UNCORRECTED PROOF

1

2

3Ž .Journal of Neuroimmunology 4315 2001 xxx45

www.elsevier.comrlocaterjneuroin6

7

8

910

Assessment of HIV-intrathecal humoral immune response in aids-related11

neurological disorders12

13

Enrico Fainardi a,), Carlo Contini b, Natale Benassi c, Andrea Bedetti d,14

Massimiliano Castellazzi a, Luca Vaghi a, Vittorio Govoni a, Ezio Paolino a,15

PierGiorgio Balboni e, Enrico Granieri a1617

a Section of Neurology, UniÕersity of Ferrara, Arcispedale S. Anna, Corso della GioÕecca 203, Ferrara 1-44100, Italy18b Section of Infectious Diseases, UniÕersity of Ferrara, Ferrara, Italy19

c Section of Virology, Azienda Ospedaliera Arcispedale S. Anna, Corso della GioÕecca 203, Ferrara 1-44100, Italy20d Unit of Infectious Diseases, Azienda Ospedaliera Arcispedale S. Anna, Corso della GioÕecca 203, Ferrara 1-44100, Italy21

e Department of Sperimental and Diagnostic Medicine, Section of Microbiology, UniÕersity of Ferrara, Õia Luigi Borsari 46, Ferrara 1-44100, Italy2223

Received 10 January 2001; received in revised form 2 May 2001; accepted 3 July 200124

25

2627

Abstract28

29Ž .Intrathecal synthesis of IgG directed to HIV antigens was investigated by antibody specific index ASI , affinity-mediated immunoblot30

Ž . Ž . Ž .AMI and Western blot WB assay in a group of 88 AIDS patients of which 28 with HIV-associated neurological disorders HAND , 1331Ž . Ž .without associated neurological disorders WAND and 47 with non-HIV-associated neurological disorders non-HAND . CD4q count32

3 Ž . 3 Ž .was above 50 cellsrmm CD4q)50 in 30 and below 50rmm CD4q-50 in 58 patients, respectively. A significantly higher33Ž . Ž .frequency for CSF complete anti-gag profile p-0.001 , and for HIV-specific oligoclonal patterns AmixedB patternsp-0.01 was34

observed in HAND as compared to patterns from the other clinical groups. A decrease in complete anti-env, anti-pol and anti-gag35

reactivity was present in CSF of patients with CD4q-50 as compared to those with CD4q)50. Our findings suggest that AIDS36

appears to be characterized by an anti-HIV intrathecal humoral immune response which is principally directed to env products with a37

prevalence of oligoclonal patterns and CSF complete anti-gag profile in HIV-associated neurological involvement. q 2001 Elsevier38

Science B.V. All rights reserved.39

40Keywords: HIV; Intrathecal IgG synthesis; Specific antibody reactivity; Viral structural proteins41

4243

44

1. Introduction45

46

Converging lines of evidence indicate that neuraxis47

represents a preferential target for Human Immunodefi-48

Ž . Ž .ciency Virus HIV Price, 1996 . A broad spectrum of49

neurological conditions has frequently been documented in50

HIV infected individuals during the various stages of the51

disease in relation to opportunistic infections induced by52

Ž .immunosuppression Price, 1996; Cohen and Berger, 1998 ,53

or as a consequence of virus attack against the nervous54

Ž . Ž .system NS Price, 1996; Gendelman et al., 1997 that55

heralds the onset of central and peripheral neurologic56

complications postulated to have a unified pathogenesis57

Ž .Tan and Guiloff, 1998 . Moreover, HIV has been isolated58

Ž .in cerebrospinal fluid CSF and brain tissue of infected5960

616263

64) Corresponding author. Tel.: q39-532-205525.6566

67

68

Ž .E-mail address: henryfai@.tin.it E. Fainardi .

69

Žpatients in the different phases of the disease McArthur et 70

.al., 1988; Ho et al., 1985; Chiodi et al., 1988a and it has 71

also been reported that the virus penetrates the intrathecal 72

Žcompartment early in the course of the infection Resnick 73

.et al., 1988; McArthur et al., 1988 . 74

A large number of CSF abnormalities including blood– 75

Ž .brain-barrier B-B-B disturbance and a gradually decreas- 76

ing intrathecal IgG production have been demonstrated 77

Žduring the entire course of the infection Resnick et al., 78

1988; McArthur et al., 1988; Elovaara et al., 1987; Luer et¨ 79.al., 1988; Marshall et al., 1988 . In this setting, the occur- 80

rence of anti-HIV antibodies restricted to CSF compart- 81

ment in asymptomatic carriers has been considered further 82

Žproof that the virus invades the brain early Elovaara et al., 83

.1987 , while in AIDS patients it may represent an active 84

virus replication producing a persistent antigenic stimula- 85

Žtion Luer et al., 1988; Chiodi et al., 1988b; Elovaara et¨ 86.al., 1993 . This HIV-specific CSF-restricted humoral im-

