Human leukocyte antigen class I and II alleles and risk of cervical neoplasia: results from a population-based study in Costa Rica.

To examine human leukocyte antigen (HLA) involvement in the development of all grades of cervical neoplasia, a nested case-control study of 10,077 women in Guanacaste, Costa Rica, was conducted. Participants had invasive cervical cancer, high-grade squamous intraepithelial lesions (HSILs; n=166), or low-grade squamous intraepithelial lesions (LSILs); were positive for human papillomavirus (HPV) with no evidence of cervical neoplasia (n=320); or were HPV negative with no evidence of cervical neoplasia but with a history of high-risk sexual behavior (n=173). Compared with women who were HPV negative, women with HLA-DRB1*1301 were associated with decreased risk for cancer/HSILs (odds ratio [OR], 0.4; 95% confidence interval [CI], 0.2-0.7) and for LSILs/HPV (OR, 0.6; 95% CI, 0.3-0.9). Women with both HLA-B*07 and HLA-DQB1*0302 had an 8.2-fold increased risk for cancer/HSILs (95% CI, 1.8-37.2) and a 5.3-fold increased risk for LSILs/HPV (95% CI, 1.2-23.7). These results support the hypothesis that multiple risk alleles are needed in order to increase risk for cervical neoplasia, but a single protective allele may be sufficient for protection.

HLA class II DR-DQ and increased risk of cervical cancer among Senegalese women.

To examine Senegalese women to confirm and extend associations between HLA class II types and cervical cancer previously observed among African-American, Caucasian, Hispanic, and Japanese ethnic populations, 55 Senegalese women with invasive cervical carcinoma were compared with age-matched (human papillomavirus) HPV-positive (n = 83) and HPV-negative (n = 107) control women. PCR-based HPV and HLA typing methods were used. Data were analyzed using a global randomization test and conditional logistic regression. Although this study failed to confirm a previously reported association between cervical cancer and DQB1*03 alleles, the DRB1*1101-DQB1*0301 haplotype was detected more frequently among cervical carcinoma cases than among controls (adjusted odds ratio, 2.6; 95% confidence interval, 1.0-7.1). Furthermore, as reported by others, we observed a negative association of borderline statistical significance between DRB1*13 and cervical carcinoma (adjusted odds ratio, 0.5; 95% confidence interval, 0.2-1.1). Observations from this study confirm earlier findings of a negative association between DRB1*13 and cervical cancer and suggest that specific DRB1-DQB1 haplotype combinations, rather than individual DQB1*03 alleles, increase the risk for cervical cancer.

Determinants of genital human papillomavirus detection in a US population.

This study investigated the association of selected demographic and behavioral characteristics with the detection of low-risk, high-risk, and uncharacterized genital human papillomavirus (HPV) in women attending clinic for routine nonreferral gynecologic health care. Cervical specimens obtained from 3863 women 18-40 years old (mean, 28 years) with no history of high-grade cervical disease were analyzed for 38 HPV types. Overall, HPV prevalence was 39.2%. The prevalence of high-risk, low-risk, and uncharacterized HPV types was 26.7%, 14.7%, and 13.0%, respectively. As expected, the characteristics most strongly associated with overall HPV detection were age and numbers of lifetime and recent sex partners. Low-risk, high-risk, and uncharacterized HPV detection increased with increasing numbers of sex partners. There was a decline in high-risk and low-risk HPV detection with increasing age but little change in uncharacterized HPV detection. These results suggest that the uncharacterized HPV types have a different natural history than either low-risk or high-risk HPV types.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men.

