Cerebrospinal Fluid Neopterin in Human Immunodeficiency Virus Type 1 Infection Bruce J. Brew, MB, FRACP," Ravi B. Bhalla, PhD,? Morris Paul, PhD,* H. Gallardo, MS,* Justin C. McArthur, MBBS,S Morton K. Schwartz, PhD,t and &chard W. Price, MD"
We evaluated cerebrospinal fluid (CSF) concentrations of neopterin, a putative marker of activated macrophages, in 97 subjects infected with human immunodeficiency virus type 1 who had a spectrum of neurological complications. The highest CSF neopterin concentrations occurred in those with neurological opportunistic infections, primary central nervous systems lymphoma, and acquired immunodeficiency syndrome (AIDS)dementia complex. In general, the CSF concentration of neopterin was independent of CSF cell count and blood-brain barrier disruption to albumin. In the patients with AIDS dementia complex, CSF neopterin concentrations correlated with severity of disease and decreased in conjunction with clinical improvement following treatment with zidovudine. These results suggest that CSF neopterin, although not disease-specific, may be useful as a surrogate marker for the presence of AIDS dementia complex and its response to antiviral therapy. Brew BJ, Bhalla FLB, Paul M, Gallardo H, McArthur JC, Schwartz MK, Price RW. Cerebrospinal fluid neopterin in human immunodeficiency virus type 1 infection. Ann Neurol 1990;28.556-560
Elevated serum and urine concentrations of neopterin (dihydroneopterin), a pteridine compound that has been considered a marker of cell-mediated immune reactions 111, have been found to correlate with the Walter Reed stage of human immunodeficiency virus type 1 (HIV-1)infection [2-41. We hypothesized that cerebrospinal fluid (CSF) levels of neopterin might similarly correlate with central nervous system (CNS) HIV-1 infection and its clinical counterpart, the acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) [ S } . Two recent reports 16, 71 documented increases in CSF levels of neopterin in patients with HIV-1 infection, but included only small numbers of patients with ADC and did not examine CSF levels of neopterin in patients with other neurological complications of AIDS or assess what effect therapy has on these concentrations. Consequently, we assayed neopterin in the CSF and serum of patients at various stages of ADC as well as those with other neurological complications of HIV- 1 infection. We also assessed the confounding influences of blood-brain barrier impairment and CSF pleocytosis as well as the effects of treatment with zidovudine (ZDV) (also azidothymidine or AZT) on CSF neopterin levels.
From the Departments of *Neurology and +Clinical Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, and the $Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD.
Subjects and Methods Subjects Three groups were studied: (1)HIV-I-infected patients with various neurological complications; (2) neurologically normal, HIV-1-infected subjects; and ( 3 ) a small group of HIV1 seronegative subjects. The first group (102 samples from 86 patients) will be described in more detail. The HIV1-infected, neurologically normal subjects (16 samples, 11 patients) are participants in a longitudinal study of the neurological manifestations of HIV-1 infection as part of the Multicenter AIDS Cohort Study (MACS) for which they have undergone semiannual neurologcal evaluation, magnetic resonance imaging (MRI) scan, and analysis of the CSF. None had neurological or neuropsychological abnormalities at the time of CSF sampling. The HIV-1 seronegative control group included 7 patients: 4 who were studied by myelography, CSF cytology, and CSF tumor markers for suspicion of metastatic cancer, but for whom the findings were normal; and 3 patients who had syringomyelia, idiopathic spastic paraparesis, and idiopathic peripheral neuropathy. HIV-1-INFECTEDNEUKOLOGICAL DISEASE GROUPS. These
patients were part of a larger population of HIV-1-infected individuals who were being prospectively followed to assess the neurological complications of HIV-1 infection. Eightynine percent had either AIDS or AlDS-related complex as
Address correspondence to Dr Price, Department of Neurology, Medical School, University of Minnesota, Box 295 UMHC, Minneapolis, MN 55455.
Received Feb 28, 1990, and in revised form Apr 26. Accepted for publication Apr 30, 1990.
