TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue.

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Cryptococcosis in AIDS patients: observations concerning CNS involvement.

The clinical course and response to therapy of seven patients with cryptococcosis and AIDS were reviewed. One patient was still in the primary stage of cryptococcosis in AIDS, i.e. the stage that is characterized by the sole cultural detection of Cryptococcus neoformans in the respiratory tract. The other six patients were in the secondary stage, where C. neoformans can be detected from the cerebrospinal fluid (CSF), blood, urine, faeces and other body sites. The main presenting features (headache, fever, nausea) were due to central nervous system involvement, although meningism and mental changes were rarely present, and CSF changes were very subtle. Treatment with amphotericin B and flucytosine was very effective, there being no more growth of fungi in cultures in most cases. Adverse reactions to the drugs used occurred frequently and consisted mainly of anaemia, hepatosis and fever. Diagnosis in the primary stage of cryptococcosis may improve the prognosis.

[Aspergillus infection in skin transplantation and its therapy]. / Aspergillus-Befall bei Hauttransplantation und Therapie

In a 10 years old girl sustaining a corrosive injury of the lower leg from sulphuric acid, in the region of a skin transplantation colonization with Aspergillus fumigatus (Fresenium) and Aspergillus niger (van Tieghem) took place. This infection endangered the attempt of transplantation and the saving of the foot. Treatment by medication with nystatin (moronal) and canesten (clotrimazol) were ineffective. Pimaricin (pimafucin, natamycin) quickly erradicated the mycotic infection and secured an undisturbed progress for the transplantation. Additionally the epidemiology of infections by Aspergillus is briefly discussed.

[Disseminated cryptococcosis with extensive cutaneous involvement in AIDS]. / Disseminierte Kryptokokkose mit ausgeprägtem Hautbefall bei AIDS.

BACKGROUND: Diagnosis of an ulceratively decaying indolent papule in the skin of an AIDS patient. CASE REPORT: In a 36-year-old HIV-positive man with a CD4 lymphocyte count of 60/microliters, a diagnosis of disseminated cryptococcosis was established based on a biopsy of an ulceratively decaying indolent papule in the skin of the left side gluteal region. In additional papulopustular skin lesions of neck, upper arm, upper thigh, and rima ani, Cryptococcus neoformans var. neoformans was detected by specific culture. The Cryptococcus neoformans antigen titre in serum was 1:160. The extensive cryptococcal skin involvement was accompanied by symptoms of meningeal cryptococcosis like hearing at an abnormal sound volume, a sensation of high pressure in the head and a change of behaviour, but absence of cephalgia and stiff neck. Attention is drawn to the importance of biopsy for the differential diagnosis of the skin lesions and to the significance of a specific cultural detection of Cryptococcus neoformans in view of the therapy and epidemiology of this mycosis. Under a combination therapy, consisting of amphotericin B, flucytosine and fluconazole, a healing of the skin lesions and disappearance of the neurological symptoms followed by negative cultural and serological tests were seen. Specific comments on the epidemiology of cryptococcosis in AIDS are made. CONCLUSION: In case of doubt, papulopustular skin lesions of HIV-positive patients should be biopsied. Cryptococcosis should also be considered.

Extracellular proteolysis by Mucoraceae in serum-albumin agar tested by the agar block method.

In a preliminary study, three strains of Absidia corymbifera (two strains from autopsy material of the lungs and one from the soil of a potted indoor plant in a hospital) and one strain of Rhizopus oryzae (from the soil of a potted plant in a hospital) were tested for secretory proteolytic activity in serum-albumin agar (SAA) with initial pH values of 5.0 and 7.0 by the agar block method and subsequent protein staining. Within an incubation period of 5 d/37 degrees C, complete proteolysis took place in all the SAA blocks with an initial pH value of 7.0. The three strains of Absidia corymbifera and the one strain of Rhizopus oryzae did not fully proteolyse the SAA blocks with an initial pH value of 5.0. On the basis of these observations, it is assumed that there are secretory proteases in the causative agents of mucormycosis (zygomycosis) whose optimal pH is not in the acid but in the neutral, perhaps in the alkaline range.

[Fungi in the home and hospital environment]. / Pilze im Wohn- und Krankenhausmilieu.

