The health of adult women in Accra, Ghana: self-reporting and objective assessments 2008-2009.

OBJECTIVES: The study provides a full description of the state of women's health in Accra, Ghana using self-reported as well as objective health measures. Using data from the Women's Health Survey of Accra, Wave 2 (WHSA-2), the authors a) examine the consistency of the objective measures of health status (anthropometry and blood pressures) with self-report measures, including the Short Form 36 indices for 8 separate domains of health; and b) describe the main socio-economic differentials in morbidity. METHODS: Cross-sectional household survey with field measurements. 2814 women aged 18 and over were interviewed and measured in their homes in late 2008 and early 2009. The physical measurements included height, weight, waist and hip measurement and 3 or more measures of resting blood pressure. RESULTS: Using the 8 domains of self-reported health captured by the Short Form 36 instrument, we find that physical health worsens more sharply with age than mental health. Social class differentials are narrow in the younger cohorts but widen amongst the elderly. The physical measurements reveal unhealthy levels of obesity and hypertension, worsening steadily with rising age. Age and the wealth of the household influence women's health more than their individual characteristics such as education. CONCLUSIONS: Younger women appear to be in good health with steady declines in physical and mental health with age. The major threat to women's health appears to be the rising levels of obesity and hypertension with mean BMIs for all women over age 45 in excess of 30, producing elevated blood pressures and associated high risks of heart attacks and stroke rising sharply amongst the elderly.

Health of urban Ghanaian women as identified by the Women's Health Study of Accra.

OBJECTIVE: The purpose of the Women's Health Study of Accra was to provide an assessment of the prevalence of communicable and non-communicable illnesses. METHOD: This was a prospective, community-based study that included an interview for medical illnesses, a comprehensive physical examination, and laboratory testing. A total of 1328 women were examined at Korle Bu Teaching Hospital, University of Ghana. RESULTS: Prevalent conditions included poor vision (66.8%), malaria (48.7%), pain (42.8%), poor dentition (41.6%), hypertension (40.2%), obesity (34.7%), arthritis (27.1%), chronic back pain (19.4%), abnormal rectal (16.0%) and pelvic examinations (12.7%), HIV in women age 24-29 (8.3%), and hypercholesterolemia (22.7%). Increasing age, lack of formal education, and low-income adversely affected health conditions. CONCLUSION: The high prevalence of preventable illnesses in this expanding urban population indicates that the health care services are obligated to develop and provide screening, preventive strategies and treatment for both general health and gynecologic health conditions.

Prevalence of obesity in women of Accra: Ghana

The Women/'s Health Study of Accra, Ghana measured the burden of obesity and obesitylinked illnesses in urban women. This is a Cross-sectional community based study. 1328 adult women, age 18 years and older, were selected as a representative sampling of the women of Accra. A comprehensive medical history, physical examination and laboratory tests were performed. Overweight and obesity status was determined by calculating the body mass index (BMI) (kg/m2). BMI measurements are available for 1237 non-pregnant women. A total of 430 women (34.8%) were obese; 340 (27.4%) were overweight; 369 (29.8%) were normal weight; and 98 (8.0%) were underweight. Risk factors for obesity include age 50 to 70 years, OR 2.12 [1.72 -2.62], p<0.001; total pregnancies > 5 (p<0.001); mean age of last delivery > 34 years (p<0.001); ownership of a television OR 1.57 [1.20-2.07], p=0.001; telephone OR 1.55 [1.22-1.98], p=0.001; or a refrigerator OR 1.55 [1.20-2.00], p=0.001. There was no significant association with socioeconomic status. Significant medical conditions associated with obesity include hypertension OR 2.97 CI [2.17-4.05], p<0.001; elevated fasting blood glucose OR 1.94 [CI 1.04 ­ 3.62], p=0.037. This study identifies an unexpected high prevalence of obesity and obesity-linked illnesses in this population. Public and professional awareness of the prevalence of obesity and the associated health risks are critical for programs designed to improve women/'s health

Secular trends in menarcheal age among Ghanaian women in Accra.

