Cervicovaginal Tissue Research Articles

Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue “demidefensin” precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1 (133.49)

Abstract Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins - 18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature stop codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature stop codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. Since retrocyclins are broadly antiviral, the ability to reawaken retrocyclin genes from their 7 million years of slumber could provide a novel way to secure enhanced resistance to several major viral pathogens. AI052017; AI065430

Durable Protection from Herpes Simplex Virus-2 Transmission Following Intravaginal Application of siRNAs Targeting Both a Viral and Host Gene

A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally, it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived, and the transfection lipid on its own unacceptably enhances transmission. Here, we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting the HSV-2 receptor nectin-1 protects for a week, but protection is delayed for a few days until the receptor is downmodulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.

Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: Potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity

BackgroundTerpenoid derivatives originating from many plants species, are interesting compounds with numerous biological effects, such as anti-HIV-1 activity. The zinc tetra-ascorbo-camphorate complex (or "C14"), a new monoterpenoid derivative was evaluated in vitro for its anti-HIV-1 activity on both R5- and X4-HIV-1 infection of primary target cells (macrophages, dendritic cells and T cells) and on HIV-1 transfer from dendritic cells to T cells.ResultsThe toxicity study was carried out in vitro and also with the New Zealand White rabbit vaginal irritation model. C14 was found to be no cytotoxic at high concentrations (CC50 > 10 μM) and showed to be a potential HIV-1 inhibitor of infection of all the primary cells tested (EC50 = 1 μM). No significant changes could be observed in cervicovaginal tissue of rabbit exposed during 10 consecutive days to formulations containing up to 20 μM of C14.ConclusionOverall, these preclinical studies suggest that zinc tetra-ascorbo-camphorate derivative is suitable for further testing as a candidate microbicide to prevent male-to-female heterosexual acquisition of HIV-1.

Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

Monitoring of Vaccine-Specific Gamma Interferon Induction in Genital Mucosa of Mice by Real-Time Reverse Transcription-PCR

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-gamma) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1(165-173) H-2D(b)-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-gamma enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-gamma mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-gamma induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.

Intravaginal Immunization of Mice with Recombinant Salmonella enterica Serovar Typhimurium Expressing Human Papillomavirus Type 16 Antigens as a Potential Route of Vaccination against Cervical Cancer

Cervical cancer, the second leading cause of cancer deaths in women, is the consequence of high-risk human papillomavirus (HPV) infections. Toward the development of therapeutic vaccines that can induce both innate and adaptive mucosal immune responses, we analyzed intravaginal (ivag) vaccine delivery of live attenuated Salmonella enterica serovar Typhimurium expressing HPV16L1 as a model antigen. Innate immune responses were examined in cervicovaginal tissues by determining gene expression patterns by microarray analysis using nylon membranes imprinted with cDNA fragments coding for inflammation-associated genes. At 24 h, a wide range of genes, including those for chemokines and Th1- and Th2-type cytokine and chemokine receptors were up-regulated in mice ivag immunized with Salmonella compared to control mice. However, the majority of transcripts returned to their steady-state levels 1 week after immunization, suggesting a transient inflammatory response. Indeed, cervicovaginal histology of immunized mice showed a massive, but transient, infiltration of macrophages and neutrophils, while T cells were still increased after 7 days. Ivag immunization also induced humoral and antitumor immune responses, i.e., serum and vaginal anti-HPV16VLP antibody titers similar to those induced by oral immunization, and significant protection in tumor protection experiments using HPV16-expressing C3 tumor cells. These results show that ivag immunization with live attenuated Salmonella expressing HPV16 antigens modulates the local mucosal gene expression pattern into a transient proinflammatory profile, elicits strong systemic and mucosal immunity against HPV16, and confers protection against HPV16 tumor cells subcutaneously implanted in mice. Examination of the efficacy with which ivag HPV16E7E6 Salmonella induces regression of tumors located in cervicovaginal tissue is warranted.

Application of Optical Coherence Tomography for Monitoring Changes in Cervicovaginal Epithelial Morphology in Macaques: Potential for Assessment of Microbicide Safety

Safety is a primary concern in the development of topical microbicides. Optical coherence tomography (OCT), a high-resolution, in-depth cross-sectional imaging modality, was utilized in conjunction with colposcopy to assess induced cervicovaginal epithelial changes that may predict product safety. OCT and colposcopic images of macaque vaginal and cervical tissues were obtained in excised tissue and in vivo under various conditions, including mechanical injury and nonoxynol-9 treatment. A scoring system was developed to categorize and quantify the OCT images based on morphologic features that indicate the presence or absence of an intact epithelial layer and inflammation. Using 3 categories (normal, mild to moderately abnormal, and severely abnormal), differences between healthy and injured tissue were apparent on OCT images. Normal images (category 1) had a bilayered structure representative of the epithelium and submucosa. Mild to moderately abnormal images (category 2) had areas of normal and abnormal epithelium. Severely abnormal images (category 3) had complete loss of the epithelium and/or inflammation, with loss of the bilayered structure on OCT. OCT is a noninvasive imaging modality complementary to colposcopy. It distinguished between normal and abnormal (or injured) tissue and thus holds promise for safety evaluations of candidate microbicides and other vaginal products.

