Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure.

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83-100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0-86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists.

HCV-RNA detection using different PCR methods in sera, cryoglobulins and peripheral blood mononuclear cells of patients with mixed cryoglobulinemia.

OBJECTIVE: The hepatitis C virus (HCV) is frequently associated with mixed cryoglobulinemia (MC), and a number of authors have reported the presence of anti-HCV antibodies and HCV-RNA in the blood of MC patients. The presence of the HCV genome in the blood cells of individuals infected by HCV may correlate with the etiopathology of MC. We investigated the presence of HCV-related sequences in the sera, cryoglobulins and peripheral blood mononuclear cells (PBMC) of patients with MC and of individuals with type C chronic active hepatitis (CAH). METHODS: 39 patients with MC, 11 non-cryoglobulinemic HCV-positive individuals with CAH, and 2 anti-HCV negative controls were included in the study. The presence of HCV-RNA was detected by nested RT-PCR and by a commercial kit. The PCR was performed by amplifying the 5'-non coding region (5'-NCR) of HCV. RESULTS: HCV-RNA was detected in the sera and cryoglobulins of about 90% of the patients; the commercial kit showed a higher sensitivity than nested PCR. One MC patient showed HCV-RNA only in the cryoglobulins. HCV-RNA was present in the PBMC of 14 of the 20 (70%) MC patients analyzed. No differences in serum and PBMC HCV-RNA positivity were found between MC patients and controls. CONCLUSION: Our results confirm the spread of HCV infection among patients with MC. HCV-RNA is present in the serum, cryoglobulins and PBMC of a large proportion of MC patients. The prevalence of HCV-RNA in the PBMC of MC patients and controls did not differ significantly; this may suggest a tropism of HCV for PBMC regardless of the presence of cryoglobulinemia.

Isoelectric focusing in immobilized pH gradients: applications in clinical chemistry and forensic analysis.

The applications of isoelectric focusing in immobilized pH gradients in clinical chemistry and forensic analysis are reviewed. Strong emphasis is given to the separation of serum proteins, in particular alpha 1-acidic glycoprotein, acid phosphatase, alkaline phosphatase, alpha 1-antitrypsin, apolipoproteins, complement component, factor B, factor XIIIB, group-specific component, lecithin:cholesterol acyltransferase, phosphoglucomutase, prealbumin, protein C and transferrin. The analysis of human parotid salivary proteins is discussed and an assessment is given of the state of the art in thalassaemia screening.

Sodium dodecyl sulphate electrophoresis of urinary proteins.

The analysis of urinary proteins and their identification are discussed, particularly in regard to the technique of sodium dodecyl sulphate electrophoresis in polyacrylamide gradient gels. Urine collection, storage and preparation are evaluated, especially in regard to problems connected with concentration and dialysis of such samples. The instrumental approach to sodium dodecyl sulphate polyacrylamide gel electrophoresis represented by the Phast System appears to be particularly valuable in routine clinical analysis of urine specimens, since no sample pretreatment is required. The following types of proteinurias are evaluated: (a) orthostatic proteinurias; (b) post-renal proteinurias; (c) Bence-Jones proteinuria; (d) lower and upper urinary tract infection (cystitis and pyelonephritis) and (e) diabetes mellitus proteinurias.

Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients.

The applications of isoelectric focusing in immobilized pH gradients to the analysis of (i) human hemoglobin mutants, (ii) animal hemoglobin mutants (from cattle, sheep, dog and mouse), and (iii) tryptic digests of alpha and beta chains, are discussed and evaluated. Immobilized pH gradients appear to be an excellent tool for screening of genetic polymorphism and for detecting "silent mutants", i.e. those substitutions involving amino acids with nonionizable side chains. At present, not even capillary zone electrophoresis, claimed to have a resolving power equivalent to 1 million theoretical plates, has shown a resolution capability comparable to that of immobilized pH gradients, at least in the field of protein separation.

Isoelectric focusing of tryptic peptides from hemoglobin subunits by immobilized pH gradients.

The technique of isoelectric focusing on immobilized pH gradients (IPG) has been applied to the analysis of tryptic digests of alpha- and beta-chains of human hemoglobin. Using peptides purified by RP-HPLC as a reference, it was possible to create a peptide map in the single IEF dimension. Unfortunately, it was not possible to find experimental conditions (medium for migration and staining) which would allow the detection of peptides of less than 10-12 residues. Almost all the bands visible on the gel could be assigned to known peptides. In order to obtain these results the IPG runs were performed in 8 M urea containing 0.5% carrier ampholytes and the gel stained with colloidal Coomassie brilliant blue G-250, in the presence of a high-salt concentration and at acidic pH.

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

Antenatal diagnosis of beta-thalassemia by isoelectric focusing in immobilized ph gradients.

A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.

Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients.

This new method for fractionation of serum alkaline phosphatase isoenzymes is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. All known forms of alkaline phosphatase are separated in an Immobiline pH 3.5-6.0 gradient, the sample being applied into pockets cast on a pH 8.0 plateau. Sharp zymogram bands are obtained by substituting alkaline-stable 5-bromo-4-chloro-3-indoxyl phosphate and tetrazolium salts for the standard 1- and 2-naphthyl phosphate-diazonium salt combinations. After hydrolysis of the phosphate group by the alkaline phosphatase the indoxyl moieties reduce tetrazolium salts to nearly insoluble and nondiffusible formazan precipitates. Normal sera show an array of about 10 isobands isoelectric between pH 3.9 and pH 4.79. In Paget's disease, two sharp isobands with pls of 4.97 and 5.09 are seen. Placental alkaline phosphatase overlaps with the higher pl bands of normal serum; however, upon heat destruction of the latter, it shows four sharp bands with the following pl's: 4.59, 4.62, 4.67 and 4.73.

Determination of glycosylated haemoglobin by isoelectric focusing in non-linear pH gradients.

A new isoelectric focusing technique for the separation and quantitation of glycosylated haemoglobin (HbA1c) is described. By using an equimolar mixture of two separators (0.2 M beta-alanine + 0.2 M 6-aminocaproic acid) a 2-pH unit Ampholine range (pH 6-8) is transformed in a shallow, 0.6-pH unit span (pH 6.7-7.3). This brings about an increment of resolution between HbA and HbA1c by a factor of about three, thus allowing proper densitometric evaluation of the trichloroacetic acid-fixed MetHb bands by conventional gel scanners. Excellent agreement is found among microchromatography, isoelectric focusing followed by densitometry in situ, and isoelectric focusing followed by band excision, elution and spectrophotometric determination. The present method also allows full resolution between HbA1c and fetal haemoglobins (F and Fac bands).