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Identification of potential antibody markers in HIV-associated dementia
Journal of Neuroimmunology 157 (2004) 120 – 125 www.elsevier.com/locate/jneuroim
Identification of potential antibody markers in HIV-associated dementiaB Steven E. Schutzera,*, Joseph R. Bergerb, Michael Brunnera a
b
Department of Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, United States Departments of Neurology and Internal Medicine, University of Kentucky College of Medicine, Lexington, KY 40536, United States Accepted 30 August 2004
Abstract Markers for HIV-associated dementia (HAD) are needed for diagnosis and management. Specific antibodies to brain and immune complexes (IC) in the cerebrospinal fluid (CSF) are potential markers. CSF IC were found in 4 of 4 HAD patients, 2 of 2 AIDS-central nervous system (CNS) lymphoma patients with dementia, 0 of 1 AIDS-CNS lymphoma patient without dementia, 0 of 1 AIDS-CNS toxoplasmosis patient without dementia, and 0 of 10 neurologic disease controls. By blinded immunoblots, antibrain antibodies in serum and CSF were found in 11 of 12 HAD cases and 7 of 19 HIV-1 patients without HAD. All 11 non-HIV-1 controls were negative. These and published data suggest antibrain antibodies and IC may serve as markers of HAD. D 2004 Published by Elsevier B.V. Keywords: AIDS; HIV dementia; Autoantibodies; Immune complexes; Anti-brain antibodies
1. Introduction The exact cause of HIV-associated dementia (HAD) is still unknown. It remains a serious manifestation in developed and developing countries. Since earlier descriptions as AIDS Dementia Complex (Navia et al., 1986a,b) there have been advances in qualitative and quantitative detection of HIV-1 and treatment of HIV-1 disease. This has altered the scope of the disease in the United States. Life expectancy, in the US, has been extended by therapies such as highly active antiretroviral therapy (HAART). The combination of longer life and improved care of HIV-1 patients may promote a more subtle form of HAD and a later appearance in the course of the disease. In circumstances where therapy is not instituted until a threshold reduction in CD4+ cells, more time may be afforded for
B
Supported in part by grants from the NIH-NINCDS (PO1 NS 25569). * Corresponding author. Tel.: +1 973 972 4872; fax: +1 801 383 8534. E-mail address: [email protected] (S.E. Schutzer). 0165-5728/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.jneuroim.2004.08.024
the factors responsible for HAD, including HIV-1 itself, to be established in the central nervous system (CNS). Further complications can occur because the HAART therapy is not efficient at penetrating the CNS. For these reasons, a reproducible laboratory marker of HAD of diagnostic or prognostic import will assist the physician in management of the patient, will assist the patient in personal decisions, and could provide objective measures for new clinical trials. The aberrancies of the immune system following HIV infection prompted us to look for specific and non-specific immunologic markers that may be associated with HAD. We looked at free antibody directed against brain components and complexed antibody in cerebrospinal fluid (CSF). Free antibody if directed specifically to the brain, or even if cross reactive with other tissues but found with the CSF could have marker potential. Because it is rare to find immune complexes in the CSF except in inflammatory, autoimmune, or infectious process, immune complexes could serve as a marker. Further, the identification of specific antibrain antibody or brain antigen within the complexes may prove a more specific marker even in the absence of free antibody.
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2. Materials and methods 2.1. Sample collection, patient population, clinical evaluation Serum and CSF from known cases of HAD were kindly provided by Drs. Price and Brew from Memorial SloanKettering Cancer Center studies. Corresponding neurological controls samples came from our banked specimens of paired serum and CSF. These included neurologic Lyme disease with cognitive dysfunction, meningitis, amyotrophic lateral sclerosis, Guillain–Barre syndrome, Herpes encephalitis, multiple sclerosis, syphilis, sciatica, transverse myelitis, lupus, Alzheimer, and back pain. Blinded serum and CSF samples came from 31 randomly selected HIV-1 positive subjects from a longitudinal, prospective study of the neurological complications of HIV-1 infection performed by one of us (JRB). Subjects had a complete clinical, history, physical and neurological examination, and extensive neuropsychological battery. The neurological examination included assessments of muscle tone, extremity dexterity, gait, balance and postural stability, and frontal release signs (snout, glabellar, involuntary grasp). A modified version of the mini mental status examination (Folstein et al., 1983) was administered. The neuropsychological battery included tests to evaluate language function, judgment in reasoning, visuo-spatial constructive abilities, auditory and visual attention, the ability to shift sets, and memory for verbal and figural material (Levin, 1994). HIV-1 serological status was assessed by ELISA and Western blot. Laboratory studies also included blood for vitamin B12 levels, VDRL and FTA-antibodies (for exposure to syphilis). Magnetic resonance image (MR) of the brain was obtained on all subjects. As part of the study design, lumbar puncture was performed on all HIV-1 positive subjects. Routine CSF analysis included cell count and differential, protein, glucose, VDRL, FTA-ABS, immunoelectrophoresis, and viral cultures on human foreskin