From the viewpoint of 40 years of experience in the field of medical mycology with the help of current topics the competence of the medical mycologist for evaluation of a presence of fungi in the home and hospital environment is commented. The topics considered are as follows: 1. The HIV-infected person and the habitat of Cr. neoformans; the predisposition for cryptococcosis and the CD4 lymphocyte count. 2. Invasive aspergillosis after heart transplantation; by exemplary cooperation between the heart surgeon and the medical mycologist it could be demonstrated that the frequently fatal invasive aspergillosis can be avoided. 3. Attention is drawn to the currently most interesting habitats of fungi in the home environment, i.e., biological waste and compost. It has been shown that among the various fungi, the causative agents of systemic mycoses may also be found.

Pathogenic fungi in human dwellings.

Airborne invasive fungal infections in various risk groups of people suffering from immunodeficiencies are an increasing problem for modern medicine. Because of the acute and rapid course of invasive infections, prevention is of principal significance. Such prevention mainly concerns the control of fungal spores in indoor air. In this connection the immediate environment offering ecological niches for growth and morphogenesis of infective particles of Aspergillus spp., Mucoraceae, Cryptococcus neoformans and some other yeast-like fungi is of main interest. The current problems of indoor air mycology, i.e. the epidemiology and ecology of pathogenic fungi in indoor air, can only be recognized and interpreted by centres routinely performing mycological diagnosis for all the various risk groups of patients. Models of specific surveillance of the most important fungal pathogens (e.g. Aspergillus spp. and Cryptococcus neoformans) in indoor air will be outlined and discussed.

The green colour effect (GCE) of the killer strain Cryptococcus laurentii CBS 139 on Staib agar.

Attention is drawn to the observation that the type strain Cryptococcus laurentii CBS 139, producing killer toxins (mycocins) directed at Cr. neoformans var. gattii, causes a green colour effect (GCE) on Staib agar (Guizotia abyssinica creatinine agar) in combination with an intense assimilation of creatinine. Five (9.6%) out of 52 strains of Cr. laurentii of various origin, showed a GCE and intense creatinine assimilation. Further research must show if all Cr. laurentii strains, characterized by a GCE similar to that of the strain CBS 139, are also capable of producing killer toxins. For further ecological and epidemiological research on strains producing killer toxins directed against species of the genus Cryptococcus, it is proposed to use Staib agar as differential culture medium indicating both colour effects, i.e. the GCE and the brown colour effect (BCE).

Sampling and isolation of Cryptococcus neoformans from indoor air with the aid of the Reuter Centrifugal Sampler (RCS) and guizotia abyssinica creatinine agar. A contribution to the mycological-epidemiological control of Cr. neoformans in the fecal matter of caged birds.

In February and March 1984, Cryptococcus neoformans was detected in the manure and ambient air of a volery in the Berlin Zoo in which a Palm Cockatoo (Probosciger aterrimus) was kept. Both times, 5 colony-forming units of Cr. neoformans could be isolated on the differential medium, Guizotia abyssinica creatinine agar, from 40 1 of air aspirated by the Reuter Centrifugal Sampler (RCS). The absence of a concurrent growth of moulds was found to be of special importance for the optimal isolation of Cr. neoformans on the above mentioned agar for purposes of epidemiological research into airborne dissemination of the fungus. The advantages and disadvantages of 0.1% biphenyl to inhibit concurring growth of moulds are discussed. The control of habitats and foci of Cr. neoformans in zoos and similar establishments is considered a necessity, to prevent inhalatory exposure of susceptible, e.g. immuno-compromised persons.

Pleural fluid as nutrient substratum for Aspergillus fumigatus and A. flavus. Submerged growth in pleural fluid and extracellular proteolysis in pleural fluid agar. A preliminary report.

Pleural fluid in liquid and agar form from a patient with adenocarcinoma of the lung was used as a nutrient medium for culturing Aspergillus fumigatus and A, flavus. The extracellular proteolytic activity of both strains could be demonstrated after an incubation period of 5 d at 37 degrees C by the agar block method using pleural fluid agar (protein content ca. 0.1-0.5% and initial pH values of 7.0 and 5.0) and protein staining with naphthalene black. In the undiluted pleural fluid, after stationary culture at 37 degrees C for 8 days, compact white granule-like colonies of a size of ca. 0.5-1.0 mm were found near the surface of the liquid and as sediment. Microscopically, these colonies consisted of radially arranged septate hyphae, similar in size and form to those seen in invasive aspergillosis.

The perfect state of Cryptococcus neoformans, Filobasidiella neoformans, on pigeon manure filtrate agar.