556 Copyright 0 1990 by the American Neurological Association
defined by systemic disease. All were examined by a neurologist, and additional diagnostic investigations were performed as indicated by the clinical setting. On the basis of these evaluations, these patients were assigned to one of the following groups: Putients with ADC. Patients were included in this group if they had: (1) characteristic neurological symptoms and signs 181, (2) compatible neuroimaging findings, and (3) exclusion of other neurological conditions. The severity of the dementia was rated on a functionally based, staging scale ranging from 0 to 4, such that stage 0 indicates no neurological disease, stage 0.5 indicates only equivocal symptoms of subclinical signs without functional impairment, stage I indicates mild disturbance but preserved ability to work with minimal limitation, stage 2 indicates moderate ADC precluding work, stage 3 indicates severe dementia such that the patient cannot perform even basic activities of daily living, and stage 4 indicates a near vegetative state [2, 93. There were 70 CSF assessments of 54 patients with ADC: 16 samples from patients with ADC stage 0.5, 27 from patients with stage l, 19 from patients with stage 2, 6 from patients with stage 3, and 2 from patients with stage 4 . As the number of samples from patients with Stage 3 or 4 was small, these groups were combined for analysis. Samples of CSF and serum that were taken from 9 patients with ADC before and after treatment (mean interval of 8 weeks) with ZDV were also analyzed. Group without ADC. This consisted of 3 subgroups of patients, namely: (1)those with primary brain lymphoma, (2) those with CNS infections, and (3) those with headache of undetermined cause. The subgroup with primary brain lymphoma comprised 6 patients, 4 of whom had histological documentation of the diagnosis. In the remaining 2, presumptive diagnosis was based on characteristic clinical and radiological findings 181; these patients additionally exhibited no response to a therapeutic trial of antitoxoplasma treatment. The subgroup with CNS infections included 14 patients with the following: cerebral toxoplasmosis (1 patient), cryptococcal meningitis (3 patients), progressive multifocal leukoencephalopathy (1 patient), herpes zoster myelitis (1 patient), cytomegalovirus (CMV) rddiculomyelitis (1 patient), probable CMV encephalitis (CMV retinitis coincident with seizures, obtundation, and unremarkable neuroimaging or CSF studies; 2 patients), and HIV-1-related aseptic meningitis (5 patients) [lo}. The group with headaches comprised 12 patients who presented with severe headaches without an identified infectious, neoplastic, or psychiatric cause and unaccompanied by CSF pleocytosis. All had equivocal cognitive complaints or minor neurological abnormalities on examination, qualifying them in classification as stage 0.5 ADC. All had been HIV-1 seropositive for at least 3 years. The headaches resolved over several weeks in all patients.
Labmutoy Methods CSF was analyzed for cell count, protein, and glucose contents, the presence of cryptococcal antigen, cytology, and VDRL along with bacterial and fungal cultures using routine methods. Neopterin concentrations in both CSF and serum were assessed by radioimmunoassay (DRG, Henning, Berlin) with particular attention to minimizing light exposure.
The previously suggested normal values for neopterin levels in CSF (1.6 +- 0.6 nmol/l) [ll] were compared to that obtained from CSF analysis of the HIV-I negative control group. The upper normal limit for neopterin levels in serum was taken to be 8.7 nmolll {127. CSF and serum values for albumin concentration were determined in order to serve as an index of the integrity of the blood-brain barrier; a CSF-toserum albumin ratio, multiplied by lo3, of greater than 6.5 was considered abnormal [l 31.
Statistics Statistical methods included analyses of variance and Student-Newman-Keuls post hoc tests in comparisons of discrete disease groups and ADC stages. Continuous variables were compared using Pearson correlations. Alpha (significance) level was established at p I0.05. Changes in neopterin concentration before and after ZDV treatment were compared using the paired t test. Those neurologically abnormal patients who had more than one CSF analysis were included in the main data set as independent observations because the CSF was repeated for assessment of clinical change. These analyses were carried out using SPSS/PC+ software (SPSS Inc, Chicago, IL).
Results As shown in the Table, the mean CSF concentration of neopterin for the HIV-1 seronegative control group was 3.5 nmol/l, somewhat higher than that found by Fredrikson and associates El 11. While significantly elevated (more than twice that of seronegatives) neopterin concentrations were noted in the seropositive, neurologically normal group, markedly higher concentrations (8 to 16 times normal) were found in the symptomatic patients; the CSF neopterin concentration was highest in those with CNS infections, followed, in turn, by those with ADC, those with lymphoma, and those with headache. The concentrations in all disease groups were different from those in the seronegative controls and concentration in the group with ADC was also different from that in the seropositive control group. In patients without confounding CNS disorders, CSF neopterin concentrations correlated with the ADC stage (Fig 1) (r = 0.54, p < 0.0001). Whereas there was no difference between those with ADC stage 0.5 and the neurologically normal seropositives ( p = 0.481), all patients with ADC stages 1 or greater had CSF neopterin concentrations outside the range of the HIV-1 seronegative group, and 89% of these patients had concentrations greater than 16.0 nmoV1, the upper limit of the 95% confidence level of the HIV-1 seropositive, neurologically normal group. In contrast to CSF levels, serum neopterin concentrations did not correlate with severity of ADC (r = 0.039). There was no discernible relationship between neopterin concentration in the CSF and the number of white blood cells in the CSF. Indeed, only 10 samples from 5 patients had CSF cell counts greater than 5 Brew et al: CSF Neopterin in HIV-1 557
CSF and Serum Neoptevin Concentrations in Different Patient Groups Neopterin (nmoV1 Patient Group
No. of Samples
Controls Seronegative HIV-1-infected, neurologically normal Disease Headache AIDS dementia complex Lymphoma CNS infection
CSF
Serum
7 16
3.5 2 0.4 8.2 t 1.0b
12 70
26.5 f 5.1b,‘
6 14
34.8
36.8 58.2
* SEMY
? ? ?
3.6b,‘ 11.5b.c 11.7b,‘
Not available 13.7 ? 1.8 33.0 40.0 35.0
44.4
&
7.0 4.1
2
10.6
?
* ll.?