fibroblasts, human embryonic lung fibroblasts, and primary monkey kidney s. Subjects with features of encephalopathy also had detailed microbiological studies of their CSF including bacterial stain and cultures, fungal cultures, India ink preparations, and cryptococcal antigens. All subjects provided informed consent. This study was approved by the institutional review. Each HAD met the definition of the Working Group of the American Academy of the Neurology AIDS Task Force (1991). Each experienced a bloss of intellectual abilities of sufficient severity to interview with social or occupational functioningQ. The criteria also included: (1) an acquired abnormality in at least two cognitive and abilities present for one or more months; (2) acquired abnormality in order function or performance end/or declining in motivation or, emotional control or change in social behavior; (3) no clouding of consciousness; and (4) no other identifiable etiology. As indicated above, all subjects were systematically screened
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for confounding factors that might contribute to their dementia. HIV-infected subjects with dementia that may have resulted from a neoplastic, nutritional, metabolic, or infectious (other than HIV) etiology were excluded from this study. Also excluded were those with a severe premorbid psychiatric disorder, chronic seizure disorder, or head trauma with residual dysfunction. 2.2. Immune complex detection 2.2.1. Raji cell assay Because the finding of any CSF immune complexes is usually indicative of an autoimmune, inflammatory, or infectious process, we performed a small study to examine its potential. We have modified the standard RIA assay to a microELISA system (Coyle et al., 1984). Briefly, flatbottomed microtiter plates were recoated with poly-llysine (l0 ug/ml) for 1 h. Plates were washed and 1106 Raji cells (in l00 ul) added to each well. Cells were sedimented, then fixed by gentle immersion in cold 0.25% glutaraldehyde-phosphate buffered saline (PBS) for 5 min. Plates were then transferred to a l% bovine serum albumin (BSA)–PBS solution for 1 h to block non-specific binding. Known amounts of aggregated human globulin (AHG) or test samples were preincubated with a complement source then used undiluted in the case of CSF or diluted 1:100 in PBS for serum and triplicate samples added to the wells for 1 h at 37 C. Plates were washed three times in PBS-Tween (0.05%), then horseradish peroxidase (HRP)-anti human IgG is added. To detect IgA or IgM complexes, HRP-anti human IgA or IgM was used. After incubation in the dark for 30 min, plates were washed three times. Enzyme substrate solution (2.3 mM o-phenylenediamine, OPD) in 0.024 M citrate, 0.5 M phosphate pH5 with fresh 0.03% H202) is added. After l5 min l00 ul from each well was transferred to a second plate and the reaction stopped with 1N H2SO4. The optical density (OD) was read at 488 nm in a microELISA reader (Dynatech Labs). The mean OD was calculated. A standard curve was plotted and samples read in ng/ml AHG, then corrected by the dilution factor. The level of sensitivity is 1 ug/ml of AHG. 2.3. Probe for antibodies reactive against brain We used a Western blot to search for the presence of free antibrain antibody in the CSF and serum samples by blotting against non-HIV infected human brain. Control antigens included HIV negative human liver and neural antigens. This has been described (Schutzer et al., 2003) but briefly. 2.3.1. Target antigens included (1)
Normal human brain prepared as described by Hall and Choppin (1981).
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(2)
(3)
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Galactocerebroside (Sigma C-8752, Type I), and ganglioside (Sigma G-2250, Type II) as positive controls in combination with the corresponding monoclonal antibody probe. HIV lysates and antigens were used to distinguish an antibody directed against brain from that against the HIV itself.
2.3.2. Biotin–avidin Western blots Brain preparation: 0.226 g was placed in a petri dish on ice with 1 ml of Laemmli gel sample reducing buffer (20% SDS, 10% 60 mM Tris–HCl (pH 6.8), 0.1 M DTT, 0.005% bromphenol blue, 1 mM PMSF). A minced solution was passed 10 through an 18-G followed by a 21-G needle. A portion was further diluted in sample buffer, and undiluted aliquots were frozen at 70 8C for later use. The diluted sample was boiled for 3 to 5 min, cooled and applied to gel. Gel electrophoresis: Twelve% SDS-polyacrylamide 78 cm mini-gels, 0.75 mm thick with stack were run using Hoeffer apparatus (Mighty small, SE 250). Five microliters of a 0.1% Methyl Green dye solution was added to each well to serve as a blotted dye front marker. Samples including SDS-PAGE biotinylated low range molecular weight standards (Bio-Rad) diluted in sample buffer containing 5% beta-mercaptoethanol and diluted brain were all boiled 3–5 min, cooled, and applied to wells of the gel. A 125 constant volts applied until samples lined up at bottom of stacking gel, and then power supply switched to 200 constant volts until dye front marker ran to bottom of gel. Electrotransfer: Gels were removed from plates, allowed to equilibrate for 15 min in transfer buffer (Tris 25 mM, Glycine 190 mM, Methanol 20%), then they were placed in a sandwich containing wetted nitrocellulose (MSI NitroBind 0.2 Am) to which protein was transferred over an hour at 100 V (approximately 0.25 A) using Hoeffer apparatus (TE 22). Membrane blots were measured for dye front migration, appropriately marked if pre-stained markers were not used, rinsed, dried. Immunoblot stain: Nitrocellulose blots were stained in a 0.2% Ponceau S solution (Sigma) which helped to align the blot with the 28 mini-well immunoblotter (Immunetics) and confirmed that approximately 1 microgram of protein/well, the lower limit of Ponceau S, was obtained during blotting. The membrane was blocked in Tris-buffered saline (TBS, 50 mM Tris–HCl, 0.2 M NaCl, 3 mM KCl, pH 7.5, 0.02% Azide) containing 1% Non-Fat Dry Milk (Carnation) and 1% normal goat serum (NGS) for 30 min in a rocking tray. After rinsing in TBS, the membrane was placed in the 28 lane miniblotter apparatus (MN28, Immunetics), and lanes aspirated dry. Sera were diluted 1:100 in Tris-buffered saline-Tween 20 (0.1% v/v)(TBS-T) containing 1% NGS; CSF was used neat or at 1:10 dilution, and positive control mouse monoclonal anti-neurofilament 68, NR-4 (Sigma) was diluted 1:500. Sixty microliters was pipetted into each lane (diluent alone in secondary reagent controls, and TBS in biotinylated
standard wells) and miniblotter was slowly rocked for 2 h for the primary incubation. Wells were aspirated and filled with TBS-T and miniblotter rocked rapidly for 5 min. This was repeated with TBS-T three more times and served as the standard wash procedure between reagent incubations. Secondary antibodies consisted of goat biotinylated antihuman IgG (gamma chain specific), biotinylated anti-human IgM (mu chain specific) which were diluted 1:250, and biotinylated horse anti-mouse IgG (H+L) which was diluted 1:500 (Vector Laboratories) in TBS-T plus 1% NGS. After washing, secondary antibodies were added and incubated with slow rocking for 30 min. During this time the avidin (reagent A) and biotinylated alkaline phosphatase (reagent B) complex was formed by adding 10 Al of reagent A and 10 Al of reagent B to one ml of TBS-T and vortexing. After standing at room temperature (RT) for 30 min it was further diluted by adding 2 ml of TBS-T. All wells of the miniblotter were washed four times, and the diluted avidin-biotinylated alkaline phosphatase complex (ABC) solution was added for a further 30 min incubation. After standard wash, the membrane was removed from the miniblotter, rinsed once with TBS and developed in a tray using Vector Kit II Alkaline phosphatase substrate in 100 mM Tris–HCl pH 9.5 buffer, washed twice with water and dried.
3. Results 3.1. CSF immune complexes in HAD patients We examined the CSF in seven AIDS patients who had neurologic symptoms of HAD and in whom no other known agent, except as noted, in the CSF was detected by conventional techniques. The microtiter Raji cell immune complexes assay was used to examine the CSF of HAD patients and controls for immune complexes. The following results were obtained and are represented as amounts of AHG per milliliter: Patient 1 (HAD) 7.8 ng/ml, Patient 2 (AIDS-CNS lymphoma) 12.3 ng/ml, Patient 3 (HAD) 9.0 ng/ml, Patient 4 (HAD) 12.5 ng/ml, Pt5 (AIDS-CNS lymphoma) 20.0 ng/ml, Patient 6 (HAD) 12.6 ng/ml. HAD refers to the syndrome alone, whereas the CNS lymphoma patients had the dementia symptoms and the tumor. An additional patient, Patient 7, with AIDS and CNS toxoplasmosis had no detectable CSF immune complexes. CSF from other neurology patients, which was previously determined to have no immune complexes, were used again as additional controls and continued to be devoid of immune complexes. Therefore, positive results were found in 4 of 4 HAD patients without other known CNS infection or tumor, 2 of 2 AIDS-central nervous system (CNS) lymphoma patients with dementia, 0 of 1 AIDS-CNS lymphoma patient without dementia, 0 of 1 AIDS-CNS toxoplasmosis patient without dementia, and 0 of 10 other neurologic disease controls. These CSF samples did not contain free anti-HIV
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antibodies, but the question of whether the antibody and/or antigen was bound remains unanswered. 3.2. Western blot demonstration of specific antibrain antibodies in serum and CSF In non-blinded studies we found immunoblot reactivity to non-HIV infected brain tissue in 6 HAD patients with their CSF and serum IgG. This involved regions between 41 and 58 kDa. The reactive regions were distinct from parallel probes against HIV lysates in these patients. Multiple sclerosis serum and CSF did not stain any of the above bands. This suggest that there are some brain reactive IgG antibodies in AIDS patients with dementia and that they are distinct from anti-HIV antibodies and are not found in a controls. In a blinded analysis of 31 patients, and 10 other disease controls, the brain material was probed with samples of serum, and cerebrospinal fluid. By Western blotting, antibrain brain reactive antibodies were detected in serum and/or CSF positive results were found in 11 of 12 (92%) of HAD cases, 7 of 19 (78%) of HIV patients without HAD. All 11 non-HIV controls were negative. There was no immunoblot reactivity to HIV negative liver, as a control tissue, in any group. Using the non-HIV subjects as the reference, positive results in the first two groups were statistically significant (Fisher Exact test). A result was considered as positive when there was immunoblot reactivity to four or more bands. The MW of the bands were 26, 31, 36, 40, 45, 55, 60, and 80–82 kDa. A representative blot displaying the range of the target of CSF antibrain antibodies from patients who also had serum brain reactive antibodies is shown in Fig. 1. The differences between HAD+ and HAD HIV+ is in the percentage who have several antibrain antibodies rather than the absolute number of bands on the blot. This may infer a possible prognostic value to the finding of such antibrain reactivity such that HAD HIV patients who have positive bands may develop clinical HAD but are not yet at that threshold.
