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Nrf2 is essential to antioxidant response element (ARE)-mediated host defense. Sulforaphane (SFN) is a phytochemical antioxidant known to affect multiple cellular targets including Nrf2-ARE pathway in chemoprevention. However, the role of SFN in non-malignant airway disorders remain unclear. To test if pre-activation of Nrf2-ARE signaling protects lungs from oxidant-induced acute injury, wild-type (Nrf2 +/+ ) and Nrf2-deficient (Nrf2 -/- ) mice were given SFN orally or as standardized broccoli sprout extract diet (SBE) before hyperoxia or air exposure. Hyperoxia-induced pulmonary injury and oxidation indices were significantly reduced by SFN or SBE in Nrf2 +/+ mice but not in Nrf2 -/- mice. SFN upregulated a large cluster of basal lung genes that are involved in mitochondrial oxidative phosphorylation, energy metabolism, and cardiovascular protection only in Nrf2 +/+ mice. Bioinformatic analysis elucidated ARE-like motifs on these genes. Transcript abundance of the mitochondrial machinery genes remained significantly higher after hyperoxia exposure in SFN-treated Nrf2 +/+ mice than in SFN-treated Nrf2 -/- mice. Nuclear factor-κB was suggested to be a central molecule in transcriptome networks affected by SFN. Minor improvement of hyperoxia-caused lung histopathology and neutrophilia by SFN in Nrf2 -/- mice implies Nrf2-independent or alternate effector mechanisms. In conclusion, SFN is suggested to be as a preventive intervention in a preclinical model of acute lung injury by linking mitochondria and Nrf2. Administration of SFN alleviated acute lung injury-like pathogenesis in a Nrf2-dependent manner. Potential AREs in the SFN-inducible transcriptome for mitochondria bioenergetics provided a new insight into the downstream mechanisms of Nrf2-mediated pulmonary protection., (Published by Elsevier Inc.)

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3 H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Despite recent advances in the conceptual understanding of the pathogenesis of ovarian cancer, it remains the foremost cause of death from gynecologic malignancies in developed countries. The main reason for such a high rate of mortality is the lack of sensitive and specific biomarkers and imaging techniques for early detection of ovarian cancer. Additional biological insights into early-stage ovarian carcinogenesis are needed to help speed the development of markers for early detection of ovarian cancer. The objective of this study was to characterize differentially expressed genes in high-grade stage I serous carcinoma of the ovary., Methods: We analyzed gene expression in macrodissected formalin-fixed, paraffin-embedded samples from 5 high-grade stage I serous carcinomas and 5 stage I borderline tumors of the ovary using the Illumina Whole Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation) corresponding to 24,000 genes. Significance Analysis of Microarrays was performed to determine differentially expressed genes in stage I serous carcinoma, and class prediction analysis was performed to determine the predictive value of differentially expressed gene sets to correctly classify serous carcinoma from borderline tumors in 3 independent data sets. Altered transcription factor pathways and biological pathways unique to stage I serous carcinoma were identified through class comparison of differentially expressed genes., Results: Unsupervised cluster analysis of gene expression correctly classified stage I serous carcinomas from serous borderline tumors. Supervised analysis identified several known, as well as novel, genes differentially expressed in stage I ovarian cancer. Using a differentially expressed gene set, class comparison prediction analysis correctly identified serous carcinomas from serous borderline tumors in 3 independent data sets at over 80% accuracy, sensitivity, and specificity. Pathway analysis demonstrated the significance of p53 and E2F pathways in serous carcinogenesis and significant involvements of cell cycle and immune response pathways in stage I serous epithelial ovarian cancer., Conclusion: We have identified differentially expressed genes associated with the carcinogenesis of high-grade stage I serous EOC. Furthermore, integrative analysis of biological and transcription pathway data contributed to the confirmation of important biological pathways and discovery of additional unique genes and pathways that may have potential importance in ovarian pathogenesis and biomarker development.

Background: Psammoma bodies in cervicovaginal cytology specimens are associated with malignant and benign conditions. Few studies have evaluated which features distinguish patients with underlying malignancy from those with benign conditions., Methods: Pathology files were searched for cervicovaginal specimens having psammoma bodies. The cytology specimen was assessed for the background, glandular atypia, squamous atypia, and presence of non-psammomatous calcifications. Clinical data was obtained from chart review., Results: Nineteen women (mean age 42.7 years) had benign outcomes. None had signs or symptoms suggesting malignancy. None had highly atypical or malignant appearing glandular cells. Twelve women had malignant neoplasms (mean age 56 years), including 6 with recurrent disease. Four women without prior malignancy had worrisome signs including bleeding or mass. All six women with prior malignancy had signs of recurrent disease. All specimens contained highly atypical or malignant glandular cells., Conclusions: The only cytologic feature predictive of outcome was the presence of highly atypical glandular cells in the specimen (P = 0.001), but these cells may be few. Women with underlying malignancy were older than those with benign outcome (P = 0.014) and more likely to be postmenopausal (P = 0.05). Women with malignancy had signs that warranted additional investigation whereas those with benign outcome were usually asymptomatic (P = 0.001).

