Detection and typing of human papillomaviruses in mucosal and cutaneous biopsies from immunosuppressed and immunocompetent patients and patients with epidermodysplasia verruciformis: a unified diagnostic approach.

AIM: To develop a unified diagnostic approach for the detection of human papillomavirus (HPV) DNA in skin and mucosal biopsies from both immunocompetent and immunosuppressed individuals using a degenerate polymerase chain reaction (PCR) method. METHODS: The sensitivity and specificity of three published degenerate primer sets (HVP2/B5 and F14/B15; MY09/MY11; CP62/69 outer and CP65/68 nested primer pairs) were evaluated in PCR reactions with serial dilutions of 12 representative cloned HPV types. This combination of primers was then used to detect HPV DNA in 49 benign and malignant lesions of cutaneous and mucosal origin from immunosuppressed, immunocompetent, and epidermodysplasia verruciformis (EV) patients, and compared with detection rates using single primer sets alone. RESULTS: The observed sensitivity of MY09/MY11 and CP62/69 + CP65/68 was high for mucosal and EV HPV types, respectively. The sensitivity of all primer sets for cutaneous types was low, but nonetheless the use of this combination of primers allowed HPV DNA detection in all of the benign warts analysed. Several mixed infections were also identified. A high prevalence of HPV DNA was similarly detected in squamous cell carcinomas from immunocompromised patients; the HPV types found were exclusively EV related. CONCLUSIONS: The use of a combined degenerate primer PCR approach considerably improves HPV DNA detection over individual primer sets and allows detection of mixed infections. The findings may help explain the discrepancies in published reports relating to HPV DNA detection in benign and malignant skin lesions. Further modifications to this method are in progress which should significantly improve comprehensive HPV detection and typing for diagnostic purposes.

Retention of low copy number human papillomavirus DNA in cultured cutaneous and mucosal wart keratinocytes.

Cultured wart keratinocytes have previously been described as having a limited proclivity to maintain episomal human papillomavirus (HPV) DNA. To investigate the nature of episome loss, and to determine keratinocyte-specific factors involved in it, we have examined a large series of anogenital and oral wart keratinocyte cultures, tracing episomal copy number with culture passage. We report that a higher proportion of oral wart keratinocytes maintain episomal HPV DNA to first passage (70% compared with 37% of anogenital wart cultures) when screened by slot blot hybridization. Furthermore, oral wart keratinocytes maintain episomal HPV copy through a greater number of passages (60% positive at passage 2 compared with 2% of anogenital wart cultures) with this technique. When anogenital cultures were examined at first passage for HPV infection by PCR with Southern blot hybridization of the product, a further 34% were found to be HPV-positive. To determine the mechanism of loss of episomal DNA from these cultures we examined the relative HPV copy number in cells which adhered to the culture vessel following passage and in those which did not adhere. Those which remained floating contained episomal HPV at high copy number whereas those which adhered were negative by slot blotting. The adherent cells, however, remained positive by PCR at subsequent passages until senescence. We conclude that a subpopulation of HPV-positive keratinocytes may be maintained in culture through serial passage until senescence.

Bladder and prostate cancer screening for human papillomavirus by polymerase chain reaction: conflicting results using different annealing temperatures.

Two sets of L1 ORF degenerative primers, GP5/6 and MYO9/11, have been used to screen for human papillomavirus (HPV) sequences in bladder tumours, cell lines and controls by polymerase chain reaction (PCR). None of the 14 bladder and prostate tumours or nine bladder cell lines contained HPV sequences when tested with L1 ORF primer pair GP5/6 at 40 degrees C annealing temperature. In contrast, use of the L1 ORF primer pair MY09/11 at this low annealing temperature consistently gave a 450 bp band, suggesting the presence of HPV. This occurred in all samples including the negative DNA controls. An increase in stringency to an annealing temperature of 55 degrees C resulted in an elimination of this band in the test and negative control samples. This finding may explain why there are contradictory reports in the literature, and further studies are in progress to clarify this issue.

Antibody-dependent cell-mediated cytotoxicity: comparison between HTLV-I and HIV-1 assays.

This study confirms the presence of detectable antibody-dependent cell-mediated cytotoxicity (ADCC) towards both HTLV-I- and HIV-1-infected cell lines, mediated by normal donor peripheral blood mononuclear cells and either by antibody from adult T-cell lymphoma and tropical spastic paraparesis patients (HTLV-I) or by antibody from sera of patients with persistent generalized lymphadenopathy, AIDS-related complex, AIDS and asymptomatic patients seropositive for HIV-1 infection. A comparison of ADCC towards these two retroviruses, under carefully controlled laboratory conditions, indicates major differences between the capacity of HTLV-I-seropositive sera and HIV-1-seropositive sera to mediate ADCC. In all cases, HIV sera showed low-titre ADCC, in contrast to the high titre (greater than 1:800,000) ADCC mediated by HTLV-I-positive sera. Both sets of sera showed the prozone phenomenon, and heat inactivation may abolish ADCC towards HIV-1-infected cells. Quantitation of surface antigen expression on HTLV-I- and HIV-1-infected cell lines indicated the presence of easily detectable amounts of virus-specific antigen. We conclude that, in contrast to some previous reports, ADCC mediated by HIV-1-specific immunoglobulin G (IgG) antibody is rather weak and of low titre when compared with HTLV-I ADCC. This is true for all cell lines and HIV-1 virus isolate combinations tested.

A search for retrovirus infection in systemic lupus erythematosus and rheumatoid arthritis.

Evidence for retroviral infection in general and human immunodeficiency virus (HIV) infection in particular was sought in freshly isolated peripheral blood T cells, B cells, and monocyte-macrophages from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and also in T cell and B cell lines established from the same source. Similar cells isolated from rheumatoid synovial membrane were also examined. The strategy used for the detection of virus was cocultivation with susceptible cell lines looking for syncytia formation, reverse transcriptase production, and nucleic acid hybridisation with HIV cDNA probes. No evidence for infection was obtained.