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4. Discussion The changing scope of HAD mandates reproducible markers of the disease. These markers may or may not be related to the etiopathogenesis of the disease. Identification of candidate markers should be considered as a first step that will require further research and validation before use. Data from our own experiments and from others indicate that antibrain reactive antibodies and CSF immune complexes may be two such markers of HAD. The immune complexes, which are in the CSF, may serve as markers by themselves or by their specific antibody and antigen content. Recent advances in genomics and proteomics will now permit identification of the specificity of these antibodies and immune complexes components. Further validation will establish their utility as markers as well as providing possible insights into the pathogenesis of the syndrome. Immune complexes by themselves are capable of causing disease such as occurs in system lupus erythematosus (SLE) where tissue deposition leads to pathology (Jones and Orlans, 1981; Theofilopoulos and Dixon, 1980). In other circumstances, they are associated with the disease. In lupus, their reappearance has correlated with disease flareups (Theofilopoulos and Dixon, 1980). Of relevance to HAD, the immune complexes contain antibody and antigen specific for the disease. For example, the antigen in the immune complexes of SLE contains DNA and anti-DNA antibody (Sano and Morimoto, 1981). In infectious diseases, the microbial antigens and reactive antibody to them can often be identified (Baughn et al., 1987; Bayer et al., 1979; Inman et al., 1982; Jones and Orlans, 1981). In certain seronegative cases, the antibody may be found only in the immune complexes form and thereby elude detection by conventional serum or CSF assays. Examples of this include measles and Lyme disease where the patients may appear to be seronegative unless immune complexes are examined (Coyle and Procyk-Dougherty, 1984; Coyle et al., 1990; Schutzer et al., 1990, 1999). Analysis of the immune complexes components, particularly in the CSF, will likely reveal components whose sequences can be identified with
Fig. 1. Western blot probe for reactive CSF IgG to non-HIV infected brain from a representative group of HIV+ HAD patients with serum positive for antibrain reactivity (CSF indices were normal indicating there was intrathecal production of the reactive antibody). MW denotes molecular weight markers. Individual patient CSF samples are shown in Lanes 1–14. Blank lanes in between the numbers represent solution controls.
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the newer proteomic approaches. Comparison of the sequence to databases will identify the antigen as that of a particular microbe (HIV, opportunistic pathogens), or host origin, such as brain. The origin of antibrain reactive antibodies in this syndrome may be purely an epiphenomen. Despite this possibility, these antibodies can function as a marker of HAD or a pre-clinical marker of the disease before it develops. There is a body of evidence associating autoimmune-like manifestations to HIV disease and a body of evidence associating autoimmunity reactivity including antibodies to CNS disease. Autoimmune phenomena have already been associated with HIV infections. This has included the presence of autoantibodies to platelets, sperm, lymphocytes, myelin basic protein (MBP), cardiolipin, and mitochondria (Pruzanski et al., 1983; Daugharty et al., 1985; Dorsett et al., 1985; Rodman et al., 1985; Rubinstein et al., 1984; Stricker et al., 1987). Some of the antibody may be cross reactive such as has occurred between lymphocytes and brain (Funke et al., 1987; Gabuzda and Hirsch, 1987; Klatzmann et al., 1984; Bresnihan et al., 1979; Solinger and Hess, 1991). An antecedent neurological infection may promote subsequent autoimmune CNS disease. This has been described with postinfectious measles encephalomyelitis (Johnson et al., 1984). Post-vaccine encephalomyelitis and polyneuritis has followed rabies vaccination. In that case, myelin basic protein (MBP) was the encephalitogen (Hemachudha et al., 1987; Johnson et al., 1984). CSF antineuronal antibodies have been linked to CNS SLE. Lupus psychosis has been associated with a specific autoantibody (Bluestein, 1987; Bonfa et al., 1987). With respect to HAD, other investigators have identified discrete antibody reactive to brain either by molecular weight or to specific components. Similar to the proportion of patients in our blinded study (Schutzer et al., 2003), Kumar et al. identified reactive serum antibrain antibody to a 45 kDa protein of human hippocampal tissue. Seventyeight percent of 18 HAD sera samples were positive and 33% of 12 samples from AIDS patients without the dementia were positive (Kumar et al., 1989). In another investigation, molecular mimicry has been described between HIV gp120 and part of its V3 loop and human brain proteins of 35, 55 and 110 kDa (Trujillo et al., 1993). Common epitopes have been found between HIV gp41 and astrocytes (Eddleston et al., 1993; Yamada et al., 1991). Some studies have not been able to show strong associations between the dementia and antibodies (Trujillo et al., 1994; Lenhardt and Wiley, 1989). These conclusions may be reconciled by expanded studies and identification of antibody specificities. In summary, there is sufficient clinical background supporting the occurrence of autoimmune phenomena in HAD and HIV disease which is characterized by immune deficiencies and perturbations. In the current paper we have not addressed possible association of antibrain
reactive antibodies as related to the pathogenesis of HAD; rather we have focused on the marker potential of the immune system to assist in diagnosis and possible prediction of HAD. Both the immunopathogenesis and the potential diagnostic and prognostic value of autoimmune markers in HAD remain understudied. The literature and recent experiments support the existence of antibrain antibodies, and possibly immune complexes, as potential markers of high utility. The scope of HAD may be changing but fortunately recent technology advances will now permit us to identify the exact antigenic target of the antibrain antibodies.
