Typing of hepatitis C virus by a new method based on restriction fragment length polymorphism.

A new restriction fragment length polymorphism (RFLP) analysis has been developed for hepatitis C virus (HCV) typing in the viral 5' non-coding region and contiguous core region. These genomic sequences were chosen for the relative nucleotide homology among different genotypes and for the presence of polymorphic sites. By employing two endonucleases (AccI and MboI) and, in some instances, a third one (EcoRII), we can unambiguously and reproducibly distinguish between genotypes and subtypes 1a, 1b, 1c, 2a, 2c, 2b, 3a, 3b, 4a, 5a and 6a. The method was applied for diagnosing two Italian groups of HCV-infected individuals reflecting a randomly collected population and a group of intravenous drug users. The accuracy of this method has been validated by comparison with INNOLiPA and by sequencing. Our approach represents an improvement over previous RFLP methods, since typing is accurate and simpler.

Recovery of long-term natural protection against reactivation of CMV retinitis in AIDS patients responding to highly active antiretroviral therapy.

OBJECTIVES: To see whether in severely immunosuppressed AIDS patients (with prior Cytomegalovirus retinal disease) who have significant increases in CD4+ lymphocytes following the initiation of highly active antiretroviral therapy (HAART) anti-Cytomegalovirus (CMV) maintenance therapy can be withdrawn with no subsequent progression of CMV retinitis. METHODS: Eight patients with AIDS and one or more previous episodes of CMV retinitis interrupted anti-CMV maintenance therapy following the successful beginning of HAART. CD4 cell counts and HIV-RNA were monitored monthly while measurement of CMV antigenemia and ophthalmoscopy were carried every 2 weeks thereafter. RESULTS: The HAART recipients in whom anti-CMV maintenance therapy had been interrupted had measureable increases of CD4+ T lymphocytes, substantial control of both HIV-RNA and CMV viraemia and did not show recurrence of retinitis during a mean follow-up of 98.4 weeks (range 78-120, SD 15.2). CONCLUSIONS: Anti-CMV maintenance therapy can be interrupted with no subsequent progression of retinal damage over a long time in patients with AIDS who successfully respond to HAART with a significant increase in CD4 cell count.

Measurement of human cytomegalovirus-associated DNA polymerase activity in patient urine as a potential diagnostic tool.

Virus-associated DNA polymerase activity has recently been proposed for the detection of human cytomegalovirus (HCMV) in urine, a method that should allow rapid and quantitative determination of the viral load. In this report, the virus-associated DNA polymerase activity recovered from the urine of a group of patients shedding HCMV was measured using a poly(dA) oligo(dT) 12-18 synthetic template after polyethylene glycol precipitation of the virions. Detection of virus-associated DNA polymerase activity was compared to the classical methods most widely used to diagnose HCMV shedding in urines such as virus culture followed by indirect immunofluorescence and pp65 gene-specific polymerase chain reaction. Although less sensitive than the polymerase chain reaction and cross-reactive with other herpesvirus DNA polymerases, the activity measured in the urine samples was correlated with the number of positive nuclei found in shell vials (r = 0.89). The diagnostic threshold of the assay could be placed between 50 and 100 fluorescent nuclei per shell with a diagnostic sensitivity of 56%. Being simple and quantitative, the measurement of virus-associated DNA polymerase activity could be of value in some clinical conditions where it is necessary to assess viral load in urine. This method is proposed as an alternative to more laborious quantitative assays and to support qualitative polymerase chain reaction.

Simultaneous polymerase chain reaction detection and restriction typing for the diagnosis of human genital papillomavirus infection.

A polymerase chain reaction method has been developed which allows the simultaneous detection of the majority of clinically relevant HPV types. Degenerate HPV-specific primers direct the one-step amplification of a DNA region spanning E1 and E7 genes. This enables an immediate distinction between the two groups of papillomaviruses, characterized by high or low oncogenic potential, simply from the size of amplified DNA. The PCR product can be subjected to a second round of amplification with internal primers, which are specific for 7 high-risk HPV types, HPV-16, -18, -31, -33, -35, -45 and -58. Precise identification of one-step or two-step amplified DNA is done by endonuclease digestion with one or two enzymes. The detection sensitivity, which has been assessed using cloned HPV genomes and HeLa and CaSki cell lines, varies from a few tens to a few hundreds of viral genome equivalents. The accuracy of the method has been confirmed by examining cervical scrapings of 44 patients.

Polymerase chain reaction amplification and restriction enzyme typing as an accurate and simple way to detect and identify human papillomaviruses.

