- Email: [email protected]
Cerebrospinal fluid tau protein is not elevated in HIV-associated neurologic disease in humans
Neuroscience Letters 254 (1998) 1–4
Cerebrospinal fluid tau protein is not elevated in HIV-associated neurologic disease in humans Ronald J. Ellis a ,*, Peter Seubert b, Ruth Motter b, Douglas Galasko a, Reena Deutsch c, Robert K. Heaton c, Melvyn P. Heyes d, J. Allen McCutchan, J. Hampton Atkinson c, Igor Grant c, HIV Neurobehavioral Research Center Group (HNRC) a
Department of Neurosciences, University of California, San Diego, CA, USA b Athena Neurosciences, San Francisco, CA, USA c Department of Psychiatry, University of California, San Diego, California, USA d Laboratory of Neurotoxicology, National Institutes of Mental Health, Bethesda, MD, USA Received 3 June 1998; received in revised form 24 June 1998; accepted 24 June 1998
Abstract We measured the concentrations of the neuron-specific protein, tau, in the cerebrospinal fluid (CSF) of 32 neurologically characterized HIV-infected (HIVpos) subjects and nine matched seronegative (HIVneg) controls using a sensitive ELISA assay. Of 32 HIVpos subjects, nine had HIV-associated neurocognitive disorders, and nine had clinically diagnosed peripheral neuropathies. CSF tau levels in subjects with HIV-associated neurocognitive disorders were similar to those in HIVneg subjects (185 ± 83 vs. 223 ± 106 pg/ml; P = 57). CSF tau levels in HIVpos subjects with peripheral neuropathies did not differ from those without neuropathies (320 ± 190 vs. 251 ± 185; P = 23). In summary, CSF tau levels were not elevated in patients with HIV-associated neurologic disease. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: HIV/AIDS; Tau protein; Cerebrospinal fluid; Neurological disorders; Dementia; Cerebrospinal fluid Markers
The microtubule-associated protein tau is abundantly expressed in axons. Tau facilitates the stability of microtubules and helps to maintain synapses through fast axonal transport. Abnormal elevations of tau in brain tissue and cerebrospinal fluid (CSF) are associated with neuronal injury in Alzheimer’s disease [13], transiently in the setting of acute ischemic stroke, and in Creutzfeldt–Jakob disease. CSF tau levels are not elevated in certain other neurodegenerative disorders, such as Parkinson’s disease [10]. Thus specific pathophysiologic mechanisms, as well as the magnitude and rate of neuronal loss, may be important in determining whether tau concentrations will be elevated in CSF. Neuronal loss, synaptic injury, and axonal damage occur in HIV-associated neurocognitive disorders and neuropathies [5,9,11], and abnormal, tangle-like structures immunoreac* Corresponding author. UCSD HNRC, 2760 Fifth Avenue, San Diego, CA 92103, USA. Tel.: +1 619 5435079; fax: +1 619 5431235; email: [email protected]
tive for tau have been observed in the brains of HIVinfected patients [12]. Despite a prior report [2] in which three of four patients with AIDS had elevated CSF tau levels, no systematic studies of HIV-associated neurological disorders have been undertaken previously. We compared CSF tau levels in subjects with HIV-associated neurological disorders (HIV-ND) to those in HIV positive (HIVpos) subjects without neurological disease and to seronegative (HIVneg) subjects. To determine the relationship of tau to other CSF markers, and to evaluate the specificity of tau elevations in CSF, we also measured CSF levels of beta-2 microglobulin (B2M) and quinolinic acid (QUIN), two markers previously shown to be associated with neurologic disease in HIV infection. All subjects were volunteers in a prospective, universitybased, longitudinal investigation of the neurobehavioral consequences of HIV-infection conducted at the San Diego HIV Neurobehavioral Research Center (HNRC). The four subject groups were: (1) nine HIV seronegative,
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00549- 7
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neurologically normal individuals (HIVneg); (2) nine patients with HIV-associated neurocognitive disorders (HAD/MCMD), (3) nine seropositive subjects with clinically latent HIV infection and no neurocognitive disorder (Latent), and (4) 14 HIV positive subjects without neurocognitive disorders, group-matched to the HAD/MCMD group for CD4+ lymphocyte counts (CD4 Match). The nine neurocognitively-impaired subjects included seven with a clinical diagnosis of HIV-associated dementia (HAD) and two with HIV-associated minor-cognitive motor disorder (MCMD). They were selected from among 515 subjects in the HNRC cohort using the following criteria in addition to neurocognitive diagnosis: (1) age 25–55 years; (2) lumbar puncture performed within 2 weeks of clinical evaluation; (3) no evidence of current or prior CNS opportunistic disease by neurological examination or brain MR imaging. The demographic and clinical characteristics of the HNRC cohort have been previously described in detail [7]. The 14 subjects in the HIVpos CD4 match group were included to control for possible effects of HIV disease stage itself on CSF tau levels, independent of neurocognitive status. Subjects were excluded if they had a history of non-HIV-related major neurological or psychiatric disorders. Informed consent was obtained for all subjects after the study’s procedures were fully explained. All subjects underwent comprehensive clinical evaluations including general physical and standardized neurological examinations, serum chemistries and hematologic and immunologic studies, including CD4 + lymphocyte counts (cells per microliter). HIV disease stage was classified by established criteria [4]. CNS opportunistic infections were excluded based on neurological