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Journal of Molecular Neuroscience Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/05/27:79–90/$30.00 DOI: 10.1385/JMN:27:01:79

ORIGINAL ARTICLE

HIV-1 Infection of Neurons Might Account for Progressive HIV-1-Associated Encephalopathy in Children Carmen Cantó-Nogués,1 Silvia Sánchez-Ramón,1 Susana Álvarez,1 César Lacruz,2 and Ma Ángeles Muñóz-Fernández*,1 1

Lab. Inmuno-Biología Molecular y 2Servicio de Anatomía Patológica, Hospital General Universitario Gregorio Marañón, Madrid, Spain Received August 25, 2004; Accepted February 8, 2005

Abstract Direct and productive infection of neurons in vivo is still a matter of debate, although in vitro experiments have demonstrated that immature neuronal cells can be productively infected by various human immunodeficiency virus (HIV) strains. To address this controversy we have analyzed, using light microscopy and in situ hybridization (ISH), HIV-1 infected cells in brain tissue from four pediatric cases of HIV-1-associated encephalopathy (EP). HIV-1 RNA-expressing cells—therefore, actively infected cells—were detected by ISH in different amounts in all brain specimens from the four children. They mainly correspond to glial cells. However, in two of the four children, who had severe progressive EP, but not in the other two, who had the static form, HIV-1infected neurons were clearly observed in the cortical brain samples. These results provide initial evidence that HIV-1 can actively infect neurons in vivo in children and show a cortical involvement of HIV brain infection in clear correlation with the clinical EP symptoms. DOI: 10.1385/JMN:27:01:79 Index Entries: Central nervous system; HIV-1; in situ hybridization; neurons; encephalopathy.

Introduction The frequency, spectrum, and severity of neurological impairment attributable to human immunodeficiency virus type-1 (HIV-1) infection are reported to be greater in children than in adults (Mintz, 1994; Tardieu et al., 2000). Human immunodeficiency virus type-1 (HIV-1) enters into the central nervous system (CNS) of children early in the course of infection, as suggested by the detection of HIV-1 and its antigens in the cerebrospinal fluid and brain tissue of patients without symptoms (Price et al., 1988; Lyman et al., 1990; Simpson, 1999). Central nervous system (CNS) infection by HIV-1 has been implicated in the pathogenesis of HIV-associated dementia in adults and

HIV-associated encephalopathy (EP) in children. Clinical symptoms of HIV-1-related progressive EP are severe neurodevelopment retardation with loss of acquired motor and cognitive milestones, cortical atrophy, and rare occurrence of opportunistic infections and neoplasms (Calvelli and Rubenstein, 1990; Belman et al., 1996). In contrast, HIV-1-associated static EP presents as fixed, nonprogressive neurological or neurodevelopmental deficits. Both progressive and static HIV-1-related EPs of childhood are different clinical entities, whose definite diagnosis requires histopathological confirmation, because of lack of specific clinical-pathological correlation. Although neuronal dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurological

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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80 disorders remains unclear. Compelling evidence from several laboratories demonstrated, by using immunolabeling (IML) of HIV-1 proteins and in situ hybridization (ISH) of HIV-1 nucleic acids, indicates that brain macrophages and microglial cells are the major HIV1 targets in the CNS of patients with HIV-1-related neurological disease (Gartner et al., 1986; Wiley et al., 1986; Vazeux et al., 1987; Kure et al., 1990; Dickson et al., 1994; Bagasra et al., 1996; An et al., 1999). Conversely, HIV-1 infections of neurons (Nuovo et al., 1994; Balluz et al., 1996; Torres-Muñóz et al., 2001; TrilloPazos et al., 2003) and astrocytes (Saito et al., 1994; Trillo-Pazos et al., 2003), which are cells of neuroectodermal origin, have been reported rarely. So far, proposed pathogenic mechanisms do not explain the differences in incidence and severity of CNS involvement in pediatric AIDS patients compared with adults (Ensoli et al., 1997). There is a clear discrepancy between the neuronal dysfunction clinically evident in pediatric patients and the inability to detect HIV-1-infected neurons in brain (Sharer et al., 1996). Arecent study of postmorten AIDS brains has demonstrated, in hippocampal neurons from paraffin-embedded microdissected tissue, that neurons in vivo contain HIV-1 DNA sequences consistent with a proviral or latent infection (Torres-Muñóz et al., 2001). However, there is no evidence of in vivo productive neuronal infection, as all IML studies have failed to disclose HIV-1 antigens within neurons and neuronal HIV-1 RNA has not been detected by standard ISH. One possible explanation for these negative results is that neither IML nor ISH techniques used until now are sensitive enough to show HIV-1 infection of low abundance or without HIV1 particle production (Takahashi et al., 1996). ISH is a useful method for the identification of cells containing only viral nucleic acid or when levels of HIV1 proteins are too low for IMLdetection. Nevertheless, the difficulty in obtaining brain samples from children with clinical manifestations of neuroAIDS and the lack of studies using sensitive and specific ISH methodology for detection of viral RNA might account for the controversy about productive or active HIV-1 infection in neurons. The purpose of this study was to employ a nonamplified ISH method, with single-stranded digoxigenin (DIG)-labeled DNA oligoprobes, to detect cells expressing HIV-1 RNA using paraffinembedded autopsy brain tissue from four pediatric cases of HIV-associated EP. Two children presented progressive EP occurring early and late in life as the first AIDS-related event, and the other two children