870165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.88

Ž .PII: S0165-5728 01 00386-1

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–000289

mune response has been reported to be markedly poly-90

Žclonal rather than oligoclonal Resnick et al., 1985; Chiodi91

.et al., 1988b . Also, the analysis of antigenic specificity of92

CSF anti-HIV antibodies has shown a progressive decrease93

in anti-pol and anti-gag reactivity in contrast to a perma-94

nent intrathecal anti-env response in asymptomatic stages95

Žas well as in AIDS-mediated conditions Chiodi et al.,96

1988b; Lolli et al., 1990; Goswami et al., 1991; Elovaara97

.et al., 1993 .98

Despite the strong correlation described between intra-99

B-B-B production of anti-HIV antibodies and virus recov-100

Ž .ery from CSF Sonneborg et al., 1989 , no relationship was¨101

detected between HIV-specific antibody response and102

Ž .HIV-associated disorders Goswami et al., 1991 . Thus,103

the recognition of a potential indicator to discriminate the104

different neurological manifestations would be helpful.105

In order to clarify the actual role of virus-specific106

humoral response in the immune deregulation which is107

generated locally in HIV-related NS involvement, we in-108

vestigated intrathecal synthesis of anti-HIV IgG and their109

antigenic specificity in a group of patients suffering from110

AIDS. Since the relationship among HIV-specific oligo-111

clonal IgG, CSF antibody reactivity profiles, clinical pic-112

tures and progression of AIDS has not previously been113

evaluated, we sought to establish whether these anti-HIV114

humoral patterns could contribute to better define some115

pathogenetic aspects of HIV-related neurological manifes-116

tations.117

118

2. Patients and methods119

120

2.1. Patient selection121

122

We conducted a prospective investigation of 88 consec-123

Žutive HIV infected patients 63 men and 25 women; mean124

.ages36.4"8.5 admitted to the Department of Infectious125

Diseases of Ferrara during the period from March 1993126

to January 1997. Enzyme-linked immunosorbent assay127

Ž . Ž .ELISA and Western blot WB analysis demonstrated128

anti-HIV-1 serology and excluded anti-HIV-2 infection.129

The affected patients enrolled in this study were 61 intra-130

venous drug addicts, 20 heterosexual partners of HIV131

infected subjects, four homosexual men and three transfu-132

sion recipients. All the patients belonged to Group IV of133

Ž .the Centers for Disease Control CDC classification of134

Ž .Atlanta CDC, 1987 : 1 to Group IVA, 19 to Group IVB,135

64 to Group IVC1, 3 to Group IVC2 and 1 to Group IVD.136

Among these patients, 28 showed HIV-associated neuro-137

Ž .logical disorders HAND representing virus-induced brain138

Ždiseases encephalopathys24 cases; peripheral neuropa-139

.thys3 cases; vacuolar myelopathys1 case , 13 were140

AIDS patients without associated neurological disorders141

Ž .WAND and 47 had non-HIV-associated neurological dis-142

Ž .orders non-HAND consisting of bacterial, viral, fungal

143

Žand protozoal opportunistic brain infections Toxoplasma 144

gondii encephalitiss11 cases; Progressive Multifocal Leu- 145

kobencepalopathys11 cases; Cryptococcoccal meningitis 146

s9 cases; Mycobacterial infectionss9 cases; Cytomega- 147

.lovirus encephalitiss4 cases; Aspergillosiss3 cases . 148

Ž 3.Peripheral CD4q cell count means92.3"226.4 mm 1493 Ž . Žwas above 50rmm CD4q)50 in 30 individuals 13 150

HAND, means227.1"166; 5 WAND, means145.2" 151

.96; 12 non-HAND, means145.5"118.5 and below 1523 Ž . Ž50rmm CD4q-50 in 58 subjects 15 HAND, mean 153

s16.5"18.3; 8 WAND, means5.4"6; 35 non-HAND, 154

.means15.4"15.2 . All patients had discontinued the 155

antiretroviral treatment for at least 3 months because of 156

intolerance or therapeutic failure in accordance with the 157

ŽState of Art Consensus Conference criteria Sande et al., 158

.1993 . Samples were obtained before the widespread use 159

of plasma HIV viral load, as a prognostic marker of 160

disease progression. None of these patients had received 161

protease inhibitors or highly active antiretroviral therapy at 162

the moment of sample collection. We also tested as HIV- 163

Žnegative and immunocompetent controls 45 patients 25 164

.women and 20 men, mean ages43q14.2 suffering from 165

Ž .other inflammatory neurological disorders OIND and 39 166

Ž .patients 19 women and 20 men, mean ages45.6q15.9 167

Ž .with non inflammatory neurological disorders NIND . 168

Research protocol was reviewed and approved by the 169

Regional Committee for Medical Ethics in Research.170

171

2.2. Routine CSF and serum examination 172

173

Paired CSF and serum samples were collected under 174

sterile conditions by atraumatic lumbar puncture per- 175

formed for the purpose of diagnosis in the absence of 176

contraindications. Specimens were frozen in aliquots at 177

y70 8C until assay. CSF and serum concentrations were 178

measured nephelometrically with the Beckman Array Pro- 179

Ž .tein System Salden et al., 1988 , while Blood–CSF-Bar- 180

Ž .rier B-CSF-B dysfunction was estimated by CSFral- 181

Ž . Ž .bumin quotient Q following Tibbling et al. 1977 .Alb182

1832.3. Anti-HIV ELISA 184

185

CSF and serum anti-HIV IgG were assayed by ELISA 186

Ž .as previously described Reiber and Lange, 1991 by com- 187

Žmercially available kit for anti-HIV quantitation Genelavia 188

Mixt, Sanofi Diagnostics Pasteur, Marnes-La-Coquette, 189

.France . Adsorbance was read at 490 nm with an auto- 190

Žmated ELISA reader Microline reader DV 920, Poli In- 191

.dustria Chimica , Milano, Italy .192

193

2.4. Calculation of HIV-specific quantitatiÕe index 194

195

Ž .According to Reiber’s formula Reiber and Lange, 1991 196

production of IgG anti-HIV was determined by antibody

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–000 3197

Ž .specific index ASI . Reference curve was generated in198

each assay using the standard serial dilutions ranging199

between 0.05 and 2.00 OD. The upper standard titer of200

Ž .each plate was considered as 100 arbitrary units AU . ASI201

means the ratio between CSF and serum AU values, with202

Ž .multiplication by the dilution factor Q and CSFrSpec203Ž .serum total IgG levels expressed as mgrdl Q inIgG204

accordance to the formula205

ASIsQ rQ 1Ž .Spec IgG206

with Q s AU rAU and Q s IgG rSpec CSF serum IgG CSF207IgG .In the case of intrathecal synthesis of total IgG,serum208Q appeared more elevated than Reiber’s hyperbolicIgG209