~
“Normal range: CSF, < 5.0 nmoul; serum, < 8.7 nmoV1. bDiffered from seronegative controls ( p < 0.001). ‘Differed from seropositive, neurologically normal controls ( p < 0.0001).
0
0.5
1
2
3-4
ADC STAGE
Fig I . CSF neopterin concentration as a function of the stage of
the AIDS dementia complex (ADC).
cells/mm3 and the neopterin values in these did not differ from those of other patients in the same ADC stage. Analysis of blood-brain barrier integrity to albumin for the whole data set, including the ADC group, failed to reveal any association with CSF levels of neopterin ( r = 0.14, p = 0.43). Furthermore, when the group with ADC was divided into those without (38 samples) and those with (32 samples) blood-brain barrier impairment to albumin, analysis revealed that the samples from patients with an intact blood-brain barrier evidenced a high correlation between CSF neopterin level and ADC stage (r = 0.64, @ = 0.0001), whereas those with an impaired blood-brain barrier to albumin had a less significant correlation ( r = 0.39, p = 0.01). Lkewise, although there was a correlation between CSF and serum neopterin levels both for all patients assessed ( Y = 0.45, @ < 0.0001) and for the patients with ADC who were considered separately ( r = 0.49, p < O.OOOl), this correlation was lost when 558 Annals of Neurology Vol 28 No 4 October 1990
PRE-ZDV
POST-ZDV
Fig 2. CSF neoptwin concentrations before and afer treatment with zidovudine CZDV).Values have been converted to a log scale in order to encompass the wide range of values and the changes with treatment.
those with blood-brain barrier dysfunction were considered separately. ZDV treatment of patients with ADC resulted in a reduction of CSF neopterin levels in each of the 9 assessed (Fig 2, p = 0.032), while serum concentrations in these patients did not significantly change (mean pretreatment level of 28.8 2 5.2 nmol/l only fell to 20.2 t 4.9 nmol/l after treatment, p = 0.23).
Discussion Our results indicate that the CSF concentration of neopterin is elevated in HIV-1-infected patients who have a variety of neurological complications. The fact that the highest CSF levels were observed in patients with CNS infections and primary CNS lymphoma is consistent with the concept of neopterin being related to “activated” cellular immunity [l}and with reports of elevated serum neopterin concentrations in systemic infections and malignancies {14, 15}. Elevation of CSF
neopterin, like other recently reported surrogate markers, such as beta-2 microglobulin 1161 and quinolinic acid C171, is thus not disease-specific. Nonetheless, the finding of greatest interest is the consistent elevation of CSF neopterin in patients with ADC and its general correlation with severity of ADC. These results, which are in keeping with those recently reported in two smaller series 16, 71,are potentially important in relation to both pathogenesis and clinical management of ADC. Activated macrophages, and to 3 much lesser extent T lymphocytes, produce neopterin, and it has been noted that interferon gamma can induce neopterin through its effect on guanosine triphosphate (GTP) cyclohydrolase, the first enzyme in the conversion of GTP to dihydroneopterin triphosphate, the precursor of neopterin 1181. Although the source of elevated CSF concentrations of neopterin in these patients was not directly studied, it is unlikely that CSF inflammatory cells alone were responsible [l 11since only a very few patients had discernible pleocytosis. Similarly, several observations suggest that alteration of the bloodbrain barrier likely did not explain the high concentration of CSF neopterin, particularly in the patients with ADC: (1) There was no correlation between CSF neopterin concentration and blood-brain-barrier impairment to albumin; (2) CSF neopterin concentration correlated with severity of ADC, while serum neopterin concentration did not; and (3) in several patients the CSF concentration of neopterin exceeded the serum concentration. A similar lack of correlation of neopterin with CSF cell count and blood-brain barrier was noted by Fuchs and colleagues [6] and Sonnerborg and coworkers 17). The CNS parenchymal cells producing neopterin are not known, but major candidates are infiltrating macrophageimononuclear cells and ontogenetically related [191 microglial cells. However, because neuropathological examination of brains from patients with ADC stage 0.5 or 1 characteristically shows only scant inflammatory or microglial infiltrates 2201, it is also possible that intrinsic neural cells, perhaps proliferating astrocytes, may produce neopterin when they are stimulated by cytokines. Indeed, these findings may provide insight into the role of “immunopathological” events in the pathogenesis of ADC. The elevation of CSF neopterin concentration with increasing severity of ADC and the decline with antiviral therapy are compatible with the view that the virus load “drives” immune-cell activation and putative CNS immunopathology [21). The plateau or possible slight reduction of neopterin in the patients with the most severe ADC (stages 3 and 4 ) may indicate a predominant role of CNS viral infection rather than immunopathology in such patients [2 11. Whether neopterin itself contributes to CNS dysfunction is highly speculative. Shen