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Cantó-Nogués et al. had static EP and died due to sepsis. The CNS tissue of these children was analyzed for HIV-1 RNA to ascertain active infection with viral transcription and not, as in the majority of studies, to detect proviral DNA and quantify the number of infected cells (Bagasra et al., 1992; Embretson et al., 1993; Nuovo et al., 1993). The sensitivity and specificity of the ISH method were already proved in two previous studies for detection of viral RNA (Cantó-Nogués et al., 2001a). It was particularly important to avoid possible conflicting interpretations of the positive signal, which might be obtained from combining gene amplification by PCR with ISH (Nuovo et al., 1994; Sharer et al., 1996; Takahashi et al., 1996). The nonamplified ISH method allowed identification of the different cell types that expressed HIV-1 RNA, as well as determining their distribution and localization in the CNS. Previous autopsy brain studies for all four cases showed cortical and subcortical atrophy as the histopathological hallmark, and the ISH studies revealed, as expected, that brain macrophages and microglial cells were the main cells expressing HIV1 RNA in both cortical and subcortical regions. Furthermore, neurons expressing HIV-1 RNA were clearly detected in cortical regions in the two children with the progressive form of EP, indicating that a direct mechanism in addition to the indirect ones contributed to neuronal injury and damage in this AIDS-related neurological disorder.

Materials and Methods Patients and Brain Tissue Selection Four children born to HIV-1-infected mothers were followed from birth at the University General Hospital Gregorio Marañón (UGHGM), Madrid, Spain. All four children were diagnosed at about 2-mo of age as HIV-1 infected on the basis of positive results in proviral DNA PCR and coculture assays, as described previously (Muñóz-Fernández et al., 1996). Two of them had a clinical diagnosis of progressive HIV-1-associated EP, whereas the other two had static EP(American Academy of Neurology, 1991). One child who died from congenital cardiopathy and had no risk factor or evidence of HIV-1 infection was studied as control to compare with the former four cases. Formalin-fixed, paraffin-embedded brain specimens from all five children were obtained from the archives of the Department of Pathology at UGHGM. For each case, two different brain block specimens Volume 27, 2005

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In Vivo HIV-1-Infected Neurons in Children were collected, one from the cortical region and the other from the subcortical area or basal ganglia; all specimens included both gray and white matter.