Single nucleotide polymorphisms (SNPs) in the human genome are DNA sequence variations that can alter an individual's response to environmental exposure. SNPs in gene coding regions can lead to changes in the biological properties of the encoded protein. In contrast, SNPs in non-coding gene regulatory regions may affect gene expression levels in an allele-specific manner, and these functional polymorphisms represent an important but relatively unexplored class of genetic variation. The main challenge in analyzing these SNPs is a lack of robust computational and experimental methods. Here, we first outline mechanisms by which genetic variation can impact gene regulation, and review recent findings in this area; then, we describe a methodology for bioinformatic discovery and functional analysis of regulatory SNPs in cis-regulatory regions using the assembled human genome sequence and databases on sequence polymorphism and gene expression. Our method integrates SNP and gene databases and uses a set of computer programs that allow us to: (1) select SNPs, from among the >9 million human SNPs in the NCBI dbSNP database, that are similar to cis-regulatory element (RE) consensus sequences; (2) map the selected dbSNP entries to the human genome assembly in order to identify polymorphic REs near gene start sites; (3) prioritize the candidate polymorphic RE containing genes by searching the existing genotype and gene expression data sets. The applicability of this system has been demonstrated through studies on p53 responsive elements and is being extended to additional pathways and environmentally responsive genes.

A symposium, Genetic Polymorphisms in DNA Repair and Cancer Risk, was presented at the 40th Annual Meeting of the Society of Toxicology, held in San Francisco, California, in March 2001. A brief report of the symposium was published (Kaiser, Science 292, 837-838, 2001). Molecular epidemiological studies have shown that polymorphic variants of genes involved in the metabolism and repair of carcinogens can act as cancer susceptibility genes. These variants of drug metabolic and DNA-repair enzymes either increase the activation of chemical carcinogens or decrease the cells' ability to detoxify/repair mutagenic damages. Although on an individual basis these variant alleles may only slightly change catalytic activity and increase cancer risk, their polymorphic frequency in the human population may contribute to a high proportion of cancer cases. Studies conducted over the past few years have identified variant alleles for a number of DNA-repair genes, some of which have been shown to change DNA-repair capacity. Identifying these genotypic alterations in DNA-repair enzymes and their association with cancer may help to elucidate the mechanisms of cancer etiology and to predict both disease risk and response to cancer therapy, since most antineoplastic treatments mediate their effects through DNA damage.

Collagen II arthritis (CIA) represents an animal model of human RA that can be induced in DBA/1J (H-2(q)) but not in C57BL/6 mice (H-2(b)). A vigorous CII specific CD4 Th1-cell response but not IgG2 anti-CII antibody or CIA could be induced in C57BL/6 mice made transgenic for the RA shared epitope DR4 (B1*0401). We developed CD4 Th1-cell clones specific for CII from these transgenic (tg) mice in order to determine if the adoptive transfer of these clones into syngeneic tg C57BL/6 recipients could induce CIA. Three bovine CII specific (bCII) CD4 Th1-cell clones and one T-cell line specific for an immunodominant region of bCII (p261-273) were generated. Among these only one clone that could up-regulate anti-CII, IgG2 antibody in the recipient mice was able to induce transient arthritis. However, this level of IgG2 anti-CII antibody was only one-third of that seen in CII immunized DBA/1J mice that develop persistent arthritis. These results confirm our previous observations that the induction of CIA requires a sustained IgG2 antibody response to CII, an effect difficult to achieve even in DR4 (B1*0401) tg mice reconstituted with CD4 Th1 cells. This suggests that a rate limiting step in the development of human RA among those individuals expressing the RA shared epitope may be the requirement to generate sustained levels of complement fixing antibody to arthritogenic antigens.