and associates E22) recently found that reduced pterins, for example neopterin and dihydroneopterin, can downregulate GTP cyclohydrolase 1 and thereby reduce the concentration of tetrahydrobiopterin, an essential cofactor in the synthesis of dopamine, serotonin, and noradrenalin. It is conceivable that some of the basal ganglion dysfunction that is characteristic of ADC might result from neopterin-induced perturbation of these neurotransmitters. Elevation of CSF neopterin concentrations in neurologically asymptomatic subjects and the further elevation in patients with headache of unknown cause await further clarification. The most likely explanation for elevation in the asymptomatic group relates to subclinical disease activity. This is consistent with other CSF abnormalities in asymptomatic patients E23-28). It also is in keeping with the hypothesis that CNS HIV-1 infection is actively “controlled” by effective immune responses early in the course of infection 1211. Headache is a common, but poorly understood, problem in HIV- 1 infected patients. Elevated CSF concentrations of neopterin in such patients may relate in part to the emergence of neurological dysfunction in this group or may suggest that headache relates to inflammatory processes not reflected in CSF pleocytosis. Additional studies are needed to resolve these questions. Whatever the mechanism of increased levels of CSF neopterin, our results suggest that assessment of this “surrogate marker” may at times prove useful in diagnosis and therapeutic monitoring of ADC. Thus, while increased CSF neopterin is clearly not disease-specific, once clinical and laboratory evaluations have eliminated other opportunistic CNS infections or tumors, assessment of this marker may be 3 useful adjunct to establishing diagnosis of ADC, and particularly its differentiation from psychiatric ( e g , anxiety and depression) and degenerative (e.g., Parkinson’s and Alzheimer’s diseases) disorders in HIV- 1-infected patients. Our present results suggest that a CSF neopterin concentration of 16.0 nmoUl might serve as a cutoff of “normal” in the HIV-1-infected patient, although further observations are needed to more precisely define: the limits of CSF neopterin in HIV-1infected, neurologically normal individuals; the possible effects of systemic infections on CSF concentrations; and the prognostic value of elevated neopterin concentrations in relation to the development of ADC. With respect to the use of CSF neopterin levels in monitoring therapy for ADC, further studies are needed to determine the clinical and pathobiological significance of the striking decline in CSF neopterin noted in our patients given ZDV. The time c o m e of this response and, particularly, its utility as an early predictor of neurological improvement need to be explored. If this proves to be a reliable surrogate marker Brew et al: CSF Neopterin in HIV-1 559
for therapeutic efficacy in the CNS, measurement of CSF neopterin concentration would then represent an important advance in evaluating new treatments for ADC.
Supported by U.S. Public Health Service research grants NS 25701, NS 26643, A1 72634, and RR 00722, and a grant from the Rudin Foundation. We thank William Wolf for help in data management and Howard Thaler and John Keilp for assistance with statistical analysis.
References 1. Fuchs D, Hausen A, Reibnegger G, et al. Neopterin as a marker of activated cell-mediated immunity. Immunol Today 1988;9:150-155 2. Abita JP, Cost H , Milstein S, et al. Urinary neopterin and biopterin levels in patients with AIDS and AIDS-related complex. Lancet 1985;2:51-52 3. Fuchs D, Jaeger H, Popescu M. Neopterin levels correlating with the Walter Reed staging classification in human immunodeficiency virus (HIV) infection. Ann Intern Med 1987;107: 784-785 4. Melmed RN, Taylor JMG, Detels R, et al. Serum neopterin changes in HIV-infected subjects: indicator of significant pathology, CD4 T cell changes and the development of AIDS. J Acq Immun Def Synd 1989;2:70-76 5. Price RW, Brew BJ. The AIDS dementia complex. J Infect Dis 1988;158:1079-1083 6. Fuchs D, Chiodi F, Albert J, et al. Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with HIV1. AIDS 1989;3:285-288 7. Sonnerborg A, von Stedingk L-K, Hansson L-0, Strannegard 0. Elevated neopterin and beta-2 microglobulin levels in blood and cerebrospinal fluid occur early in HIV-1 infection. AIDS 198913~277-283 8. Brew BJ, Sidtis JJ, Petito CK, Price RW. The neurologic complications of AIDS and human immunodeficiency virus infection. In: Plum F, ed. Advances in contemporary neurology. Contemporary neurology series. Philadelphia: FA Davis, 1988: 1-49 9. Sidtis JJ, Price RW. Early HIV-1 infection and the AIDS dementia complex. Neurology 1989;40:323-326 10. Hollander H, Stringari S. Human immunodeficiency virusassociated meningitis: clinical course and correlations. Am J Med 1987;83:813-816 11. Fredrikson S, Eneroth P, Link H. Intrathecal production of neopterin in aseptic meningoencephalitis and multiple sclerosis. Clin Exp Immunol 1987;67:76-81 12. Werner ER, Bichler A, Daxenbichler G, et al. Determination of neopterin in serum and urine. Clin Chem 1987;33:62-66
560 Annals of Neurology Vol 28 No 4 October 1990