examination, CSF studies, and magnetic resonance brain scans. The clinical diagnosis of peripheral neuropathy required at least two of the following: complaints of sensory dysfunction (pain, paresthesiae, sensory loss); objective sensory abnormalities (increase in pin or vibration threshold in a stocking/glove or dermatomal pattern on examination by a neurologist); and loss or diminution of deep tendon reflexes. In four cases, nerve conduction studies were done to confirm the peripheral neuropathy. Lumbar punctures (LPs) were performed according to standard techniques and fresh CSF was analyzed for leukocyte and red cell numbers, and for protein, albumin and glucose. CSF samples containing .500 red blood cells per microliter were rejected. After storage at −70°C, CSF aliquots were batch-shipped to the tau assay laboratory. All subjects completed a detailed neuropsychological (NP) test battery consisting of measures of verbal functioning, abstraction, complex perceptual-motor skills, attention, learning, memory, motor skills and sensory functioning. A senior neuropsychologist (RKH), blinded to HIV serostatus, assigned clinical impairment ratings based on age, education and gender-adjusted normalized score distributions. Performance on a test was considered impaired when a subject’s score fell more than 1 standard deviation below the normative mean. ‘Global’ impairment indicated deficits
in two or more separate domains. All available clinical and laboratory data were reviewed by a team comprising a neurologist, neuropsychologist, clinical nurse and psychiatrist to assign a neurocognitive diagnosis, based on published consensus clinical criteria for HIV-associated minor cognitive-motor disorder (MCMD) and HIV-associated dementia [1]. The diagnosis of MCMD required (1) at least mild global neuropsychological impairment, (2) symptoms of cognitive and motor difficulties, and (3) mild-to-moderate disability (such as impaired performance at work) for at least 1 month, not attributable to comorbidities. The diagnosis of HIV-associated dementia (HAD) indicated moderate-to-severe neuropsychological test deficits and marked functional disability not attributable to comorbidities. Subjects without NP impairment were designated neurocognitively normal (NL). Tau in cerebrospinal fluid was measured using a double monoclonal antibody-based, ‘sandwich’ ELISA assay previously described in detail [13]. The reference standard used for the assay was human brain tau. The assay detection limit was 25 pg/ml, and quantitation was unaffected by tau phosphorylation state. In previous studies, the normal upper limit for CSF tau in neurologically healthy elderly controls is approximately 300 pg/ml [6,13]. b-2 microglobulin (B2M) in CSF (mg/l) was measured using a standard enzyme immunoassay (Pharmacia Diagnostics, Fairfield, NJ, USA). CSF quinolinic acid levels (nM) were measured by electron capture, negative chemical ionization mass spectrometry, with [13C7]-QUIN as an internal standard [8]. All CSF assays were performed blinded to the results of clinical evaluations. Because the distribution of CSF tau values in the sample was positively skewed, we used nonparametric Kruskal– Wallis one-way analysis of variance (ANOVA) for multiple group comparisons and the Wilcoxon rank sum test for pairwise comparisons. Corresponding parametric tests were used to compare normally distributed variables. Bivariate correlations were analyzed using Spearman’s rho. CSF quinolinic acid measurements were logtransformed. P-Values , 0.05 were considered significant. Data presented in the tables and text are group means and standard deviations. The clinical and demographic characteristics of the subjects are given in Table 1. Of those with HIV-associated neurocognitive disorders, seven had frank dementia and two had MCMD. The groups were similar in age, gender, and education. As expected, HIVpos subjects had significantly lower CD4+ lymphocyte counts than HIVneg controls, and CD4+ counts in the HAD/MCMD group were lower than in HIVpos subjects without AIDS. By design, CD4+ counts in the remaining HIVpos CD4 Match group were similar to those of subjects with HAD/MCMD. About one-third of subjects in each of the HAD/MCMD and CD4 Match groups had clinical AIDS. HIVpos subjects with neurocognitive disorders had elevated levels of the CSF markers B2M and quinolinic acid compared to HIVneg con-
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R.J. Ellis et al. / Neuroscience Letters 254 (1998) 1–4 Table 1 Demographic and medical characteristics of the study subjects
Age (years) Men (n, %) Education (years) Blood CD4+ count (cells/mm3) AIDS opportunistic disease (n, %) Log10 CSF quinolinic acid (nM) CSF beta-2 microglobulin (mg/l)
Sero-negative (n = 9)
HIV + latent (n = 9)
CD4 match (n = 14)
HAD or MCMD (n = 9)
ANOVA P
34.2 (7.9) 9 (100) 14.3 (3.7) 982 (298) – 1.1 (0.24) 0.87 (0.28)
33.4 (8.1) 9 (100) 13.6 (2.0) 548 (206)a 0 (0.0) 1.3 (0.25) 1.5 (1.4)
36.0 (6.4) 14 (100) 13.5 (1.6) 274 (213)a,b 4 (28.6)c 1.4 (0.28) 1.7 (0.82)
37.4 (9.5) 8 (89) 14.3 (2.4) 227 (206)a,b 3 (33.3)c 1.6 (0.51)d 2.2 (1.1)d
n.s. n.s. n.s. 0.0001 0.0001 0.02 0.04
Values for age and CD4+ cells are mean (±SD). Values for gender and CDC category are n (percent). CSF, cerebrospinal fluid. aSignificantly different from HIVneg controls, P , 0.001, by Tukey-Kramer HSD test. bSignificantly different from HIVpos latent infection, P , 0.05, by TukeyKramer HSD test. cCompared to HIVpos latent infection: 0.10 , P , 0.15, one-sided Fisher’s exact test. dSignificantly different from HIVneg controls, P , 0.05. CSF B2M value missing for one subject with HIV-associated dementia. All other group differences were not significant (P>0.15).
