Perianal infection of highly oncogenic human papilloma virus: how to prevent from cancer? Case report.

The presence of oncogenic types of human papilloma virus (HPV) in location of the anus is related to anal carcinoma. However, there is little knowledge about the natural history of such infections in patients outside risk groups, its relation to cervical cancer, the risk of anal cancer development as well as any way to prevent it. There are no standard procedures in the case of finding of HPV-associated anal intraepithelial neoplasia in the perianal area. Case report describes an incidental finding of a highly oncogenic type of HPV discovered in a histopathological assessment of a 48-year old woman after a haemorrhoidectomy. This paper presents the approach taken for this patient in terms of diagnosis, treatment and methods of prevention of anal and cervical cancer development.

Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O.

BACKGROUND: Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O. METHODS: To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO-positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells. RESULTS: GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-ß-cyclodextrin. CONCLUSIONS: Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.

Apolipoprotein E genotype and LRP1 polymorphisms in patients with different clinical types of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a severe, neurodegenerative, metabolic disease which is caused by deficient activity of arylsulfatase A (ARSA). Sulfatides and other substrates of ARSA are stored in central and peripheral nervous systems, and in some other organs. Accumulated sulfatides are especially toxic to oligodendrocytes and Schwann cells leading to progressive demyelination. The kind of apolipoprotein E (apoE) isoform is of essential significance for the modulation of sulfatide quantity in the brain as apoE4 contains more sulfatides than apoE3. Taking into consideration the fact that apoE4 leads to the loss of sulfatides in CSF of Alzheimer's disease patients, we examined if apoE isoforms display any impact on clinical outcome in patients with different forms of MLD in whom sulfatides accumulate. The significant association of age at the onset of MLD symptoms with APOE ε2/ε3/ε4 and LRP1 c.766C>T polymorphisms was shown in multivariate stepwise regression analysis, in which other factors known to affect age at onset of the disease, i.e. clinical type of MLD, family connection of the patient and sex were also analyzed. As expected, the clinical type of MLD explained about 80% of the variance of the dependent variable. The impact of both polymorphisms on age of onset of the disease was considerably lower: 2.0% in the case of APOE polymorphism and 1.0% in the case of LRP1 polymorphism. Thus, the clinical outcome in MLD patients is related principally to the genotype of the ARSA gene, while the polymorphisms in the APOE and LRP1 genes are only slightly modifying factors.

Diagnostic difficulties in Krabbe disease: a report of two cases and review of literature.

Globoid cell leukodystrophy (GLD, also known as Krabbe disease), whose pathophysiology is still not completely elucidated, is an inherited, metabolic, and neurodegenerative disease, caused by the deficiency of ß-galactocerebrosidase (GALC) or in very rare cases by lack of active saposin A. We describe two patients, in whom first MRI changes were not suggestive of GLD. Additionally, in Patient 1, the residual ß-galactocerebrosidase activity was rather high leading to difficulties in the diagnosing process. Molecular analysis of the GALC and PSAP genes in Patient 1, and of the GALC gene in Patient 2 confirmed the diagnosis of Krabbe disease. We have detected a novel mutation in the GALC gene in Patient 2, a deletion in exon 16, leading to the STOP codon (c.1851delT, p.Y617X). This deletion interrupts the reading frame prematurely: codon 617 is replaced by a STOP codon. A careful clinical description of presented patients is followed by a discussion of radiological, biochemical, genetic, and neuropathological studies. It concludes with a discussion of the potential difficulties encountered when diagnosing patients with rare diseases. In Patient 1 the postmortem examination of CNS revealed the presence of globoid cells grouped in multiple clusters seen in the white matter near the vessels. We would like to emphasize that proper clinical-radiological-biochemical co-operation and exchange of information between parents and specialists is a key issue in the diagnosis of rare and difficult neurological diseases, in particular, if the clinical picture is inconclusive.

[Genetically based states of elevated quantity of foetal haemoglobin (Hb F) in healthy individuals and patients]. / Genetycznie uwarunkowane stany zwiekszonej ilosci hemoglobiny plodowej (Hb F) u ludzi zdrowych i chorych.

During human development, the switch from foetal gamma- to adult beta-globin on chromosome 11p is not complete resulting in the residual gamma-globin expression in a modest subpopulation of erythrocytes termed "F-cells". Genetic determinants that are responsible for higher level of Hb F include various mutations within the beta-globin gene cluster as well as singular nucleotide polymorphisms in other loci associated with Hb F quantitative trait, and also epigenetic mechanisms. All these molecular conditions may drive to hereditary persistence of foetal haemoglobin (HPFH). Taking into account a morphologic criterion, HPFH is called pancellular (p-) HPFH, if the increased Hb F is distributed in a uniform fashion among all of the red cells. When the Hb F distribution is restricted, the syndrome is referred to as heterocellular (h-) HPFH. The Hb F persistence rarely occurs in healthy adults or accompanies certain hemoglobinopathies changing substantially phenotypic diversity of sickle cell disease and beta-thalassaemia.

Different RBC aggregating properties of the Aalpha2Bbeta2gammaA2 and Aalpha2Bbeta2gammaAgamma' subpopulations of human fibrinogen.

Human fibrinogen (TF) has been separated into two fractions: F1 - homodimers with respect to the gamma chain, and F2 - heterodimers composed of gammaA and gamma' polypeptides. Their rouleaux-inducing properties were as follows: (1) both, at the same concentration of 0.8%, were less effective than TF; (2) F1 produced larger rouleaux even under static conditions of a hemocytometer where F2 was silent; (3) F2 induced the process when a suspension was gently sheared between microscopic slides. Since the synthetic peptide gamma'(414-427) inhibited the rouleau formation in a mixture with F2, the C-terminal amino acids of the gamma' polypeptide probably bind the molecule to the cell. The inhibition was feebly visible in the native ratio of F1/F2, implicating a compensatory effect of F1.

Are there two functionally distinguished Neu5Gc pools with respect to rouleau formation on the bovine red blood cell?

Bovine red blood cells (RBCs) do not exhibit any aggregation tendency in autologous plasma and, therefore, all bovine rouleaux obtained in vitro are regarded as artificial. The present study reports the bovine RBC rouleau formation by either bovine or human fibrinogen and Ca2+ ions. The phenomenon was induced through two-step cell incubation: in 0.9% NaCl and 1% bovine albumin at 37 degrees C for 30 min followed by 20 hrs incubation at 30 degrees C in the fresh solution supplemented with 10 mM glucose. Its mechanism is unknown. During the incubation the number of N-glycolylneuraminic acid molecules per cell decreased from 48.1 to 44.9 amoles, which accounted for 7%. The treatment of RBCs with V. cholerae sialidase under the same conditions resulted in a 94% drop in the Neu5Gc quantity and did not induce the rouleau formation in the same fibrinogen preparation. The preliminary results rise a question whether the bulk of sialic acid is required in the aggregation of bovine erythrocytes under static conditions. Only a minor pool of Neu5Gc seems to be responsible for suppression of the phenomenon.