13. Eeg-Olofsson 0, Link H , Wigertz A. Concentrations of CSF proteins as a measure of blood brain barrier function and synthesis of IgG within the CNS in “normal” subjects from the age of 6 months to 30 years. Acta Paediatr Scand 1981;70:167-170 14. Reibnegger G, Fuchs D, Grubauer G, et al. Neopterin excretion during incubation period, clinical manifestations and reconvalescence of viral infection. In: Pfleiderer W, Wachter H , Curtius HC, eds. Biochemical and clinical aspects of pteridines. New York: de Gruyter, 1984:433-447 15. Rokos K, Rokos H, Frisius H, et al. Pteridines in cancer and other diseases. In: Blair JA, .ed. Chemistry and biology of pteridines. New York: de Gruyter, 1983:141-155 16. Brew BJ, Bhalla RB, Fleisher M, et al. Cerebrospinal fluid pz microglobulin in patients infected with human immunodeficiency virus. Neurology 1989;39(6):830-834 17. Heyes MP, Brew BJ, Martin A, et al. Quinolinic acid concentrations are increased in plasma and cerebrospinal fluid in AIDS and correlate with AIDS dementia complex. Presented at the V International Conference on AIDS, Montreal, June 8, 1989. 18. Schoenden G , Troppmair J, Adolf G, et al. Interferon-gamma enhances biosynthesis of pterins in peripheral blood mononuclear cells by induction of GTF-cyclohydrolase I activity. J Interferon Res 1986;6:697-703 19. Dolman CL Microglia. In: Davis RL,Robertson DM, eds. Textbook of neuropathology. Baltimore: Williams and Wilkins, 1985:117-137 20 Navia BA, Cho ES, Petito CK, Price RW. The AIDS dementia complex: 11. Neuropathology. Ann Neurol 1986;19:525-535 21 Price RW, Brew BJ, Rosenblum M. The AIDS dementia complex and HIV-1 infection: a pathogenetic model of virusimmune interaction. In: Waksman BH, ed. Immunologic mechanisms in neurologic and psychiatric disease. New York: Raven Press, 1990:269-290 22. Shen R, Alam A, Zhang Y. Inhibition of GTP cyclohydrolase 1 by pterins. Biochim Biophys Acta 1988;965:9-15 23. Elovaara I, Iivanainen M, Sirkka-Liisa V, et al. CSF protein and cellular profiles in various stages of HIV infection related to neurological manifestations. J Neurol Sci 1987;78:33 1-342 24. Resnick L, Berger JR, Shapshak P, Tourtellotte WW. Early penetration of the blood-brain barrier by HIV. Neuroiogy 1988; 38~9-14 25. Goudsmit J, de Wolf F, Paul DA, et al. Expression of human immunodeficiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet 1986; 2:177-180 26. McArthur JC, Cohen BA, Farzadegan H, et al. Cerebrospinal fluid abnormalities in homosexual men with and without neuropsychiatric findings. Ann Neurol 1988;23(suppl):S34-S37 27. Hollander H , Levy JA. Neurologic abnormalities and recovery of human immunodeficiency virus from cerebrospinal fluid. Ann Intern Med 1987;106:692-695 28. Sonnerborg AB, Ehrnst AC, Bergdahl SKM, et al. HIV ixtlation from cerebrospinal fluid in relation to immunological deficiency and neurological symptoms. AIDS 1988;2:89-93
We evaluated cerebrospinal fluid (CSF) concentrations of neopterin, a putative marker of activated macrophages, in 97 subjects infected with human immunodeficiency virus type 1 who had a spectrum of neurological complications. The highest CSF neopterin concentrations occurred in those with neurological opportunistic infections, primary central nervous systems lymphoma, and acquired immunodeficiency syndrome (AIDS)dementia complex. In general, the CSF concentration of neopterin was independent of CSF cell count and blood-brain barrier disruption to albumin. In the patients with AIDS dementia complex, CSF neopterin concentrations correlated with severity of disease and decreased in conjunction with clinical improvement following treatment with zidovudine. These results suggest that CSF neopterin, although not disease-specific, may be useful as a surrogate marker for the presence of AIDS dementia complex and its response to antiviral therapy. Brew BJ, Bhalla FLB, Paul M, Gallardo H, McArthur JC, Schwartz MK, Price RW. Cerebrospinal fluid neopterin in human immunodeficiency virus type 1 infection. Ann Neurol 1990;28.556-560
Elevated serum and urine concentrations of neopterin (dihydroneopterin), a pteridine compound that has been considered a marker of cell-mediated immune reactions 111, have been found to correlate with the Walter Reed stage of human immunodeficiency virus type 1 (HIV-1)infection [2-41. We hypothesized that cerebrospinal fluid (CSF) levels of neopterin might similarly correlate with central nervous system (CNS) HIV-1 infection and its clinical counterpart, the acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) [ S } . Two recent reports 16, 71 documented increases in CSF levels of neopterin in patients with HIV-1 infection, but included only small numbers of patients with ADC and did not examine CSF levels of neopterin in patients with other neurological complications of AIDS or assess what effect therapy has on these concentrations. Consequently, we assayed neopterin in the CSF and serum of patients at various stages of ADC as well as those with other neurological complications of HIV- 1 infection. We also assessed the confounding influences of blood-brain barrier impairment and CSF pleocytosis as well as the effects of treatment with zidovudine (ZDV) (also azidothymidine or AZT) on CSF neopterin levels.
From the Departments of *Neurology and +Clinical Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, and the $Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD.