trols (Table 1), but the differences between the three HIVpos subgroups were not statistically significant. CSF tau values in the overall sample ranged from 65 to 951 pg/ml. The distribution of tau concentrations was positively skewed, with a median of 235 (interquartile range, 158 to 301). Tau values were unrelated to age, to CSF protein or albumin concentrations, or to the numbers of red blood cells or leukocytes in CSF (Spearman rank correlations; all P . 0.05). CSF tau levels in HIVpos subjects did not differ significantly from those of HIVneg controls (mean ± s.d., 270 ± 186 pg/ml vs. 223 ± 106; P . 0.50). Among HIVpos subjects, there was no relationship between CSF tau levels and blood CD4+ lymphocyte counts (rho = −0.04; P . 0.50), or disease stage (P . 0.25). Tau levels were not related to CSF protein (rho = 0.27; P . 0.10) or quinolinic acid (rho = −0.01; P . 0.50). CSF tau and beta-2 microglobulin (B2M) were moderately, but nonsignificantly correlated in HIVpos subjects (rho = 0.31; P = 0.065). The overall Kruskall–Wallis ANOVA for the four subject groups (HIVneg, Latent, CD4 Match and MCMD/HAD) showed a non-significant trend (P = 0.08) (Fig. 1). Posthoc pairwise comparisons showed that CSF tau levels in the neurocognitively impaired group did not differ from those of the HIVneg subjects (185 ± 83 vs. 223 ± 106; P . 0.50). None of the subjects in the HAD/MCMD group had a CSF tau level that exceeded the 95th percentile for the seronegative control group. CSF tau levels in HAD/ MCMD subjects were similar to those in the CD4 match group as well (304 ± 205; P = 0.10). Nine (29%) of the 32 HIVpos subjects had clinicallydiagnosed peripheral nerve abnormalities. These included four (44%) of the nine patients in the HIVpos HAD/ MCMD group, and five (36%) of the 14 patients in the HIVpos CD4 match group. The mean CD4+ count of the nine subjects with peripheral neuropathies was 146 (±168) per microliter. Nerve conduction studies confirmed the presence of peripheral nerve dysfunction in four patients, but were not performed in the other five. Six (67%) of the neu-
ropathy cases had a distal, symmetric, sensory-predominant neuropathy affecting principally the feet. The remaining three included one case each of cervical radiculopathy, unilateral S1 radiculopathy, and multifocal mononeuropathy. CSF tau levels among the nine HIVpos subjects with peripheral nerve disorders did not differ significantly from those of HIVneg controls (320 ± 190 vs. 223 ± 105; P . 0.25), nor did they differ from those of HIVpos subjects without neuropathy (251 ± 185; P . 0.20). Two ‘outlier’ CSF tau values were found among the HIVpos subjects. One (CSF tau = 951 pg/ml) was an asymptomatic man with a CD4+ count of 403, who was neurocognitively normal. The other (CSF tau = 767) was an HIV positive man with a CD4+ count of 96 and subacute onset of extremity numbness, urinary frequency and unsteady gait beginning approximately 4 months prior to examination. Neurologic examination showed diffusely diminished deep tendon reflexes, absent ankle jerks and difficulty with the Romberg test. Muscle strength and sensation on a bedside examination were normal. However, a quantitative sensory examination revealed diminished
Fig. 1. Tau in cerebrospinal fluid by ELISA assay (pg/ml) according to subject group. CSF tau levels of subjects with HIV-associated neurocognitive disorders (dementia, HAD; minor cognitive-motor disorder, MCMD) did not differ from those of uninfected controls (HIVneg), subjects with latent stage HIV infection (Latent) or HIV positive subjects matched for CD4+ lymphocyte counts (CD4 match).