The Women's Health Study of Accra is a population-based cross-sectional survey that was conducted between March and September 2003 to assess the burden of disease in women in Accra. In addition to data relating to general health and living conditions, data on age at first menstruation was collected during the survey. A retrospective cohort analysis of the reported age at menarche was conducted using data from 2,644 women aged between 18 and 100 years. The median age of first menstruation of the entire cohort was 15.5 years and the median age of first menstruation among those aged <20 was 14.5 years. There was a statistically significant difference in median age at menstruation among the different age and socioeconomic groups. Multiple linear regression showed a significant decline of 0.2 years per decade in the mean age at menarche among Ghanaian women.

American ginseng transcriptionally activates p21 mRNA in breast cancer cell lines.

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p < or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p < or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.

American ginseng and breast cancer therapeutic agents synergistically inhibit MCF-7 breast cancer cell growth.

BACKGROUND AND OBJECTIVES: American ginseng (Panax quinquefolius L.) purportedly alleviates menopause symptoms because of putative estrogenicity. METHODS: Using a standardized American ginseng (AG) extract in MCF-7 breast cancer cells, the objectives were to evaluate the ability of AG to induce the estrogen- regulated gene pS2 by Northern blot analysis, determine the effect on cell growth using the MTT assay, and evaluate the cell cycle effects by flow cytometry. RESULTS: AG and estradiol equivalently induced RNA expression of pS2. AG, in contrast to estradiol, caused a dose-dependent decrease in cell proliferation (P < 0.005). AG had no adverse effect on the cell cycle while estradiol significantly increased the proliferative phase (percent S-phase) and decreased the resting phase (G(0)-G(1) phase) (P < 0.005). Concurrent use of AG and breast cancer therapeutic agents resulted in a significant (P < 0.005) suppression of cell growth for most drugs evaluated. CONCLUSIONS: In vitro use of AG and breast cancer therapeutics synergistically inhibited cancer cell growth.

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo.

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.

Skin-sparing mastectomy and immediate reconstruction: oncologic risks and aesthetic results in patients with early-stage breast cancer.

Skin-sparing mastectomy has been advocated as an oncologically safe approach for the management of patients with early-stage breast cancer that minimizes deformity and improves cosmesis through preservation of the skin envelope of the breast. Because chest wall skin is the most frequent site of local failure after mastectomy, concerns have been raised that inadequate skin excision could result in an increased risk of local recurrence. Precise borders of the skin resection have not been well established, and long-term local recurrence rates after skin-sparing mastectomy are not known. The purpose of this study was to evaluate the oncologic safety and aesthetic results for skin-sparing mastectomy and immediate breast reconstruction with a latissimus dorsi myocutaneous flap and saline breast prosthesis. Fifty-one patients with early-stage breast cancer (26 with ductal carcinoma in situ and 25 with invasive carcinoma) undergoing primary mastectomy and immediate reconstruction with a latissimus flap were studied from 1991 through 1994. For 32 consecutive patients, skin-sparing mastectomy was defined as a 5-mm margin of skin designed around the border of the nipple-areolar complex. After the mastectomy, biopsies were obtained from the remaining native skin flap edges. Patients were followed for 44.8 months. Histologic examination of 114 native skin flap biopsy specimens failed to demonstrate breast ducts in the dermis of any of the 32 consecutive patients studied. One of 26 patients with ductal carcinoma in situ had metastases to the skin of the lateral chest wall and back. Four other patients, one with stage I disease and three with stage II-B disease, had recurrent breast carcinoma. The stage I patient had a local recurrence in the subcutaneous tissues near the mastectomy specimen. Two patients suffered axillary relapse, and one had distant metastases to the spine. The findings of this study support the technique of skin-sparing mastectomy as an oncologically safe one, based on an absence of breast ductal epithelium at the margins of the native skin flaps and a local recurrence rate of 2 percent after 45 months of follow-up. Although these results need to be confirmed with greater numbers of patients and longer follow-up, skin-sparing mastectomy and immediate breast reconstruction may be considered an excellent alternative treatment to breast conservation for patients with ductal carcinoma in situ and early-stage invasive breast cancer.

Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state.

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using a C57BL/6 mouse model, OK-432 was found to induce multiple cytokines including the Th1 polarizing cytokine IL-12. Expression of IL-12 protein by murine splenocytes was restricted to macrophages and B cells and led to high levels of IFN-gamma production from both CD4+ and CD8+ T cells. Of the Th2 cytokines IL-4 and IL-10, only IL-10 protein was detected and originated primarily from the adherent cell population. Its expression was delayed relative to IL-12. A similar pattern of cytokine induction was observed from human PBMCs. OK-432-driven IFN-gamma production was inhibited by anti-IL-12 Ab, anti-IL-2 Ab, anti-TNF-alpha Ab, and anti-IL-2R alpha Ab, suggesting that IFN-gamma production from Th1 cells is induced by the cooperation action of these cytokines through the IL-2R alpha pathway. When compared with another widely used immunopotentiator bacillus Calmette-Guérin (BCG), OK-432 was a stronger IL-12 and IFN-gamma inducer. Furthermore, the mechanism of IFN-gamma induction by OK-432 differed from BCG in that coincident granulocyte-macrophage CSF and IL-1 expression played little to no role. These results suggest that OK-432 is a potent multicytokine inducer, specifically a strong inducer of IL-12, and that OK-432 may exert its antitumor effect by promoting a Th1-dominant state.

pS2 expression induced by American ginseng in MCF-7 breast cancer cells.

BACKGROUND: Alternative medicines are frequently used by patients with breast cancer for general health benefits. American ginseng, an herbal remedy, purportedly alleviates treatment-induced postmenopausal symptoms. METHODS: Estrogenic potential of American ginseng root extract to induce the expression of pS2, an estrogen-regulated gene, was evaluated in breast cancer cell lines MCF-7, T-47D, and BT-20 by Northern and Western blot analysis. Competitive studies were performed with ginseng in combination with tamoxifen. Cell proliferation assays were performed using the tetrazolium dye procedure and direct cell count. RESULTS: Ginseng and estradiol induce the expression of pS2 RNA and protein in MCF-7 cells, whereas tamoxifen suppresses expression. Neither ginseng nor estradiol induced increased pS2 expression in T-47D or BT-20 cell lines. Although estradiol exhibited a proliferative effect and tamoxifen had an inhibitory effect, ginseng demonstrated no significant effect on cell proliferation. CONCLUSIONS: The results of this study suggest that ginseng may exhibit estrogenlike effects on estrogen receptor-positive breast cancer cells by inducing pS2 expression and that the effect of ginseng may be mediated in part through the estrogen receptor. Because ginseng does not exhibit a proliferative effect, it may play a protective role against breast cancer rather than serve as a mitogen.

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-gamma, which inhibits B16F10 melanoma cell growth in vitro.

Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-gamma (IFN gamma) was the most potent single agent (IC50 approximately 50 pg/ml). Tumor necrosis factor alpha was substantially weaker (IC50 > 10 ng/ml) but provided synergy with IFN gamma. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INF gamma production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFN gamma concentration and was completely blocked by neutralizing antibody to IFN gamma. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFN gamma, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation.

Germ-line BRCA1 mutations in Jewish and non-Jewish women with early-onset breast cancer.