Ž . Ž .discrimination line Q Reiber and Felgenhauer, 1987 .Lim210Q represented the CSF total IgG fraction completelyLim211blood-derived calculated from the individual Q of aAlb212single patient. Therefore, owing to the introduction of this213

barrier-related correction of the Q , the ratio becameIgG214between Q and Q in agreement with the formulaSpec Lim215Ž .Reiber, 1996216

ASIsQ rQ 2Ž .Spec Lim217

with218

2 y6 y3(Q s0.93 Q q6P10 y1.7P10 .Ž .Lim Alb219

Ž . ŽTherefore, to avoid false-negative results Eq. 1 Q )IgG220.Q is used when no significant intrathecal total IgGLim

221Ž . Ž .synthesis occurs, while Eq. 2 Q -Q was pre-IgG Lim 222

ferred in case of intense brain-derived total IgG production 223

in CSF. 224

HIV-specific intrathecal IgG synthesis was assumed for 225

values of ASI greater than 1.5.226

227

2.5. HIV antigen preparation228

229

Preparation of HIV strain HXBC2 antigens stock was 230

Ž .carried out as described by Destrosiers 1990 . Briefly, 231

Ž 6.CEM-SS cells 1=10 were infected with HIV at a 232

Ž .multiplicity of infection MOI of 0.001 TCID rcell;50 233every week media were collected by centrifugation at 3000 234

rpm for 10 min from virus producing cell cultures. The 235

supernatant was then centrifuged at 17.000 rpm for 3 h at 236

4 8C. Virus pellets were resuspended in PBS-a containing 237

Triton X-100 0.5% to obtain a complete viruses lysis. 238

Antigen stocks were stored aty70 8C. At every collection 239

Ž 7.of media uninfected cells 1=10 were added to virus- 240

producing cell cultures to ensure high levels of virus 241

production. The strain HXBC2, which is HIV-1 IIIB de- 242

rived, has been shown a high affinity for CD4 receptor of 243

T lymphocytes. Thus, its antigens were chosen because 244

representative of all variants, including CD4-indipendent 245

isolates of HIV, and implicated in pathologic effects in 246

Ž .humans La Branche et al., 1999 .

247248

Ž .Fig. 1. Affinity-mediated immunoblot AMI patterns: Asnormal; Bsmirror; Cs local synthesis; Dsmixed; Espatient with multiple sclerosis used249as negative control for non-specific binding. CSFscerebrospinal fluid; Ssserum.

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–0004250251

( )2.6. HIV-specific affinity-mediated immunoblotting AMI252

253

Ž .HIV-specific IgG oligoclonal bands OCB were de-254

tected by AMI according to the protocol of Dorries et al.¨255Ž .1989 using a commercially available isoelectric focusing256

Žkit Titan Gel IgG IEF, Helena Laboratories, Gateshead,257

.Tyne and Wear, UK with some modifications. Briefly, 5258

ml of paired undiluted CSF and 1:300 diluted serum259

Ž .samples were applied to the agarose gel pH range 3–10260

and were focused for 85 min at 10 8C in a multiphor unit261

Ž .Isolab, Akron, OH, USA . After IEF run, the gel was262

Ž .blotted onto a nitrocellulose sheet NC previously coated263

overnight at room temperature with HIV antigen solution264

Žin Tris-buffered saline TBS, 50 mM Tris, 200 NaCl, pH265

.7.5 . HIV antigen solution total protein were measured by266

Ž .Markwell procedure Markwell et al., 1981 and were267

diluted with TBS to obtain the optimal concentration of268

100 mgrml per 10 cm3 of NC area. After incubation with269

HIV antigen, the NC membranes were washed in TBS and270

non-specific binding sites were blocked for 1 h in TBS271

Ž .containing 5% bovine serum albumin BSA . The affinity-272

mediated capillary blotting was performed under a uniform273

weight of 1 kg for 30 min at room temperature. After274

washing with PBS, NC papers were incubated for 30 min275

at room temperature with goat anti-human IgG antiserum276

diluted 1:1000 in TBS. The immunoblots were further277

washed with TBS and incubated for 30 min at room278

temperature with peroxidase conjugated rabbit anti-goat279

IgG diluted 1:1000 in TBS. After another washing cycle280

with TBS, the blots were stained using peroxidase conju-281

gated substrate of the kit according to the manufacturer’s282

instructions. The immunoblotting specificity was evaluated283

Žin accordance to the above mentioned protocol Dorries et¨284.al., 1989 . No cross-reactivity to HIV was detected. Ac-285

Žcording to the CSF European Consensus criteria Anders-286

.son et al., 1994 , we identified four different AMI IgG287

banding patterns when comparing CSF and parallel serum288

Ž . Ž .from the same patient Fig. 1 : 1 AnormalBsdiffuse289

polyclonal IgG background in CSF and serum, without any290

Ž .detectable IgG OCB; 2 Alocal synthesisBs two or more291

CSF restricted IgG OCB and absent in the corresponding292

Ž .serum; 3 AmixedBsCSF restricted IgG OCB with addi-293

Ž .tional identical IgG OCB in both CSF and serum; 4294

AmirrorBs identical IgG OCB located at the same isoelec-295

tric point in both CSF and serum. The results were exam-296

ined by two independent investigators. Only the detection297

of AMI profiles 2 and 3 was considered suggestive of298

intrathecal synthesis of oligoclonal anti-HIV IgG.299

300

( )2.7. Western blot WB assay301

302

WB analysis was carried out using a commercially303

Žavailable system Novapath HIV-1 Immunoblot Assay,304

.Bio-Rad Laboratories, CA, USA according to the manu-305

facturer’s instructions. Briefly, 30 ml of undiluted CSF

306

specimen was dispensed onto NC strips containing the 307

electrophoresed and transferred viral proteins env, pol and 308

gag. After incubation on a rocking platform for 30 min at 309

room temperature and subsequent two washing cycles, the 310

NC membranes were incubated with secondary antibody 311

for 30 min at room temperature on a rocking platform. 312

After two washing cycles, colour reaction was developed 313

as described in the protocol of the kit. The staining intensi- 314

ties of the bands corresponding to HIV proteins were 315

scored visually by two independent investigators as fol- 316

Ž . Ž . Ž .lows: 1 absent; 2 weak; 3 strong. An immunostaining 317

type 2 or 3 was recorded as positive for each single HIV

318319

Ž .Fig. 2. Western blot WB profiles: Lane AsCSF complete anti-gag 320Ž .pattern arrows ; Lane BsCSF gp160qgp120 anti-env pattern; Lane 321