Polymorphic products of genes in the HLA region contributing to variability in the course of human immunodeficiency virus type 1 (HIV-1) infection were identified by screening 375 Caucasian seroconverters who were aggregated from 3 cohorts. AIDS-free time was related to numerous (15) class I alleles, alone or in conjunction with transporter protein variants, to homozygosity at the A or B locus, and to alleles of two class II haplotypes. A prognostic scoring algorithm derived from the 3 cohorts captured multiple HLA contributions to protection or to risk (relative hazard=0.57-60 per unit increase in score, all P<<.001). The impact of HLA was strong and appeared independent of the effects of chemokine receptor/ligand polymorphisms and antiretroviral treatment. The algorithm also predicted divergent rates of CD4+ cell decline in 2 other groups, totaling 227 seropositive persons (P=.06 - <.001). Confirmation of these relationships should encourage investigation of HIV-1 antigen processing and presentation mediated by polymorphisms in the HLA region.

Familial and sporadic cases of nasopharyngeal carcinoma in Taiwan.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has a striking geographic/ethnic distribution, with especially high rates among southern Chinese. Previous studies have indicated that a family history of NPC is associated with increased risk and noted familial clustering in low-risk populations. MATERIALS AND METHODS: We investigated differences between sporadic and familial cases of NPC in a case-control study of 375 histologically confirmed NPC cases (99% response rate) and 328 age-, sex-, and geographically-matched controls (88% response rate). All participants answered a detailed risk factor interview and donated blood for EBV and CYP 2E1 testing. RESULTS: Subjects with a first degree relative with NPC had on odds ratio (OR) of 7.6 (95% confidence interval (CI) = 2.3-25), while those with a family history of any other cancer had only a slightly elevated risk of disease (OR = 1.4; 95% CI = .93-2.2). Of the cases, 25 (6.7%) were familial--having at least one first degree relative with NPC. No significant difference was seen between familial and sporadic cases with respect to sex, age, ethnicity, histology or stage. There was a nonsignificant (p = 0.16) increase in T1N2 tumors among familial cases, suggesting a more aggressive tumor. Family history of other cancers, EBV serologies, or the distribution of the RsaI c2 form of the allele of cytochrome P450 2E1 were also not significantly different between the two groups. CONCLUSIONS: In conclusion, while genetic factors are likely to play an important role in NPC pathogenesis, our results provide little evidence that a familial form of NPC exists with characteristics notably distinct from sporadic cases.

Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

Genetic evaluation of suspected cases of transient HIV-1 infection of infants.

Detection of human immunodeficiency virus-type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1 env sequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.

Comparison of human leukocyte antigen DR-DQ disease associations found with cervical dysplasia and invasive cervical carcinoma.

BACKGROUND: Human leukocyte antigen (HLA) molecules, whose biological role is in the regulation of immune responses to foreign antigens and in discrimination of self from non-self antigens, are encoded by a series of closely linked genetic loci found on chromosome 6. Although the evidence for a link between HLA and cervical cancer has been controversial, it has been recently reported that certain HLA class II haplotypes (linked class II alleles) are positively associated with invasive cervical cancer, while other class II haplotypes are negatively associated or protective. Since HLA associations between human papillomavirus type 16 (HPV16)-mediated cancer cases and non-HPV16-mediated cancer cases have been found to be different, this suggests that specific HLA class II haplotypes may influence the immune response to HPV infection and may affect the risk of acquiring invasive cervical carcinoma. PURPOSE: This study was conducted to determine if the same HLA class II haplotypes that are associated with invasive cervical carcinoma are also associated with cervical dysplasia (presumed precursors of invasive cervical cancer). METHODS: We have examined HLA DR-DQ haplotypes among 128 Hispanic women from New Mexico with biopsy-confirmed cervical dysplasia in a case-control study using sensitive DNA-based polymerase chain reaction amplification and sequence-specific oligonucleotide probe hybridization methods to detect the presence and type of HPV and to detect allelic polymorphism in the HLA DRB1 and DQB1 loci. RESULTS: Dysplasia cases were divided into two groups for comparison to controls: severe dysplasia/carcinoma in situ (CIS), and slight/moderate dysplasia. The frequency distribution of HLA class II haplotypes among the HPV16-positive severe dysplasia/CIS cases had a statistically significant (two-tailed P < .005) difference compared with controls, whereas haplotypes among the severe dysplasia/CIS cases containing HPV types other than HPV16 did not show statistically significant frequency differences. DR-DQ haplotypes previously found to be associated with HPV16-invasive cervical carcinomas were also associated with HPV16-positive severe dysplasia/CIS. However, no statistically significant haplotype frequency difference was observed between slight/moderate dysplasia cases and controls. In addition, we noted a DQA1-DQB1 haplotype negatively associated with severe dysplasia/CIS but not with invasive cervical cancers. CONCLUSIONS: Our results strongly suggest that certain HLA haplotypes confer an increased risk for severe cervical dysplasia and invasive cervical carcinoma following HPV16 infection. IMPLICATIONS: Further molecular studies are needed to identify HLA alleles or haplotypes that may provide increased susceptibility to HPV-associated cervical disease.

HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity.

Cervical carcinoma is now known to be associated with human papillomaviruses (HPV), but the evidence for a link with specific HLA loci is controversial. The role of genetic variation at the HLA class II loci and among HPV types in cervical carcinoma was investigated by PCR DNA amplification and oligonucleotide probe typing of paraffin-embedded invasive cervical cancer tissue from Hispanic patients and of cervical swabs from Hispanic controls. Certain HLA class II haplotypes (such as DRB1*1501-DQB1*0602) were associated significantly, while DR13 haplotypes were negatively associated with cervical carcinoma. These associations are HPV16-type specific. These results suggest that specific HLA class II haplotypes may influence the immune response to specific HPV-encoded epitopes and affect the risk of cervical neoplasia.

Two new HLA DRB1 alleles found in African Americans: implications for balancing selection at positions 57 and 86.

Two new DRB1 alleles have been identified (DRB1*0303 and DRB1*0805) in African Americans that differ from known DRB1 alleles only by a glycine to valine exchange at position 86. The novel DRB1*0303 allele, found in one individual, has the same DQA1*0401-DQB1*04 haplotype as DRB1*0302, suggesting that it may be a recent diversification of *0302. The novel DRB1*0805 allele, identified in 4 individuals, was found on two haplotypes, sharing a DQA1*0501-DQB1*0301 haplotype with DRB1*0804, and a DQA1*0102-DQB1*0602 haplotype found with DRB1*0801 in some African populations. DRB1*0805 differs from *0804 only at position 57 and differs from *0801 only at position 86. Assuming that DRB1*0801 and DRB1*0802 are ancestral, based on their distribution in various human populations, DRB1*0805 may have been generated twice by two independent mutations or gene conversion events at each of these positions. Alternatively, DRB1*0805 may have arisen from a single gene conversion event (or mutation) and recombined to generate multiple DR-DQ haplotypes. These findings increase the number of DRB1 allelic pairs that differ only at position 86 to 9, suggesting strong balancing selection at this position. A number of DRB1 alleles for DR8 and DR4 also differ only at position 57, a site previously postulated to be strongly influenced by balancing selection in DQB1 alleles by phylogenetic analysis.

T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.

Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells.

Cationization of BSA generates a molecule that mounts antibody responses of increased magnitude and duration and induces T cell proliferation at concentrations 500 times less than native BSA (nBSA). To explain the alteration in immunogenic properties of this Ag, the uptake of nBSA and cationized BSA (cBSA) by splenic APC has been investigated. T cell proliferation assays were conducted with nBSA and cBSA preparations with varying degrees of substitution. An inverse correlation between the degree of cationization and the amounts of Ag needed for optimal T cell reactivity was observed. To determine whether affinity for APC resulted in an increased uptake of cBSA, splenic APC were incubated with nBSA or cBSA for varying amounts of time. Comparisons were made at each time point between untreated Ag-pulsed APC (Ag uptake) and paraformaldehyde-fixed Ag-pulsed APC (processed Ag). Proliferation of T cells primed with nBSA or cBSA increased in proportion to the amount of time of APC exposure to high concentrations of nBSA, first appearing after a 2-h pulse and peaking at 8 h. Conversely, untreated APC needed only a 30-min cBSA exposure to induce either nBSA- or cBSA-primed T cell proliferation, indicating a rapid uptake of cBSA. Comparisons with proliferation induced by paraformaldehyde-fixed cBSA APC indicate that nBSA T cells recognize a lag phase-processed form of cBSA, whereas a majority of cBSA T cells recognize either a rapidly processed form of cBSA, or a membrane-processed cBSA molecule without a classical lag phase processing event. When monensin was used as an inhibitor of fluid phase pinocytosis in splenic APC, the presentation of nBSA was inhibited by 85%, but the presentation of cBSA was inhibited by only 20%. These results imply that nBSA enters the cell by fluid phase pinocytosis, whereas cBSA enters by a nonspecific adsorptive mechanism. The different modes of cellular entry for the two molecules, nBSA and cBSA, resulting in a rapid uptake of cBSA, may have important ramifications on T cell activation and immunoregulation.

