Urinary cyclic nucleotides and the cytopathology of human uterine cervical dysplasias.

Blinded urinary assays for cyclic guanosine 3':5'-monophosphate (cGMP) and cyclic adenosine 3':5'-monophosphate (cAMP) were performed on 49 subjects with documented abnormal cervical cytology and 21 control subjects with normal cytology. A significant difference in the mean cGMP:cAMP ratios between the case and control groups was found. A significantly greater proportion of women with cytological abnormalities had a cGMP:cAMP ratio above the 0.2 level (p less than 0.001). Cases treated surgically for severe dysplasia or carcinoma in situ of the cervix revealed a significant postsurgical fall in the cGMP:cAMP ratios (p less than 0.025). The possibility of utilizing urinary ratios of cyclic nucleotides as an objective index in the detection, monitoring of progression, and therapy of preneoplastic cervical lesions is discussed.

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.

Antibody to folic acid: increased specificity and sensitivity in ELISA by using epsilon-aminocaproic acid modified BSA as the carrier protein.

For raising high titre and specific antibody to haptens or drugs, epsilon-aminocaproic acid modified bovine serum albumin (epsilon-ACA-BSA) was prepared for use as a carrier protein. Folic acid (FA) was coupled to epsilon-ACA-BSA, Imj.BSA and BSA for raising antibodies in rabbits. Enhancement of FA immunogenicity with FA-ACA-BSA was observed. Apart from determination of titre by indirect ELISA, dose-response behaviour and specificity of these antisera were also compared. FA-ACA-BSA antibody showed high sensitivity and specificity. Using this antibody, an ELISA method for the determination of FA was developed. The study provides a simple approach to raise highly specific and high titre antibody against small molecules.

Direct microtitre plate enzyme immunoassay of folic acid without heat denaturation of serum.

A new and simple method for enzyme immunoassay of folic acid (FA) has been developed, which does not require extraction or heat denaturation of serum. FA-free serum for standards was prepared by a new immunosorbent technique as conventional methods were unsuccessful. The detection limit of the assay is 0.05 ng/ml. Intra- and interassay variabilities ranged between 5-13.3%. Analytical recoveries obtained after spiking with different amounts of FA ranged between 93-110%. We eliminated the interference of endogenous folate binding protein--a major problem in direct FA assay by incubating serum samples (or standards) with FA-HRP conjugate in antibody coated plates at 50 degrees C. Comparison of our data with results obtained by microbiological assay and also by heating samples in alkaline buffer showed good correlation.

Sex difference in cellular retinol- and retinoic acid-binding proteins in human colon adenocarcinomas.

Human colon adenocarcinomas and adjacent non-cancerous, normal colon from the same patient were assayed for the presence and amounts of cellular binding proteins for retinol (CRBP) and retinoic acid (CRABP) by sucrose gradient analysis. In male patients, the mean concentrations of both CRBP and CRABP in the colon cancers were statistically significantly higher than in the adjacent normal colon. By contrast, in female colon cancers, the mean levels for both binding proteins were reduced approximately 2-fold, compared to the concentrations in the adjacent normal colon. These findings reveal an unexpected sex difference in the binding proteins for retinol and retinoic acid in human colon malignancies.

Oncogenic human papillomavirus (HPV) infection and uterine cervical cancer: a screening strategy in the perspective of rural India.

The predominance of cervical cancer in India can mostly be attributed to the lack of early screening. The objective of the present study has been, therefore, to determine a cost-effective oncogenic human papillomavirus (HPV)-based cervical cancer screening plan for rural Indian women. The results showed that in normal women, highest prevalence of HPV 16/18 infection was in the age group < or =23 years and lowest in > or =44 years with an insignificant change in between. HPV 16/18 infection was significantly associated with cervical erosion at age < or =23 years, but not with cytology or visual inspection with acetic acid testing at any age. The low-grade cytological lesions, however, increased only with increase in age. Fourteen per cent of the cervical malignancy was also found to be present in the age group 24-33 years with an 87% HPV infection. Here we proposed a cost-effective screening scheme in which HPV testing must be performed in women (a) < or =23 years with cervical erosion and (b) 24-43 years, as an adjunct to Pap smears (both HPV and cytology were prevalent in this group). For women > or =44 years, HPV testing might not be useful, since abnormal cytology was more prominent over the viral infection. We infer that by not performing HPV test in the group < or =23 years, approximately 76% of the high-risk HPV-infected individuals potentially "at risk" for developing cervical cancer might be missed.

Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis.

We have developed an improved method, using 96-well microtiter plates, for the microbiological assay of folic acid. With this method, the tedium of conventional microbiological analysis is substantially decreased. Culture volumes have been reduced 33-fold, and pipetting procedures have been simplified. Assay time has been reduced to 14 h, and sensitivity has increased 10-fold (0.1 ng/mL). Analytical recoveries range from 98 to 104%. Intra-assay and interassay variabilities are less than 11%. The assay does not require extensive manipulation of inoculum. Day-to-day variability has been minimized by using saline aliquots of the bacterial suspension stored at 4 degrees C. The procedure is accurate, selective, and useful for direct measurement of folic acid in multivitamin formulations.

A comparative profile of the prevalence and age distribution of human papillomavirus type 16/18 infections among three states of India with focus on northeast India.

Human papillomavirus (HPV) DNA in cervical scrape samples of married women from Manipur (n= 692) and Sikkim (n= 415) in northeast India was determined and compared with that of women from West Bengal (n= 1112) in eastern India by polymerase chain reaction. HPV prevalence was lower in Manipur (7.4%) than in Sikkim (12.5%), which was closely followed by West Bengal (12.9%). HPV18 was predominant in Manipur (2.03%) and strikingly lower (0.2%) in Sikkim and West Bengal (0.9%), while the reverse was true for HPV16. The proportion of HPV16/18 infections in Manipur (3.3%, 22/672) and Sikkim (3.89%, 14/359) were comparable and significantly lower compared to that in West Bengal (7.8%, 79/1007) among women having normal cervical cytology. Such prevalence was similar among all age groups in Manipur: increased with age for women in Sikkim and dropped with age for those in West Bengal similar to that reported previously. At age < or =30 years, HPV16/18 prevalence in Manipur (3.3%) and Sikkim (2.5%) was comparable but was significantly lower (P < 0.05) in contrast to that in West Bengal (8.8%). Among abnormal cytologic lesions, HPV16/18 infections were significantly higher than in normals (P= 0.000) both in Sikkim (14.3%) and West Bengal (20.9%) and absent in Manipur. Such prevalence was noted among women in Sikkim aged >30 years and equally among those in West Bengal aged < or =30 or >30 years. Thus, women from northeast India, particularly from Manipur, appear less susceptible to HPV16/18 infection and related cervical lesions compared to those from West Bengal, where such proneness was prominently evident at age < or =30 years.

Biosocial factors influencing women to become prostitutes in India.

Based on materials collected during a study of risk factors associated with cervix cancer among prostitutes in Domjur, Howrah, West Bengal, this report explores various biosocial factors which may lead women to prostitution. When family and marital ties fail or disintegrate for various reasons, the life situations which a woman encounters may themselves generate a "process" of anchorlessness. The failure of family support along with the lack of ability to provide for themselves due to poverty and illiteracy were key factors why the women in this study had adopted prostitution.

PIP: In India, interviews with 33 female prostitutes in Domjur, Howrah District, West Bengal, were conducted to understand the processes by which women become prostitutes. 84.8% were Hindus, 72.7% of whom were from lower castes. 31 prostitutes were illiterate. The prostitutes tended to have most recently worked as domestic workers, casual laborers, or farm laborers. The main reason for leaving their last occupation was obligation to provide sex services. 21 had been married. More than 50%, who had been married before age 18, became prostitutes before age 25 and were older than 30. 66% did not engage in illicit sex before becoming prostitutes. About 20% had been prostitutes for more than 15 years. Most prostitutes earned about Rs. 1000/month. Most prostitutes send remittances to their families. About 50% still had friendly relations with their native places. The prostitutes took care of each other. 66% had a maximum number of 5 clients/day. Three prostitutes had as many as 7-8 clients/day. Life events and the prostitutes' reactions to these events that led them to become prostitutes belonged to two categories: 1) women were either widowed (17 women) or abused by husbands and in-laws (4 women), leaving them with no social or economic support and 2) women chose prostitution as an easy means to support themselves (9 women) or because they had sexual urges or were curious (3 women). The prostitutes tended not to use health care services due to perceived need to hide their identities, time constraints, and overly expensive health care. They were referred to the State General Hospital, where their profession was known. They received adequate treatment (e.g., for sexually transmitted diseases) and were happy to be respected and asked the right questions. 70% wanted to quit the profession. These findings show that key factors for these women adopting this type of life style were lack of family support and inability to provide for themselves due to poverty and illiteracy.