CsCSF complete anti-env, anti-pol and anti-gag patterns. Numbers 322represent molecular weight standars.

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–000 5323

protein. For each HIV gene, we evaluated the occurrence324

Ž . Ž .of Fig. 2 : a IgG directed to the total of the gene325

Ž .products; b profiles obtained by the combination of IgG326

directed to each single gene product.327

328

2.8. Statistical analysis329

330

The patient group percentages were compared using the331

Ž .Log-Likelihood Ratio Test G . A difference was consid-332

ered significant at p-0.05.333

334

3. Results335

336

3.1. ELISA tests337

338

ASI values above the normal limit were found in 66r88339

Ž . Ž .75% of total AIDS patients. Of these, 21r28 75% were340

Ž . Ž .HAND, 10r13 76.9% were WAND, 35r47 74.5%341

were non-HAND. CD4q values above or below 50rmm3342

Ž . Ž .were found in 23r30 76.7% and in 43r58 74.1%343

patients, respectively, without any statistically significant344

difference among the various groups studied. No signifi-345

cant differences were also observed when the larger sub-346

groups of HAND and non-HAND were compared, since347

Ž .ASI values greater than 1.5 was present in 20r24 83.3%348

Ž . Ž .of HIV encephalopathy HIVE , in 8r11 72.2% of Toxo-349

Ž .plasma gondii encephalitis TE and Progressive Multifo-350

Ž . Ž .cal Leukoencepalopathy PML , in 7r9 77.8% of Cryp-351

Ž .tococcoccal meningitis CM and Mycobacterial infections352

Ž .MI . None of OIND NIND patients displayed an intrathe-353

cal synthesis of anti-HIV antibodies by ASI.354

355

3.2. AMI technique356

357

As summarized in Table 1, in all clinical categories358

examined we detected a relative low frequency of AMI359

oligoclonal IgG profiles indicative of HIV local synthesis

360361

Table 1362Ž .Distribution of affinity-mediated immunoblot AMI intrathecal synthesis363

profiles into 88 total AIDS patients with or without neurological disorders364aITS Local synthesis Mixed

b )Ž . Ž . Ž .HAND 12r28 42% 3r28 10.7% 9r28 32.2% 8c Ž . Ž . Ž .WAND 2r13 15.4% 2r13 15.4% 0r13 0%

d Ž . Ž . Ž .Non-HAND 9r47 19.2% 7r47 19.2% 2r47 14.9%e Ž . Ž . Ž .Total 23r88 26.1% 12r88 13.6% 11r88 12.5%

365

Ž .Mixed profile was significantly more frequent in HAND 8p-0.01 than366in WAND and non-HAND. ITS was significantly more consistent in367

Ž) .HAND p-0.05 than in non-HAND.368a Ž .Intrathecal synthesis patterns local synthesisqmixed .369bHIV-associated neurological disorders.370cWithout associated neurological disorders.371dNon-HIV-associated neurological disorders.372eTotal patients.

373374

Table 2 375Ž .Distribution of affinity-mediated immunoblot AMI intrathecal synthesis 376

Ž .profiles into AIDS patients with HIV encephalopathy HIVE , Toxo- 377Ž .plasma gondii encephalitis TE , Progressive Multifocal Leukoen- 378

Ž . Ž .cepalopathy PML , Cryptococcoccal meningitis CM and Mycobacterial 379Ž .infections MI 380

aITS Local synthesis Mixed') 'Ž . Ž . Ž .HIVE 12r24 45.8% 3r24 12.5% 8r24 33.3% 8

Ž . Ž . Ž .TE 2r11 18.2% 2r11 18.2% 0r11 0%Ž . Ž . Ž .PML 1r11 9.1% 1r11 9.1% 0r11 14.9%Ž . Ž . Ž .CM 1r9 11.1% 1r9 11.1% 0r9 12.5%Ž . Ž . Ž .MI 1r9 11.1% 1r9 11.1% 0r9 12.5%

381

Mixed profile was significantly more frequent in HIVE than in TE, PML, 382Ž . Ž' .MI 8p-0.01 and CM p-0.02 ; ITS was significantly more consis- 383

Ž) . Ž' .tent in HIVE than in PML, CM, MI p-0.05 and TE p-0.02 . 384a Ž .Intrathecal synthesis patterns local synthesisqmixed .