BACKGROUND: Mutations in a germ-line allele of the BRCA1 gene contribute to the familial breast cancer syndrome. However, the prevalence of these mutations is unknown in women with breast cancer who do not have the features of this familial syndrome. We sought BRCA1 mutations in women who were given a diagnosis of breast cancer at an early age, because early onset is characteristic of a genetic predisposition to cancer. METHODS: Clinical information and peripheral-blood mononuclear cells were obtained from 418 women from the Boston metropolitan area in whom breast cancer was diagnosed at or before the age of 40. A comprehensive BRCA1 mutational analysis, involving automated nucleotide sequencing and a protein-truncation assay, was undertaken in 30 of these women, who had breast cancer before the age of 30. In addition, the BRCA1 mutation 185delAG, which is prevalent in the Ashkenazi Jewish population, was sought with an allele-specific polymerase-chain-reaction assay in 39 Jewish women among the 418 women who had breast cancer at or before the age of 40. RESULTS: Among 30 women with breast cancer before the age of 30, 4 (13 percent) had definite, chain-terminating mutations and 1 had a missense mutation. Two of the four Jewish women in this cohort had the 185delAG mutation. Among the 39 Jewish women with breast cancer at or before the age of 40, 8 (21 percent) carried the 185delAG mutation (95 percent confidence interval, 9 to 36 percent). CONCLUSIONS: Germ-line BRCA1 mutations can be present in young women with breast cancer who do not belong to families with multiple affected members. The specific BRCA1 mutation known as 185delAG is strongly associated with the onset of breast cancer in Jewish women before the age of 40.

Recombinant BCG therapy suppresses melanoma tumor growth.

BACKGROUND: Melanoma is the fastest rising cancer in the United States. Bacillus Calmette-Guerin (BCG) has been genetically engineered to actively express and secrete the cytokine interleukin-2 (IL-2). Both BCG and IL-2 have known potent antitumor and immunomodulatory properties. METHODS: This recombinant BCG (rBCG 3A) has been tested as an intratumoral injection and a vaccine therapy in conjunction with irradiated tumor cells against melanoma in the murine B16 melanoma model. RESULTS: The transfection process did not adversely alter the function of the wild-type (WT) BCG. rBCG 3A and WT BCG are equally effective intratumoral and vaccine therapies against melanoma when compared with normal saline control groups. Tumor burdens were significantly smaller (p < or = 0.01 and 0.05) for the treatment groups for both intratumoral and vaccine administration of therapy. Immunization with rBCG 3A and WT BCG 14 days before a B16 challenge resulted in an approximately 45% smaller tumor burden when compared with controls. CONCLUSIONS: Novel therapies based on the immunogenic properties of melanoma combined with molecular technologies may offer promise for an effective and safe treatment of melanoma.

Local recurrence after conservative surgery and radiation therapy for ductal carcinoma in situ: Possible importance of family history.

PURPOSE: The optimal treatment of ductal carcinoma in situ is controversial. Traditionally, women with this disease have been treated with mastectomy with excellent results, but recently the need for such extensive surgery has been questioned. Long-term data on the use of conservative surgery and radiation therapy for treatment are limited. A retrospective analysis was performed to assess treatment outcome and prognostic factors for patients with ductal carcinoma in situ treated with conservative surgery and radiotherapy. PATIENTS AND METHODS: From 1976 to 1990, 76 women with ductal carcinoma in situ were treated with conservative surgery followed by radiation therapy. The median age at diagnosis was 48 years. Seventeen patients had a positive family history of breast cancer in a first-degree (n=8) or second-degree (n=9) relative. Median follow-up interval was 74 months for the 71 survivors. In 54 patients, the carcinoma was detected by mammography alone; in 13 patients, by mammography and physical examination; and in 4 patients, by physical examination with a normal mammogram; and in 5 patients, by physical examination alone without mammography. Fifty patients had re-excision after initial biopsy. Final margins were positive in 11, close in 11, negative in 34, and unknown in 20. The median volume of excised tissue was 60 cm3. The axilla was surgically staged in 30 patients (39%) and all were negative. The whole breast was irradiated to a dose of 45 to 50 Gy in all patients. Seventy-two patients also received a boost to the primary site. The median total radiation dose to the primary site was 61 Gy (range, 46 to 71). RESULTS: Seven patients had a recurrence in the treated breast at 16, 18, 41, 63, 72, 83, and 104 months after treatment. The 5- and 10-year actuarial rates of local recurrence were 4% and 15%, respectively. Six of seven recurrences occurred in the vicinity of the original lesion. Four local recurrences were invasive, and three were ductal carcinoma in situ. Two patients developed a contralateral invasive carcinoma. The 5- and 10-year cause-specific survival rates were 100% and 96%, respectively. The 10-year actuarial rate of local recurrence was 25% in the group with a total excision volume less than 60 cm3, as compared with 0% in those with 60 cm3 or more excised (P=0.04). In patients with a positive family history, the 10-year actuarial rate of local recurrence was 37%, as compared with 9% in patients with a negative family history (P=0.008). Of the 17 patients with a positive family history, four developed either an ipsilateral or contralateral invasive breast cancer, whereas 1 of the 58 patients without a family history developed a subsequent invasive breast cancer (P=0.008). CONCLUSION: These results suggest that patients with ductal carcinoma in situ treated with conservative surgery and radiotherapy (including a boost to the primary site) appear to benefit from wide, rather than limited, resection. These results also suggest that family history may be an important prognostic factor for progression of disease.