Subjects and Methods Subjects Three groups were studied: (1)HIV-I-infected patients with various neurological complications; (2) neurologically normal, HIV-1-infected subjects; and ( 3 ) a small group of HIV1 seronegative subjects. The first group (102 samples from 86 patients) will be described in more detail. The HIV1-infected, neurologically normal subjects (16 samples, 11 patients) are participants in a longitudinal study of the neurological manifestations of HIV-1 infection as part of the Multicenter AIDS Cohort Study (MACS) for which they have undergone semiannual neurologcal evaluation, magnetic resonance imaging (MRI) scan, and analysis of the CSF. None had neurological or neuropsychological abnormalities at the time of CSF sampling. The HIV-1 seronegative control group included 7 patients: 4 who were studied by myelography, CSF cytology, and CSF tumor markers for suspicion of metastatic cancer, but for whom the findings were normal; and 3 patients who had syringomyelia, idiopathic spastic paraparesis, and idiopathic peripheral neuropathy. HIV-1-INFECTEDNEUKOLOGICAL DISEASE GROUPS. These
patients were part of a larger population of HIV-1-infected individuals who were being prospectively followed to assess the neurological complications of HIV-1 infection. Eightynine percent had either AIDS or AlDS-related complex as
Address correspondence to Dr Price, Department of Neurology, Medical School, University of Minnesota, Box 295 UMHC, Minneapolis, MN 55455.
Received Feb 28, 1990, and in revised form Apr 26. Accepted for publication Apr 30, 1990.
556 Copyright 0 1990 by the American Neurological Association
defined by systemic disease. All were examined by a neurologist, and additional diagnostic investigations were performed as indicated by the clinical setting. On the basis of these evaluations, these patients were assigned to one of the following groups: Putients with ADC. Patients were included in this group if they had: (1) characteristic neurological symptoms and signs 181, (2) compatible neuroimaging findings, and (3) exclusion of other neurological conditions. The severity of the dementia was rated on a functionally based, staging scale ranging from 0 to 4, such that stage 0 indicates no neurological disease, stage 0.5 indicates only equivocal symptoms of subclinical signs without functional impairment, stage I indicates mild disturbance but preserved ability to work with minimal limitation, stage 2 indicates moderate ADC precluding work, stage 3 indicates severe dementia such that the patient cannot perform even basic activities of daily living, and stage 4 indicates a near vegetative state [2, 93. There were 70 CSF assessments of 54 patients with ADC: 16 samples from patients with ADC stage 0.5, 27 from patients with stage l, 19 from patients with stage 2, 6 from patients with stage 3, and 2 from patients with stage 4 . As the number of samples from patients with Stage 3 or 4 was small, these groups were combined for analysis. Samples of CSF and serum that were taken from 9 patients with ADC before and after treatment (mean interval of 8 weeks) with ZDV were also analyzed. Group without ADC. This consisted of 3 subgroups of patients, namely: (1)those with primary brain lymphoma, (2) those with CNS infections, and (3) those with headache of undetermined cause. The subgroup with primary brain lymphoma comprised 6 patients, 4 of whom had histological documentation of the diagnosis. In the remaining 2, presumptive diagnosis was based on characteristic clinical and radiological findings 181; these patients additionally exhibited no response to a therapeutic trial of antitoxoplasma treatment. The subgroup with CNS infections included 14 patients with the following: cerebral toxoplasmosis (1 patient), cryptococcal meningitis (3 patients), progressive multifocal leukoencephalopathy (1 patient), herpes zoster myelitis (1 patient), cytomegalovirus (CMV) rddiculomyelitis (1 patient), probable CMV encephalitis (CMV retinitis coincident with seizures, obtundation, and unremarkable neuroimaging or CSF studies; 2 patients), and HIV-1-related aseptic meningitis (5 patients) [lo}. The group with headaches comprised 12 patients who presented with severe headaches without an identified infectious, neoplastic, or psychiatric cause and unaccompanied by CSF pleocytosis. All had equivocal cognitive complaints or minor neurological abnormalities on examination, qualifying them in classification as stage 0.5 ADC. All had been HIV-1 seropositive for at least 3 years. The headaches resolved over several weeks in all patients.
Labmutoy Methods CSF was analyzed for cell count, protein, and glucose contents, the presence of cryptococcal antigen, cytology, and VDRL along with bacterial and fungal cultures using routine methods. Neopterin concentrations in both CSF and serum were assessed by radioimmunoassay (DRG, Henning, Berlin) with particular attention to minimizing light exposure.
The previously suggested normal values for neopterin levels in CSF (1.6 +- 0.6 nmol/l) [ll] were compared to that obtained from CSF analysis of the HIV-I negative control group. The upper normal limit for neopterin levels in serum was taken to be 8.7 nmolll {127. CSF and serum values for albumin concentration were determined in order to serve as an index of the integrity of the blood-brain barrier; a CSF-toserum albumin ratio, multiplied by lo3, of greater than 6.5 was considered abnormal [l 31.
