Spinal and sacroiliac assessment and treatment techniques used by osteopathic physicians in the United States

Background

Without systematic exposure to biomedical research concepts or applications, osteopathic medical students may be generally under-prepared to efficiently consume and effectively apply research and evidence-based medicine information in patient care. The academic literature suggests that although medical residents are increasingly expected to conduct research in their post graduate training specialties, they generally have limited understanding of research concepts. With grant support from the National Center for Complementary and Alternative Medicine, and a grant from the Osteopathic Heritage Foundation, the University of North Texas Health Science Center (UNTHSC) is incorporating research education in the osteopathic medical school curriculum. The first phase of this research education project involved a baseline assessment of students' understanding of targeted research concepts. This paper reports the results of that assessment and discusses implications for research education during medical school.

Methods

Using a novel set of research competencies supported by the literature as needed for understanding research information, we created a questionnaire to measure students' confidence and understanding of selected research concepts. Three matriculating medical school classes completed the on-line questionnaire. Data were analyzed for differences between groups using analysis of variance and t-tests. Correlation coefficients were computed for the confidence and applied understanding measures. We performed a principle component factor analysis of the confidence items, and used multiple regression analyses to explore how confidence might be related to the applied understanding.

Results

Of 496 total incoming, first, and second year medical students, 354 (71.4%) completed the questionnaire. Incoming students expressed significantly more confidence than first or second year students (F = 7.198, df = 2, 351, P = 0.001) in their ability to understand the research concepts. Factor analyses of the confidence items yielded conceptually coherent groupings. Regression analysis confirmed a relationship between confidence and applied understanding referred to as knowledge. Confidence scores were important in explaining variability in knowledge scores of the respondents.

Conclusion

Medical students with limited understanding of research concepts may struggle to understand the medical literature. Assessing medical students' confidence to understand and objectively measured ability to interpret basic research concepts can be used to incorporate competency based research material into the existing curriculum.     

Treatment of specific macrovascular beds in patients with diabetes mellitus

Background

A ubiquitous dilemma in medical education continues to be whether and how to integrate research competencies into the predoctoral curriculum. Understanding research concepts is imbedded in the six core competencies for physicians, but predoctoral medical education typically does not explicitly include research education. In an effort to quickly report academic research findings to the field, this is the second in a series of articles reporting the outcomes of a research education initiative at one college of osteopathic medicine. The first article described the competency model and reported baseline performance in applied understanding of targeted research concepts. This second article reports on the learning outcomes from the inaugural year of a course in basic biomedical research concepts.

Methods

This course consisted of 24 total hours of classroom lectures augmented with web-based materials using Blackboard Vista, faculty moderated student presentations of research articles, and quizzes. To measure changes in applied understanding of targeted research concepts in the inaugural year of the course, we administered a pretest and a posttest to second year students who took the course and to first year students who took an informatics course in the same academic year.

Results

We analyzed 154 matched pretests and posttests representing 56% of the 273 first and second year students. On average, the first year (53) and second year students (101) did not differ in their mean pretest scores. At posttest the second year students showed significant improvement in their applied understanding of the concepts, whereas the first year students' mean posttest score was lower than their mean pretest score.

Conclusions

This biomedical research course appears to have increased the second year students' applied understanding of the targeted biomedical research concepts. This assessment of learning outcomes has facilitated the quality improvement process for the course, and improved our understanding of how to measure the benefits of research education for medical students. Some of the course content and methods, and the outcome measures may need to be approached differently in the future to more effectively lay the foundation for osteopathic medical students to utilize these concepts in the clinical setting.     

Integration of neuroscience and endocrinology in hybrid PBL curriculum

At the University of Missouri-Columbia, the medical school employs a problem-based learning curriculum that began in 1993. Since the curriculum was changed, student performance on step 1 of the United States Medical Licensing Examination has significantly increased from slightly below the national average to almost one-half a standard deviation above the national mean. In the first and second years, classes for students are organized in classes or blocks that are 8 wk long, followed by 1 wk for evaluation. Initially, basic science endocrinology was taught in the fourth block of the first year with immunology and molecular biology. Student and faculty evaluations of the curriculum indicated that endocrinology did not integrate well with the rest of the material taught in that block. To address these issues, basic science endocrinology was moved into another block with neurosciences. We integrate endocrinology with neurosciences by using the hypothalamus and its role in neuroendocrinology as a springboard for endocrinology. This is accomplished by using clinical cases with clear neuroscience and endocrinology aspects such as Cushing's disease and multiple endocrine neoplastic syndrome type 1.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Mitogenic regulation of normal and malignant breast epithelium

The multiple roles of both estrogenic and polypeptide regulators of mammary epithelial cell growth are reviewed in this article. Effects of both steroidal and peptide hormones are complex and involve multiple interactions with malignant cells and non-malignant host components. Initial carcinogenesis and progression of mammary epithelium to cancer probably require both proliferative stimuli (estrogen, polypeptide growth factors) and genetic damage. This condition may lead to qualitatively different hormonal responses (hormone-responsive cancer). Estrogens can be shown to induce growth-regulatory polypeptide growth factors and interact with them in hormone-dependent breast cancer. Progression of hormone-dependent (estrogen-responsive) breast cancer to hormone independence probably involves multiple mechanisms, including oncogene activation, loss of the estrogen receptor, or loss of hormone responsivity of other gene products. One direction for further therapies may be blockade of hormonal stimulation and interference with necessary activated or induced components of malignant progression such as oncogenes or polypeptide growth factor-receptor systems.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.     