Rapamycin reduces CCR5 mRNA levels in macaques: potential applications in HIV-1 prevention and treatment

G1 cytostatic drugs reduce CCR5 co-receptor expression and enhance the antiviral activity of a CCR5 antagonist in vitro. The administration of rapamycin, a G1 cytostatic agent, to three cynomolgous macaques led to decreased CCR5 messenger RNA expression in peripheral blood mononuclear cells and cervicovaginal tissue. These results support further clinical evaluation of G1 cytostatic agents such as rapamycin targeting the downregulation of CCR5 expression as a strategy for both the prevention and treatment of HIV infection.

The retrocyclin analogue RC‐101 prevents human immunodeficiency virus type 1 infection of a model human cervicovaginal tissue construct

Retrocyclins are cyclic antimicrobial peptides that exhibit potent activity towards a broad range of primary and laboratory-adapted strains of human immunodeficiency virus type 1 (HIV-1) in vitro. The current study shows that RC-101, an analogue of retrocyclin, prevented HIV-1 infection in an organ-like construct of human cervicovaginal tissue and retained full activity in the presence of vaginal fluid. The peptide remained within the cervicovaginal tissues throughout the 9-day incubation period without altering tissue viability, inducing damage or inducing the release of inflammatory cytokines. Collectively, these data support the potential development of RC-101 as a topical microbicide to prevent HIV-1 infection and transmission.

Preclinical Evaluation of Synthetic −2 RANTES as a Candidate Vaginal Microbicide To Target CCR5

A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1alpha, and MIP-1beta, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (-2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated -2 RANTES was released from both vehicles and retained anti-HIV-1 activity. Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of -2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that -2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.

Detection of chromosomal aberrations by fluorescence in situ hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol in utero

Epidemiologic studies have associated estrogens with human neoplasms such as those in the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17beta-estradiol [17beta-E(2)]) and synthetic (diethylstilbestrol [DES]) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17beta-E(2) treatment of mice results in increased nuclear DNA content of cervicovaginal epithelium that precedes histologically evident neoplasia. In order to determine whether this effect was associated with chromosomal changes in humans, the frequencies of trisomy of chromosomes 1, 7, 11, and 17 were evaluated by the fluorescence in situ hybridization (FISH) technique in cervicovaginal tissue from 19 DES-exposed and 19 control women. The trisomic frequencies were significantly elevated in 4 of the 19 (21%) DES-exposed patients. One patient presented with trisomy of chromosomes 1, 7, and 11, while trisomy of chromosome 7 was observed in one patient. There were two patients with trisomy of chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was not observed in the cervicovaginal tissue taken from control patients. These data suggest that DES-induced chromosomal trisomy may be an early event in the development of cervicovaginal neoplasia in humans.

Retrocyclins: Novel Circular Peptides Active Against HIV-1

Heterosexual transmission through mucosal surfaces is one of the most common routes of HIV-1 spread. Topical microbicides are self-applied prophylactic agents, used to prevent vaginal and other mucosal transmission of HIV-1, which have the advantage of empowering vulnerable receptive partners to take effective measures for their own protection. In a search for candidate topical microbicides we discovered that retrocyclin, a unique θ-defensin synthetically constructed based on sequence data from its pseudogene, can potently prevent infection of CD4+ cells by both X4 and R5 HIV-1. While many studies have utilized simulants of mucosal fluid to test compounds, we are studying how whole human vaginal fluid and its functional components affect the activity and stability of peptide-based microbicides. We explored a novel, physiologically relevant approach to assess the ability of a candidate retrocyclin microbicide, RC-101, to inhibit HIV-1 infection of immunocompetent human cervicovaginal tissue. We revealed that 1) using a novel proteomic approach, human vaginal fluid contained at least 20 different cationic (poly)peptides with purported roles in innate host defense, 2) the cationic polypeptide fraction of vaginal fluid was required for innate anti-HIV-1 activity, 3) RC-101 retained full anti-HIV-1 activity in the presence of whole human vaginal fluid, 4) when applied apically to organotypical cervicovaginal epithelium, RC101 was retained in the tissue, and 5) RC-101 prevented HIV-1 infection of immunocompetent organotypic cervicovaginal epithelium. Collectively, we have characterized the innate antiviral host defense factors within vaginal fluid, and developed a highly relevant ex vivo vaginal model suitable for peptide-based microbicide evaluation. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Preclinical Evaluation of Anti-HIV-1 Polybiguanide Vaginal Microbicides in a Murine Model of Toxicity and Inflammation

The use of vaginal microbicides has gained support as a strategy for the protection of women against HIV-1 and other sexually transmitted disease (STD) pathogens. The preclinical Swiss Webster murine model has been developed to specifically measure cervicovaginal tissue integrity and inflammation following application of candidate vaginal microbicides, when potential exposure to an STD pathogen may occur. This model demonstrates both mechanistic and temporal differences in inflammatory responses following microbicide exposure. Currently, specific markers of inflammation, including pro-inflammatory cytokines, are being evaluated in the cervicovaginal mucosa. Safety profiles of polybiguanides (PBGs), which demonstrated significant in vitro efficacy against HIV-1, are being assessed in vivo. Intravaginal application of PEHMB (1%) resulted in little or no cervicovaginal toxicity after shortor long-term exposure. Collectively, these studies support the Swiss Webster model as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides. Furthermore, these results strongly support further development of polybiguanide derivatives as vaginal microbicidal agents. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid

Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.