Rheumatoid arthritis (RA) is an autoimmune disease that is genetically associated with the MHC class II molecule HLA-DRbeta1*0401 (DR4). In order to determine if this MHC can influence the immune response to the candidate autoantigen type II collagen (CII), we have studied collagen induced arthritis (CIA) resistant C57BL/6 mice, made transgenic (Tg) for human DR4. These DR4 Tg mice exhibited a strong T cell proliferative response to CII and its DR4 restricted peptide p261-273 after immunization with these antigens that was not seen in the C57BL/6 wild type mice. DR4 Tg mice also exhibited an increase in IFN-gamma production in response to CII, indicating the activation of Th1 cells. While these Tg mice produced IgM anti-CII antibodies, they failed to produce a detectable level of IgG2a (Th1 type) anti-bCII antibody and did not develop CIA. This study shows that a Th1 type T cell response to CII can be established in CIA non-susceptible mice by introducing the human transgene, DR4. This T cell response, however, is not sufficient to induce an antibody isotype switch to IgG2a, nor is it sufficient for the induction of CIA. These results may help to explain why many individuals expressing HLA-DRbeta1*0401 do not develop RA., (Copyright 2002 Elsevier Science Ltd.)

Objective: Matrix metalloproteinase-9 (MMP-9) can degrade gelatin and type IV collagen and is known to play an important role in tumor cell invasion across the basement membrane. The tissue inhibitor of metalloproteinase-1 (TIMP-1) is able to prevent activation of pro-MMP-9 and forms a 1:1 complex with the active form of MMP-9. The aim of the present study was to investigate the expression of MMP-9 and TIMP-1 in benign, borderline, and invasive epithelial ovarian tumors., Materials and Methods: A total of 90 patients with epithelial ovarian tumor were treated at the Brigham and Women's Hospital and were used as the study population. Immunohistochemistry and in situ hybridization were performed to detect protein and mRNA expression of MMP-9 and TIMP-1., Results: In the 90 epithelial ovarian tumors tested, MMP-9 expression in tumor cells was found to be significantly enhanced in serous and mucinous ovarian carcinomas compared with benign and borderline tumors. We also observed the immunostaining of MMP-9 in stromal cells of benign, borderline, and invasive epithelial ovarian tumors. Moreover, the expression levels of TIMP-1 in tumor cells were significantly higher in borderline and invasive ovarian tumors than in benign tumors., Conclusion: Using an in situ hybridization technique, we disclosed a direct correlation between the presence of mRNA and protein expression for both MMP-9 and TIMP-1. The present data suggest that high levels of MMP-9 protein in invasive epithelial ovarian carcinoma are strongly associated with tumor cell invasion. Enhanced expression of TIMP-1 protein in borderline and invasive tumors indicates that endogenous TIMP-1 protein may play a paradoxical role in ovarian tumor progression., (Copyright 2000 Academic Press.)

Objective: The ras genes are a well-studied family of proto-oncogenes whose involvement in many cancers has been delineated. However, K-ras mutations have not previously been examined in papillary serous carcinoma of the peritoneum (PSCP), a tumor which resembles serous epithelial ovarian carcinoma (SEOC) both in histology and epidemiology. In this study we examine the role of the K-ras oncogene in PSCP compared to SEOC., Methods: Using single-strand conformational polymorphism analysis and cycle sequencing protocols, we evaluated our collection of 51 cases of PSCP for K-ras mutations and compared these findings with the experience in SEOC. We then examined 5 cases of PSCP for activation of ras proteins and MAP kinase to evaluate the potential involvement of the ras pathway in PSCP tumorigenesis., Results: We found only one K-ras mutation in our 51 cases (2%) of PSCP compared to three mutations in 46 cases (6.5%) of high-grade, late-stage SEOC. This was not significantly different (P > 0.10). In the single PSCP case with a K-ras mutation, the mutation was found in only one of five tumor sites tested. All four mutations involved a single nucleotide alteration in codon 12 (GGT to GTT, Gly to Val). To evaluate the ras pathway in PSCP, we used the known activated ras binding domain on Raf-1 to perform an assay to test for activated ras. We identified ras activation in 4 of 5 PSCP cases tested and, to confirm that the activation was functional, we tested and found similar activation of MAP kinase, a downstream mediator for K-ras expression., Conclusions: K-ras mutations occur at low rates in both PSCP and high-grade, late-stage SEOC, and therefore K-ras mutations are not involved in the development of these two diseases. Finding the mutation in only one of multiple tumor sites in the PSCP case supports growing evidence for a multifocal origin of PSCP. Our findings of ras activation in four of five cases of PSCP suggest that ras activation by mechanisms other than genetic mutation is important for PSCP tumorigenesis., (Copyright 2000 Academic Press.)

