondrial electron transport and prolonged considerably the time required for phase transition at the same temperatures. A virulent strain, if treated with unsaturated fatty acid under condition in which no heat shock was detectable, lost its virulence probably as a consequence of decreased ability to adapt to the new living condition present in the host.

Effects of an experimental diet on parotid saliva and dental plaque pH in institutionalized children.

Sixty-six children aged 6-12, permanent residents of a children's home, were placed on a diet during a 45-day experimental period to measure salivary flow-rate, pH of saliva and dental plaque, total concentrations of salivary proteins, inorganic phosphate, bicarbonate, calcium and amylase. The total caloric content, as well as the proportional nutrient and calorie distribution of the foods, were determined and compared with those of the previous habitual diet. After the experimental period, stimulated parotid salivary flow, increased by 40 per cent over the pre-experimental values. Total proteins of saliva and pH of both saliva and dental plaque increased significantly, whereas inorganic phosphate concentration decreased. Concentrations of bicarbonate, calcium and amylase did not differ from those found pre-experimentally. The findings appear to derive from lesser retention and increased hardness of the foods in the experimental diet.

A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

Unraveling the secrets of Histoplasma capsulatum. A model to study morphogenic adaptation during parasite host/host interaction.

Early in the developmental period of microbiology, Pasteur first observed the phenomenon of dimorphism in fungi when he noticed that the bread mold Mucor grew as a filamentous mold aerobically on the surface of broth cultures but at the bottom of the flask where the environment was anaerobic it reproduced as budding yeast cells. Several infectious fungal pathogens of humans, namely Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenkii, and Coccidioides immitis change from a multicellular filamentous form to an unicellular morphology when they invade tissues. The ability of pathogenic fungi to assume a different shape is referred to as dimorphism. This phenomenon has intrigued clinicians, and medical mycologists since its discovery at the turn of the century. The ability of pathogens to initiate infection, invade host tissues and survive in mammalian hosts is critically linked to the induction of specific gene products. In dimorphic fungi, developmentally regulated gene expression is particularly important, since they may exist in phylogenetically distinct hosts with different body temperatures. Using Histoplasma capsulatum as a model to study parasite-host interactions at the biochemical and molecular level, my laboratory has attempted to relate the clinical spectrum of disease to natural variations in the characteristics of this organism and to adaptations it must make as a saprobe and a parasite. Histoplasma capsulatum is the etiologic agent of histoplasmosis, a respiratory infection that is world-wide in distribution. As a saprobe in soil it is mycelial, but it becomes a budding yeast as a parasite in susceptible hosts. These morphological phases can be reversibly reproduced in vitro by shifting the temperature from 25 degrees C, at which it is mycelial, to 37 degrees C, when it becomes a budding yeast. The process of mycelial-to-yeast conversion is of particular interest since it is triggered by an increase in temperature and conversion to virulence. Viable mycelial fragments and conidia become airborne and enter the pulmonary tract by inhalation after which the fungus rapidly disseminates to other organs. Progressive disseminated histoplasmosis along with candidiasis, cryptococcosis, and invasive aspergillosis are opportunistic fungal infections in patients who are immunosuppressed or otherwise debilitated. Importantly, they are diagnostic hallmarks of acquired immunodeficiency disease syndrome (AIDS). The clinical features of these infections and the genetic characteristics of the etiologic agents present unique parasite-host interactions that make them valuable research models to study. In the infected host, Histoplasma capsulatum encounters various environmental stresses to which it adapts by regulating the expression of specific genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphologic features of the root canal system of the maxillary fourth premolar and the mandibular first molar in dogs.

Root canal morphology was evaluated in 72 maxillary fourth premolar and 59 mandibular first molar teeth. An apical delta was present in all roots (n = 334). The apical delta represented approximately 12-18% of the total root length for all roots. Non-apical ramifications from the main canal were observed in 25.1% of roots. Secondary canal(s) were present in 20.6% of roots, while lateral canal(s) were present in 6.9% of roots. The distal root of the maxillary fourth premolar had the highest incidence (47.2%) of non-apical ramifications. Overall, 68.0 and 20.4% of maxillary fourth premolar and mandibular first molar teeth had non-apical ramifications, respectively. The prevalence and location of non-apical ramifications may influence clinical decision making when performing endodontic therapy.

Study on gutta-percha points using scanning electron microscopy and analysis with electron microprobe.

A comparative study of the apical morphology and contour was performed with the scanning electron microscope at 45x, 70x and 300x, while the chemical composition of eight gutta-percha point brands was surveyed with an EDAX 9100 electron microprobe. They were classified according to their apical morphology as conical (25%) and truncate (75%), based on their contour as regular (50%) and irregular (50%) and according to their surface as without defects (50%) and with defects (50%). The analysis of inorganic compounds revealed the presence of varying proportions of Zn, Ba, Si, Mg. Ca, P, Cl and Al, possibly implying that the presence of Ba and Al could interfere with postendodontic repair or at least irritate the periapical area if inadvertently overfilled.

Presence of heat shock protein in chronic periapical pathology of pulpal origin.

In order to establish whether tissues damaged by Chronic Periapical Pathology (CPP) of endodontic origin produced heat shock protein (HSP) capable of attracting reactive lymphocytes, paraffin sections of samples from the oral cavity of 10 patients with CPP were incubated with commercially available anti-HSP monoclonal antibodies. Antibodies were evidenced employing the alkaline phosphatase immunoenzymatic method (DAKO). HSP was found in the lamina propria infiltrated by lymphocytes, in six of the ten samples. These results suggest that HSP may be one of the lymphocyte recruiting factors in the damaged area and opens new possibilities for further research.