Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining.

In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI).

Mitochondrial alterations with mitochondrial DNA depletion in the nerves of AIDS patients with peripheral neuropathy induced by 2'3'-dideoxycytidine (ddC).

The 2'3'-dideoxycytidine (ddC), a nonazylated dideoxynucleoside analog used for the treatment of AIDS, causes a dose-dependent, painful, sensorimotor axonal peripheral neuropathy in up to 30% of the patients. To investigate the cause of the neuropathy, we performed morphological and molecular studies on nerve biopsy specimens from well-selected patients with ddC-neuropathy and from control subjects with disease, including patients with AIDS-related neuropathy never treated with ddC. Because ddC, in vitro, inhibits the replication of mitochondrial DNA (mtDNA), we counted the number of normal and abnormal mitochondria in a 0.

Mitochondrial and cellular toxicity induced by fialuridine in human muscle in vitro.

Fialuridine (FIAU), when used experimentally in the treatment of patients with chronic hepatitis B, caused irreversible acute hepatic failure, myopathy, myoglobinuria, severe lactic acidosis, neuropathy, and death. To investigate the primary cellular elements involved in the neuromuscular toxicity of FIAU, we examined its effects on human myotubes in cultures and searched for signs of recovery. From a total of 75 flasks of normal myotubes prepared from human muscle biopsies, 63 were exposed to various concentrations of FIAU (0.

Inclusion body myositis in HIV-1 and HTLV-1 infected patients.

Sporadic inclusion body myositis (IBM) is the most common inflammatory myopathy affecting patients over the age of 50 years. Dysimmune and degenerative aetiologies have been postulated, but viral infections have not been associated with the disease. Two HIV-I (human immunodeficiency virus type 1) infected men and one woman infected with HTLV-1 (human T cell leukaemia virus type 1) developed progressive proximal muscle weakness unrelated to antiretroviral therapy.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family.

Exons 16 and 17 of the amyloid precursor protein gene in familial inclusion body myopathy.

Accumulation of beta-amyloid protein (A beta) occurs in some muscle fibers of patients with inclusion body myopathy and resembles the type of amyloid deposits seen in the affected tissues of patients with Alzheimer's disease and cerebrovascular amyloidosis. Because mutations in exons 16 and 17 of the beta-amyloid precursor protein (beta APP) gene on chromosome 21 have been identified in patients with early-onset familial Alzheimer's disease and Dutch-type cerebrovascular amyloidosis, we searched for mutations of the same region in patients with familial inclusion body myopathy. Sequencing of both alleles in 8 patients from four unrelated families did not reveal any mutations in these exons.

Expression of poliovirus receptor in human spinal cord and muscle.

Because a prerequisite for infection of a cell with the poliovirus is the presence of poliovirus receptor (PVR), we examined its tissue localization in the human muscle, spinal cord, and muscle cultures using a specific monoclonal antibody against PVR in immunocytochemical studies on serial sections. We found weak expression of PVR in the motor neurons but not the axons. In normal muscle, PVR was expressed at the end plate as confirmed by immunolocalization in serial sections with alpha-bungarotoxin.

Detection of poliovirus antibodies and poliovirus genome in patients with the post-polio syndrome.

To investigate the role of poliovirus (PV) infection in the development of the post-polio syndrome (PPS), we studied the serum, spinal fluid, peripheral blood lymphocytes, and muscle from 47 patients with PPS. We found high titers of IgM PV antibodies (up to 1:250) in the serum of 6 patients, compared to very low titers (less than 1:50) in normal subjects or disease controls. By polymerase chain reaction, using primers of the replicase PV gene, we amplified PV sequences in the peripheral blood lymphocytes in 7 of 37 patients and in the CSF in 4 of 40 patients, but in none of the controls.

The effect of L-carnitine on the AZT-induced destruction of human myotubes. Part II: Treatment with L-carnitine improves the AZT-induced changes and prevents further destruction.

Background: Zidovudine (AZT) as used in the treatment of AIDS causes a mitochondrial myopathy characterized by enzymatic defects in the respiratory chain system, accumulation of lipid droplets, and carnitine deficiency. Human myotubes treated with AZT demonstrate abnormal mitochondria, accumulation of lipid, and increased lysosomes. Because L-carnitine plays a major role in the transport of long chain fatty acids across the inner mitochondrial membrane and facilitates the beta-oxidation of fatty acids, we examined whether L-carnitine can enhance the recovery of the affected myotubes after withdrawal of AZT and can improve the structural changes of the myotubes while AZT treatment continues.