The carcinogenic potential of dichloromethane (DCM) has been linked to its metabolism to formaldehyde by glutathione-S-transferase theta 1 (GSTT1). GSTT1 is polymorphic in humans. The frequency of the GSTT1 homozygous null genotype ranges from 10 to 60% in different ethnic and racial populations around the world. We investigated how varying GSTT1 genotype frequencies would impact cancer risk estimates for DCM by the application of Monte Carlo simulation methods in combination with physiologically based pharmacokinetic (PBPK) models. The PBPK model was used to estimate the DNA-protein cross links (DPX) caused by metabolism of DCM based on an earlier model. Cancer potency of DCM was obtained by the application of the estimated DPX amounts to the results of a carcinogenicity study by National Toxicology Program in B6C3F(1) mice. Human risks were estimated based on the carcinogenic potency of DCM to mice and PBPK-predicted amounts of DPX formed in humans. The Monte Carlo simulations were used to provide distributions of risk estimates for a sample of 1000 PBPK runs, each run representing a collection of biochemical and physiological parameters for a single person (with and without polymorphism included in the model). Our results show that average and median risk estimates were 23-30% higher when GSTT1 polymorphism was not included at inhalation DCM doses of 1000, 100, 10, and 1 ppm. This increase in risk was significantly reduced when it was based on the 95th percentile measure for all the doses. The specific effect of this polymorphism on population risk was further investigated by varying the probability that an individual may have a nonfunctional form of the enzyme at a constant dose level of 10 ppm of DCM. Higher values of this probability resulted in a corresponding decrease in risk. Again, this drop in population risk was not as significant when the 95th percentile measure was used.

We investigated song sharing and dispersion of song types in the wild in a colour-marked population of the non-migratory Nuttall's white-crowned sparrow, Zonotrichia leucophrys nuttalli. The songs of fathers, their male progeny (sons), and the neighbours of the sons at recruitment sites were analysed spectrographically and compared qualitatively and quantitatively. To determine whether a son's song more closely matched that of his father or a neighbour at the site settled, we subjected frequency and temporal characteristics of songs within each father-son-neighbour triad to multivariate cluster analysis. The songs of 14 of 16 sons clustered with their neighbours' rather than their fathers' songs, confirming that song matching of neighbours is an integral component of territory settlement by juveniles. Principal components analysis of frequency and temporal measurements of song within a dialectal area show that songs group into neighbourhoods and are non-randomly distributed. Multivariate analysis suggests that sons may entrain on frequency and temporal characteristics of a neighbour's song without matching phrases or complex syllables. Implications for models of instructive versus selective learning are discussed. The timing of closure of the sensitive phase, the length of the silent interval between the sensory phase and plastic song stage, and the time to song crystallization remain open questions in song ontogeny. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Results obtained from both epidemiologic studies and experimental animal model systems have shown a wide range of phenotypic variation in the ability of individuals to metabolize drugs and environmental chemicals. Several studies have noted correlations between specific metabolic phenotypes and the incidence of disease, suggesting that certain allelic forms of drug metabolic enzymes can render the individual either more sensitive or resistant to the toxic or therapeutic effects of exogenous drugs and chemicals. While some of this variation can be attributed to different environmental exposures, it has become clear that genetic factors also play an important role in determining the response of the individual organism to exogenous agents. Recent advances in molecular biological techniques have begun to allow scientists to correlate observed phenotypic differences with the actual differences in genetic sequence at the gene level. This has allowed a correlation between gene structure and function, thus providing a mechanistic basis to explain the interaction between genetic background and individual response to environmental exposures. Results presented at this symposium discussed how genetic polymorphisms for both Phase I and Phase II metabolic enzymes in the human population modulate the response to environmental toxicants.

Metabolism of dichloromethane (DCM) to formaldehyde (HCHO) via a glutathione S-transferase (GST) pathway is thought to be required for its carcinogenic effects in B6C3F1 mice. In humans, this reaction is catalyzed primarily by the protein product of the gene GSTT1, a member of the Theta class of GST, and perhaps to a small extent by the protein product of the gene GSTM1. Humans are polymorphic with respect to both genes. Since HCHO may bind to both DNA and RNA forming DNA-protein crosslinks (DPX) and RNA-formaldehyde adducts (RFA), respectively, these products were determined in isolated hepatocytes from B6C3F1 mice, F344 rats, Syrian golden hamsters, and humans to compare species with respect to the production of HCHO from DCM and its reaction with nucleic acids. Only mouse hepatocytes formed detectable amounts of DPX, the quantities of which corresponded well with quantities of DPX formed in the livers of mice exposed to DCM in vivo [Casanova, M., Conolly, R.B., and Heck, H. d'A. (1996). Fundam. Appl. Toxicol. 31, 103-116]. Hepatocytes from all rodent species and from humans with functional GSTT1 and GSTM1 genes formed RFA. No RFA were detected in human cells lacking these genes. Yields of RFA in hepatocytes of mice were 4-fold higher than in those of rats, 7-fold higher than in those of humans, and 14-fold higher than in those of hamsters. The RFA:DPX ratio in mouse hepatocytes incubated with DCM was approximately 9.0 +/- 1.4, but it was 1.1 +/- 0.3 when HCHO was added directly to the medium, indicating that HCHO generated internally from DCM is not equivalent to that added externally to cells and that it may occupy separate pools. DPX were not detected in human hepatocytes even at concentrations equivalent to an in vivo exposure of 10,000 ppm; however, the possibility that very small amounts of DPX were produced from DCM cannot be excluded, since HCHO was formed in human cells. Maximal amounts of DPXliver that might be formed in humans were predicted from the amounts in mice and the relative amounts of RFA in hepatocytes of both species. With predicted DPXliver as the dosimeter, the unit risk, the upper 95% confidence limit on the cancer risk, and the margin of exposure were calculated at several concentrations using the linearized multistage and benchmark dose methods. Since the actual delivered dose is smaller than that predicted, the results suggest that DCM poses at most a very low risk of liver cancer to humans.