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vibratory sensation at the ankles. Electrophysiological studies were remarkable for delayed sural nerve conduction (latency 10.5 s) and diminished amplitude (3.2 mV). CSF protein (30 mg/dl), glucose (63 mg/dl), and cell counts (one lymphocyte per microliter) were normal. Clinically, this patient was believed to have a developing sensory neuronopathy (dorsal root ganglionitis). In summary, we found no consistent elevations of CSF tau protein in subjects with HIV-ND. CSF tau levels in patients with HAD and MCMD were similar to those of neurocognitively normal, seronegative, age-matched control subjects. Subjects with peripheral neuropathies also did not show elevated CSF tau levels, with one exception occurring in a subject with a clinical diagnosis of subacute sensory neuronopathy. Dorsal root ganglion neuron dropout may have contributed to the elevated CSF tau levels seen in this subject. A second subject with an elevated CSF tau level had a normal neurological examination. Due to the relatively small number of subjects in each group, and because of limited statistical power, a true difference between the groups may have been missed. However, none of the subjects with neurocognitive disorders had a CSF tau concentration that exceeded the 95th percentile for our seronegative control subjects. Nor did any of these values exceed the previously-defined normal range for elderly controls without neurological disease (,300 pg/ ml) [6,13]. For comparison, the mean ± s.d. CSF tau level for a group of patients with early Alzheimer disease in a previous study was 509 ± 255. Although the sample size in the present study was small, other markers of CNS involvement (b-2 microglobulin and quinolinic acid) were elevated among neurocognitively impaired subjects, as reported previously [3]. Accordingly, if a meaningful group difference in tau levels had been present, it likely would have been detected. We conclude that CSF tau levels are not markedly elevated in HIV-associated neurocognitive disorders or peripheral nerve abnormalities. These findings may have implications for the pathogenesis and treatment of HIVassociated neurological disorders. If CSF tau protein levels are elevated due to pronounced neuronal loss, then normal tau levels in HIV-associated neurological disorders are consistent with less severe neuronal loss. Neuronal dysfunction, rather than cell death, may be the principal underlying cause for the clinical abnormalities, and HIV-related neurodegeneration may involve reversible alterations in dendritic arborization and synaptic function. If these speculations are correct, then therapeutic strategies designed to protect or rescue surviving neurons may bear significant promise in the treatment of HIV-associated neurologic disorde>rs. Supported by a center grant from NIMH (MH45294), and by Athena Neurosciences. The San Diego HIV Neurobehavioral Research Center (HNRC) group includes: I. Grant, J.H. Atkinson, T.D. Marcotte, J.L. Chandler, M.R. Wallace,
J.A. McCutchan, S.A. Spector, R.K. Heaton, T. Jernigan, J. Hesselink, E. Masliah, R.J. Ellis, D.R. Masys, I. Abramson and J. Nelson. [1] AAN. Nomenclature and research case definitions for neurologic manifestations of human immunodeficiency virus-type 1 (HIV-1) infection. Report of a working group of the American academy of neurology AIDS task force, Neurology, 41 (1991) 778–785. [2] Arai, H., Terajima, M., Miura, M., Higuchi, S., Muramatsu, T., Machida, N., Seiki, H., Takase, S., Clark, C.M., Lee, V.M., Trojanowski, J.Q. and Sasaki, H., Tau in cerebrospinal fluid: a potential diagnostic marker in Alzheimer’s disease, Ann. Neurol., 38 (1995) 649–652. [3] Brew, B.J., Bhalla, R.B., Paul, M., Sidtis, J.J., Keilp, J.J., Sadler, A.E., Gallardo, H., McArthur, J.C., Schwartz, M.K. and Price, R.W., Cerebrospinal fluid beta 2-microglobulin in patients with AIDS dementia complex: an expanded series including response to zidovudine treatment, AIDS, 6 (1992) 461–465. [4] CDC, Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Morbidity Mortality Weekly Report, 41 (1992) 1–19. [5] Everall, I.P., Hudson, L., Al-Sarraj, S., Honavar, M., Lantos, P. and Kerwin, R., Decreased expression of AMPA receptor messenger RNA and protein in AIDS: a model for HIV-associated neurotoxicity, Nat. Med., 1 (1995) 1174–1178. [6] Galasko, D., Clark, C., Chang, L., Miller, B., Green, R.C., Motter, R. and