Colocalisation of human immunodeficiency virus and human cytomegalovirus infection in brain autopsy tissue from AIDS patients.

We have examined 26 human AIDS brains obtained at post mortem for infection by human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV), and for dual infection of cells by both viruses. The techniques used were enzyme-linked immunocytochemistry for HCMV and in situ hybridisation using a cDNA probe for HIV. Using these techniques, HCMV infection was detected in 14 brains, HIV infection in 14 brains, and coinfection with HIV and HCMV in 7 brains. Four case of dual HIV/HCMV infection were found where no colocalisation could be detected. In randomly chosen dually infected areas 19.2% of infected cells were coinfected with both viruses. Although cells identified morphologically as macrophages were the most common infected cell type, astrocytes and neurons were both singly and doubly infected with HIV and HCMV. Complete clinical data were available for 4 of the 7 cases with coinfection and each had AIDS dementia complex.

Correlation of the ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine in the brain and cerebrospinal fluid of the pig: implications for the determination of this methylation ratio in human brain.

1. Pigs were maintained in air or in an atmosphere of nitrous oxide which dramatically changes the S-adenosyl-L-methionine to S-adenosyl-L-homocysteine ratio in neural tissues. Samples of cerebrospinal fluid, cortex, cerebellum and spinal cord were then extracted and analysed for S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Regression analyses were carried out on values obtained in cerebrospinal fluid and in neural tissues. 2. Highly significant correlations were obtained between levels of S-adenosyl-L-homocysteine (r2 = 0.42-0.69; P less than 0.001) and S-adenosyl-L-methionine/S-adenosyl-L-homocysteine ratios (r2 = 0.56-0.65; P less than 0.001) in cerebrospinal fluid and levels and ratios in cortex, cerebellum and spinal cord. The levels of S-adenosyl-L-methionine did not show a significant correlation. 3. We conclude that the ratio of these metabolites in the cerebrospinal fluid may reflect the ratio in the central nervous system and we suggest that this may also be true in human tissues. This finding will permit the determination of the probable methylation ratio in the central nervous system in human conditions, such as vitamin B12 deficiency and acquired immune deficiency syndrome, where a similar myelopathy occurs to that seen in the nitrous oxide-treated pig. All three myelopathies may arise from an inhibition of methyltransferases involved in the synthesis of myelin that would occur when the methylation ratio is reduced.

Evidence of brain methyltransferase inhibition and early brain involvement in HIV-positive patients.

The myelopathy associated with human immunodeficiency virus (HIV) infection closely resembles that in subacute combined degeneration, a disorder of vitamin B12 metabolism. To investigate whether the disorders share a pathogenetic mechanism, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured in the cerebrospinal fluid (CSF) of 20 HIV-seropositive patients and 30 HIV-negative patients who were undergoing lumbar puncture for other medical reasons. The HIV-seropositive patients had significantly lower CSF concentrations of SAM (mean 77 [SD 25] vs 131 [35] nmol/l; p less than 0.001) and significantly higher concentrations of SAH (30.5 [6.8] vs 19.0 [7.1] nmol/l; p less than 0.001) than the controls. There was therefore a significant difference between the groups in the SAM/SAH (methylation) ratio (HIV 2.7 [1.0] vs control 7.6 [3.4]; p less than 0.001). There were no correlations between SAM or SAH concentrations or methylation ratio and age or sex in both groups, or serum B12 and folate concentrations, CSF folate, serum or CSF methylmalonic acid, risk factors, body mass index, specific drug treatment received, or disease stage in the HIV group. This finding suggests that HIV affects the brain from a very early stage of the infection. We suggest that, as in the pig, the CSF methylation ratio closely reflects that in the brain. In HIV-infected patients a reduced brain methylation ratio would inhibit methyltransferase enzymes, which would lead to hypomethylation in the central nervous system and ultimately to neurological lesions. In a pig model of subacute combined degeneration and in vitamin-B12-deficient human beings, the primary cause of the low methylation ratio is impaired recycling of SAH back to SAM, a process which requires vitamin-B12-dependent methionine synthase. The HIV patients in this study were vitamin B12 and folate replete, which suggests a different cause for the low methylation ratio.

Structural and biochemical changes in a spinal myelinopathy in twelve English foxhounds and two harriers.