To enable studies of the dependence of Cryptococcus neoformans and its perfect and imperfect states upon bird manure as a habitat of this pathogen, a nutrient medium closely resembling natural conditions was prepared. As sole nutrient, the water soluble ingredients of manure from pigeons (Columbia livia) were used. There was no heat sterilization of the manure filtrate. Using a standard pair of C. neoformans strains for mating, it could be demonstrated that the perfect state of the fungus developed on this so called pigeon manure filtrate agar within 48 h at 26 degrees C. This medium is supposed to help in the elucidation of the epidemiological significance of the perfect and imperfect states of this pathogen.

[Mycoses caused by fungal spores in indoor air]. / Mykosen durch Pilzsporen in der Raumluft.

Fungal spores in indoor air may cause mycoses and allergies in man. The following different types of exposure are interesting from the epidemiological view-point: (a) the occupational exposure (work done with fungi-contaminated material), (b) the accidental exposure (formation and growing of fungi by constructive defects), and (c) the inconspicuous constant exposure (as part of lifestyle, e.g. by plants or small pets). It is stressed that the only way to characterize the indoor air quality with regard to its content of fungal spores is by uncontestable mycological diagnostics.

[Mycoses caused by fungal spores in indoor air]. / Mykosen durch Pilzsporen in der Raumluft.

Airborne fungal spores may cause mycoses and allergies. From the epidemiological angle, various modes of exposure are possible, viz. 1. Professional (Work with material contaminated with fungi); 2. Accidental (Growth or presence of fungi owing to shortcomings of construction); and 3. Discrete, constant (Household-associated activities such as keeping of potted plants and pets). To enable an evaluation of indoor air quality in respect of its content of fungal spores, a reliable mycological diagnosis is required.

Ecological and epidemiological aspects of aspergilli pathogenic for man and animal in Berlin (West).

A summary of five years of experimental and clinical mycological work in Berlin (West) is presented. The subjects of this research include: Soil of potted indoor plants as a habitat for aspergilli; exposure of sensitive patients suffering from different basic diseases to Aspergillus spores; polyene-producing streptomycetes beside aspergilli in the rhizosphere of potted plants and their role; and ecological and clinical significance of atypical Aspergillus strains isolated from clinical specimens. Reference is also made to the role of nuts as source of infection and as a baiting technique.

Air aspirator for the detection of Aspergillus flavus in foodstuffs.

The principle of aspiration as a sampling method for the detection of Aspergillus flavus in foodstuffs was used. The importance of the type and quality of the nutrient medium to be used for the isolation of A. flavus in this connection is discussed.

Effect of purines on the formation of the perfect state of Cryptococcus neoformans, Filobasidiella neoformans.

A comparative study of growth and mating behaviour of C. neoformans strains grown on nutrient media containing a variety of organic nitrogen sources revealed striking differences of both parameters. Although growth was sparse on an adenine-containing medium, mating was excellent. Attention is drawn to the possible role of adenine in the morphogenesis of F. neoformans.

Chlamydospore formation on Staib agar. Observations made before Candida dubliniensis was described.

When routinely using Staib agar to detect Cryptococcus neoformans in AIDS patients by the brown colour effect of its colonies, rough-looking colonies of a questionable variety of Candida albicans were also found. Microscopically, these colonies consisted of pseudohyphae with abundant masses of chlamydospores. However, the colonies of C. albicans were smooth-edged and formed by round-oval blastospores only. Such observations were made during the mycological supervision of 36 cryptococcosis cases during the 1987-94 period. All these questionable cultures of Candida spp. were discarded. However, because the corresponding photographs of and records on such strains were found to be identical with those recently published by molecular biologists under the title 'Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis' [Staib, P. & Morschhäuser, J. (1999) Mycoses 42, 521-524], the present communication presents a report on such observations in a representative and exemplary case of an AIDS patient.

Brown-red pigment formation by the mycelial phase of a clinical isolate of Histoplasma capsulatum on Staib agar. A preliminary report.

In an HIV-positive patient, the suspected diagnosis of histoplasmosis capsulatum (being the first opportunistic infection indicating AIDS) on the basis of histopathological findings in biopsy material could be proved by culture on Staib agar (syn. Guizotia abyssinica creatinine agar, bird seed agar, etc.). On Staib agar, after 4 weeks at 26 degrees C, there was a cockade-like colony growth, consisting of a white centre, followed by a brown-red pigmented zone surrounded by a border of submerged mycelial growth of tan to brownish colour. Morphologically, there was a moderate formation of tuberculate macroconidia and a heavy formation of microconidia. On neutral Sabouraud's dextrose agar, there was a colony formation without pigment (albino type) free of tuberculate macroconidia and microconidia. Proposals for further investigation of these preliminary observations are made.