Seubert, P., Assessment of CSF levels of tau protein in mildly demented patients with Alzheimer’s disease, Neurology, 48 (1997) 632–635. [7] Heaton, R., Grant, I., Butters, N., White, D., Kirson, D., McCutchan, J., Taylor, M., Kelly, M., Wolfson, T., Velin, R., Marcotte, T., Atkinson, J., Ellis, R., Hesselink, J., Jernigan, T., Wallace, M., Abramson, I. and Group, H., The HNRC 500: neuropsychology of HIV infection at different disease stages, J. Int. Neuropsychol. Soc., 1 (1995) 231–251. [8] Heyes, M.P. and Markey, S.P., [18O]-Quinolinic acid: its esterification without back exchange for use as internal standard in the quantification of brain and CSF quinolinic acid, Biomed. Environ. Mass Spectrom., 15 (1988) 291–293. [9] Masliah, E., Ge, N., Achim, C.L., Hansen, L.A. and Wiley, C.A., Selective neuronal vulnerability in HIV encephalitis, J. Neuropathol. Exp. Neurol., 51 (1992) 585–593. [10] Molina, J.A., Benito-Leon, J., Jimenez-Jimenez, F.J., OrtiPareja, M., Berbel, A., Tallon-Barranco, A., deBustos, F. and Hernanz, A., Tau protein concentrations in cerebrospinal fluid of non-demented Parkinson’s disease patients, Neurosci. Lett., 238 (1997) 139–141. [11] Rizzuto, N., Cavallaro, T., Monaco, S., Morbin, M., Bonetti, B., Ferrari, S., Galiazzo-Rizzuto, S., Zanette, G. and Bertolasi, L., Role of HIV in the pathogenesis of distal symmetrical peripheral neuropathy, Acta Neuropathol. (Berlin), 90 (1995) 244–250. [12] Stanley, L.C., Mrak, R.E., Woody, R.C., Perrot, L.J., Zhang, S., Marshak, D.R., Nelson, S.J. and Griffin, W.S., Glial cytokines as neuropathogenic factors in HIV infection: pathogenic similarities to Alzheimer’s disease, J. Neuropathol. Exp. Neurol., 53 (1994) 231–238. [13] Vigo-Pelfrey, C., Seubert, P., Barbour, R., Blomquist, C., Lee, M., Lee, D., Coria, F., Chang, L., Miller, B., Lieberburg, I. and Schenk, D., Elevation of microtubule-associated protein tau in the cerebrospinal fluid of patients with Alzheimer’s disease, Neurology, 45 (1995) 788–793.
Cerebrospinal fluid tau protein is not elevated in HIV-associated neurologic disease in humans Ronald J. Ellis a ,*, Peter Seubert b, Ruth Motter b, Douglas Galasko a, Reena Deutsch c, Robert K. Heaton c, Melvyn P. Heyes d, J. Allen McCutchan, J. Hampton Atkinson c, Igor Grant c, HIV Neurobehavioral Research Center Group (HNRC) a
Department of Neurosciences, University of California, San Diego, CA, USA b Athena Neurosciences, San Francisco, CA, USA c Department of Psychiatry, University of California, San Diego, California, USA d Laboratory of Neurotoxicology, National Institutes of Mental Health, Bethesda, MD, USA Received 3 June 1998; received in revised form 24 June 1998; accepted 24 June 1998
Abstract We measured the concentrations of the neuron-specific protein, tau, in the cerebrospinal fluid (CSF) of 32 neurologically characterized HIV-infected (HIVpos) subjects and nine matched seronegative (HIVneg) controls using a sensitive ELISA assay. Of 32 HIVpos subjects, nine had HIV-associated neurocognitive disorders, and nine had clinically diagnosed peripheral neuropathies. CSF tau levels in subjects with HIV-associated neurocognitive disorders were similar to those in HIVneg subjects (185 ± 83 vs. 223 ± 106 pg/ml; P = 57). CSF tau levels in HIVpos subjects with peripheral neuropathies did not differ from those without neuropathies (320 ± 190 vs. 251 ± 185; P = 23). In summary, CSF tau levels were not elevated in patients with HIV-associated neurologic disease. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: HIV/AIDS; Tau protein; Cerebrospinal fluid; Neurological disorders; Dementia; Cerebrospinal fluid Markers
The microtubule-associated protein tau is abundantly expressed in axons. Tau facilitates the stability of microtubules and helps to maintain synapses through fast axonal transport. Abnormal elevations of tau in brain tissue and cerebrospinal fluid (CSF) are associated with neuronal injury in Alzheimer’s disease [13], transiently in the setting of acute ischemic stroke, and in Creutzfeldt–Jakob disease. CSF tau levels are not elevated in certain other neurodegenerative disorders, such as Parkinson’s disease [10]. Thus specific pathophysiologic mechanisms, as well as the magnitude and rate of neuronal loss, may be important in determining whether tau concentrations will be elevated in CSF. Neuronal loss, synaptic injury, and axonal damage occur in HIV-associated neurocognitive disorders and neuropathies [5,9,11], and abnormal, tangle-like structures immunoreac* Corresponding author. UCSD HNRC, 2760 Fifth Avenue, San Diego, CA 92103, USA. Tel.: +1 619 5435079; fax: +1 619 5431235; email: [email protected]