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Identification of potential antibody markers in HIV-associated dementiaB Steven E. Schutzera,*, Joseph R. Bergerb, Michael Brunnera a
b
Department of Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, United States Departments of Neurology and Internal Medicine, University of Kentucky College of Medicine, Lexington, KY 40536, United States Accepted 30 August 2004
Abstract Markers for HIV-associated dementia (HAD) are needed for diagnosis and management. Specific antibodies to brain and immune complexes (IC) in the cerebrospinal fluid (CSF) are potential markers. CSF IC were found in 4 of 4 HAD patients, 2 of 2 AIDS-central nervous system (CNS) lymphoma patients with dementia, 0 of 1 AIDS-CNS lymphoma patient without dementia, 0 of 1 AIDS-CNS toxoplasmosis patient without dementia, and 0 of 10 neurologic disease controls. By blinded immunoblots, antibrain antibodies in serum and CSF were found in 11 of 12 HAD cases and 7 of 19 HIV-1 patients without HAD. All 11 non-HIV-1 controls were negative. These and published data suggest antibrain antibodies and IC may serve as markers of HAD. D 2004 Published by Elsevier B.V. Keywords: AIDS; HIV dementia; Autoantibodies; Immune complexes; Anti-brain antibodies
1. Introduction The exact cause of HIV-associated dementia (HAD) is still unknown. It remains a serious manifestation in developed and developing countries. Since earlier descriptions as AIDS Dementia Complex (Navia et al., 1986a,b) there have been advances in qualitative and quantitative detection of HIV-1 and treatment of HIV-1 disease. This has altered the scope of the disease in the United States. Life expectancy, in the US, has been extended by therapies such as highly active antiretroviral therapy (HAART). The combination of longer life and improved care of HIV-1 patients may promote a more subtle form of HAD and a later appearance in the course of the disease. In circumstances where therapy is not instituted until a threshold reduction in CD4+ cells, more time may be afforded for
B
Supported in part by grants from the NIH-NINCDS (PO1 NS 25569). * Corresponding author. Tel.: +1 973 972 4872; fax: +1 801 383 8534. E-mail address: [email protected] (S.E. Schutzer). 0165-5728/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.jneuroim.2004.08.024
the factors responsible for HAD, including HIV-1 itself, to be established in the central nervous system (CNS). Further complications can occur because the HAART therapy is not efficient at penetrating the CNS. For these reasons, a reproducible laboratory marker of HAD of diagnostic or prognostic import will assist the physician in management of the patient, will assist the patient in personal decisions, and could provide objective measures for new clinical trials. The aberrancies of the immune system following HIV infection prompted us to look for specific and non-specific immunologic markers that may be associated with HAD. We looked at free antibody directed against brain components and complexed antibody in cerebrospinal fluid (CSF). Free antibody if directed specifically to the brain, or even if cross reactive with other tissues but found with the CSF could have marker potential. Because it is rare to find immune complexes in the CSF except in inflammatory, autoimmune, or infectious process, immune complexes could serve as a marker. Further, the identification of specific antibrain antibody or brain antigen within the complexes may prove a more specific marker even in the absence of free antibody.
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2. Materials and methods 2.1. Sample collection, patient population, clinical evaluation Serum and CSF from known cases of HAD were kindly provided by Drs. Price and Brew from Memorial SloanKettering Cancer Center studies. Corresponding neurological controls samples came from our banked specimens of paired serum and CSF. These included neurologic Lyme disease with cognitive dysfunction, meningitis, amyotrophic lateral sclerosis, Guillain–Barre syndrome, Herpes encephalitis, multiple sclerosis, syphilis, sciatica, transverse myelitis, lupus, Alzheimer, and back pain. Blinded serum and CSF samples came from 31 randomly selected HIV-1 positive subjects from a longitudinal, prospective study of the neurological complications of HIV-1 infection performed by one of us (JRB). Subjects had a complete clinical, history, physical and neurological examination, and extensive neuropsychological battery. The neurological examination included assessments of muscle tone, extremity dexterity, gait, balance and postural stability, and frontal release signs (snout, glabellar, involuntary grasp). A modified version of the mini mental status examination (Folstein et al., 1983) was administered. The neuropsychological battery included tests to evaluate language function, judgment in reasoning, visuo-spatial constructive abilities, auditory and visual attention, the ability to shift sets, and memory for verbal and figural material (Levin, 1994). HIV-1 serological status was assessed by ELISA and Western blot. Laboratory studies also included blood for vitamin B12 levels, VDRL and FTA-antibodies (for exposure to syphilis). Magnetic resonance image (MR) of the brain was obtained on all subjects. As part of the study design, lumbar puncture was performed on all HIV-1 positive subjects. Routine CSF analysis included cell count and differential, protein, glucose, VDRL, FTA-ABS, immunoelectrophoresis, and viral cultures on human foreskin fibroblasts, human embryonic lung fibroblasts, and primary monkey kidney s. Subjects with features of