385

within the CSF compartment. AMI oligoclonal patterns 386

supporting an anti-HIV IgG synthesis restricted to brain 387

were slight statistically more frequent in HAND than in 388

Ž .non-HAND p-0.05 and were more represented even in 389

HAND than in WAND, although this difference was not 390

statistically significant. Moreover, HAND had statistically 391

higher percentage of AmixedB profile when compared to 392

Ž .WAND and non-HAND p-0.01 . There were no signif- 393

icant differences for AMI patterns among the different 394

clinical pictures and between CD4q)50 and CD4q- 395

50 lymphocyte count groups. On the other hand, patterns 396

suggestive of systemic oligoclonal IgG production trans- 397

Ž .ferred into brain AmirrorB and AmixedB displayed more 398

Ž .prominent values in HAND 16r28; 57.1% compared to 399

Ž . Ž .WAND 4r13; 30.8% and non-HAND 13r47; 27.6% 400

with a statistically significant difference between HAND 401

Ž .and non-HAND p-0.02 . Finally, as shown in Table 2, 402

the strong discrepancy found between HAND and non- 403

HAND for HIV-specific CSF-restricted oligoclonal re- 404

sponse was confirmed by the evaluation of their larger 405

subgroups. No HIV-specific oligoclonal bands were identi- 406

fied in CSF and serum of OIND and NIND. 407

408

3.3. WB anti-HIV reactiÕity 409

410

3.3.1. CSF total anti-HIV reactiÕity 411

The occurrence of CSF total anti-env, anti-pol and 412

anti-gag response was listed in Table 3. CSF total anti-env 413

specific staining was recognized more frequently than 414

anti-pol and anti-gag IgG bands in the various groups 415

examined with a slight statistically significant prevalence 416

when total anti-pol reactivity of CD4q)50 were com- 417

Ž .pared to CD4q-50 p-0.05 . No anti-HIV specific 418

staining was detected in OIND and NIND. 419

420

3.3.2. CSF anti-HIV patterns 421

In CSF, complete gp160qgp120qgp41 and gp160q 422

gp120 anti-env patterns were remarkably prevalent rather

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–0006423424

Table 3425Ž .Frequencies of CSF Western Blot WB total anti-HIV reactivity ob-426

served in 88 AIDS patients divided into various categories427

Anti-env Anti-pol Anti-gaga Ž . Ž . Ž .HAND 28r28 100% 13r28 46.4% 13r28 46.4%b Ž . Ž . Ž .WAND 13r13 100% 6r13 46.1% 6r13 46.1%

c Ž . Ž . Ž .Non-HAND 46r47 97.9% 25r47 53.2% 23r47 48.9%d )Ž . Ž . Ž .CD4q)50 30r30 100% 20r30 66.7% 18r30 60%e Ž . Ž . Ž .CD4q-50 56r58 96.5% 24r58 41.1% 24r58 41.4%

f Ž . Ž . Ž .Total 87r88 98.9% 44r88 50% 42r88 47.7%428

CSF total anti-pol profile was significantly more frequent in CD4q)50429Ž) .p-0.05 than in CD4q-50.430

aHIV-1-associated neurological disorders.431bWithout associated neurological disorders.432cNon-HIV-1-associated neurological disorders.433dPatients with CD4q cell count above 50rmm3.434ePatients with CD4q cell count below 50rmm3.435f Total patients.

436

than those found in all AIDS groups. Significantly more437

increased values were found for CSF complete anti-env438

pattern in CD4q)50 than in CD4q-50 and for gp160439

qgp120 anti-env profile in CD4q-50 than in CD4q440

Ž . Ž .)50 p-0.001 Fig. 3a . In addition, the frequency of

441

CSF complete p65qp32 pattern was significantly more 442

Ž .elevated in CD4q)50 than in CD4q-50 p-0.01 443

Ž .Fig. 3b . When the distinct CSF anti-gag profiles were 444

tested, statistically more pronounced values for complete 445

p55qp24q18 pattern was calculated in HAND in com- 446

Ž .parison to WAND and non-HAND p-0.001 and in 447

Ž . ŽCD4q)50 with respect to CD4q-50 p-0.01 Fig. 448

.3c . The striking predominance of CSF complete anti-gag 449

profile found in HAND than in non-HAND was corrob- 450

orated by significantly greater values obtained in HIVE 451

Ž . Ž10r24; 41.7% with respect to TE and PML 1r11; 9.1%; 452

. Ž .p-0.05 , CM and MI 0r9; 0%; p-0.01 . 453

454

4. Discussion 455

456

The aim of present study was to provide further insights 457

into the effective relevance of anti-HIV IgG restricted 458

production within the brain in a large series of AIDS 459

patients, and to distinguish the different neurological com- 460

plications occurring in the course of AIDS. 461

ŽAccording to prior investigations Resnick et al., 1988; 462

Elovaara et al., 1987; Luer et al., 1988; Chiodi et al.,¨

463464

Fig. 3. Panel A shows the distribution of CSF complete gp160q120q41 and gp160q120 anti-env patterns in patients with CD4q cell count above465Ž . Ž . 3CD4q)50 and below CD4q-50 50rmm with greater rates for complete anti-env profile in CD4q)50 than in CD4q-50 and for466

Ž) .gp160q120 in CD4q-50 than CD4q)50 p-0.001 . Panel B indicates the distribution of CSF complete p65q32 anti-pol pattern in CD4q-50467Ž .and CD4q-50 patients with higher rates in CD4q-50 than CD4q-50 8p)0.01 . Panel C illustrates the distribution of CSF complete468

Ž . Ž .p55q24q18 anti-gag profile in HIV-1-associated neurological disorders HAND , without associated neurological disorders WAND , non-HIV-1-asso-469Ž .ciated neurological disorders non-HAND , CDq)50 and CD4q-50 with more prominent rates in HAND than in non-HAND and WAND470

Ž) . Ž .p-0.001 and in CD4q)50 than in CD4q-50 8p-0.01 .