Estrogen and progesterone receptor concordance between primary and recurrent breast cancer.

Estrogen and progesterone receptor status in breast cancer can determine therapeutic options and may provide prognostic information. The purpose of this study is to compare the concordance of the primary breast cancer steroid hormone receptor status to that of the recurrent breast cancer and to determine whether the type of second lesion (local recurrence, second primary, or metastatic lesion) and adjuvant therapy received changed the receptor concordance. The records of eighty-three patients with estrogen receptor (ER) analysis available for primary (p) and recurrent (r) breast cancer for 1976-1990 were reviewed. In addition, 32 of these patients also had available progesterone receptor (PR) values for primary and recurrent breast cancers. Statistical evaluation was performed by chi-square, Student's t-test, and Wilcoxon signed-rank test. ER concordance (primary/recurrent, p/r) was identified in 59/83 (71%) patients; PR concordance was identified in 18/32 (56%) patients. Whether the second lesion was a local recurrence, second primary, or a metastatic lesion did not affect ER concordance or PR concordance. Adjuvant chemotherapy, hormonal therapy, or radiation therapy, either alone or in combination, did not affect ER or PR concordance. The disease-free survival (DFS) for patients with ER (p+)/(r-) (primary receptor positive/recurrent receptor negative) was significantly shorter than those with ER (p-)/(r+)(27.6 +/- 7.4 months versus 50.6 +/- 7.6 mo, P = 0.04). The DFS for PR (p+)/(r-) patients was 28.8 +/- 7.9 months compared to the DFS of 46.8 +/- 11.8 months for PR (p-)/(r+) patients (P = NS). A significantly shorter DFS for ER (p+)/(r-) patients compared to ER (p-)/(r+) patients and a trend towards a shorter DFS for PR (p+)/(r-) patients compared to PR (p-)/(r+) patients may reflect a loss of hormonal regulation or an increase in cancer aggressiveness.

The relationship between microscopic margins of resection and the risk of local recurrence in patients with breast cancer treated with breast-conserving surgery and radiation therapy.

