Identification of shared populations of human immunodeficiency virus type 1 infecting microglia and tissue macrophages outside the central nervous system.

Infection of microglia and other cells of the macrophage/monocyte lineage in the central nervous system (CNS) by human immunodeficiency virus type I (HIV-1) underlies the development of giant cell encephalitis (GCE). It is currently unknown whether GCE depends on the emergence of virus populations specifically adapted to replicate in cells of the monocyte/macrophage lineage and whether this also leads to the specific targeting of macrophages in other nonlymphoid tissues. Autopsy samples from lymph node, brain (frontal region), lung, and full-thickness colon sections were obtained from nine study subjects with GCE and from nine without. The two groups showed no significant differences in CD4 counts, disease progression, or treatment history before death. Genetic relatedness between variants recovered from lymph node and nonlymphoid tissues was assessed by sequence comparison of V3 and p17(gag) regions using a newly developed method that scores the sample composition at successive nodes in a neighbor-joining tree. The association index enabled objective, numerical comparisons on the degree of tissue compartmentalization to be made. High proviral loads and p24 antigen expression in the brain were confined to the nine individuals with GCE. GCE was also associated with significantly higher proviral loads in colon samples (median of the GCE(+) group: 1,010 copies/10(6) cells; median of GCE(-) group, 10/10(6) cells; P = 0.006). In contrast, there were no significant differences in proviral load between the GCE(+) and GCE(-) groups in lymph node or lung samples, where HIV infection was manifested predominantly by infiltrates of lymphoid cells. V3 sequences from brain samples of individuals with GCE showed the greatest compartmentalization from those of lymph node, although samples from other tissues, particularly the colon, frequently contained variants phylogenetically related to those found in brain. The existence of shared, distinct populations of HIV specifically distributed in cells of the monocyte/macrophage lineage was further indicated by immunocytochemical detection of CD68(+), multinucleated giant cells expressing p24 antigen in samples of lung and colon in two individuals with GCE. This study provides the basis for future investigation of possible phenotypic similarities that underline the shared distributions of HIV variants infecting microglia and tissue macrophages outside the CNS.

Influence of risk group and zidovudine therapy on the development of HIV encephalitis and cognitive impairment in AIDS patients.

OBJECTIVE: To determine the associations between HIV encephalitis and other central nervous system (CNS) pathology, viral burden, cognitive impairment, zidovudine therapy and risk group in AIDS patients. DESIGN: Planned autopsy study in AIDS patients evaluated prospectively for numerous clinical parameters. SETTING: Regional academic centre for clinical care and pathology examination of patients with HIV infection. PATIENTS: Edinburgh cohort of HIV-positive patients prospectively assessed for cognitive impairment, immunosuppression and clinical course. Unbiased series of consecutive autopsies in 27 homosexual men and 39 drug-using patients with AIDS. INTERVENTIONS: Zidovudine therapy monitored in all patients. MAIN OUTCOME MEASURES: Determination of CNS viral burden and pathology including immunocytochemically confirmed HIV encephalitis in injecting drug users (IDU) versus homosexual AIDS patients with known CD4 counts and cognitive function. RESULTS: HIV encephalitis was present in 59% of IDU and 15% of homosexuals: 88% of patients with encephalitis had displayed cognitive impairment. HIV encephalitis was strongly associated with a high viral load and HIV p24 immunopositivity. Opportunistic infections and lymphomas were more common in homosexuals (63%) than in IDU (31%) and were associated with the degree of immunosuppression before death. Within both groups, prolonged zidovudine treatment was associated with a lower incidence of HIV encephalitis. CONCLUSIONS: This study documents two separate CNS outcomes in AIDS patients in that HIV encephalitis occurs independently of opportunistic infections and lymphomas and shows different associations with risk group, immunosuppression and antiviral treatment before death.

In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage-tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS.

Redistribution of HIV outside the lymphoid system with onset of AIDS.

The basis for many of the symptoms and pathological changes found in patients with the acquired immunodeficiency syndrome (AIDS) remains poorly understood. We have used a quantitative polymerase chain reaction technique to investigate the extent to which direct infection with human immunodeficiency virus (HIV) produces the disease manifestations of AIDS. In five patients who died with AIDS-defining illnesses (Centers for Disease Control and Prevention class IV), we found variable, but in many cases extensive, infection by HIV at various sites, including brain, lung, colon, and liver. By contrast, in three HIV-positive subjects who died without HIV-related disease (CDC class II), we found no evidence of significant infection of any non-lymphoid organ. In both groups of patients there were high levels of infection in cells of the spleen, lymph nodes, and peripheral blood. Pathological examination of tissues from the AIDS patients revealed many abnormalities, of which some, such as giant-cell encephalitis in the brain, were specifically associated with the presence of high levels of HIV infection. These findings suggest that spread of HIV outside cells of the immune system is a late event in HIV infection and is extremely sensitive to the degree of immunosuppression in the patient.

PCR analysis of the upstream regulatory region of human papillomavirus genomes in cervical intraepithelial neoplasia and cervical carcinoma.