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–000 7471

1988b; Lolli et al., 1990; Goswami et al., 1991; Elovaara472

.et al., 1993 , we observed a high frequency of ASI positive473

values, suggesting an intrathecal synthesis of HIV-specific474

IgG in AIDS clinical groups examined, without any statis-475

tical discrimination among them.476

ŽIn agreement with previous observations Chiodi et al.,477

.1988b; Goswami et al., 1991 and in contrast with others478

Ž .Dorries et al., 1989; Grimaldi et al., 1988 , a small¨479

percentage of AIDS patients was found to be producer of480

CSF restricted oligoclonal IgG bands. However, AMI pro-481

files indicative of anti-HIV intrathecal IgG synthesis, par-482

ticularly AmixedB pattern, were principally associated with483

HAND rather than with the other clinical pictures. Further-484

more, a more elevated frequency of AMI patterns reflect-485

ing blood-derived CSF oligoclonal IgG secretion was486

shown in HAND as compared to non-HAND and, to a487

lesser extent, WAND.488

Antigenic specificity evaluation of CSF and serum anti-489

HIV IgG confirmed the broad spectrum of antibody pro-490

Žduction previously described Ellis et al., 1997; Lolli et al.,491

1990; Goswami et al., 1991; Elovaara et al., 1993; Grimaldi492

.et al., 1988 involving several virus structural proteins as493

targets which are most likely imputable to a different494

Ž .processing of their precursors Chiodi et al., 1988a . Ac-495

cording to some prior studies, we detected a CSF antibody496

response which was mainly directed to env products such497

Žas gp160, gp120 Chiodi et al., 1988a; Lolli et al., 1990;498

.Goswami et al., 1991; Grimaldi et al., 1988 and, to a499

Ž .lesser extent, to gp41 Elovaara et al., 1993 , while the500

high IgG reactivity against pol and gag antigens previously501

Ždocumented in CSF Chiodi et al., 1988a; Goswami et al.,502

.1991; Elovaara et al., 1993 was not found. By examining503

patterns of antibody reactivity to HIV structural proteins,504

we found a greater frequency for CSF complete anti-gag505

profile in HAND than WAND and non-HAND. Generally,506

a slight decrease in CSF anti-pol total response, a strong507

decline in CSF complete env, pol and gag patterns and an508

evident increase in CSF gp160qgp120 reactivity were509

found in patients with advanced AIDS.510

Taken together, these findings support the hypothesis511

that the later stages of the disease are characterized by a512

relatively intense HIV-specific intra-B-B-B antibody re-513

sponse which is predominantly directed toward gp160 and514

Žgp120 env products Chiodi et al., 1988a; Lolli et al.,515

.1990; Goswami et al., 1991; Grimaldi et al., 1988 and516

probably occur in the context of a polyspecific intrathecal517

reaction triggered by immune network interferences and518

persisting even in the absence of the corresponding antigen519

ŽSindic et al., 1994; Conrad et al., 1994; Reiber et al.,520

.1998 .521

This most likely reflects a continual antigenic stimula-522

tion sustained by an active intrathecal virus replication523

ŽLuer et al., 1988; Sonneborg et al., 1989; Elovaara et al.,¨ ¨524.1993 . The low oligoclonal IgG production observed in our525

series may be referred to an overturning of regulatory526

mechanisms which modulate clonal expansion of HIV-

527

Ž .specific antibody producing B cells Chiodi et al., 1988b . 528

This profound dysregulation of brain humoral response 529

may account for the relatively scarce detection of anti-pol 530

and anti-gag IgG bands in comparison to elevated anti-env 531

reactivity shown in CSF by AIDS patients, as well as for 532

the changes occurring to antigenic specificity of CSF 533

anti-HIV IgG with the progression of AIDS. 534

The main result of this study is, however, represented 535

by the remarkable prevalence of AMI AmixedB pattern and 536

CSF complete anti-gag profile in HAND compared to the 537

other clinical groups. In fact, the appearance of this type of 538

response seems to be very specific to recognize HIV-re- 539

lated neurological syndromes due to the influence of virus 540

neuroinvasion indicating potential implications for the in- 541

terpretation of intrathecal immunological events during 542

AIDS. 543

In conclusion, our data underline the possibility that an 544

oligoclonal response directed against the virus, when pre- 545

sent in AIDS patients with neurological disorders induced 546

by HIV, is frequently both intrathecal and systemic. More- 547

over, as previously demonstrated for Toxoplasma gondii 548

Ž .encephalitis Contini et al., 1998 , these findings further 549

confirm that ASI as well as AMI may represent powerful 550

tools for evaluating the specificity of humoral intrathecal 551

immune response in AIDS-related diseases. In addition, an 552

increase in oligoclonal IgG release in HIV-associated neu- 553

rological manifestations seems to corroborate the Aclonal 554

dominance theoryB suggesting that the shift from the nor- 555

mal polyclonal to a restricted anti-HIV response could 556

induce the failure of viral infection control by the immune 557

system with the consequent development of virus variants 558

Ž .escaping antibody reaction Kohler et al., 1992 . 559

The cellular sources of HIV antigenic epitops within the 560

CSF compartment remain to be identified. According to 561

Žobservations recently reported for HIV-1 RNA McArthur 562

.et al., 1997; Ellis et al., 1997, 2000 , we hypothesize that 563

structural virus proteins and their precursors originate both 564

intrathecally and systemically from colonization and de- 565

struction of relatively long-lived host cells as macrophages, 566

microglia and astrocytes by HIV. In addition, the presence, 567

in the brain, of these long-lived infected cells could ac- 568

count for the slow rate of decrease in intrathecal antigenic 569

stimulation despite systemic immunosuppression. 570

Further studies are required to prove whether this ap- 571

proach may contribute to clarify immunological aspects of 572

the pathogenesis of the disease. 573

574

Acknowledgements 575

576

We thank Dr. Tiziana Bellini and Professor Franco 577

Dallocchio of the Biochemistry Department of University 578

of Ferrara for performing total protein measurements of 579

HIV-1 antigen solution, for technical assistance, for help- 580

ful discussions and for substantial intellectual support. The 581

study was supported by a grant of M.U.R.S.T. 1997.

