Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification.

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.

Lack of granzyme expression in T lymphocytes indicates poor cytotoxic T lymphocyte activation in human papillomavirus-associated cervical carcinomas.

Changes in major histocompatibility complex (MHC) class I expression in neoplastic cells are frequently observed in human papillomavirus type 16 (HPV16)-associated cervical carcinomas. In order to investigate whether this affects cytotoxic T-cell (CTL) activation, 20 HPV16-positive cervical carcinomas with variable MHC expression were analyzed immunohistochemically for the expression of granzyme B and interleukin-2 receptor (IL-2R). The results revealed that most carcinomas show strong CD3+ T-lymphocyte infiltrates. In nine of these cases CD8+ cells outnumbered the CD4+ cells, whereas in the remaining cases equal amounts of CD4+ and CD8+ cells were found. Double staining revealed that CD3+ granzyme B+ cells were not detected in 19 out of 20 cervical lesions, whereas in one carcinoma an occasional cluster of granzyme B+ T cells was observed. Positive controls, including genital warts and renal allograft rejections, showed granzyme B+ T cells. In agreement with this observation was the extremely low frequency of IL-2R+ T cells in the carcinomas tested, while warts contained IL-2R+ lymphocytes. The data indicate that cytotoxic (CD8+) T cells in cervical carcinomas are not activated, as demonstrated by the lack of granzyme B and IL-2R expression in the T cells.

Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression.

The development of cervical carcinoma is strongly associated with specific types of human papillomaviruses (HPVs). A role for cellular immunity in cervical disease is supported by the increased occurrence of HPV-associated lesions in immunosuppressed individuals. Upon viral infection or malignant transformation, ensuing alterations in gene expression result in the generation of novel sets of peptides which can form complexes with specific HLA class I heavy chains and beta 2-microglobulin. These are then expressed at the cell surface as potential targets for specific T cells. In this study of 100 carcinomas HLA-A and -B class I expression by the tumour cells was down-regulated at one or more alleles in at least 73% of cervical carcinomas. Interference with the transporter associated with antigen presentation (TAP), which translocates cytosolic peptides from endogenously synthesised proteins (e.g. viral) into the lumen of the endoplasmic reticulum was found in 38% of the HLA class I down-regulated tumours. Loss of expression for common HLA class I alleles ranged from 36% to 71%, and such changes might be expected to influence specific immunogenic peptide presentation and consequent immune recognition. These results underline the importance of single as well as multiple allelic loss in cervical neoplasia and have important implications for attempts to intervene immunologically in cervical cancer.

Differences in MHC and TAP-1 expression in cervical cancer lymph node metastases as compared with the primary tumours.

In previous studies we have shown down-regulation of class I major histocompatibility complex (MHC) expression in a significant proportion of primary cervical carcinomas, which was found to be strongly correlated with loss of expression of the transporter associated with antigen presentation (TAP). By contrast, class II MHC expression was frequently up-regulated on neoplastic keratinocytes in these malignancies. In order to investigate whether these changes are associated with biological behaviour of the tumours, 20 cervical carcinomas were analyzed for MHC (HLA-A, HLA-B/C, HLA-DR) and TAP-1 expression in the primary tumours and in lymph node metastases by immunohistochemistry. The results showed a significant increase in the prevalence of HLA-A and HLA-B/C down-regulation in metastasised neoplastic cells as compared with the primary tumour (P = 0.01). In all cases this was accompanied by loss of TAP-1 expression. Up-regulated HLA-DR expression was found exclusively in primary tumours and was absent in the corresponding metastases (P = 0.002). These data are consistent with the hypothesis that loss of TAP-1 and the consequent down-regulation of class I MHC expression provides a selective advantage for neoplastic cervical cells during metastasis. Furthermore, the lack of class II MHC expression in metastasised cells either reflects a different local lymphokine production or indicates that these cells may have escaped CD4+ cytotoxic T-lymphocyte (CTL)-mediated killing.

Loss of transporter protein, encoded by the TAP-1 gene, is highly correlated with loss of HLA expression in cervical carcinomas.

Malignant tumor cells can escape CD8+ cytotoxic T cell killing by downregulating class I major histocompatibility complex (MHC) expression. Stable class I MHC surface expression requires loading of the heavy chain/light chain dimer with antigenic peptide, which is delivered to class I MHC molecules in the endoplasmic reticulum by the presumed peptide transporter, encoded by the transporter associated with antigen presentation (TAP) 1 and 2 genes. We have investigated whether loss of class I MHC expression frequently observed in different cancers could result from interference with TAP function. A polyclonal antiserum, raised against a bacterial glutathione S-transferase/human TAP-1 fusion protein, was used for the immunohistochemical analysis of TAP-1 expression in 76 cervical carcinomas. Results showed loss of TAP-1 expression in neoplastic cells in 37 out of 76 carcinomas. Immunohistochemical double staining procedures in combination with HLA-specific antibodies revealed congruent loss at the single cell level of TAP-1 and HLA-A/B expression in 28 out of 37 carcinomas. The remaining samples expressed HLA(-A) in the absence of TAP-1 (n = 6) or showed loss of HLA(-A/B) while TAP-1 was expressed (n = 3). These data strongly indicate that inhibition of peptide transport by downregulation of TAP-1 is a potential strategy of malignant cells to evade immune surveillance.