AIMS: To test whether human papillomavirus (HPV) variants with large scale sequence alterations to the upstream regulatory region are present in cervical intraepithelial neoplasias (CIN) and cervical carcinomas. METHODS: New PCR based assays were designed specifically to detect large scale insertion/deletion alterations in the upstream regulatory region of HPV 16 and 18. The assays were applied to 24 cases of CIN and 34 cases of cervical carcinoma previously shown to contain these two high risk HPV types. RESULTS: No large scale sequence alterations were found in any of the HPV containing CIN or carcinomas. CONCLUSIONS: These negative findings suggest that major upstream regulatory region variants of HPV 16 and 18 do not contribute to most cervical neoplasms.

Human immunodeficiency virus and the brain: investigation of virus load and neuropathologic changes in pre-AIDS subjects.

Brain tissue was examined for evidence of human immunodeficiency virus (HIV) infection in 23 intravenous drug users who died suddenly some years after seroconversion but while still in presymptomatic stages of infection. None showed giant cell encephalitis, but 14 showed T cell lymphocytic leptomeningitis and 3 showed other significant neuropathologic features. Quantitative polymerase chain reaction for HIV was applied to 13 of the 23 with negative results in 6 and very low positive results in the other 7, a finding consistent with contamination by residual infected blood in the brain tissue. This contrasted with findings in AIDS-infected tissue, in which substantial amounts of provirus were found. It is concluded that significant infection in brain tissue does not occur in presymptomatic stages of HIV infection and that invasion of the central nervous system may be delayed until the transition to symptomatic AIDS.

A PCR approach to discriminate between integrated and episomal HPV DNA in small clinical specimens.

HPV infection has long been implicated in the development of cervical carcinoma. There is strong evidence for association of high-risk HPV types 16 and 18 with cervical intraepithelial neoplasia (CIN) grades 2 and 3, and integration of viral DNA of these types into the host genome has been suggested to play an important role in progression to invasive cervical carcinoma. However, the existing techniques for detection of integrated DNA in clinical specimens are time-consuming and require large amounts of template DNA, often unavailable for small premalignant CIN lesions. In this study, a novel, two stage PCR assay was designed to discriminate between integrated and episomal HPV 16 DNA. The first stage was designed to determine whether intact HPV genomes were present. This initial PCR analysis of the entire viral genome in eight segments was successfully applied to authentic human cervical cancers. The second stage consisted of an ANCHOR-PCR-based analysis, developed specifically to discriminate between integrated and episomal HPV DNA, that was successfully tested with cloned HPV containing plasmids used to mimic both episomal and integrated viral DNA. Further optimization and validation will be required for application of the second stage to clinical specimens. The entire assay was developed to be applicable to small colposcopic biopsies or cervical scrape samples, representative of those acquired in routine clinical investigation of CIN 2 or CIN 3, in which determination of the physical state of HPV DNA may provide prognostically valuable information.

Human papillomavirus type 18 associates with more advanced cervical neoplasia than human papillomavirus type 16.

A type-specific, sensitive, polymerase chain reaction-based assay for human papillomavirus (HPV) types 6b, 11, 16, 18, and 33 was applied to 47 cervical carcinomas, 60 cases of cervical intraepithelial neoplasia (CIN), and 24 samples of histologically normal cervix. As expected, the combined incidence of the common high-risk genital HPVs (types 16 and 18) was high in carcinomas (79%) and CIN 2/3 (60%), low in CIN 1 (25%), and nonexistent in the normal controls. Analysis of the data by viral type and pathology revealed statistically significant differences that consistently pointed to an association of HPV 18 with more advanced disease than HPV 16. This was exemplified by calculation of the relative HPV frequency in squamous cancers and CIN 2/3 lesions, which gave cancer to CIN prevalence ratios of 1.2 for HPV 16 and 2.3 for HPV 18, a twofold difference suggesting the possibility that there is a greater risk of progression or a more rapid transition to malignancy associated with HPV 18. Furthermore, HPV 16 was associated with 2.5-fold more cancers showing squamous differentiation (58%) than HPV 18 (23%), but both types showed an identical prevalence of 41% in the clinically more sinister adenocarcinomas, indicating that there may be an association between HPV type and cancer cell differentiation.

HPV in full thickness cervical biopsies: high prevalence in CIN 2 and CIN 3 detected by a sensitive PCR method.

A new type-specific, sensitive, non-radioactive assay is described for the detection of human papillomavirus (HPV) DNA in tissues. Sequences within the E6 gene were amplified by the polymerase chain reaction (PCR), using primer pairs which clearly distinguish HPV types, including those with close sequence homology such as 6b and 11. The amplified DNA products were identified by non-radioactive oligonucleotide hybridization and restriction endonuclease mapping, and the method was sufficiently sensitive to detect between 3 and 5 SiHa cells (each containing 1-2 copies of HPV 16 DNA) amongst 10,000 non-HPV-containing cells. Frozen and archival paraffin sections were equally acceptable substrates for the reaction. The assay was applied to frozen sections of full thickness cervical epithelium from 60 cases of cervical intraepithelial neoplasia (CIN) and 24 normal cervical controls. HPV DNA was detected in 60 per cent of cases of CIN 3 and CIN 2, in 25 per cent of cases of CIN 1, and in none of the normal controls. Prevalence of HPV 16 was similar (approximately 50 per cent) in both CIN 2 and CIN 3, and in the whole series HPV 16 was almost five-fold more common than HPV 18. Low-risk HPV types were present in 5 per cent of CIN 1, but 0 per cent of CIN 2 and CIN 3 biopsies. The data emphasize the biological similarity of CIN 2 and CIN 3 lesions, and their divergence from CIN 1.