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( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–0008582

583

References584

585Andersson, M., Alvarez-Cermeno, J., Bernardi, G., Cogato, I., Fredman,˜586

P., Frederiksen, J., Fredriksonn, S., Gallo, P., Grimaldi, L.M.,587Grønning, M., Keir, G., Lamers, K., Link, H., Magalhaes, A., Mas-˜588

¨saro, A.R., Ohman, S., Reiber, H., Ronnback, L., Schluep, M.,¨ ¨589Shuller, E., Sindic, C.J.M., Thompson, E.J., Trojano, M., Wurster, U.,¨5901994. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a591consensus report. J. Neurol., Neurosurg. Psychiatry 57, 897–902.592

Centers for Disease Control, 1987. Revision of the CDC surveillance case593definition for acquired immunodeficiency syndrome. MMWR 36594Ž .Suppl. 1 , 3–15.595

Chiodi, F., Sonneborg, A., Albert, J., Gaines, H., Norkrans, G., Hagberg,¨596˚ ¨L., Asjo, B., Strannegard, O., Fenyo, E.M., 1988a. Human immunod-¨ ˚ ¨597

eficiency virus infection of the brain: I. Virus isolation and detection598of HIV specific antibodies in the cerebrospinal fluid of patients with599varying clinical conditions. J. Neurol. Sci. 85, 245–257.600

Chiodi, F., Norkrans, G., Hagberg, L., Sonneborg, A., Gaines, H.,¨601Frøland, S., Fenyo, E.M., Morrby, E., Vandvik, B., 1988b. Human¨602immunodeficiency virus infection of the brain: II. Detection of in-603trathecally synthesized antibodies by enzyme linked immunosorbent604assay and imprint immunofixation. J. Neurol. Sci. 87, 37–48.605

Cohen, B.A., Berger, J.R., 1998. Neurologic opportunistic infections in606AIDS. In: Gendelman, H.E., Lipton, S.A., Epstein, L., Swindells, S.607Ž .Eds. , The Neurology of AIDS. Chapman & Hall, New York, pp.608303–332.609

Conrad, A.J., Chiang, E.Y., Andeen, L.E., Avolio, C., Walker, S.M.,610Baumhefner, R.W., Tourtellotte, W.W., 1994. Quantitation of in-611trathecal measles virus IgG antibody synthesis rate: subacute scleros-612ing panencephalitis and multiple sclerosis. J. Neuroimmunol. 54,61399–108.614

Contini, C., Fainardi, E., Cultrera, R., Canipari, R., Peyron, F., Delia, S.,615Paolino, E., Granieri, E., 1998. Advanced laboratory techniques for616diagnosing Toxoplasma gondii encephalitis in AIDS patients: signifi-617cance of intrathecal production and comparison with PCR and ECL-618Western blotting. J. Neuroimmunol. 92, 29–37.619

Destrosiers, R.C., 1990. Virus purification, preparation of infectious virus620Ž .stock and virus storage. In: Aldovini, A., Walker, B.D. Eds. ,621

Techniques in HIV Research. Stockton Press, New York, pp. 121–127.622Dorries, R., Kaiser, R., ter Meulen, V., 1989. Human immunodeficiency¨623

virus infection: affinity-mediated immunoblot detects intrathecal syn-624thesis of oligoclonal IgG specific for individual viral protein. AIDS625Res. Hum. Retroviruses 5, 303–310.626

Ellis, R.J., Hsia, K., Spector, S.A., Nelson, J.A., Heaton, R.K., Wallace,627M.R., Abramson, I., Atkinson, J.H., Grant, I., McCutchan, J.A., 1997.628Cerebrospinal fluid human immunodeficiency virus type 1 RNA629levels are elevated in neurocognitively impaired individuals with630acquired immunodeficiency syndrome. Ann. Neurol. 42, 679–688.631

Ellis, R.J., Gamst, A.C., Capparelli, E., Spector, S.A., Hsia, K., Wolfson,632T., Abramson, I., Grant, I., Mc Cutchan, J.A., 2000. Cerebrospinal633fluid HIV RNA originates from both local CNS and systemic sources.634Neurology 54, 927–936.635

Elovaara, I., Iivanainen, M., Valle, S.L., Suni, J., Tervo, T., Lahdevirta,¨636J., 1987. CSF protein and cellular profiles in various stages of HIV637infection related to neurological manifestation. J. Neurol. Sci. 78,638331–342.639

Elovaara, I., Albert, P.S., Ranki, A., Krohn, K., Seppala, I., 1993. HIV-1¨ ¨640specificity of cerebrospinal fluid and serum IgG, IgM and IgG1-G4641antibodies in relation to clinical disease. J. Neurol. Sci. 117, 111–119.642

Gendelman, H.E., Persidsky, Y., Ghorpade, A., Limoges, J., Stims, M.,643Fiala, M., Morrisett, R., 1997. The neuropathogenesis of the AIDS644

Ž .dementia complex. AIDS 11 Suppl. A , S35–S45.645Goswami, K.K., Kaye, S., Miller, R., Mc Allister, R., Tedder, R., 1991.646

Intrathecal IgG synthesis and specificity of oligoclonal IgG in patients647infected with HIV-1 do not correlate with CNS disease. J. Med. Virol.64833, 106–113.649

Grimaldi, L.M.E., Roos, R.P., Devare, S.G., Robey, W.G., Casey, J.M.,

650Gurney, M.E., Apatoff, B.R., Lazzarin, A., 1988. Restricted hetero- 651geneity of antibody to gp120 and p24 in AIDS. J. Immunol. 141, 652114–117. 653

Ho, D.D., Rota, T.R., Schooley, R.T., Kaplan, J.K., Groopman, J.E., 654Resnick, L., Felsenstein, D., Andrews, C.A., Hirsh, M.S., 1985. 655Isolation of HTLV-III from cerebrospinal fluid and neural tissue of 656patients with neurologic symptoms related to the acquired immunode- 657ficiency syndrome. New Engl. J. Med. 313, 1493–1497. 658

Kohler, H., Goudsmit, J., Nara, P., 1992. Clonal dominance: cause for a 659limited and failing immune response to HIV-1 infection and vaccina- 660tion. J. Acquired Immune Defic. Syndr. 5, 1158–1168. 661