Statistics Statistical methods included analyses of variance and Student-Newman-Keuls post hoc tests in comparisons of discrete disease groups and ADC stages. Continuous variables were compared using Pearson correlations. Alpha (significance) level was established at p I0.05. Changes in neopterin concentration before and after ZDV treatment were compared using the paired t test. Those neurologically abnormal patients who had more than one CSF analysis were included in the main data set as independent observations because the CSF was repeated for assessment of clinical change. These analyses were carried out using SPSS/PC+ software (SPSS Inc, Chicago, IL).
Results As shown in the Table, the mean CSF concentration of neopterin for the HIV-1 seronegative control group was 3.5 nmol/l, somewhat higher than that found by Fredrikson and associates El 11. While significantly elevated (more than twice that of seronegatives) neopterin concentrations were noted in the seropositive, neurologically normal group, markedly higher concentrations (8 to 16 times normal) were found in the symptomatic patients; the CSF neopterin concentration was highest in those with CNS infections, followed, in turn, by those with ADC, those with lymphoma, and those with headache. The concentrations in all disease groups were different from those in the seronegative controls and concentration in the group with ADC was also different from that in the seropositive control group. In patients without confounding CNS disorders, CSF neopterin concentrations correlated with the ADC stage (Fig 1) (r = 0.54, p < 0.0001). Whereas there was no difference between those with ADC stage 0.5 and the neurologically normal seropositives ( p = 0.481), all patients with ADC stages 1 or greater had CSF neopterin concentrations outside the range of the HIV-1 seronegative group, and 89% of these patients had concentrations greater than 16.0 nmoV1, the upper limit of the 95% confidence level of the HIV-1 seropositive, neurologically normal group. In contrast to CSF levels, serum neopterin concentrations did not correlate with severity of ADC (r = 0.039). There was no discernible relationship between neopterin concentration in the CSF and the number of white blood cells in the CSF. Indeed, only 10 samples from 5 patients had CSF cell counts greater than 5 Brew et al: CSF Neopterin in HIV-1 557
CSF and Serum Neoptevin Concentrations in Different Patient Groups Neopterin (nmoV1 Patient Group
No. of Samples
Controls Seronegative HIV-1-infected, neurologically normal Disease Headache AIDS dementia complex Lymphoma CNS infection
CSF
Serum
7 16
3.5 2 0.4 8.2 t 1.0b
12 70
26.5 f 5.1b,‘
6 14
34.8
36.8 58.2
* SEMY
? ? ?
3.6b,‘ 11.5b.c 11.7b,‘
Not available 13.7 ? 1.8 33.0 40.0 35.0
44.4
&
7.0 4.1
2
10.6
?
* ll.?
~
“Normal range: CSF, < 5.0 nmoul; serum, < 8.7 nmoV1. bDiffered from seronegative controls ( p < 0.001). ‘Differed from seropositive, neurologically normal controls ( p < 0.0001).
0
0.5
1
2
3-4
ADC STAGE
Fig I . CSF neopterin concentration as a function of the stage of
the AIDS dementia complex (ADC).
cells/mm3 and the neopterin values in these did not differ from those of other patients in the same ADC stage. Analysis of blood-brain barrier integrity to albumin for the whole data set, including the ADC group, failed to reveal any association with CSF levels of neopterin ( r = 0.14, p = 0.43). Furthermore, when the group with ADC was divided into those without (38 samples) and those with (32 samples) blood-brain barrier impairment to albumin, analysis revealed that the samples from patients with an intact blood-brain barrier evidenced a high correlation between CSF neopterin level and ADC stage (r = 0.64, @ = 0.0001), whereas those with an impaired blood-brain barrier to albumin had a less significant correlation ( r = 0.39, p = 0.01). Lkewise, although there was a correlation between CSF and serum neopterin levels both for all patients assessed ( Y = 0.45, @ < 0.0001) and for the patients with ADC who were considered separately ( r = 0.49, p < O.OOOl), this correlation was lost when 558 Annals of Neurology Vol 28 No 4 October 1990
PRE-ZDV
POST-ZDV
Fig 2. CSF neoptwin concentrations before and afer treatment with zidovudine CZDV).Values have been converted to a log scale in order to encompass the wide range of values and the changes with treatment.
those with blood-brain barrier dysfunction were considered separately. ZDV treatment of patients with ADC resulted in a reduction of CSF neopterin levels in each of the 9 assessed (Fig 2, p = 0.032), while serum concentrations in these patients did not significantly change (mean pretreatment level of 28.8 2 5.2 nmol/l only fell to 20.2 t 4.9 nmol/l after treatment, p = 0.23).