encephalopathy also had detailed microbiological studies of their CSF including bacterial stain and cultures, fungal cultures, India ink preparations, and cryptococcal antigens. All subjects provided informed consent. This study was approved by the institutional review. Each HAD met the definition of the Working Group of the American Academy of the Neurology AIDS Task Force (1991). Each experienced a bloss of intellectual abilities of sufficient severity to interview with social or occupational functioningQ. The criteria also included: (1) an acquired abnormality in at least two cognitive and abilities present for one or more months; (2) acquired abnormality in order function or performance end/or declining in motivation or, emotional control or change in social behavior; (3) no clouding of consciousness; and (4) no other identifiable etiology. As indicated above, all subjects were systematically screened
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for confounding factors that might contribute to their dementia. HIV-infected subjects with dementia that may have resulted from a neoplastic, nutritional, metabolic, or infectious (other than HIV) etiology were excluded from this study. Also excluded were those with a severe premorbid psychiatric disorder, chronic seizure disorder, or head trauma with residual dysfunction. 2.2. Immune complex detection 2.2.1. Raji cell assay Because the finding of any CSF immune complexes is usually indicative of an autoimmune, inflammatory, or infectious process, we performed a small study to examine its potential. We have modified the standard RIA assay to a microELISA system (Coyle et al., 1984). Briefly, flatbottomed microtiter plates were recoated with poly-llysine (l0 ug/ml) for 1 h. Plates were washed and 1106 Raji cells (in l00 ul) added to each well. Cells were sedimented, then fixed by gentle immersion in cold 0.25% glutaraldehyde-phosphate buffered saline (PBS) for 5 min. Plates were then transferred to a l% bovine serum albumin (BSA)–PBS solution for 1 h to block non-specific binding. Known amounts of aggregated human globulin (AHG) or test samples were preincubated with a complement source then used undiluted in the case of CSF or diluted 1:100 in PBS for serum and triplicate samples added to the wells for 1 h at 37 C. Plates were washed three times in PBS-Tween (0.05%), then horseradish peroxidase (HRP)-anti human IgG is added. To detect IgA or IgM complexes, HRP-anti human IgA or IgM was used. After incubation in the dark for 30 min, plates were washed three times. Enzyme substrate solution (2.3 mM o-phenylenediamine, OPD) in 0.024 M citrate, 0.5 M phosphate pH5 with fresh 0.03% H202) is added. After l5 min l00 ul from each well was transferred to a second plate and the reaction stopped with 1N H2SO4. The optical density (OD) was read at 488 nm in a microELISA reader (Dynatech Labs). The mean OD was calculated. A standard curve was plotted and samples read in ng/ml AHG, then corrected by the dilution factor. The level of sensitivity is 1 ug/ml of AHG. 2.3. Probe for antibodies reactive against brain We used a Western blot to search for the presence of free antibrain antibody in the CSF and serum samples by blotting against non-HIV infected human brain. Control antigens included HIV negative human liver and neural antigens. This has been described (Schutzer et al., 2003) but briefly. 2.3.1. Target antigens included (1)
Normal human brain prepared as described by Hall and Choppin (1981).
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(2)
(3)
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Galactocerebroside (Sigma C-8752, Type I), and ganglioside (Sigma G-2250, Type II) as positive controls in combination with the corresponding monoclonal antibody probe. HIV lysates and antigens were used to distinguish an antibody directed against brain from that against the HIV itself.
2.3.2. Biotin–avidin Western blots Brain preparation: 0.226 g was placed in a petri dish on ice with 1 ml of Laemmli gel sample reducing buffer (20% SDS, 10% 60 mM Tris–HCl (pH 6.8), 0.1 M DTT, 0.005% bromphenol blue, 1 mM PMSF). A minced solution was passed 10 through an 18-G followed by a 21-G needle. A portion was further diluted in sample buffer, and undiluted aliquots were frozen at 70 8C for later use. The diluted sample was boiled for 3 to 5 min, cooled and applied to gel. Gel electrophoresis: Twelve% SDS-polyacrylamide 78 cm mini-gels, 0.75 mm thick with stack were run using Hoeffer apparatus (Mighty small, SE 250). Five microliters of a 0.1% Methyl Green dye solution was added to each well to serve as a blotted dye front marker. Samples including SDS-PAGE biotinylated low range molecular weight standards (Bio-Rad) diluted in sample buffer containing 5% beta-mercaptoethanol and diluted brain were all boiled 3–5 min, cooled, and applied to wells of the gel. A 125 constant volts applied until samples lined up at bottom of stacking gel, and then power supply switched to 200 constant volts until dye front marker ran to bottom of gel. Electrotransfer: Gels were removed from plates, allowed to equilibrate for 15 min in transfer buffer (Tris 25 mM, Glycine 190 mM, Methanol 20%), then they were placed in a sandwich containing wetted nitrocellulose (MSI NitroBind 0.2 Am) to which protein was transferred over an hour at 100 V (approximately 0.25 A) using Hoeffer apparatus (TE 22). Membrane blots were measured for dye front migration, appropriately marked if pre-stained markers were not used, rinsed, dried. Immunoblot stain: Nitrocellulose blots were stained in a 0.2% Ponceau S solution (Sigma) which helped to align the blot with the 28 mini-well immunoblotter (Immunetics) and confirmed that approximately 1 microgram of protein/well, the lower limit of Ponceau S, was obtained during blotting. The membrane was blocked in Tris-buffered saline (TBS, 50 mM Tris–HCl, 0.2 M NaCl, 3 mM KCl, pH 7.5, 0.02% Azide) containing 1% Non-Fat Dry Milk (Carnation) and 1% normal goat serum (NGS) for 30 min in a rocking tray. After rinsing in TBS, the membrane was placed in the 28 lane miniblotter apparatus (MN28, Immunetics), and lanes aspirated dry. Sera were diluted 1:100 in Tris-buffered saline-Tween 20 (0.1% v/v)(TBS-T) containing 1% NGS; CSF was used neat or at 1:10 dilution, and positive control mouse monoclonal anti-neurofilament 68, NR-4 (Sigma) was diluted 1:500. Sixty microliters was pipetted into each lane (diluent alone in secondary reagent controls, and TBS in biotinylated