tive for tau have been observed in the brains of HIVinfected patients [12]. Despite a prior report [2] in which three of four patients with AIDS had elevated CSF tau levels, no systematic studies of HIV-associated neurological disorders have been undertaken previously. We compared CSF tau levels in subjects with HIV-associated neurological disorders (HIV-ND) to those in HIV positive (HIVpos) subjects without neurological disease and to seronegative (HIVneg) subjects. To determine the relationship of tau to other CSF markers, and to evaluate the specificity of tau elevations in CSF, we also measured CSF levels of beta-2 microglobulin (B2M) and quinolinic acid (QUIN), two markers previously shown to be associated with neurologic disease in HIV infection. All subjects were volunteers in a prospective, universitybased, longitudinal investigation of the neurobehavioral consequences of HIV-infection conducted at the San Diego HIV Neurobehavioral Research Center (HNRC). The four subject groups were: (1) nine HIV seronegative,
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00549- 7
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R.J. Ellis et al. / Neuroscience Letters 254 (1998) 1–4
neurologically normal individuals (HIVneg); (2) nine patients with HIV-associated neurocognitive disorders (HAD/MCMD), (3) nine seropositive subjects with clinically latent HIV infection and no neurocognitive disorder (Latent), and (4) 14 HIV positive subjects without neurocognitive disorders, group-matched to the HAD/MCMD group for CD4+ lymphocyte counts (CD4 Match). The nine neurocognitively-impaired subjects included seven with a clinical diagnosis of HIV-associated dementia (HAD) and two with HIV-associated minor-cognitive motor disorder (MCMD). They were selected from among 515 subjects in the HNRC cohort using the following criteria in addition to neurocognitive diagnosis: (1) age 25–55 years; (2) lumbar puncture performed within 2 weeks of clinical evaluation; (3) no evidence of current or prior CNS opportunistic disease by neurological examination or brain MR imaging. The demographic and clinical characteristics of the HNRC cohort have been previously described in detail [7]. The 14 subjects in the HIVpos CD4 match group were included to control for possible effects of HIV disease stage itself on CSF tau levels, independent of neurocognitive status. Subjects were excluded if they had a history of non-HIV-related major neurological or psychiatric disorders. Informed consent was obtained for all subjects after the study’s procedures were fully explained. All subjects underwent comprehensive clinical evaluations including general physical and standardized neurological examinations, serum chemistries and hematologic and immunologic studies, including CD4 + lymphocyte counts (cells per microliter). HIV disease stage was classified by established criteria [4]. CNS opportunistic infections were excluded based on neurological examination, CSF studies, and magnetic resonance brain scans. The clinical diagnosis of peripheral neuropathy required at least two of the following: complaints of sensory dysfunction (pain, paresthesiae, sensory loss); objective sensory abnormalities (increase in pin or vibration threshold in a stocking/glove or dermatomal pattern on examination by a neurologist); and loss or diminution of deep tendon reflexes. In four cases, nerve conduction studies were done to confirm the peripheral neuropathy. Lumbar punctures (LPs) were performed according to standard techniques and fresh CSF was analyzed for leukocyte and red cell numbers, and for protein, albumin and glucose. CSF samples containing .500 red blood cells per microliter were rejected. After storage at −70°C, CSF aliquots were batch-shipped to the tau assay laboratory. All subjects completed a detailed neuropsychological (NP) test battery consisting of measures of verbal functioning, abstraction, complex perceptual-motor skills, attention, learning, memory, motor skills and sensory functioning. A senior neuropsychologist (RKH), blinded to HIV serostatus, assigned clinical impairment ratings based on age, education and gender-adjusted normalized score distributions. Performance on a test was considered impaired when a subject’s score fell more than 1 standard deviation below the normative mean. ‘Global’ impairment indicated deficits