Polymyositis in patients infected with human T-cell leukemia virus type I: the role of the virus in the cause of the disease.

To investigate the mechanism of polymyositis in human T-cell leukemia virus type I (HTLV-I) infection, we studied 6 HTLV-I-positive patients, 3 with polymyositis and 3 with adult T-cell leukemia but without clinical signs of muscle disease, by (a) quantitative single or double immunocytochemistry on serial 4-microns-thick muscle biopsy sections using antibodies to lymphocyte subsets, major histocompatibility complex (MHC) antigens, and HTLV-I proteins; (b) polymerase chain reaction using HTLV-I primers in the RNA and DNA extracted from 50 micrograms of muscle tissue or from serial 5-microns-thick fresh-frozen tissue sections; and (c) cocultures of the patients' HTLV-I-positive peripheral blood lymphocytes with their homologous muscles searching for replication of HTLV-I within the myotubes. In the muscle of patients with HTLV-I-associated myopathy, the predominant endomysial cells surrounding healthy muscle fibers were CD8+ cells followed by CD4+ cells and macrophages. MHC-I antigens were ubiquitous in the muscles of all 6 patients, even in those without endomysial inflammation.

Effect of L-carnitine on the zidovudine-induced destruction of human myotubes. Part I: L-carnitine prevents the myotoxicity of AZT in vitro.

Background: Zidovudine (AZT) as used in the treatment of AIDS, causes a mitochondrial myopathy characterized by depletion of mitochondrial DNA, enzymatic defects in the respiratory chain system, and accumulation of lipid droplets. Most of these changes are also seen in normal human myotubes treated with AZT. Because L-carnitine plays a major role in the transport of long chain fatty acids across the inner mitochondrial membrane and facilitates the beta-oxidation of fatty acids, we examined the effect of L-carnitine in preventing the destructive effect of AZT on the mitochondria and the myotubes of human muscle in tissue culture.

Zidovudine-induced mitochondrial myopathy is associated with muscle carnitine deficiency and lipid storage.

The use of zidovudine (AZT) for the treatment of acquired immunodeficiency syndrome (AIDS) induces a DNA-depleting mitochondrial myopathy, which is histologically characterized by the presence of muscle fibers with "ragged-red"-like features, red-rimmed or empty cracks, granular degeneration, and rods (AZT fibers). Because dysfunctioning muscle mitochondria may lead to defects of beta-oxidation of fatty acids, we examined the degree of neutral fat accumulation and muscle carnitine levels in the muscle biopsy specimens from 21 patients with AZT-induced myopathic symptoms of varying severity. Six patients with no AZT fibers had normal endomyofibrillar lipid deposits and muscle carnitine levels; 7 patients with fewer than 5 AZT fibers per field had a mild (+) to moderate (++) increase in lipid droplets, and reduced muscle carnitine levels (3 patients); and 8 patients with more than 5 AZT fibers had severe muscle changes, a ++ to marked ( ) increase in lipid droplets, and reduced muscle carnitine levels (6 patients).

Search for HIV proviral DNA and amplified sequences in the muscle biopsies of patients with HIV polymyositis.

We searched for the presence of human immunodeficiency virus (HIV) in fresh-frozen muscle biopsy specimens from 10 patients with HIV-associated polymyositis (HIV-PM) using (a) 35S-labeled HIV-RNA transcript of the virus and in situ hybridization, and (b) polymerase chain reaction and slot-blot hybridization utilizing primers amplifying sequences from the gag and pol genes of the HIV genome. With in situ hybridization, positive signals were detected in sparse lymphoid cells surrounding the muscle fibers, but not within the muscle fibers, in up to two consecutive sections in 6 of the 10 specimens. By the polymerase chain reaction, amplified HIV-specific sequences were noted in 2 specimens, but in only 2 of 8 consecutive sections, implying infection of lymphoid cells rather than muscle fibers.

[Serologic activity against retrovirus in patients with Sjögren syndrome].

Background: The primary Sjögren's syndrome (SS) is a systemic disease which destroys the exocrine glands by autoimmune mechanisms. The etiology of this syndrome is unknown although different virus are involved in its genesis.

Methods: The presence of serologic reactivity IgG and IgM versus the human immunodeficiency virus (HIV-1), HIV-2, HTLV-I and HTLV-II were studied in 14 patients with SS and in 15 controls.