Autoimmune thrombocytopenia has been attributed to the presence of antiplatelet autoantibodies which mediate platelet destruction. The derivation of these autoantibodies is presently unknown. While normal B cells do not produce these autoantibodies in vivo, it has been demonstrated in vitro by somatic cell hybridization that the B lymphocytes of nonthrombocytopenic individuals have the potential to produce antiplatelet autoantibodies. Antigen specificities of these antibodies are similar to those seen in autoimmune thrombocytopenic purpura and the lupus anticoagulant syndrome. The immunoglobulin V region genes encoding two such human monoclonal antiplatelet antibodies, an anti-GP IIb (STO 171) and an anti-phospholipid antibody (STO 103) derived from tonsillar lymphocytes of a non-thrombocytopenic male, have now been sequenced. These antiplatelet antibodies were found to be encoded by unmutated germline VH and VK genes. The third complementarity determining region (CDR3) of the genes encoding both of these antibodies have unique D regions with evidence of N-nucleotide additions, and the light chain genes show VK-JK junctional diversity. STO 103 is encoded by the VH4 V71-2 germline gene and a truncated JH4 gene. The light chain gene showed closest homology with the VK4 Humk18 gene and JK2 gene. STO 171 showed closest homology with the VH4.18 germline gene and had a complete germline JH6 gene. The light chain of STO 171 is encoded by the VK3 Humkv325 germline gene, which is also used by some rheumatoid factors and cold agglutinins, and a JK4 gene. Although these antibodies were not derived from circulating B cells or found to be actively producing antibody at the time they were harvested, it is possible that naturally occurring antibody producing B cells, similar to those represented here, are recruited for the development of pathogenic autoantibodies in immune thrombocytopenia.

The prognostic significance of cellular DNA content of epithelial ovarian cancer as determined by flow-cytometric analysis of paraffin-embedded tumor blocks was investigated in 87 patients. Seventy-five percent of tumors were DNA aneuploid and 25% were DNA diploid. The survival at median follow-up for patients with DNA diploid tumors (68%) was significantly longer than for DNA aneuploid tumors (49%; P = 0.003). The other prognostic factors which significantly affected survival were stage (P < 0.0001), tumor grade (P < 0.006), and residual disease at completion of initial surgery (P = 0.0005). When patients were separated into low-stage and advanced-stage disease, DNA content was a significant prognostic variable for survival in Stage I and II patients (P = 0.05). In Stage III and IV patients, DNA content had no independent prognostic significance. There were 33 patients who underwent second-look surgery. Seven of 15 patients (47%) with negative second-look surgery were DNA aneuploid, whereas 17 of 18 patients (94%) with positive second-look surgery were DNA aneuploid. Therefore, there was a much higher likelihood of positive second-look in the DNA aneuploid group (17/24) compared to the DNA diploid group (1/9) (P = 0.003). In addition for those patients with negative second-look surgery, none (0/8) of the DNA diploid tumors recurred; however, 3 of 7 (43%) of the DNA aneuploid tumors recurred and died. Cox proportional/hazards analysis showed that DNA content is an independent prognostic factor for survival in epithelial ovarian cancer. Aneuploid DNA content in ovarian tumors is also correlated with more aggressive biologic behavior, and therefore, a worse clinical course.