Preparation of DIG-Labeled DNA Probes for ISH Single-stranded DNA probes labeled with DIG were prepared using asymmetric nested PCR, as described previously (Cantó-Nogués et al., 2001a, 2001b). Briefly, two pairs of oligonucleotide primers were used to amplify, by PCR, regions of the HIV-1 gag (HG1214N, 5’-GGT ACA TCA GGC CAT ATC ACC-3’; HG1686C, 5’-ACC GGT CTACAT AGT CTC3’) and env (HE6539N, 5’-GAG GAT ATA ATC AGT TTA TGG-3’; HE6976C, 5’-AAT TCC ATG TGT ACA TTG TAC TG-3’) genes. After purification and quantification of PCR products, approx 60 ng from the first-round amplification was transferred to a second round of 40 cycles of amplification, containing a single oligonucleotide primer and a dNTP mix with DIGlabeled dUTP (Roche Molecular Biochemicals). Two single-stranded probes were prepared from each product of the first-round amplification. Probes that were identical to sequences within HIV-1 transcripts—normal probes or N-probes—were generated by using primers HG1407N (5’-GAG GAA GCT GCA GAA TGG G-3’) and HE6560N (5’-GAT CAA AGC CTA AAG CCA TG-3’). Probes that were complementary to sequences of HIV-1 transcripts— complementary probes or C-probes—were obtained by using primers HG1646C (5’-GGT CCT TGT CTT ATG TCC AGAATG CTG-3’) and HE6876C (5’-CAA TAA TGT ATG GGA ATT GG-3’). Incorporation of DIG-labeled dUTP into each final product was assessed by end-point dilution using reagents supplied in the DIG nucleic acid detection Kit (Roche Molecular Biochemicals). ISH Sections of 5 m from wax-embedded brain blocks were mounted onto capillary gap slides (75 m) coated with an adhesive (Fisher HealthCare). Prior to hybridization, sections were dewaxed, rehydrated, and permeabilized with proteinase K before refixing and dehydrating in ethanol. Hybridization with DIG-labeled probes was performed using the MicroProbe Staining Station (Fisher Scientific, Pittsburgh, USA). Two different mixes of labeled probes were prepared and diluted up to 1:70 in hybridization buffer, as reported previously (Cantó-Nogués et al., 2001a,b): one mix with the two N-probes and other with the two C-probes. Consecutive sections from each specimen block were Journal of Molecular Neuroscience

81 incubated separately and allowed to hybridize with the N-probe mix or C-probe mix for 2 h at 37°C. Following hybridization, slides were washed twice in TBS buffer containing Triton X-100 and, finally, in TBS. The location of DIG-labeled probes was visualized using an alkaline phosphatase–conjugated sheep anti-DIG antibody (Roche Molecular Biochemicals) and NBT/BCIP solution (GIBCO-BRL, Life Technologies) as chromogenic substrate, following conditions recommended by the manufacturers. Cells with positive ISH were stained in dark brown–purple, corresponding to actively infected cells expressing HIV-1 RNA. No detectable hybridization was obtained in any brain section using hybridization buffer mix with N-probes (data not shown), confirming the specificity of signal obtained with C-probes. Five different experiments were performed for each brain region (cortical or subcortical) of every child to confirm the reproducibility of the results obtained by the ISH method. Figure 3 shows representative fields of each patient. To exclude the possibility of false-negative and positive results, a known positive control of HIV-1infected 8E5 cells and a negative control from a child who had no evidence of HIV-1 infection were included in each ISH experiment. All analyses of ISH experiments were performed by technicians without prior knowledge of specimen source.

Results Postmortem brain tissues from two different CNS regions, cortical and subcortical, from four children with the diagnosis of HIV-1-associated EP were studied by a nonamplified ISH method to identify which cells on the brain were actively infected in vivo with HIV-1 and to determine their relative localization within the CNS. One HIV-1 seronegative child who died from congenital cardiopathy within 2 mo was also studied by ISH as uninfected control and to compare with the four children with HIV-1-associated EP at the histopathological examination.

Clinical and Pathological Data The most relevant clinical characteristics are shown in Table 1. The choice of therapy for children, which was antiretroviral combined therapy and monotherapy with nucleoside analogs, was dependent on the Volume 27, 2005

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5 yr, 6 mo

Hypertonia, 6 mo exaltation of deep tendon reflexes, aquileal clonus

Hypertonia, 4 mo mild development retardation, mnicrocephalia

Abnormalgait, psychomotor regression

3 mo

Static HIVassociated EP

Static HIVassociated EP

Progressive HIVassociated EP

Progressive HIVassociated EP

Diagnosis

Others

N.P.

N.P.