MHC class I expression in HPV 16 positive cervical carcinomas is post-transcriptionally controlled and independent from c-myc overexpression.

Squamous cell carcinomas of the uterine cervix (n = 23) were selected for the presence of human papillomavirus type 16 (HPV 16) using the polymerase chain reaction (PCR). Localization of transcripts coding for the E7 protein was demonstrated in neoplastic cells with RNA in situ hybridization. Consecutive tissue sections were investigated for expression of the major histocompatibility complex class I (MHC-I) and c-myc using immunohistochemical double staining procedures, since a role has been suggested for the c-myc protein in MHC-I down-regulation and c-myc overexpression has been described in cervical carcinomas. Reduced expression of class I heavy chains was observed in neoplastic cells from 18 out of 23 carcinomas (78%). Varying levels of c-myc overexpression were observed in 12 carcinomas (52%), from which four showed positive MHC-I expression in c-myc overexpressing cells. In the remaining eight c-myc overexpressing carcinomas MHC-I down-regulation was observed. Additional RNA in situ hybridization with class I heavy chain locus-specific RNA-probes revealed presence of class I mRNAs in those neoplastic cells that show negative staining for MHC-I protein. These data strongly indicate that MHC-I down-regulation in cervical carcinomas involves post-transcriptional mechanisms, not directly related to E7 transcription and overexpression of c-myc.

Analysis of MHC class I and II expression in relation to presence of HPV genotypes in premalignant and malignant cervical lesions.

Cervical intraepithelial neoplasia (CIN) grades I to III lesions (n = 94) and squamous cell carcinomas of the uterine cervix (n = 27) were analysed for MHC class I and II expression and presence of HPV genotypes. MHC class I and II expression was studied by immunohistochemistry and HPV typing was performed by general primer- and type-specific primer mediated PCR (GP/TS PCR). Both techniques were performed on paraffin embedded tissue sections. Results show disturbed MHC class I heavy chain expression in CIN I to CIN III, as well as in cervical carcinomas. Upregulated MHC class II expression on dysplastic epithelial cells was also found in the different CIN groups and carcinomas. Prevalence of HPV genotypes increased with the severity of the lesion, mainly due to the contribution of the HPV types 16 and 18. No correlation could be established between the presence of specific HPV genotypes and any MHC expression pattern in the different CIN groups or cervical carcinomas. In some cases these data were confirmed by RNA in situ hybridisation showing HPV 16 E7 transcripts in the same dysplastic/neoplastic cells from which MHC status was determined. The results indicate that local differences may exist in the type of cellular immune response to HPV induced lesions.

Prevalence and expression of human papillomavirus in tonsillar carcinomas, indicating a possible viral etiology.

The presence of human papillomavirus (HPV) DNA was assessed in biopsies of tonsillar carcinomas (n = 10) and cases of tonsillitis (n = 7), serving as controls, by general-primer-mediated PCR (GP-PCR). All carcinomas appeared HPV-positive, whereas all cases of tonsillitis were HPV-negative. Additional type-specific PCR for HPV 6, 11, 16, 18, 31 and 33 revealed that 4 carcinomas contained HPV 16 DNA, 4 contained HPV 33 DNA and 1 contained an HPV 16/33 double infection. False positivity was excluded by additional Southern blot analysis of type-specific PCR-positive samples (n = 4). Further characterization of GP-PCR products by sequence analysis revealed that 2 carcinomas contained still uncharacterized HPV genotypes; one of these also contained HPV 33 DNA and one was negative by type-specific PCR. Application of RNA PCR revealed expression of HPV 16 or HPV 33 E7 encoding spliced E6*1 transcripts in all tonsillar carcinomas (n = 4) examined. Additional non-radioactive RNA in situ hybridization performed on 3 biopsies revealed the presence of HPV 16 or HPV 33 E7 transcripts exclusively localized within the carcinoma cells, whereas stroma stained negative. These findings strongly support a role for certain HPV types in the pathogenesis of tonsillar carcinomas.

Nonradioactive RNA in situ hybridization detection of human papillomavirus 16-E7 transcripts in squamous cell carcinomas of the uterine cervix using confocal laser scan microscopy.

Paraffin-embedded squamous cell carcinomas of the uterine cervix selected for the presence of human papillomavirus (HPV) genotype 16 (n = 19) by polymerase chain reaction, were studied for transcription of the early open reading frame E7 (ORF E7). Nonradioactive RNA in situ hybridization (RISH) was performed using in vitro generated biotinylated probes. Hybrids were visualized by streptavidin gold and silver enhancement staining in combination with confocal laser scan microscopy. Quality of mRNA was verified by detection of beta-actin gene transcripts before E7 expression was studied. In all carcinomas containing HPV 16 DNA and showing beta-actin mRNA signals (n = 13), clear E7 ORF transcription could be found. Additional RNA-PCR on purified cytoplasmic RNA of snapfrozen tissue of identical carcinomas (n = 7) showed E6-E7 specific transcripts in all E7 RISH positive samples. These results indicate continuous expression of E7 ORF in all cervical carcinomas containing HPV 16 DNA and support an active role of the E7 ORF in the pathogenesis of cervical cancer.

Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function.

Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.