La Branche, C.C., Hoffman, T.L., Romano, J., Haggarty, B.S., Edwards, 662T.G., Matthews, T.J., Doms, R.W., Hoxie, J.A., 1999. Determinants 663of CD4 indipendence for a human immunodeficiency virus type 1 664variant map outside regions required for coreceptor specificita. J.` 665Virol. 73, 10310–10319. 666

Lolli, F., Colao, M.G., De Maio, E., Di Pietro, M., Galli, E., Grippo, A., 667Mazzotta, F., Pinto, F., 1990. Intrathecal synthesis of anti-HIV anti- 668bodies in AIDS patients. J. Neurol. Sci. 99, 281–289. 669

Luer, W., Poser, S., Weber, T., Jurgens, S., Eichenlaub, D., Pohle, H.D.,¨ ¨ 670Felgenhauer, K., 1988. Chronic HIV encephalitis: 1. Cerebrospinal 671fluid diagnosis. Klin. Wochenschr. 66, 21–25. 672

Markwell, M.A.K., Haas, S.M., Tolbert, N.E., Bieber, L.L., 1981. Protein 673determination in membrane and lipoprotein samples: manual and 674automated procedures. Methods Enzymol. 72, 296–300. 675

Marshall, D.W., Brey, R.L., Cahill, W.T., Houk, R.W., Zajac, R.A., 676Boswell, R.N., 1988. Spectrum of cerebrospinal fluid findings in 677various stages of human immunodeficiency virus infection. Arch. 678Neurol. 45, 954–958. 679

McArthur, J.C., Cohen, B.A., Farzedegan, H., Cornblath, D.R., Selnes, 680O.A., Ostrow, D., Johnson, R.T., Phair, J., Polk, B.F., 1988. Cere- 681brospinal fluid abnormalities in homosexual men with and without 682

Ž .neuropsychiatric findings. Ann. Neurol. 23, S34–S37 suppl . 683McArthur, J.C., McClernon, D.R., Cronin, M.F., Nance-Sproson, T.E., 684

Saah, A.J., Clair, M.S., Lanier, E.R., 1997. Relationship between 685human immunodeficiency virus-associated dementia and viral load in 686cerebrospinal fluid and brain. Ann. Neurol. 42, 689–698. 687

Price, R.W., 1996. Neurological complications of HIV infection. Lancet 688348, 445–452. 689

Reiber, H., 1996. Evaluation of blood cerebrospinal fluid barrier function 690and quantification of the humoral immune response within the central 691

Ž .nervous system. In: Thompson, E.J., Trojano, M., Livrea, P. Eds. , 692Cerebrospinal Fluid Analysis in Multiple Sclerosis. Springer, Milano, 693pp. 51–72. 694

Reiber, H., Felgenhauer, K., 1987. Protein tranfer at the blood cere- 695brospinal fluid barrier and the quantitation of the humoral immune 696response within the central nervous system. Clin. Chem. Acta 163, 697319–328. 698

Reiber, H., Lange, P., 1991. Quantification of virus-specific antibodies in 699cerebrospinal fluid and serum: sensitive and specific detection of 700antibody synthesis in brain. Clin. Chem. 37, 1153–1160. 701

Reiber, H., Ungefehr, S., Jacobi, C., 1998. The intrathecal, polyspecific 702and oligoclonal immune response in multiple sclerosis. Mult. Scler. 4, 703111–117. 704

Resnick, L., Di Marzo-Veronese, F., Schupbach, J., Tourtellotte, W.W.,¨ 705Ho, D.D., Muller, F., Shapshak, P., Vogt, M., Groopman, J.E.,¨ 706Markham, P.D., Gallo, R.C., 1985. Intra-blood–brain-barrier synthe- 707sis of HTLV-III-specific IgG in patients with neurological symptoms 708associated with AIDS or AIDS-related complex. N. Engl. J. Med. 709313, 1498–1504. 710

Resnick, L., Berger, J.R., Shapshak, P., Tourtellotte, W.W., 1988. Early 711penetration of the blood–brain-barrier by HIV. Neurology 38, 9–14. 712

Salden, H.J.M., Bas, B.M., Hermas, J.T.H., Janson, P.C.V., 1988. Analyt- 713ical performance of three commercially available nephelomethers 714compared quantifying protein in serum and cerebrospinal fluid. Clin. 715Chem. 34, 1594–1596. 716

Sande, M.A., Carpenter, C.C.J., Cobbs, C.G., Holmes, K.K., Sanford,

UNCORRECTED PROOF

( )E. Fainardi et al.rJournal of Neuroimmunology 00 2001 000–000 9717

J.P., 1993. Antiretroviral therapy for adult HIV-infected patients.718Recommendation from a State-of-the-Art Conference. JAMA 270,7192583–2589.720

Sindic, C.J.M., Monteyne, P., Laterre, E.C., 1994. The intrathecal synthe-721sis of virus-specific oligoclonal IgG in multiple sclerosis. J. Neuroim-722munol. 54, 75–80.723

Sonneborg, A.B., von Sydow, M.A.E., Forsgren, M., Strannegard, O.O.,¨ ˚7241989. Association between intrathecal anti-HIV-1 immunoglobulin G

725synthesis and occurrence of HIV-1 in cerebrospinal fluid. AIDS 3, 726701–705. 727

Tan, S.V., Guiloff, R.J., 1998. Hypothesis on the pathogenesis of vacuo- 728lar myelopathy, dementia, and peripheral neuropathy in AIDS. J. 729Neurol. Neurosurg. Psychiatry 65, 23–28. 730

¨Tibbling, G., Link, H., Ohman, S., 1977. Principles of albumin and IgG 731analyses in neurological disorders: I. Establishment of reference value. 732Scand. J. Clin. Lab. Invest. 37, 385–390.733