Cortical atrophy of frontal predominance, leukoencephalopathy

Neuropathology findings

2 yr, 8 mo Major cortical atrophy, severe neuronal loss, perivascular calcification in basal ganglia

Age of exitus

ZDV

2 yr, 10 mo Major atrophy predominantly at white matter

ZDV, ddI 9 yr, 6 mo Major cortical atrophy, low neuronal density, perivascular micronodular calcification in basal ganglia

ZDV

Antiretroviral theraphy

Chronic otitis, ZDV, ddI, 1 yr, 4 mo Major atrophy pneumonia from P. d4T, predominantly at carinii, hepatitis B, ritonavir white matter enterococcal sepsis

CMV, hepatitis B and C, HIV-related cardiomyopathy, enterococcal sepsis

Poor postnatal growth, pneumonia from P. Carinii

Basal Cachectic ganglia with marked calcification, weight-growth cortical atrophy, delay, otitis ventricular with C. albicans, dilation CMV

Cranial CT

ZDV, zidovudine; CMV, cytomegalovirus; ddI, didesoxiadenosine; d4T, stavudine; N.P., not performed.

F

4

M

2

Hypertonia, irritability, psychomotor delay

Age at first neurological symptoms

01:11 pm

M

M

1

First neurological symptoms

01/07/2005

3

Sex

Case no.

Table 1 Pathological and Clinical Data of HIV-Associated EP in Study Subjects

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Fig. 1. The most frequently reported abnormality of pediatric AIDS is calcification of the basal ganglia and deep cerebral white matter. (A) Cranial CT of case 1, with bilateral microcalcifications in the lenticular nuclei. (B) Perivascular micronodular calcifications in a section of basal ganglia of case 2 brain tissue, counterstained with heamatoxylin and eosin (bar  50 m).

current guidelines when they were born, and both regimens have shown acceptable blood–brain barrier penetration (Groothuis and Levy, 1997; McGuire and Marder, 2000). Neuropathological findings at autopsy in the two cases with progressive EP disclosed evident neuronal loss with universal symmetrical ventricular dilation, widened sulci, and narrowed convolutions. These features were not so apparent in the two cases with static EP. All four showed vascular congestion and cerebral edema, as well as ischemic diffuse changes. Cranial computed tomography (CT) performed previously on case 1 demonstrated bilateral microcalcifications in the

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lenticular nuclei and a pattern of dilation of the ventricular system and cisternal spaces (Fig. 1a); with case 2, there was cortical atrophy of frontal predominance and leukoencepalopathy.

Histopathological Studies Histopathological examination of the brain sections of all four children revealed a lower density of neurons and some demyelinization with respect to the uninfected control. Neither microglial nodules nor multinucleated giant cells, characteristic features of adult neuroAIDS, were observed in the histopathological study with hematoxylin- and Volume 27, 2005

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Cantó-Nogués et al.

Fig. 2. Comparison of the binding of normal (A) and complementary (B) single-stranded DIG-labeled DNA oligoprobes to formalin-fixed HIV-1-infected 8E5 cells (bar  25 m).

eosin-stained slides. Perivascular micronodular calcifications were observed in sections from the basal ganglia of case 2. (Fig. 1B).

ISH A nonamplified ISH protocol was established to localize HIV-1 RNA-expressing cells within brain tissue. This protocol detects viral transcripts and viral RNAand not double-stranded HIV-1 DNA. The probes were tested, prior to the analyses of brain sections, on formalin-fixed HIV-1-infected 8E5 cells. No detectable hybridization was obtained on 8E5 cells using N-probes (Fig. 2A), whereas a strong hybridization signal on a large number of cells was obtained with C-probes (Fig. 2B).

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In situ hybridization (ISH)-positive stained cells, which reflected HIV viral transcription or active infection, were found in all brain specimens from the four HIV-1-infected children (Fig. 3A–D). Morphology of the positive cells revealed that the majority of infected cells were brain macrophages or microglial cells. They were detected in both cortical and basal ganglia brain specimens. No positive stained cells were detected using C-probes in brain sections from the uninfected child (Fig. 3E). Intriguingly, the two children with progressive EP (cases 1 and 2) had HIV-1-infected neurons in the gray matter of cortical specimens (Fig. 3A,B.1,B.2), but not in basal ganglia specimens (data not shown). Not all positive neurons had the same stained appearance;

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85 some showed cytoplasmic dark brown–purple dots in a paranuclear location (Fig. 3A), whereas others presented their cytoplasm partly or almost completely stained (Fig. 3B.1,B.2), indicating different levels of HIV-1 RNA expression. Similar patterns of staining were found on ISH-positive brain macrophages and microglial cells (Fig. 3A,C). In the two cases with static EP, mild neurological symptoms and minimal pathological evidence of neurological disease (cases 3 and 4) were noted, and no HIV-1-positive neurons were detected (Fig. 3C,D).