in two or more separate domains. All available clinical and laboratory data were reviewed by a team comprising a neurologist, neuropsychologist, clinical nurse and psychiatrist to assign a neurocognitive diagnosis, based on published consensus clinical criteria for HIV-associated minor cognitive-motor disorder (MCMD) and HIV-associated dementia [1]. The diagnosis of MCMD required (1) at least mild global neuropsychological impairment, (2) symptoms of cognitive and motor difficulties, and (3) mild-to-moderate disability (such as impaired performance at work) for at least 1 month, not attributable to comorbidities. The diagnosis of HIV-associated dementia (HAD) indicated moderate-to-severe neuropsychological test deficits and marked functional disability not attributable to comorbidities. Subjects without NP impairment were designated neurocognitively normal (NL). Tau in cerebrospinal fluid was measured using a double monoclonal antibody-based, ‘sandwich’ ELISA assay previously described in detail [13]. The reference standard used for the assay was human brain tau. The assay detection limit was 25 pg/ml, and quantitation was unaffected by tau phosphorylation state. In previous studies, the normal upper limit for CSF tau in neurologically healthy elderly controls is approximately 300 pg/ml [6,13]. b-2 microglobulin (B2M) in CSF (mg/l) was measured using a standard enzyme immunoassay (Pharmacia Diagnostics, Fairfield, NJ, USA). CSF quinolinic acid levels (nM) were measured by electron capture, negative chemical ionization mass spectrometry, with [13C7]-QUIN as an internal standard [8]. All CSF assays were performed blinded to the results of clinical evaluations. Because the distribution of CSF tau values in the sample was positively skewed, we used nonparametric Kruskal– Wallis one-way analysis of variance (ANOVA) for multiple group comparisons and the Wilcoxon rank sum test for pairwise comparisons. Corresponding parametric tests were used to compare normally distributed variables. Bivariate correlations were analyzed using Spearman’s rho. CSF quinolinic acid measurements were logtransformed. P-Values , 0.05 were considered significant. Data presented in the tables and text are group means and standard deviations. The clinical and demographic characteristics of the subjects are given in Table 1. Of those with HIV-associated neurocognitive disorders, seven had frank dementia and two had MCMD. The groups were similar in age, gender, and education. As expected, HIVpos subjects had significantly lower CD4+ lymphocyte counts than HIVneg controls, and CD4+ counts in the HAD/MCMD group were lower than in HIVpos subjects without AIDS. By design, CD4+ counts in the remaining HIVpos CD4 Match group were similar to those of subjects with HAD/MCMD. About one-third of subjects in each of the HAD/MCMD and CD4 Match groups had clinical AIDS. HIVpos subjects with neurocognitive disorders had elevated levels of the CSF markers B2M and quinolinic acid compared to HIVneg con-
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R.J. Ellis et al. / Neuroscience Letters 254 (1998) 1–4 Table 1 Demographic and medical characteristics of the study subjects
Age (years) Men (n, %) Education (years) Blood CD4+ count (cells/mm3) AIDS opportunistic disease (n, %) Log10 CSF quinolinic acid (nM) CSF beta-2 microglobulin (mg/l)
Sero-negative (n = 9)
HIV + latent (n = 9)
CD4 match (n = 14)
HAD or MCMD (n = 9)
ANOVA P
34.2 (7.9) 9 (100) 14.3 (3.7) 982 (298) – 1.1 (0.24) 0.87 (0.28)
33.4 (8.1) 9 (100) 13.6 (2.0) 548 (206)a 0 (0.0) 1.3 (0.25) 1.5 (1.4)
36.0 (6.4) 14 (100) 13.5 (1.6) 274 (213)a,b 4 (28.6)c 1.4 (0.28) 1.7 (0.82)
37.4 (9.5) 8 (89) 14.3 (2.4) 227 (206)a,b 3 (33.3)c 1.6 (0.51)d 2.2 (1.1)d
n.s. n.s. n.s. 0.0001 0.0001 0.02 0.04
Values for age and CD4+ cells are mean (±SD). Values for gender and CDC category are n (percent). CSF, cerebrospinal fluid. aSignificantly different from HIVneg controls, P , 0.001, by Tukey-Kramer HSD test. bSignificantly different from HIVpos latent infection, P , 0.05, by TukeyKramer HSD test. cCompared to HIVpos latent infection: 0.10 , P , 0.15, one-sided Fisher’s exact test. dSignificantly different from HIVneg controls, P , 0.05. CSF B2M value missing for one subject with HIV-associated dementia. All other group differences were not significant (P>0.15).