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.

A myasthenia gravis plasma immunoglobulin reduces miniature endplate potentials at human endplates in vitro.

A particular myasthenia gravis (MG) plasma Ig has previously been shown to block a single alpha-bungarotoxin (alpha-BuTx) binding site on embryonic rat muscle acetylcholine receptor (AChR). We have investigated its effect on embryonic/denervated and adult human AChR both in extracts and in situ. Plasma Ig blocked 125I-alpha-BuTx binding by greater than 85% to the AChR extracted from denervated muscle, but only by 55% to AChR extracted from normal human muscle. Incubation of intact human muscle fibers with the plasma Ig reduced 125I-alpha-BuTx binding to the endplate AChRs by 63%, and substantially decreased the amplitude of miniature endplate potentials. We conclude that anti-alpha-BuTx site antibodies, when present, can be important in the pathophysiology of the disease.

Nosocomial epidemic of active tuberculosis among HIV-infected patients.

In an investigation of a nosocomial outbreak of tuberculosis, 18 HIV-infected inpatients were found to have been exposed to Mycobacterium tuberculosis; active tuberculosis developed in 8, 7 within 60 days of diagnosis of the index case. The patients with lower total lymphocyte and CD4 lymphocyte counts were more likely to get the disease than were those with higher counts. A low score on multiple antigen skin testing was also associated with the development of active tuberculosis. 4 of the 18 patients had a positive tuberculin skin test before exposure to M tuberculosis; none of them subsequently got the disease.

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides.

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.

Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.

We have synthesized a tetradecapeptide corresponding to residues 354-367 of the delta-subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg-Arg-Ser-Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP-dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP-dependent protein kinase. Antibodies elicited against peptide 354-367 were shown to cross-react with native AChR and to bind specifically to the delta- and gamma-subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP-dependent phosphorylation of both the delta- and gamma-subunits. This suggests that the phosphorylation sites in the delta- and gamma-subunits are highly cross-reactive, and is in agreement with the demonstration that an endogenous cAMP-dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta-subunit isolated from phosphorylated AChR yields a single 25-kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354-367 within this phosphorylated fragment.

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit.

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.

Antigenic specificity of acetylcholine receptor in developing muscle. Studies with monoclonal antibodies.

Monoclonal antibodies (mcAbs) elicited against the nicotinic acetylcholine receptor (AChR) from Torpedo, were used to follow antigenic changes in AChR during muscle development. Newborn rat muscle and denervated mouse muscle were used as sources of extrajunctional AChR; adult innervated rat and mouse muscle were used as sources of junctional AChR. Most of the mcAbs tested reacted preferably, but not exclusively with extrajunctional AChR (EJR), as compared to junctional AChR (JR). None was found to react with only one of the two forms of AChR. We conclude that the anti-AChR monoclonal antibodies used in this study detect antigenic determinants which are shared by EJR and JR, but which probably undergo structural changes during muscle development.

An anti-acetylcholine receptor monoclonal antibody cross-reacts with phosvitin.

Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.

Thymic involvement in myasthenia gravis. Study by immunofluorescent and immunoperoxidase staining.

Changes in the thymus are often encountered in myasthenia gravis (MG), together with neuromuscular pathology. In the present study, the cell population of 28 thymuses from patients with MG were analyzed by immunofluorescent and 49 thymuses by immunoperoxidase techniques. We were able to show the presence of sIg receptor positive, SpA reactive medium and large B lymphocytes of light specific density in the majority of thymuses, and to demonstrate a characteristic pattern of distribution in the gland. PAP analysis showed that the infiltrating B cells appeared in the interlobular septa, then reached the medulla, occasionally the cortex which mostly revealed signs of atrophy. These findings are consistent with an autoimmune reaction occurring against a thymic antigen; such an antigen could be acetylcholine receptor (AchR) of the thymic structures, since anti-AchR antibody or serum from patients with MG will react with 60% thymocytes and some epithelial cells and Hassall's corpuscles.

Peripheral neuropathy associated with immunoglobulin disorders an immunological and ultrastructural study.

Light-, electron microscopic and immunopathological findings in a nerve biopsy of a patient with peripheral neuropathy associated with monoclonal gammopathy are reported. Loss of fibers, Wallerian-like degeneration and segmental demyelination were the most important features observed in light microscopy. In E.M. widening of the peripheral lamellae of myelin sheaths and occasional aspects of hypermyelination were seen. Immunoperoxidase study showed binding of IgM (k light chain) on the myelin sheath. The possible pathogenetic implications of these findings are discussed.