HIV binding, penetration, and primary infection in human cervicovaginal tissue

We have developed human cervicovaginal organ culture systems to examine the initiating events in HIV transmission after exposure to various sources of HIV infectivity, including semen. Newly infected cells were detected in the cervical submucosa 3-4 days after exposure to a primary HIV isolate. At earlier times, extensive and stable binding occurred when cervical surfaces were exposed to virions or seminal cells. Cervical mucus provided some protection for the endocervical surface, by physically trapping virions and seminal cells. Confocal microscopy combined with 3D surface reconstruction revealed that virions could both bind to the external surface of the cervical epithelium and actually penetrate beneath the epithelial surface. In quantitative assays, pretreatment with a blocking antibody directed against beta1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract.

CD8+T-Lymphocyte Response to Major Immunodominant Epitopes after Vaginal Exposure to Simian Immunodeficiency Virus: Too Late and Too Little

In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is "too late and too little" to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission.

Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus.

In the current global AIDS pandemic, more than half of new human immunodeficiency virus type 1 (HIV-1) infections are acquired by women through intravaginal HIV exposure. For this study, we explored pathogenesis issues relevant to the development of effective vaccines to prevent infection by this route, using an animal model in which female rhesus macaques were exposed intravaginally to a high dose of simian immunodeficiency virus (SIV). We examined in detail the events that transpire from hours to a few days after intravaginal SIV exposure through week 4 to provide a framework for understanding the propagation, dissemination, and establishment of infection in lymphatic tissues (LTs) during the acute stage of infection. We show that the mucosal barrier greatly limits the infection of cervicovaginal tissues, and thus the initial founder populations of infected cells are small. While there was evidence of rapid dissemination to distal sites, we also show that continuous seeding from an expanding source of production at the portal of entry is likely critical for the later establishment of a productive infection throughout the systemic LTs. The initially small founder populations and dependence on continuous seeding to establish a productive infection in systemic LTs define a small window of maximum vulnerability for the virus in which there is an opportunity for the host, vaccines, or other interventions to prevent or control infection.

Histopathologic Correlation of Atypical Parakeratosis Diagnosed on Cervicovaginal Cytology

To retrospectively examine the histopathologic findings in women with the isolated finding of atypical parakeratosis (PK) on a Pap test. The cytology files (1999-2001) were searched for cervicovaginal Pap tests interpreted as atypical PK. Cases were included for study only if there was a subsequent cervicovaginal tissue sample within 1 year of the cytologic interpretation and there was no diagnosis of squamous intraepithelial lesion (SIL) within the previous five years. Of 355 patients with atypical PK, 109 (aged 14-69 years, mean 31.5) met the inclusion criteria of the study. The interval between the cytologic interpretation and cervicovaginal tissue examination ranged from 0.5 to 12 months (mean, 2.5). Sixty-one patients underwent both endocervical curettage (ECC) and cervical biopsy (CBx), 20 patients underwent ECC only, 19 patients underwent CBx only, and the remainder underwent other procedures. Histopathologic findings on the tissue samples included: no significant pathologic change but no squamous epithelium present for evaluation (n = 10, 9.2%), benign changes other than hyperkeratosis and/or PK (n = 50, 45.9%), hyperkeratosis and/or PK (n = 8, 7.3%), low grade SIL (n = 33, 30.3%), high grade SIL (n = 6, 5.5%) and invasive squamous cell carcinoma (n = 2, 1.8%). The isolated finding of atypical PK on a Pap test correlated with the presence of an underlying SIL or invasive carcinoma in approximately 40% of patients. Of these, 80% had low grade SIL. The cytologic finding of atypical PK warrants further investigation in order to exclude SILs and/or carcinoma. We suggest that atypical PK be routinely included in the category of atypical squamous cells of undetermined significance.

Cytologic and Histologic Diagnosis and Significance of Controversial Squamous Lesions of the Uterine Cervix

The interpretation and precise classification of abnormal squamous cell changes in cervicovaginal smears (Pap tests) and tissue samples are challenging and controversial. The cytologic category atypical squamous cells of undetermined significance and the newly described squamous lesions atypical immature metaplasia, papillary immature metaplasia, and transitional cell metaplasia are representative. Their current status is reviewed, and future directions aimed at resolving some of the controversy and challenge are suggested.