Discussion Our results indicate that the CSF concentration of neopterin is elevated in HIV-1-infected patients who have a variety of neurological complications. The fact that the highest CSF levels were observed in patients with CNS infections and primary CNS lymphoma is consistent with the concept of neopterin being related to “activated” cellular immunity [l}and with reports of elevated serum neopterin concentrations in systemic infections and malignancies {14, 15}. Elevation of CSF
neopterin, like other recently reported surrogate markers, such as beta-2 microglobulin 1161 and quinolinic acid C171, is thus not disease-specific. Nonetheless, the finding of greatest interest is the consistent elevation of CSF neopterin in patients with ADC and its general correlation with severity of ADC. These results, which are in keeping with those recently reported in two smaller series 16, 71,are potentially important in relation to both pathogenesis and clinical management of ADC. Activated macrophages, and to 3 much lesser extent T lymphocytes, produce neopterin, and it has been noted that interferon gamma can induce neopterin through its effect on guanosine triphosphate (GTP) cyclohydrolase, the first enzyme in the conversion of GTP to dihydroneopterin triphosphate, the precursor of neopterin 1181. Although the source of elevated CSF concentrations of neopterin in these patients was not directly studied, it is unlikely that CSF inflammatory cells alone were responsible [l 11since only a very few patients had discernible pleocytosis. Similarly, several observations suggest that alteration of the bloodbrain barrier likely did not explain the high concentration of CSF neopterin, particularly in the patients with ADC: (1) There was no correlation between CSF neopterin concentration and blood-brain-barrier impairment to albumin; (2) CSF neopterin concentration correlated with severity of ADC, while serum neopterin concentration did not; and (3) in several patients the CSF concentration of neopterin exceeded the serum concentration. A similar lack of correlation of neopterin with CSF cell count and blood-brain barrier was noted by Fuchs and colleagues [6] and Sonnerborg and coworkers 17). The CNS parenchymal cells producing neopterin are not known, but major candidates are infiltrating macrophageimononuclear cells and ontogenetically related [191 microglial cells. However, because neuropathological examination of brains from patients with ADC stage 0.5 or 1 characteristically shows only scant inflammatory or microglial infiltrates 2201, it is also possible that intrinsic neural cells, perhaps proliferating astrocytes, may produce neopterin when they are stimulated by cytokines. Indeed, these findings may provide insight into the role of “immunopathological” events in the pathogenesis of ADC. The elevation of CSF neopterin concentration with increasing severity of ADC and the decline with antiviral therapy are compatible with the view that the virus load “drives” immune-cell activation and putative CNS immunopathology [21). The plateau or possible slight reduction of neopterin in the patients with the most severe ADC (stages 3 and 4 ) may indicate a predominant role of CNS viral infection rather than immunopathology in such patients [2 11. Whether neopterin itself contributes to CNS dysfunction is highly speculative. Shen
and associates E22) recently found that reduced pterins, for example neopterin and dihydroneopterin, can downregulate GTP cyclohydrolase 1 and thereby reduce the concentration of tetrahydrobiopterin, an essential cofactor in the synthesis of dopamine, serotonin, and noradrenalin. It is conceivable that some of the basal ganglion dysfunction that is characteristic of ADC might result from neopterin-induced perturbation of these neurotransmitters. Elevation of CSF neopterin concentrations in neurologically asymptomatic subjects and the further elevation in patients with headache of unknown cause await further clarification. The most likely explanation for elevation in the asymptomatic group relates to subclinical disease activity. This is consistent with other CSF abnormalities in asymptomatic patients E23-28). It also is in keeping with the hypothesis that CNS HIV-1 infection is actively “controlled” by effective immune responses early in the course of infection 1211. Headache is a common, but poorly understood, problem in HIV- 1 infected patients. Elevated CSF concentrations of neopterin in such patients may relate in part to the emergence of neurological dysfunction in this group or may suggest that headache relates to inflammatory processes not reflected in CSF pleocytosis. Additional studies are needed to resolve these questions. Whatever the mechanism of increased levels of CSF neopterin, our results suggest that assessment of this “surrogate marker” may at times prove useful in diagnosis and therapeutic monitoring of ADC. Thus, while increased CSF neopterin is clearly not disease-specific, once clinical and laboratory evaluations have eliminated other opportunistic CNS infections or tumors, assessment of this marker may be 3 useful adjunct to establishing diagnosis of ADC, and particularly its differentiation from psychiatric ( e g , anxiety and depression) and degenerative (e.g., Parkinson’s and Alzheimer’s diseases) disorders in HIV- 1-infected patients. Our present results suggest that a CSF neopterin concentration of 16.0 nmoUl might serve as a cutoff of “normal” in the HIV-1-infected patient, although further observations are needed to more precisely define: the limits of CSF neopterin in HIV-1infected, neurologically normal individuals; the possible effects of systemic infections on CSF concentrations; and the prognostic value of elevated neopterin concentrations in relation to the development of ADC. With respect to the use of CSF neopterin levels in monitoring therapy for ADC, further studies are needed to determine the clinical and pathobiological significance of the striking decline in CSF neopterin noted in our patients given ZDV. The time c o m e of this response and, particularly, its utility as an early predictor of neurological improvement need to be explored. If this proves to be a reliable surrogate marker Brew et al: CSF Neopterin in HIV-1 559
for therapeutic efficacy in the CNS, measurement of CSF neopterin concentration would then represent an important advance in evaluating new treatments for ADC.
Supported by U.S. Public Health Service research grants NS 25701, NS 26643, A1 72634, and RR 00722, and a grant from the Rudin Foundation. We thank William Wolf for help in data management and Howard Thaler and John Keilp for assistance with statistical analysis.
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