standard wells) and miniblotter was slowly rocked for 2 h for the primary incubation. Wells were aspirated and filled with TBS-T and miniblotter rocked rapidly for 5 min. This was repeated with TBS-T three more times and served as the standard wash procedure between reagent incubations. Secondary antibodies consisted of goat biotinylated antihuman IgG (gamma chain specific), biotinylated anti-human IgM (mu chain specific) which were diluted 1:250, and biotinylated horse anti-mouse IgG (H+L) which was diluted 1:500 (Vector Laboratories) in TBS-T plus 1% NGS. After washing, secondary antibodies were added and incubated with slow rocking for 30 min. During this time the avidin (reagent A) and biotinylated alkaline phosphatase (reagent B) complex was formed by adding 10 Al of reagent A and 10 Al of reagent B to one ml of TBS-T and vortexing. After standing at room temperature (RT) for 30 min it was further diluted by adding 2 ml of TBS-T. All wells of the miniblotter were washed four times, and the diluted avidin-biotinylated alkaline phosphatase complex (ABC) solution was added for a further 30 min incubation. After standard wash, the membrane was removed from the miniblotter, rinsed once with TBS and developed in a tray using Vector Kit II Alkaline phosphatase substrate in 100 mM Tris–HCl pH 9.5 buffer, washed twice with water and dried.
3. Results 3.1. CSF immune complexes in HAD patients We examined the CSF in seven AIDS patients who had neurologic symptoms of HAD and in whom no other known agent, except as noted, in the CSF was detected by conventional techniques. The microtiter Raji cell immune complexes assay was used to examine the CSF of HAD patients and controls for immune complexes. The following results were obtained and are represented as amounts of AHG per milliliter: Patient 1 (HAD) 7.8 ng/ml, Patient 2 (AIDS-CNS lymphoma) 12.3 ng/ml, Patient 3 (HAD) 9.0 ng/ml, Patient 4 (HAD) 12.5 ng/ml, Pt5 (AIDS-CNS lymphoma) 20.0 ng/ml, Patient 6 (HAD) 12.6 ng/ml. HAD refers to the syndrome alone, whereas the CNS lymphoma patients had the dementia symptoms and the tumor. An additional patient, Patient 7, with AIDS and CNS toxoplasmosis had no detectable CSF immune complexes. CSF from other neurology patients, which was previously determined to have no immune complexes, were used again as additional controls and continued to be devoid of immune complexes. Therefore, positive results were found in 4 of 4 HAD patients without other known CNS infection or tumor, 2 of 2 AIDS-central nervous system (CNS) lymphoma patients with dementia, 0 of 1 AIDS-CNS lymphoma patient without dementia, 0 of 1 AIDS-CNS toxoplasmosis patient without dementia, and 0 of 10 other neurologic disease controls. These CSF samples did not contain free anti-HIV
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antibodies, but the question of whether the antibody and/or antigen was bound remains unanswered. 3.2. Western blot demonstration of specific antibrain antibodies in serum and CSF In non-blinded studies we found immunoblot reactivity to non-HIV infected brain tissue in 6 HAD patients with their CSF and serum IgG. This involved regions between 41 and 58 kDa. The reactive regions were distinct from parallel probes against HIV lysates in these patients. Multiple sclerosis serum and CSF did not stain any of the above bands. This suggest that there are some brain reactive IgG antibodies in AIDS patients with dementia and that they are distinct from anti-HIV antibodies and are not found in a controls. In a blinded analysis of 31 patients, and 10 other disease controls, the brain material was probed with samples of serum, and cerebrospinal fluid. By Western blotting, antibrain brain reactive antibodies were detected in serum and/or CSF positive results were found in 11 of 12 (92%) of HAD cases, 7 of 19 (78%) of HIV patients without HAD. All 11 non-HIV controls were negative. There was no immunoblot reactivity to HIV negative liver, as a control tissue, in any group. Using the non-HIV subjects as the reference, positive results in the first two groups were statistically significant (Fisher Exact test). A result was considered as positive when there was immunoblot reactivity to four or more bands. The MW of the bands were 26, 31, 36, 40, 45, 55, 60, and 80–82 kDa. A representative blot displaying the range of the target of CSF antibrain antibodies from patients who also had serum brain reactive antibodies is shown in Fig. 1. The differences between HAD+ and HAD HIV+ is in the percentage who have several antibrain antibodies rather than the absolute number of bands on the blot. This may infer a possible prognostic value to the finding of such antibrain reactivity such that HAD HIV patients who have positive bands may develop clinical HAD but are not yet at that threshold.