DNA ploidy status, as determined by flow cytometry, has been useful in predicting survival in certain malignancies. Between January 1975 and January 1985, 85 cases that met the inclusion criteria-pathologic stage I endometrial adenocarcinomas, primary surgical therapy, endometrioid cell type, and adequate paraffin-block samples-were analyzed by DNA flow cytometry. Depth of myometrial invasion, tumor grade, and presence of lymphatic vascular space invasion were established in all cases. Ten of the 85 tumors (12%) were aneuploid. The median follow-up was 9 years with a range of 2 to 16 years. There were 9 recurrences with 5 deaths from cancer, and 8 patients died of intercurrent disease. The relative hazards for recurrence associated with aneuploidy, S-phase fraction, grade 3 histology, and any myometrial invasion were 3.1, 3.18, 3.2, and 4.9, respectively, but did not reach statistical significance. Because of the low recurrence and mortality rate in this homogeneous group, a larger sample will be necessary to fully establish a role for DNA ploidy status in identifying a poor prognostic subset.

Thirty-nine patients underwent primary surgery for epithelial ovarian tumors of low malignant potential at the Massachusetts General Hospital between 1970 and 1980. Eighty-five percent of patients were found to have Stage I disease and 15% were found to have Stage III disease. Fifty-four percent of patients had a tumor with serous histology, 39% had a tumor with mucinous histology, and the remainder of patients had tumors with an endometrioid or mixed-cell type. Second malignancies and benign ovarian tumors were frequently found concomitantly with the borderline tumors or in follow-up. Gastrointestinal and endometrial adenocarcinomas were the most common second malignancies and were frequently found associated with a borderline tumor of serous histology. Follow-up was available in all 39 patients (100%). Mean time of follow-up was 11.8 years. Sixty-nine percent of patients are clinically without evidence of disease with a mean follow-up of 14.7 years, 23% died of other causes, 5% died of disease, and 3% died with disease and sepsis. All patients dying with disease did so within 7.3 years of their primary surgery. Seven patients underwent conservative surgery, defined as preservation of some ovarian tissue. Six of 7 patients are clinically free of disease with a mean follow-up of 14.6 years; 1 patient died of other causes. No patients treated conservatively had a recurrence of their disease.

Three hundred sixty consecutive patients followed at the Massachusetts General Hospital Colposcopy Clinic had HPV typing performed from exfoliated cervical cells by a commercially available dot blot hybridization assay. This assay tests for three groups of HPV types: 6/11, 16/18, and 31/33/35. Of this group, 171 patients were referred because of a Papanicolaou (Pap) smear showing squamous atypia. Of the 171 patients referred for squamous atypia, 17 (10%) had histologic evidence of CIN. Thirty-three of the 171 (19%) had detectable HPV DNA, and 28 of the 33 had high-risk HPV types (16/18, 31/33/35). Of the 17 women with CIN, only 6 (35%) had high-risk types and 11 (65%) had no HPV DNA detected. Of the 28 women with atypia and high-risk HPV types, only 6 (21%) had CIN. This study demonstrates that commercially available HPV typing when used in patients with Pap smears showing squamous atypia is not clinically useful in identifying patients for colposcopic referral.

Human-human hybridomas obtained from the separate fusion of tonsillar lymphoid cells from three different normal individuals to the lymphoblastoid cell line GM 4672 were screened by ELISA for the presence of autoantibody to Ro(SS-A). Those anti-Ro(SS-A) reactive hybridomas were then cloned by limiting dilution. Nineteen monoclonal IgM anti-Ro(SS-A) antibodies were obtained, which showed specificity to Ro(SS-A) by ELISA and Western blotting (60 kDa). Some of these monoclonal anti-Ro(SS-A) antibodies showed reactivity to DNA (2/19), cardiolipin (9/19), Sm/RNP (15/19) by ELISA, and to IgG (12/19) and La(SS-B) (19/19) by ELISA and Western blotting. None showed reactivity to the unrelated proteins casein and BSA, nor to RNA. Inhibition studies revealed that the binding to Ro(SS-A) of both IgM hybridoma monoclonal and SLE serum polyclonal IgM anti-Ro(SS-A) antibodies was inhibited with Ro(SS-A), La(SS-B) and Sm/RNP but not with IgG, DNA, RNA and BSA. These data indicate that (1) normal humans have the genetic potential to express antibodies to Ro(SS-A) and (2) the normally derived monoclonal and SLE serum IgM anti-Ro(SS-A) antibodies share similar antigen binding properties and therefore may possibly originate from a common pool of precursor B cells.