Idiotype regulation of the anti-bovine serum albumin response. I. Generation of an idiotype-specific Lyt-1-2+ suppressor T cell.

A monoclonal antibody (3C-7) specific for a determinant localized on the carboxy-terminus of the BSA molecule (P505-582) has been shown to cause suppression of the multispecific BSA antibody response if given i.v. before immunization. The fine binding specificity and the isotype subclass are not responsible for the suppression generated. Administration of 3C-7 i.v. results in the generation of a suppressor T cell that, when transferred into reconstituted irradiated mice, results in a diminished anti-BSA response. Suppression can be eliminated by panning T cells on idiotype (3C-7) coated plates, but not by panning on BSA, polyclonal anti-BSA antibodies, or MOPC 21. The action of the cell is antigen (BSA) specific. Idiotype-binding T cells reconstitute suppression and appear to be Lyt-1-2+. These observations demonstrate that a limited set of monoclonal antibodies directed against a single determinant on a protein molecule have the capacity to regulate the immune response to a multiplicity of determinants present on the same protein. These data support the concept of antibody-induced regulation by the induction of suppressor cells through idiotype recognition.

Peptic fragments of bovine serum albumin bind antigen-specific T suppressor cells from orally tolerized mice.

The antigenic structures capable of binding immunoregulatory T cells have been investigated. The functional properties (suppression or help) of BSA-specific T cells from primed or orally tolerized mice with capacity to adhere to bovine serum albumin or its peptic fragments were examined in reconstitution experiments in which splenic T-cell populations together with naive B cells were transferred into irradiated syngeneic recipients. Antigen-specific T suppressor cells isolated from mice tolerized to BSA adhered to peptic fragments of BSA as well as to the intact antigen. BSA-specific T helper cells adhered only to the intact antigen. Our data suggest that the preferential activation of T suppressor cells following administration of peptic fragments may be due to their ability to adhere to such fragments. These findings offer a novel approach of separation and identification of T suppressor cells and may be useful in further studies of immunosuppression.

Cationization of protein antigens. I. Alteration of immunogenic properties.

We have shown that a cationized form of bovine serum albumin (BSA) produced by substituting anionic side chain carboxylic groups with aminoethylamide groups possesses unique immunologic properties. The two forms of antigen, native (nBSA) and cationized (cBSA), cross-react at the level of the B cell, as evidenced by the ability of antibody raised against one form to react with the other and by inhibition assays using ELISA. T cell cross-reactivity was also observed in proliferation assays, but the amount of cBSA required for stimulation was 500 times less than the amount of native protein needed. In vivo, cBSA produced responses which, at their optimal levels, were at least double the response to nBSA and which showed a different kinetic pattern, peaking later and lasting longer than the response to the native molecule. Moreover, antibodies were produced in response to administration of cBSA but not nBSA when given i.v. in saline, without an adjuvant. Although a mechanism for these phenomena is not yet clear, we speculate that the cBSA may have a greater affinity for antigen-presenting cells or for the T cell receptor, or that the altered structure may enhance recognition of the molecule by APC and/or helper T cells.