BACKGROUND: The relationships among the involvement of tumor at the final margins of resection, the presence of an extensive intraductal component (EIC), and the risk of local recurrence are important considerations in patients treated with conservative surgery and radiation therapy for early stage breast cancer but have not been defined adequately. METHODS: Between 1982 and 1985, 885 patients were treated for clinical Stage I or II invasive breast cancer. The study population was limited to 181 patients with an infiltrating ductal carcinoma who received a radiation dose to the surgical site of 60 Gy or greater, whose final microscopic margins of resection were evaluable, and who had at least 5 years of follow-up. A positive margin was defined as tumor present at the inked margin of resection, a close margin as tumor within 1 mm of the inked margin, and a negative margin as no tumor within 1 mm of the inked margin. A focally positive margin was defined as tumor at the margin in three or fewer low-power fields. In 157 patients (87%), the tumor was evaluable for the presence or absence of an EIC. The median follow-up was 86 months. RESULTS: In 12 of 181 patients (7%), a recurrence developed at or near the primary site (true recurrence/marginal miss [TR/MM]) within 5 years. The 5-year rate of TR/MM (with 95% confidence intervals) among patients with negative, close, focally positive, and more than focally positive margins was 0% (0-4%), 4% (0-20%), 6% (1-17%) and 21% (10-37%), respectively. Patients with positive margins also were more likely to have a distant failure within 5 years (14%, 8%, 25%, and 32% in the four groups, respectively). However, patients with positive margins more often had positive axillary lymph nodes than patients with negative or close margins (59% vs. 38%, P < 0.02). The 5-year rate of TR/MM was 20% for patients with an EIC-positive tumor and 7% for patients with an EIC-negative tumor. However, among the 127 patients with an EIC-negative tumor, the 5-year rate of TR/MM was less than 10% in all margin groups. Among the 30 patients with an EIC-positive tumor, the 5-year rate of TR/MM was 0% when margins were negative or close but 50% when margins were more than focally positive. CONCLUSIONS: These results provide support for the use of breast-conserving surgery and breast irradiation in all patients with uninvolved margins, whether the tumor is EIC-positive or EIC-negative. This study suggests that breast-conserving therapy (including a radiation boost to the primary site) also may be a reasonable option for some patients with an EIC-negative tumor and margin involvement.

Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes.

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.

Growth factor receptor and related oncogene determination in mesenchymal tumors.

BACKGROUND: Sarcoma are rare malignant neoplasms that originate from a mesenchymal cell line. The epidermal growth factor receptor (EGF-R) has been identified in these malignant neoplasms by immunohistochemical techniques. METHODS: This investigation has evaluated the gene amplification and expression of EGF-R and the homologous oncogene c-erbB-2 in soft tissue and osseous sarcomas by Southern and northern blot analysis. RESULTS: Amplification of EGF-R and c-erbB-2 was identified in 2 of 117 (1.7%) and 6 of 105 (5.7%) of the sarcomas, respectively. Increased expression of EGF-R and c-erbB-2 was identified in 21 of 43 (49%) and 35 of 94 (37%) sarcomas, respectively. CONCLUSIONS: The expression of these two genes in sarcomas appears to occur independently and not be associated with tumor histologic characteristics, grade, size, DNA content, or proliferative activity.

Detection of c-erbB-2 oncoprotein expression in breast tissue by multiparameter flow cytometry.

A novel technique in multiparameter flow cytometry (FCM) using dual laser excitation of three fluorescent dyes has been developed to differentiate breast epithelial cells from stromal components. This technique has been applied to determine the expression of the oncoprotein c-erbB-2 in breast epithelial tumors. SK-BR-3, a breast cancer cell line with c-erbB-2 overexpression, can be identified by FCM from a mixed cell suspension using a monoclonal anti-human cytokeratin antibody conjugated with fluorescein isothiocyanate. Using the mouse monoclonal anti-c-erbB-2 antibody, TA-1 (4.8 +/- 1.0 x isotype control), the c-erbB-2 oncoprotein overexpression in breast epithelial cells can be detected as an increase in indirect blue fluorescence by aminomethyl coumarin. MCF-7, a breast cancer cell line with normal c-erbB-2 expression, has baseline blue fluorescence (1.0 +/- 0.5 x isotope control). Twenty-one fresh breast specimens have been examined by FCM. Overexpression of c-erbB-2, defined by blue fluorescence ratio of TA-1/isotype control > or = 1.5 (> 3 SD from baseline), is detected in 0 of 3 patients with Stage I cancer, 5 of 14 patients with Stage II and III cancer, and 3 of 4 patients with proliferative disease. Patients with elevation of oncoprotein detected by FCM have corresponding RNA overexpression detected by Northern blot hybridization and increased gene amplification detected by Southern blot hybridization. FCM allows for the simultaneous identification of breast epithelial cells and the selective examination of these cells for the expression of c-erbB-2 oncoprotein, thus minimizing stromal contamination. This represents a novel application of FCM with potential for wide clinical applicability.