Preoperative serum CA-125 levels were evaluated in 38 patients who underwent primary surgery for epithelial ovarian tumors of borderline malignancy at the Brigham and Women's Hospital and Massachusetts General Hospital between 1981 and 1990. Surgical staging was Stage I in 25 (66%) patients, Stage II in 2 (5%) patients, Stage III in 10 (26%) patients, and Stage IV in 1 (3%) patient. The mean sizes of mucinous and serous ovarian tumors were 21.9 and 10.3 cm, respectively (P = 0.0002). All 13 patients (100%) with mucinous tumors had Stage I disease, while 12 (50%) of 24 patients with serous tumors were Stage I. Combining all cell types, 10 (40%) of 25 patients with Stage I disease had an elevated preoperative CA-125 level, while 2 (100%) of 2, 9 (90%) of 10, and 1 (100%) of 1 patient with Stage II, III, and IV disease, respectively, had increased preoperative levels. Among patients with serous tumors, 3 (25%) of 12 Stage I patients had an elevated preoperative CA-125 level, while 11 (92%) of 12 Stage II-IV tumors had elevated levels (P less than 0.001). These data suggest that preoperative CA-125 level correlates with stage of disease in patients with serous borderline ovarian tumors.

A panel of human:human hybridomas secreting monoclonal anti-Sm/RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to the lymphoblastoid B cell line GM4672. The specificity of these antibodies was studied by direct binding and competitive inhibition in enzyme linked immunosorbent assays (ELISAs), and by immunoblotting. The stable subclones of these hybridoma monoclonal anti-Sm/RNP antibodies could be classified into four groups according to ELISA. Group I bound Sm/RNP only, group II bound Sm-RNP, Ro/SS-A and La/SS-B, group III bound Sm/RNP and ssDNA, and group IV bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. When antibodies from each of the groups were tested by immunoblotting, the following pattern of reactivity emerged. Group II and IV antibodies reacted with U1RNP-A, Sm-B'/B, Sm-D and Sm-E proteins, as well as the Ro/SS-A and La/SS-B proteins. In contrast, group I and III antibodies did not bind to any individual protein components of Sm/RNP,Ro/SS-A or La/SS-B antigens, but recognized their conformational epitopes. These results, therefore, directly demonstrate for the first time that normal-derived B cells have the genetic potential, revealed here by somatic cell hybridization, to produce anti-Sm/RNP antibody responses which are ordinarily only associated with systemic lupus erythematosus (SLE) and related connective tissue diseases.

Paraffin-embedded tissue from 38 patients with early- and advanced-stage juvenile granulosa cell tumor (JGCT) of the ovary was analyzed by flow cytometry to investigate whether the DNA content is related to the histologic features, stage, or clinical outcome. Thirty-three cases were suitable for analysis: twenty-seven Stage Ia, two Stage Ic, and four Stage III. Eighteen (55%) tumors were DNA diploid and fifteen (45%) tumors were DNA aneuploid with a mean S-phase fraction (SPF) of 13.6% and a range of DNA indices from 1.0 to 2.2. Neither the DNA ploidy nor the SPF was associated with the stage of the disease. An analysis of the relation between DNA content and histopathologic features revealed that aneuploidy was associated with high mitotic rates and to a lesser extent with high-grade nuclear atypia. DNA aneuploidy was not associated with aggressive behavior of Stage Ia JGCTs. However, among the four patients with Stage III tumors, the two with diploid, low-SPF tumors were alive and well, whereas the two with aneuploid, high-SPF tumors developed recurrences or died. These data suggest that further studies on the prognostic significance of flow cytometric analysis of DNA content in advanced-stage JGCTs are warranted.

Nucleosomes released spontaneously in short-term culture from murine spleen cells have a significant immunoproliferative effect in vitro, including the stimulation of anti-DNA antibody responses. The present studies show that during short-term cultures, tonsil lymphoid cells also undergo spontaneous apoptosis revealed morphologically (electron microscopy) by the appearance of changes in nuclear chromatin, typical of apoptosis and similar to morphologic changes of apoptosis in cultured normal splenic lymphocytes. This process is followed by the release, in the greater than 30-kDa cell free supernatant fraction, of core histones (H2A, H2B, H3, H4) and low molecular weight DNA (approx 160 bp) constituents of nucleosomes. The greater than 30-kDa tonsil lymphocyte cell-free supernatant material containing the constituents of core nucleosomes, as well as the greater than 30-kDa supernatant fractions of tonsil cell lysates harvested at the same time, had a significant immunoproliferative effect on human or murine lymphocytes, increasing both DNA and immunoglobulin synthesis (protein A plaque-forming cells). Thus the release of immunoproliferative nucleosomes form dying human lymphoid cells provides an autocrine lymphocyte stimulatory network which may be important in immunoproliferative disorders and in normal cell turnover. Apoptosis in vivo may also provide a potential source for the circulating nucleosomal DNA identified in plasma in some systemic lupus erythematosus patients as well as contributing to increased polyclonal B lymphocyte stimulation and autoantibody responses in this disorder.