Apoptotic changes in cervical intraepithelial neoplasia.

We examined apoptosis in paraffin-embedded archival cervical tissues from cervical intraepithelial neoplasia (CIN) cases to determine whether the apoptotic process is involved in the cellular changes of CIN. Apoptotic bodies, determined by the Tunel method, were largely found at the surface epithelium in a few tissue specimens with no significant abnormality and in most tissues with CIN. Apoptotic bodies were also found within the stratified epithelium of CIN lesions and were altogether absent in specimens with no significant abnormalities. The apoptotic index was significantly associated with the severity of CIN and not with either age or human papillomavirus infection. Based on the findings of increased numbers of apoptotic bodies and their presence within the stratified epithelium in CIN tissues, we hypothesize that dysregulation in the exfoliation of apoptotic cells and resistance towards apoptosis may be prerequisites for the pathogenesis of CIN.

Chemoprevention of cervix cancer: Phase I-II: A feasibility study involving the topical vaginal administration of retinyl acetate gel.

A Phase I-II clinical trial has been conducted with a retinyl acetate (RA) gel applied cervicovaginally in women having a histopathologic lesion diagnosed as mild or moderate dysplasia. With informed consent, volunteer subjects were observed and followed with Pap smears and colposcopy in a standardized protocol involving a self-administered 7-day treatment course for three successive menstrual cycles. Varying dosages of RA including placebo, 3, 6, 9, and 18 mg per 6 g of an inert vehicle were employed. A total of 50 subjects were monitored for local and systemic side effects. No intolerable side effects or disturbing toxicity was reported or detected at any of these doses. With the 18-mg dosage, significant discomfort was reported by all women. Despite associated side effects and a considerable patient effort involved in carrying out the self-administration of the gel, a high level of compliance was achieved in this trial. It has been established that women will voluntarily participate in an intervention clinical trial to determine whether retinyl acetate is an alternative method of therapy of presumed precancerous lesions of the cervix. The analysis of the side effects of the gel at the various dosage concentrations favors the selection of the 9-mg dosage for a multicenter Phase III clinical trial to determine efficacy.

Are Muslim women less susceptible to oncogenic human papillomavirus infection? A study from rural eastern India.

Muslim women are known to have lower incidences of cervical cancer and/or human papillomavirus (HPV) infection. Here we aim to determine any association that may be present between the oncogenic HPV16/18 infections and abnormal cytological lesions along with demographic and other attributes among Indian Muslim women (n = 478) and compare with the neighboring Hindus (n = 534) from a prospective cohort study. Agewise distribution of both subject-groups is similar. HPV16/18 infection is present in 9.6% Muslims and 7.5% Hindu women. Jointly atypical cells of undetermined significance (a typical cells of undetermined significance) and HPV16/18 are present in seven Muslim and two Hindu women. No high squamous intraepithelial lesions or cervical cancer is detected at the baseline. HPV16/18 infections show trends that varied with age, a nonlinear trend among Muslim women. In Hindu women the prevalence is highest at age < or =24 years, which linearly drops with increasing age. Abnormal cytology increases significantly in both religion-groups with increasing age. The data show that these Indian Muslim women are equally susceptible to HPV16/18 infection and for the development of abnormal cytology. There is a paucity in epidemiological data, which justifies the need to screen women of all religions for cervical cancer (that includes oncogenic HPV testing).

Alterations in erythrocyte glutathione metabolism associated with cervical dysplasias and carcinoma in situ.

The study was designed to test the hypothesis whether cervical dysplasias of the more severe grades are associated with elevated erythrocyte glutathione levels. Subjects were women who obtained Pap tests and were subsequently found (1) not to have any cervical lesions or (2) to have colposcopically visualized, biopsy-confirmed cervical abnormalities histopathologically diagnosed as mild, moderate, severe dysplasias, or carcinoma in situ (CIS). The erythrocyte levels of reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase (6PGD) were analyzed from coded peripheral venous blood samples. GSH and GR concentrations increased with increasing severity of dysplasia. Exploratory data analysis and multiple pairwise comparisons suggested comparable levels of the glutathione-related variables between these histopathological pairs: (1) mild and moderate dysplasias or (2) severe dysplasia and CIS. We suggest that the changes in erythrocyte glutathione-related indices in conjunction with histopathological diagnosis may have the potential to distinguish between low- and high-grade cervical dysplastic lesions.