Discussion Identification of the specific cells susceptible to HIV-1 infection in the CNS has been a matter of controversy for a long time. In this study we aimed to avoid the risk of false-positive cells from amplification of the DNA or RNA target and the possible diffusion of amplicon with resulting inaccurate localization of ISH-positive signal (Nuovo et al., 1994; Sharer et al., 1996; Takahashi et al., 1996). On the other hand, with the direct ISH method, false negatives could arise because of insufficient or no RNA within cells of brain specimens owing to degradation by postmortem autolysis or tissue processing. However, other studies suggest

Fig. 3. HIV-1 active infection detected by ISH in the cortical brain tissue of four children with HIV-1-related EP. Cells that hybridized to a mix of two single-stranded, DIG-labeled oligoprobes, complementary to regions of the HIV-1 gag and env genes, were stained brown-purple by the enzymatic conversion of the NBT/BCIP chromogenic substrate. Brain tissue was counterstained with neutral red. Solid arrowheads point to positive neurons; open arrowheads point to positive brain macrophages or microglial cells. (A) Case 1: dark brown–purple dots on the cytoplasm in paranuclear location of positive neurons indicate HIV-1 viral transcription. (B) Case 2: positive neurons by ISH showing different degrees of staining that might indicate different levels of HIV-1 expression; (B.1) cytoplasm partly stained with dark brown–purple, or (B.2) almost completely stained. (C) Case 3: ISH-positive brain macrophages and microglial cells are detected showing different level of HIV-1 expression but no staining of neurons. (D) Case 4: a few ISH-positive microglial cells. (E) Uninfected control from a child who had no evidence of HIV-1 infection: No positive stained cells were detected using C-probe hybridization mix. All micrographs are at the same magnification (bar  25 m).

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86 that low-molecular-weight nucleic acids in the form of RNA are well preserved in paraffin-embedded tissue samples (Delabie et al., 1996). The specificity of the ISH signal was confirmed by absence of positive ISH cells in the uninfected brain tissue and in all specimens that were incubated with the N-probe hybridization mixture. The majority of HIV-1 infected-cells that were detected by direct ISH method in the brain samples of four children with HIV-1-associated EP corresponded to macrophages and microglial cells, as has been described by several other laboratories in biopsies from HIV-1-infected adults. The remarkable finding of this work is that the two children with progressive HIV-1-related EP, defined by marked cortical atrophy, perivascular brain calcification, and demyelinization, had a small but reproducible number of neurons positive for HIV-1 RNA, clearly suggestive of active infection in those cells. To determine that HIV was in neurons, we e performed an immunocytochemistry study with specific neuronal and glial markers on sequential slices of the same cerebral regions used in ISH. The results obtained showed that positive HIV signal was both in glial cells and in neurons (data not shown). It is tempting to speculate that this fact might contribute to the clinical manifestations and the brain dysfunction observed in these two children who died because of HIV-1-related EP. Thus, the active infection of neurons might be a factor in HIV-1-induced neuropathogenesis in children, especially in the development of the progressive form of EP, the most devastating and severe complication in childhood AIDS. We have found infected neurons in two children representing early and late neurological manifestations in life, probably reflecting in utero and intrapartum infection, respectively, which indicate more similarities in progressive HIV-1associated EP for infants and children than reported previously (Tardieu et al., 2000). It is remarkable that the two cases in which no infected neurons were detected and only a few brain macrophages and microglial cells were HIV-1 RNA positive corresponded to children with diagnosis of the static form of EP. Thus, our results suggest that the rate of HIV-1-infected cells in the CNS (Nuovo et al., 1994), together with the presence of actively infected neurons, might therefore correlate with the degree of clinical manifestations of HIV-1-associated EP and its presentation. Human immune deficiency virus type-1 (HIV1)-associated EP is the most common major CNS complication in children, having devastating

