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. Sequencing of the amplified product confirmed that it belonged to PV type 1 with a 99.3% homology. We conclude that some patients with PPS have in the serum high titers of IgM anti-PV antibodies, implying an ongoing antibody response to a viral antigen. The presence of PV-RNA in the CSF or lymphocytes suggests possible persistence of mutated virus or defective PV particles. The significance of these findings in the pathogenesis of PPS remains to be determined.

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. In neurogenic conditions and in myopathies, PVR was found in occasional denervated muscle fibers and in several regenerating ones. Human myotubes expressed PVR and were susceptible to the poliovirus infection. We conclude that PVR is present at the motor end-plate that can serve as one of the routes of entry of the virus to the motor neurons. The presence of PVR in the regenerating muscle fibers is in accord with clinical observations that muscle injuries can predispose patients to paralytic poliomyelitis.

Anxiogenesis induced by nitric oxide synthase inhibition and anxiolytic effect of melanin-concentrating hormone (MCH) in rat brain.

In this study, the involvement of nitric oxide (NO) in the mechanism of anxiety was investigated. The rats received an intraamygdaline or intrahippocampal injection of the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine (L-NOARG), and were then tested in the plus-maze test. L-NOARG induced a decrease in the time spent by rats in the open arms. Conversely, the administration of the melanin-concentrating hormone (MCH) into these structures increased the number of entries into the open arms as well as the time spent on them. MCH injected in rats pretreated with L-NOARG also was able to revert the anxiogenic effects of L-NOARG in amygdala.

Melanin-concentrating hormone (MCH) modifies memory retention in rats.

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.

Response to novelty after i.c.v. injection of melanin-concentrating hormone (MCH) in rats.

Some behavioral response of rats to spatial novelty after i.c.v. administration of melanin-concentrating hormone (MCH) were evaluated. To this purpose, an open-field test was used, as well as an elevated plus-maze to study the possible anxiolytic effect of this peptide. In the open field, the frequency of exploratory components (locomotion and rearing) increased after MCH administration in comparison to controls. Moreover, in the plus-maze, MCH increased the number of entries into the open arms as well as the time spent on them, whereas no changes in the number of entries onto the closed arms were found. The data indicate that MCH exerts an anxiolytic effect, and suggests a physiological role for this.

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. EXPERIMENTAL DESIGN: Myotubes, prepared from human muscle biopsies, were exposed to 250 microM of AZT for 3 to 6 weeks. After 3 weeks of AZT treatment, the cultures were treated with L-carnitine or medium for 3 weeks, while AZT treatment was either withdrawn or continued for 3 more weeks. The cultures were evaluated with: (a) light microscopy; (b) immunocytochemistry, to count the number of myotubes stained with antibodies to Leu-19; (c) oil red O stain, to assess the lipid droplet accumulation; and (d) electron microscopy, to count all the organelles within representative sections of the myotubes, at x24,000, and to calculate the volumetric density (Vvi) of each organelle per unit volume of tissue. RESULTS: In the post-AZT-treated cultures, L-carnitine increased the number of Leu-19-positive myotubes from 3.83 +/- 1.23 to 23 +/- 1.5 per field, normalized their mitochondria, decreased the lipid droplets, and increased the Vvi of the myofibrils. In the cultures treated with 3 weeks of L-carnitine while AZT treatment continued for 3 more weeks, the number of myotubes increased from 3.3 +/- 0.74 to 6.87 +/- 1.35; the absolute number of the mitochondria increased from 1.65 +/- 0.35 to 9.02 +/- 1.11 and their Vvi from 3.67 +/- 0.83 to 6.57 +/- 0.78 (p < 0.05); the Vvi of the myofibrils increased from 2.50 +/- 0.52 to 5.37 +/- 0.76 (p < 0.05); and the Vvi of the lipid droplets decreased from 5.06 +/- 1.44 to 2.72 +/- 0.72 (p < 0.05). In the AZT-treated cultures that did not receive L-carnitine, the mitochondria demonstrated extensive vacuolation, abnormal cristae, and paracrystalline inclusions; in contrast, in the L-carnitine-treated cultures, the mitochondria had substantially improved in spite of continuation of AZT. CONCLUSIONS: L-carnitine enhances the pace and degree of recovery of the AZT-associated destruction of human myotubes, restores and preserves the structure of mitochondria, mobilizes the endomyotubular fat, and allows the regeneration of myofibrils, even if AZT treatment continues. The findings may have potential clinical implications in improving the myotoxicity of AZT in patients with AIDS when the administration of AZT treatment must continue.

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. EXPERIMENTAL DESIGN: Myotubes, prepared from human muscle biopsies, were exposed to various concentrations of AZT for up to 3 weeks. One-third of the flasks were treated with AZT alone, another third with AZT plus L-carnitine and another third were untreated. The cultures were evaluated with: (a) immunocytochemistry counting the number of myotubes stained with antibodies to Leu-19; (b) enzyme histochemistry for NADH reaction and oil-red-O stain to assess mitochondrial enzymatic activity and lipid droplet accumulation; and (c) electron microscopy counting all the organelles within representative sections of the myotubes, at x24,000, and calculating the volumetric density of each organelle/unit volume of tissue. RESULTS: AZT, at concentrations 250 microM and above, caused depopulation of the Leu-19-positive myotubes, destructive changes in the mitochondria consisting of swelling, lamellar inclusions and multiple concentric cristae, accumulation of lipid droplets, and increase lysosomes. L-Carnitine increased the number of Leu-19-positive myotubes from 3.4 +/- 0.6 to 9.4 +/- 1.2, preserved the morphology of the mitochondria, increased their volumetric density from 2.5 +/- 0.4 to 6.0 +/- 0.7, and reduced the volumetric density of the lipid droplets from 12.2 +/- 4.9 to 1.4 +/- 0.7 and of the lysosomes from 15.6 +/- 3.6 to 3.9 +/- 1.4 (p < 0.001). CONCLUSIONS: L-Carnitine, used concurrently with AZT, prevents the human myotubes from the AZT-associated destruction, preserves the structure and volume of mitochondria and prevents the accumulation of lipids. The findings may have potential clinical implications in preventing the myotoxicity of AZT in patients with AIDS.

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). Serial sections showed lipid globules often within "cracks" or vacuoles of the abnormal muscle fibers. We conclude that the muscle mitochondrial impairment caused by AZT results in (1) accumulation of lipid within the muscle fibers owing to poor utilization of long-chain fatty acids, (2) reduction of muscle carnitine levels probably due to decreased carnitine uptake by the muscle, and (3) depletion of energy stores within the muscle fibers. The findings may have potential therapeutic implications in the treatment of AZT-induced myopathic symptoms using oral carnitine supplementation.

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. Restriction endonuclease maps of PNG-1 proviral DNA differed from that of a prototype strain of HTLV-I (MT-2), but, as verified by polymerase chain reaction, PNG-1 was definitely HTLV-I, not HTLV-II. Nucleotide sequencing and further molecular genetic studies of this variant may provide insights into the origin and evolution of HTLV-I.

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. Although antibodies against Melanesian variant-specific env gene products and variant-specific env gene sequences were detected by Western blot analysis and polymerase chain reaction, respectively, in all 11 HTLV-I Western blot-positive Melanesians, none of the 27 seroindeterminate Melanesians had such variant-specific antibodies or HTLV-I proviral sequences. In addition, attempts to isolate HTLV-I from seroindeterminate individuals were unsuccessful. These data indicate that HTLV-I infection is not the cause of the indeterminate Western blot reactivity seen in Melanesia.