Influence of vitamin A on cervical dysplasia and carcinoma in situ.

A case-control study was undertaken to determine the dietary intake of vitamin A in women having abnormal uterocervical cytology. The study groups (87 cases and 82 controls) were drawn from a population of women who received a screening Pap test in the ambulatory health care section of a large municipal hospital center. A subset of cases (with abnormal cytology) were matched to controls for age, ethnicity, socioeconomic status, and parity. Nutrient intake and retinol binding protein concentrations were determined; epidemiological data were also obtained. It was found that the subset of cases with severe dysplasia or carcinoma in situ (CIS) were more likely to have a total dietary vitamin A intake below the pooled median (3,450 IU) and/or a beta-carotene intake below the pooled median (2,072 IU) than were normal controls (p less than 0.05 and p less than 0.025, respectively). Odds ratios revealed approximately a 3-fold greater risk for severe dysplasia or CIS in women with lowered vitamin A or beta-carotene intake. In addition, retinol binding protein was either absent or undetectable in 78.8% of the dysplastic tissue samples, versus 23.5% of the normal tissue samples (p less than 0.005).

Retinoids and the prevention of cervical dysplasias.

Women with abnormal cytology were matched with normal control subjects for age, parity, ethnicity, and socioeconomic class and participated in a blind case-control study focused on the role of nutrition in cervical dysplasia. Sucrose gradient ultracentrifugation studies for determination of the presence and concentration of the binding proteins for retinol and retinoic acid were performed on colposcopic biopsy tissue specimens. The nutritional survey revealed statistically significant differences for vitamins A and C and beta carotene. Retinol binding protein was absent or minimally detectable and inversely related to the severity of the dysplasia. It is proposed that a double-blind clinical trial be conducted to evaluate whether retinoids may pharmacologically inhibit, arrest, or reverse cervical dysplasia.

Dietary vitamin C and uterine cervical dysplasia.

A case-control study of women with cervical abnormalities identified through Pap smears, was conducted in the Bronx, New York, to explore the relationship between nutritional intake and cervical dysplasia. Nutrient intake was estimated from computer analysis of three-day food records and 24-hour recall for 169 study participants (87 cases, 82 controls), including a subset of 49 pairs matched for age, race and parity. Mean vitamin C intake per day from three-day food record for controls was 107 mg, compared to 80 mg for cases (p less than 0.01). Analysis of matched pairs showed similar results; 29% of cases compared to 3% of controls in matched subset had vitamin C intake less than 50% of the recommended daily allowance, yielding a ten-fold increase in risk of cervical dysplasia as estimated by odds ratio (p less than 0.05). Younger age, greater frequency of sexual intercourse and younger age at first intercourse were associated with higher risk of cervical dysplasia. Multiple logistic analyses indicated that low vitamin C intake is an independent contributor to risk of severe cervical dysplasia when age and sexual activity variables are controlled. Approximately 35% of US women in their reproductive years have daily vitamin C intake below 30 mg, and 68% have vitamin C intake below 88 mg. If other studies confirm these findings, it may be important to explore a possible protective role of supplementary vitamin C for women at high risk of cervical cancer.

Plasma vitamin C and uterine cervical dysplasia.

Plasma concentrations of vitamin C were determined in a case-control study of women (n = 80) who had sought a Papanicolaou test in the Bronx Municipal Hospital Center. Controls (n = 34) were women having negative cytologic tests, negative colposcopic findings, and no known gynecologic dysfunction. Cases (n = 46) were defined as women who had either one positive or two consecutive suspicious Papanicolaou smears in a 12-month period. The mean concentration of vitamin C in the plasma was significantly lower in the cases than in the controls (0.36 versus 0.75 mg/dl, p less than 0.0001). Cases were further stratified according to the histopathologic diagnosis. The data direct attention to a possible etiologic association of vitamin C in human cervical epithelial abnormalities. A clinical trial with vitamin C intervention is suggested.