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Cantó-Nogués et al. consequences for neuropsychological development and being life-threatening (Tardieu et al., 2000). Differences between pediatric and adult neuroAIDS might depend on potential HIV interactions with both the developing immune and nervous systems in children (Muñóz-Fernández and Fresno, 1998). Immature neuronal and glial cells, which are present during fetal development and early postnatal life (Ensoli et al., 1997), might be more susceptible to HIV-1 infection than their mature counterparts. In addition, the higher viral load titers usually present in infants could favor latent, as well as productive, infection of immature cells of the CNS (Gurbindo et al., 1999). Neuronal injury of HIV-1-related neurological disorders might arise from neurotoxins released from activated or infected monocytes and macrophages or from exposure to cytotoxic HIV-1 proteins (gp120, tat, vpr), or it might be secondary to HIV-1-related astrocyte dysfunction (Lipton, 1991; Nath and Geiger, 1998; Brack-Werner, 1999; Kaul et al., 2001). However, direct HIV infection of neurons is another potential mechanism for neuronal injury or death. Support for this hypothesis lies in the fact that fetal human neurons, as well as neuronal cell lines, show a transient productive HIV infection in vitro (Li et al., 1990; Ensoli et al., 1995; Obregon et al., 1999; Álvarez Losada et al., 2002) and that human brain neurons express high levels of HIV chemokine coreceptors in vivo (Lavi et al., 1997; Rottman et al., 1997). Taking into account the general uniformity in the clinical picture of HIV-associated EP in children, the topographical differences (cortical vs. subcortical) in the location of HIV-1-infected neurons found in this study could reflect a true neurotropism of HIV-1, which might add important clues to understanding HIV-1 neuropathogenesis. Our data might also indicate a greater susceptibility of cortical neurons to HIV-1 in the developing CNS than neurons in other locations, as HIV-1 is widespread and encountered throughout the brain in the monocyte-macrophage and microglia cell lineage. This permissiveness for viral entry into a certain group of neurons might be related with a different level of expression of CXCR4 and CCR5 HIV chemokine coreceptors that might confer a differential vulnerability to these cells. Clinical and neuropathological data indicate that HIV dementia in adults is principally subcortical in origin. Cerebrovascular changes and atrophy are frequently seen in basal ganglia, both in adult and

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In Vivo HIV-1-Infected Neurons in Children pedriatic neuroAIDS. Although it is difficult to ascertain cortical from subcortical dementia at neurological examination in children, motor aphasia, language disturbances, and loss of cognitive functions are characteristic cortical signs, whereas the involvement of the pyramidal pathway might result from a disruption at any point between motor cortex and anterior spinal horn cells. The clinical presentation of pediatric EP usually shows a cortical pattern. The present study indi´ pattern of HIV-1 infeccates a different topographic tion in the brains of children to the previously reported subcortical pattern found in adults. Moreover, in both children with progressive EP, a predominantly cortical involvement was observed both clinically and by neuroimaging. The selective cortical location of active HIV-1 infection in neurons indicates a direct effect of HIV-1, which might contribute to cortical dysfunction, in addition to indirect mechanisms. Our findings are consistent with data reported previously on correlation between cerebrospinal fluid viral load and cortical atrophy measured by neuroimaging (Brouwers et al., 2000).

Conclusions In conclusion, we have clearly demonstrated the presence of HIV-1 RNA in neurons as an unequivocal sign of viral transcription and active infection. Also, actively infected neurons were specifically localized in cortical areas of the CNS in children with progressive HIV-1-related EP, which indicates direct neuronal involvement in the clinical picture. This evidence of active HIV-1infected neurons in vivo needs to be followed by further studies on the possible role of neurons in the pathogenesis of HIV-1-related neurological disorders.

Authors’ Contributions C. C. N. conceived of the study and performed most of the work and the image analysis; S. S. R. assisted (substantially) in writing the manuscript and participated in image analysis; S. A. contributed substantially to the experiments and discussion of the results; C. L. contributed in the discussion of the results; and M. A. M. F. participated in the design and coordination of the study, performing the final data analyses.

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Acknowledgments We are indebted to José Manuel Lara for providing brain sections, Lola Garcia for excellent technical assistance, and Dr. David Hockley for critical review of the manuscript. We are grateful to Drs.Dolores Gurbindo and Caridad Garzo for follow-up of children. This work was supported by grants from Fondo de Investigación Sanitaria (00/0207), Programa Nacional de Salud (SAF 0309209), Comunidad de Madrid (08.5/0019/98), “Red Temática Cooperativa de Investigación en SIDA” (RIS G03/173) del FIS, Fundación para la Investigación y la Prevención del SIDA en España (36365/02).

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