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4. Discussion The changing scope of HAD mandates reproducible markers of the disease. These markers may or may not be related to the etiopathogenesis of the disease. Identification of candidate markers should be considered as a first step that will require further research and validation before use. Data from our own experiments and from others indicate that antibrain reactive antibodies and CSF immune complexes may be two such markers of HAD. The immune complexes, which are in the CSF, may serve as markers by themselves or by their specific antibody and antigen content. Recent advances in genomics and proteomics will now permit identification of the specificity of these antibodies and immune complexes components. Further validation will establish their utility as markers as well as providing possible insights into the pathogenesis of the syndrome. Immune complexes by themselves are capable of causing disease such as occurs in system lupus erythematosus (SLE) where tissue deposition leads to pathology (Jones and Orlans, 1981; Theofilopoulos and Dixon, 1980). In other circumstances, they are associated with the disease. In lupus, their reappearance has correlated with disease flareups (Theofilopoulos and Dixon, 1980). Of relevance to HAD, the immune complexes contain antibody and antigen specific for the disease. For example, the antigen in the immune complexes of SLE contains DNA and anti-DNA antibody (Sano and Morimoto, 1981). In infectious diseases, the microbial antigens and reactive antibody to them can often be identified (Baughn et al., 1987; Bayer et al., 1979; Inman et al., 1982; Jones and Orlans, 1981). In certain seronegative cases, the antibody may be found only in the immune complexes form and thereby elude detection by conventional serum or CSF assays. Examples of this include measles and Lyme disease where the patients may appear to be seronegative unless immune complexes are examined (Coyle and Procyk-Dougherty, 1984; Coyle et al., 1990; Schutzer et al., 1990, 1999). Analysis of the immune complexes components, particularly in the CSF, will likely reveal components whose sequences can be identified with
Fig. 1. Western blot probe for reactive CSF IgG to non-HIV infected brain from a representative group of HIV+ HAD patients with serum positive for antibrain reactivity (CSF indices were normal indicating there was intrathecal production of the reactive antibody). MW denotes molecular weight markers. Individual patient CSF samples are shown in Lanes 1–14. Blank lanes in between the numbers represent solution controls.
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the newer proteomic approaches. Comparison of the sequence to databases will identify the antigen as that of a particular microbe (HIV, opportunistic pathogens), or host origin, such as brain. The origin of antibrain reactive antibodies in this syndrome may be purely an epiphenomen. Despite this possibility, these antibodies can function as a marker of HAD or a pre-clinical marker of the disease before it develops. There is a body of evidence associating autoimmune-like manifestations to HIV disease and a body of evidence associating autoimmunity reactivity including antibodies to CNS disease. Autoimmune phenomena have already been associated with HIV infections. This has included the presence of autoantibodies to platelets, sperm, lymphocytes, myelin basic protein (MBP), cardiolipin, and mitochondria (Pruzanski et al., 1983; Daugharty et al., 1985; Dorsett et al., 1985; Rodman et al., 1985; Rubinstein et al., 1984; Stricker et al., 1987). Some of the antibody may be cross reactive such as has occurred between lymphocytes and brain (Funke et al., 1987; Gabuzda and Hirsch, 1987; Klatzmann et al., 1984; Bresnihan et al., 1979; Solinger and Hess, 1991). An antecedent neurological infection may promote subsequent autoimmune CNS disease. This has been described with postinfectious measles encephalomyelitis (Johnson et al., 1984). Post-vaccine encephalomyelitis and polyneuritis has followed rabies vaccination. In that case, myelin basic protein (MBP) was the encephalitogen (Hemachudha et al., 1987; Johnson et al., 1984). CSF antineuronal antibodies have been linked to CNS SLE. Lupus psychosis has been associated with a specific autoantibody (Bluestein, 1987; Bonfa et al., 1987). With respect to HAD, other investigators have identified discrete antibody reactive to brain either by molecular weight or to specific components. Similar to the proportion of patients in our blinded study (Schutzer et al., 2003), Kumar et al. identified reactive serum antibrain antibody to a 45 kDa protein of human hippocampal tissue. Seventyeight percent of 18 HAD sera samples were positive and 33% of 12 samples from AIDS patients without the dementia were positive (Kumar et al., 1989). In another investigation, molecular mimicry has been described between HIV gp120 and part of its V3 loop and human brain proteins of 35, 55 and 110 kDa (Trujillo et al., 1993). Common epitopes have been found between HIV gp41 and astrocytes (Eddleston et al., 1993; Yamada et al., 1991). Some studies have not been able to show strong associations between the dementia and antibodies (Trujillo et al., 1994; Lenhardt and Wiley, 1989). These conclusions may be reconciled by expanded studies and identification of antibody specificities. In summary, there is sufficient clinical background supporting the occurrence of autoimmune phenomena in HAD and HIV disease which is characterized by immune deficiencies and perturbations. In the current paper we have not addressed possible association of antibrain
reactive antibodies as related to the pathogenesis of HAD; rather we have focused on the marker potential of the immune system to assist in diagnosis and possible prediction of HAD. Both the immunopathogenesis and the potential diagnostic and prognostic value of autoimmune markers in HAD remain understudied. The literature and recent experiments support the existence of antibrain antibodies, and possibly immune complexes, as potential markers of high utility. The scope of HAD may be changing but fortunately recent technology advances will now permit us to identify the exact antigenic target of the antibrain antibodies.
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