A collaborative study was performed to compare the expression of a series of idiotypes defined on human anti-DNA and other autoantibodies. Three panels of human monoclonal antibodies were tested: eight derived from patients with systemic lupus erythematosus (SLE); 13 from an individual with lepromatous leprosy; and 38 from normal subjects. The following rabbit anti-idiotype sera were used: one (RId16/6) raised against the lupus-derived monoclonal anti-DNA antibody 16/6, four (RId8E7, RId4G7, RId4D5 and RIdTH9) against leprosy-derived monoclonal antibodies of various specificities, and one (anti-4.6.3) against a normal-derived anti-DNA monoclonal (KIM 4.6). In addition, two other anti-idiotypes were used--one a murine monoclonal (3I), the other a rabbit polyclonal (RIdD)--which had been raised against polyclonal anti-DNA antibodies from lupus serum. Further experiments were performed with immunoabsorbed fractions of RId8E7. Direct-binding and competition assays were used. All of the anti-idiotypes produced different patterns of positivity among the three panels of human monoclonal antibodies, with the exception of RId8E7 and RId4G7, which showed considerable concordance. There was a tendency towards anti-idiotypes being disease- or group-specific: thus anti-4.6.3 failed to bind to any of the lupus or leprosy-derived monoclonals, while RId16/6 and RId8E7 bound most strongly to the lupus- and leprosy-derived antibodies respectively. KIM 4.6 itself was bound only weakly by RId16/6, while 16/6 was not recognized by anti-4.6.3; 16/6 was, however, bound by 3I, while KIM 4.6 was not. 3I bound to several other monoclonals but RIdD, which has been shown to be specific for the anti-DNA fraction of lupus serum, did not bind to any of them. These results indicate that the majority of these anti-idiotype preparations recognize largely separate sets of determinants. The monoclonal antibodies which bind to DNA may be only partly representative of anti-DNA antibodies in the serum of lupus patients.

The circulating peripheral blood of 13/28 patients with definite or classical rheumatoid arthritis (RA) had increased numbers of spontaneous in vivo active immunoglobulin-producing B lymphocytes detected by a reverse hemolytic PFC assay (mean = 3000 (1310-7920) Ig PFC/10(6) B cells) compared to an age/sex-matched control population (mean = 550 (300-900) Ig PFC/10(6) B cells). Among the remaining 16 RA patients who had normal numbers (less than 900 Ig PFC/10(6)) of such circulating B cells, 5 patients had increased numbers of activated B cells in the synovial fluid and 6 patients had no increase. Extraarticular features (nodules and vasculitis) in 11/13 patients and advanced but relatively inactive synovitis characterized those RA patients with increased numbers of active circulating B cells. In contrast, extraarticular features were seldom observed (1/16) among the remaining patients with normal numbers of active circulating B cells. Among these patients, more active generalized synovitis characterized those patients with increased numbers of active synovial fluid B cells compared to those patients with normal numbers. These studies imply that in RA patients, whose disease is primarily articular, active Ig synthesis is limited to the synovial compartment, while in those with extraarticular features active Ig-producing B cells also appear in the circulation.

The effect of pregnancy on patients with CCA of the vagina and cervix was evaluated based upon a review of 503 cases. Eighty-five had been pregnant (24 at diagnosis), 408 had never been pregnant, and the pregnancy history was unavailable in 10 cases. All of the 24 patients pregnant at diagnosis were over 16 years of age; 14 were in the first trimester, 6 in the second, and 4 in the third trimester. By FIGO criteria, 15 were stage I, 7 stage II, and 2 were stage III. The 24 pregnant and 408 never pregnant (age corrected) cases were compared with regard to maternal hormone history, symptoms, stage, location, predominant histologic or cell type, greatest tumor diameter, surface area, depth of invasion, grade, and number of mitoses. No significant differences were detected. Twelve of the 24 pregnant patients had radical hysterectomy with or without irradiation (9 stage I, 3 stage II); of the 7 treated by local therapy (5 stage I, 2 stage II), 3 required additional therapy due to persistent disease; 4 had radiotherapy alone (1 stage I, 2 stage II, and 1 stage III); one had teletherapy followed by exenteration (stage III). Six of the 24 died 2 to 12 years after diagnosis (1 stage I, 3 stage II, 2 stage III). Eighteen are alive at 1 to 17 years. The overall 5 and 10 year actuarial survival rates (age adjusted) for the group pregnant at diagnosis (86 and 68%) do not differ significantly from the never pregnant group. Pregnancy does not seem to adversely affect the outcome of CCA. Guidelines are presented to treat pregnant patients with CCA.