Degenerative lesions in the spinal cord white matter of 12 English Foxhounds and two Harriers between 3 and 6 years old were associated with a diet composed mainly of ruminant stomachs. Lesions were present throughout the length of the spinal cord and were more severe in ventral and lateral columns than in dorsal columns. Degenerate fibers were accompanied by astrocytic proliferation. Changes suggestive of a primary myelinopathy included vacuolated myelin sheaths around apparently intact axons and thick-thin transitions in myelin sheath thickness. Mixed sensory and motor peripheral nerves and muscle histochemical fiber type profiles appeared normal. Similarities were noted with the changes described in subacute combined degeneration of the spinal cord in human beings, a neuropathy caused by methionine and methylation deficiency in patients with vitamin B12 deficiency. Mean serum methionine levels were significantly lower (P greater than 0.01) and mean liver methionine synthetase levels were significantly greater (P greater than 0.01) in affected dogs restored to a balanced diet than in age-matched controls maintained on the balanced diet. The elevated methionine synthetase levels possibly reflected compensatory reactions to the associated dietary change.

Effect of thiouracil in modifying folate function in severe vitamin B12 deficiency.

The effects of thiouracil in correcting defects in folic acid function produced by B12 deficiency were studied. Addition of the thyroid inhibitor, thiouracil, to a low methionine diet containing B12, increased the oxidation of [2-14C]histidine to carbon dioxide, and increased liver folate levels. Addition of 10% pectin to the diet accentuated B12 deficiency as evidenced by a greatly decreased rate of histidine oxidation (0.19%) and an increased excretion of methylmalonic acid. Addition of thiouracil to the diet restored folate function as measured by increased histidine oxidation and increased liver folate levels similar to that produced by addition of methionine to a B12-deficient diet. Thiouracil decreased methylmalonate excretion, and increased hepatic levels of B12 in animals on both B12-deficient and -supplemented diets. Hepatic methionine synthase was increased by thiouracil, which may be the result of the elevated B12 levels. S-Adenosylmethionine and the enzyme methionine adenosyltransferase were also increased by thiouracil. Thus it is possible that the effect of thiouracil in increasing folate function consists both in the effect of thiouracil in decreasing levels of methylenetetrahydrofolate reductase, and also in its action in increasing S-adenosylmethionine which exerts a feedback inhibition of this enzyme.

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency.

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.

Folic acid: effect on zinc absorption in humans and in the rat.

The effect of folic acid on zinc absorption and utilization was studied in humans and in rats. Serum Zn response curves after a 25-mg oral dose of Zn was measured for 4 h with and without 10 mg folic acid. The increase in serum Zn expressed as AUC (area under the curve) was 57 mumol/L for Zn alone and 65 mumol/L with folate. Zn intragastric intubation experiments with rats showed that adding 4.4 or 176 micrograms of folate to test meals containing 13 micrograms Zn did not affect the bioavailability of Zn as measured by the retention of 65Zn by the liver and kidney. A 3-wk feeding test carried out with diets containing either 6 or 12 mg Zn/kg diet showed that adding either 2 or 160 mg of folic acid had no effect on growth or Zn uptake by the femur. These results do not indicate any inhibition of Zn utilization by folic acid.

Demonstration of methionine synthetase in intestinal mucosal cells of the rat.

Methionine synthetase was measured in the mucosal cells of the rat duodenum, jejunum and ileum by a previously employed method for mucosal cell isolation. No activity was found in these cells. When a dual buffer system for the isolation of villous and crypt cell population was substituted, however, methionine synthetase was found to be active in the duodenum, jejunum and ileum, both in the villous and crypt cell populations. The activity was significantly higher in the crypt cells than in the villous cells throughout the intestine, and higher levels were found in the ileum than in the duodenum or jejunum. As had been previously reported for the rat liver, nitrous oxide in vivo reduced the enzyme activity in both the villous and crypt cell populations, suggesting a role in vivo for the enzyme. We conclude that methionine synthetase is both present and active in the small intestinal mucosal cells of the rat.

The effect of ethanol consumption on folate polyglutamate biosynthesis in the rat.

Contrary to previous studies, the folate polyglutamate chain length in the rat liver appears to be unaffected by ethanol ingestion for periods of 2-13 weeks. The appearance of short chain length folates after 13 weeks has been shown to arise as a result of increased in vitro folate polyglutamate breakdown during extraction due to a greater release of lysosomal conjugase in these animals.