Absence of persistent infection with enteroviruses in muscles of patients with inflammatory myopathies.

We searched for enteroviral nucleic acid sequences using the polymerase chain reaction and slot-blot hybridization in coded muscle biopsy specimens from 39 patients with active inflammatory myopathies (polymyositis, dermatomyositis, and inclusion-body myositis) and from 16 patients with other neuromuscular diseases, including patients with postpolio syndrome. For primers, we used sequences of the noncoding region at the 5' end of the viral RNA. We failed to detect specific enteroviral nucleic acid sequences in the muscle biopsy specimens.

Failure to isolate human T cell lymphotropic virus type I and to detect variant-specific genomic sequences by polymerase chain reaction in Melanesians with indeterminate western immunoblot.

The controversy over the endemicity of human T cell lymphotropic virus type I (HTLV-I) in Melanesia has been settled recently by the isolation of genetically distinct, highly divergent sequence variants of HTLV-I from unrelated inhabitants of Papua New Guinea and the Solomon Islands. Still at issue, however, is the significance of the high frequency of indeterminate HTLV-I Western blots (defined as reactivity to only gag-encoded proteins) among Melanesians. To investigate whether this indeterminate seroreactivity reflects specific reactivity to the Melanesian HTLV-I variants, 27 seroindeterminate Melanesians from Papua New Guinea and the Solomon Islands were studied for evidence of HTLV-I infection.

Regenerating and denervated human muscle fibers and satellite cells express neural cell adhesion molecule recognized by monoclonal antibodies to natural killer cells.

The monoclonal antibodies anti-Leu-19 and anti-NKH-1 recognize the CD56 differentiation antigen expressed on natural killer (NK) cells and on a T-cell subset. Because CD56 is an isoform of neural cell adhesion molecule (N-CAM), we examined its expression on human muscle using antibodies to Leu-19, NKH-1, and purified N-CAM in an immunohistochemical, immunoblot, and immunoprecipitation study on 70 muscle biopsy specimens from various muscle diseases and on human muscle in tissue culture. Anti-Leu-19, anti-NKH-1, and anti-N-CAM had identical immunoreactive patterns.

Neutralizing antibody responses in patients with AIDS with neurologic complications.

Although the human immunodeficiency virus type 1 (HIV-1) is frequently isolated from the cerebrospinal fluid of infected patients, only a small percentage of patients are found to have clinical dementia or neuropathies (or both). The reasons for this remain unclear. In our study, serum neutralizing antibody titers against the human T cell leukemia virus-IIIB isolate of HIV-1 were tested in 10 patients with acquired immunodeficiency syndrome (AIDS) with neurologic complications and 20 patients with HIV infection without neurologic complications.

Characterization of a variant of human T-lymphotropic virus type I isolated from a healthy member of a remote, recently contacted group in Papua New Guinea.

We report the characterization of a variant of human T-lymphotropic virus type I (HTLV-I) isolated from an interleukin 2-dependent, CD8+ T-cell line derived from peripheral blood mononuclear cells of a healthy member of a remote, recently contacted hunter-horticulturalist group (Hagahai) in Madang province of Papua New Guinea. Antigenic characterization of this variant, designated PNG-1, by immunofluorescence, indicated no expression of gag-encoded proteins p19 and p24 (even after incubation with 5-bromo-2'-deoxyuridine), using monoclonal and polyclonal antibodies against HTLV-I gag gene products. Virus-specific proteins of 15, 19, 46, 53, and 61/68 kDa were demonstrated by Western blot analysis, using sera from patients with serologically and/or virologically confirmed HTLV-I myeloneuropathy, sera from HTLV-I-infected rabbits, and antibodies prepared against the C terminus of the major envelope glycoprotein gp46.

In situ hybridization detection of HTLV-I RNA in peripheral blood mononuclear cells of TSP/HAM patients and their spouses.

This is the first report of the direct detection of HTLV-I RNA in uncultured peripheral blood mononuclear cells (PBMNC's) of patients with tropical spastic paraparesis and HTLV-I-associated myelopathy (TSP/HAM) and their spouses, using the technique of in situ hybridization. Twenty-one Colombian patients were tested, all of whom had antibodies to HTLV-I; the presence of HTLV-I proviral DNA in their PBMNC's was confirmed by the polymerase chain reaction technique. Of the 21 patients 15 had a clinical diagnosis of tropical spastic paraparesis (TSP/HAM), 5 were asymptomatic relatives, and 1 patient had leukemia.