trols (Table 1), but the differences between the three HIVpos subgroups were not statistically significant. CSF tau values in the overall sample ranged from 65 to 951 pg/ml. The distribution of tau concentrations was positively skewed, with a median of 235 (interquartile range, 158 to 301). Tau values were unrelated to age, to CSF protein or albumin concentrations, or to the numbers of red blood cells or leukocytes in CSF (Spearman rank correlations; all P . 0.05). CSF tau levels in HIVpos subjects did not differ significantly from those of HIVneg controls (mean ± s.d., 270 ± 186 pg/ml vs. 223 ± 106; P . 0.50). Among HIVpos subjects, there was no relationship between CSF tau levels and blood CD4+ lymphocyte counts (rho = −0.04; P . 0.50), or disease stage (P . 0.25). Tau levels were not related to CSF protein (rho = 0.27; P . 0.10) or quinolinic acid (rho = −0.01; P . 0.50). CSF tau and beta-2 microglobulin (B2M) were moderately, but nonsignificantly correlated in HIVpos subjects (rho = 0.31; P = 0.065). The overall Kruskall–Wallis ANOVA for the four subject groups (HIVneg, Latent, CD4 Match and MCMD/HAD) showed a non-significant trend (P = 0.08) (Fig. 1). Posthoc pairwise comparisons showed that CSF tau levels in the neurocognitively impaired group did not differ from those of the HIVneg subjects (185 ± 83 vs. 223 ± 106; P . 0.50). None of the subjects in the HAD/MCMD group had a CSF tau level that exceeded the 95th percentile for the seronegative control group. CSF tau levels in HAD/ MCMD subjects were similar to those in the CD4 match group as well (304 ± 205; P = 0.10). Nine (29%) of the 32 HIVpos subjects had clinicallydiagnosed peripheral nerve abnormalities. These included four (44%) of the nine patients in the HIVpos HAD/ MCMD group, and five (36%) of the 14 patients in the HIVpos CD4 match group. The mean CD4+ count of the nine subjects with peripheral neuropathies was 146 (±168) per microliter. Nerve conduction studies confirmed the presence of peripheral nerve dysfunction in four patients, but were not performed in the other five. Six (67%) of the neu-
ropathy cases had a distal, symmetric, sensory-predominant neuropathy affecting principally the feet. The remaining three included one case each of cervical radiculopathy, unilateral S1 radiculopathy, and multifocal mononeuropathy. CSF tau levels among the nine HIVpos subjects with peripheral nerve disorders did not differ significantly from those of HIVneg controls (320 ± 190 vs. 223 ± 105; P . 0.25), nor did they differ from those of HIVpos subjects without neuropathy (251 ± 185; P . 0.20). Two ‘outlier’ CSF tau values were found among the HIVpos subjects. One (CSF tau = 951 pg/ml) was an asymptomatic man with a CD4+ count of 403, who was neurocognitively normal. The other (CSF tau = 767) was an HIV positive man with a CD4+ count of 96 and subacute onset of extremity numbness, urinary frequency and unsteady gait beginning approximately 4 months prior to examination. Neurologic examination showed diffusely diminished deep tendon reflexes, absent ankle jerks and difficulty with the Romberg test. Muscle strength and sensation on a bedside examination were normal. However, a quantitative sensory examination revealed diminished
Fig. 1. Tau in cerebrospinal fluid by ELISA assay (pg/ml) according to subject group. CSF tau levels of subjects with HIV-associated neurocognitive disorders (dementia, HAD; minor cognitive-motor disorder, MCMD) did not differ from those of uninfected controls (HIVneg), subjects with latent stage HIV infection (Latent) or HIV positive subjects matched for CD4+ lymphocyte counts (CD4 match).
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vibratory sensation at the ankles. Electrophysiological studies were remarkable for delayed sural nerve conduction (latency 10.5 s) and diminished amplitude (3.2 mV). CSF protein (30 mg/dl), glucose (63 mg/dl), and cell counts (one lymphocyte per microliter) were normal. Clinically, this patient was believed to have a developing sensory neuronopathy (dorsal root ganglionitis). In summary, we found no consistent elevations of CSF tau protein in subjects with HIV-ND. CSF tau levels in patients with HAD and MCMD were similar to those of neurocognitively normal, seronegative, age-matched control subjects. Subjects with peripheral neuropathies also did not show elevated CSF tau levels, with one exception occurring in a subject with a clinical diagnosis of subacute sensory neuronopathy. Dorsal root ganglion neuron dropout may have contributed to the elevated CSF tau levels seen in this subject. A second subject with an elevated CSF tau level had a normal neurological examination. Due to the relatively small number of subjects in each group, and because of limited statistical power, a true difference between the groups may have been missed. However, none of the subjects with neurocognitive disorders had a CSF tau concentration that exceeded the 95th percentile for our seronegative control subjects. Nor did any of these values exceed the previously-defined normal range for elderly controls without neurological disease (,300 pg/ ml) [6,13]. For comparison, the mean ± s.d. CSF tau level for a group of patients with early Alzheimer disease in a previous study was 509 ± 255. Although the sample size in the present study was small, other markers of CNS involvement (b-2 microglobulin and quinolinic acid) were elevated among neurocognitively impaired subjects, as reported previously [3]. Accordingly, if a meaningful group difference in tau levels had been present, it likely would have been detected. We conclude that CSF tau levels are not markedly elevated in HIV-associated neurocognitive disorders or peripheral nerve abnormalities. These findings may have implications for the pathogenesis and treatment of HIVassociated neurological disorders. If CSF tau protein levels are elevated due to pronounced neuronal loss, then normal tau levels in HIV-associated neurological disorders are consistent with less severe neuronal loss. Neuronal dysfunction, rather than cell death, may be the principal underlying cause for the clinical abnormalities, and HIV-related neurodegeneration may involve reversible alterations in dendritic arborization and synaptic function. If these speculations are correct, then therapeutic strategies designed to protect or rescue surviving neurons may bear significant promise in the treatment of HIV-associated neurologic disorde>rs. Supported by a center grant from NIMH (MH45294), and by Athena Neurosciences. The San Diego HIV Neurobehavioral Research Center (HNRC) group includes: I. Grant, J.H. Atkinson, T.D. Marcotte, J.L. Chandler, M.R. Wallace,
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