Acquired resistance to selective estrogen receptor modulators (SERMs) in clinical practice (tamoxifen & raloxifene) by selection pressure in breast cancer cell populations

Tamoxifen, a pioneering selective estrogen receptor modulator (SERM), has long been a therapeutic choice for all stages of estrogen receptor (ER)-positive breast cancer. The clinical application of long-term adjuvant antihormone therapy for the breast cancer has significantly improved breast cancer survival. However, acquired resistance to SERM remains a significant challenge in breast cancer treatment. The evolution of acquired resistance to SERMs treatment was primarily discovered using MCF-7 tumors transplanted in athymic mice to mimic years of adjuvant treatment in patients. Acquired resistance to tamoxifen is unique because the growth of resistant tumors is dependent on SERMs. It appears that acquired resistance to SERM is initially able to utilize either E₂ or a SERM as the growth stimulus in the SERM-resistant breast tumors. Mechanistic studies reveal that SERMs continuously suppress nuclear ER-target genes even during resistance, whereas they function as agonists to activate multiple membrane-associated molecules to promote cell growth. Laboratory observations in vivo further show that three phases of acquired SERM-resistance exists, depending on the length of SERMs exposure. Tumors with Phase I resistance are stimulated by both SERMs and estrogen. Tumors with Phase II resistance are stimulated by SERMs, but are inhibited by estrogen due to apoptosis. The laboratory models suggest a new treatment strategy, in which limited-duration, low-dose estrogen can be used to purge Phase II-resistant breast cancer cells. This discovery provides an invaluable insight into the evolution of drug resistance to SERMs, and this knowledge is now being used to justify clinical trials of estrogen therapy following long-term antihormone therapy. All of these results suggest that cell populations that have acquired resistance are in constant evolution depending upon selection pressure. The limited availability of growth stimuli in any new environment enhances population plasticity in the trial and error search for survival.     

Mechanisms of normal and malignant breast epithelial growth regulation

In this presentation we review information highlighting the multiple roles of both steroidal and polypeptide regulators of mammary epithelial cell growth with some additional emphasis on the work of our laboratory. The effects of both classes of hormones are complex and involve multiple interactions with epithelial components (malignant or normal) and the stromal compartment. Estrogens induce growth regulatory polypeptide growth factors which are responsible for many of the induced phenotypic effects in hormone-dependent breast cancer. Progression of hormone-dependent breast cancer to hormone independence probably involves multiple genetic mechanisms of oncogene activation, loss of the estrogen receptor, or loss of hormone responsivity of other gene products. Initial carcinogenesis and progression of mammary epithelium to cancer probably also requires both proliferative stimuli (estrogen, polypeptide growth factors) and genetic damage, leading to qualitatively different hormonal responses (hormone responsive cancer). New therapeutic strategies based on these biological considerations are emerging, including a variety of approaches which interfere at multiple points with ability of ligand to induce receptor signaling.     

Estrone sulfate-sulfatase and 17β-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence

The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E₁ S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E₁ S to E₂ very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E₁ to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E₂) in hormone-dependent cells, but oxidative (E₂ → E₁) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E₁ to E₂. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E₂; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E₁ S to E₂. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease.     

Successful Estrogen Therapy for Post-Adrenalectomy Relapses of Breast Cancer

Four postmenopausal women are described in whom breast cancer responded both to bilateral adrenalectomy and bilateral oophorectomy, and subsequently, after relapse, to estrogen therapy. This paradoxical finding demonstrates the complexity of the response of breast carcinoma to hormone manipulations. Simple estrogen dependence and estrogen suppression of pituitary mammotrophins are seen to be inadequate explanations of this phenomenon. Seven fundamental observations are listed that have to be accounted for by hypotheses concerning the endocrinology of breast cancer. It is suggested that in the past we have perhaps overlooked (1) the difficulty of extrapolating observations on experimental animal tumours to spontaneous human neoplasms, (2) the fact that there may be more than one type of breast cancer, and (3) the important role that must be played by the different tissues bearing the metastases.     

A combined histological, immunocytochemical and biochemical approach in the evaluation of estrogen receptors in breast carcinomas

Correlation of structural and functional data might lead to better identification of hormone-dependent tumors. Sixty breast cancer specimens, sent to the biochemistry laboratory for estrogen receptor (ER) analysis, were studied here by a combined morpho-functional approach. Histological examination of needle biopsies on frozen tissue blocks showed that 12 cases (10%) were free of tumor cells; these cases mostly proved ER negative. On the other 48 cases, an immunocytochemical reaction was performed on the biopsy sections with a monoclonal antibody directed against p 29, an estrogen receptor related antigen. The staining values for p 29 and the biochemical ER findings were significantly correlated. A combined histological, immunocytochemical study seems to offer advantages in the selection of patients for hormonal therapy.     

Synthesis,antiproliferative and pro-apoptotic activity of 2-phenylindoles

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC₅₀ values of 2.71 μM and 1.86 μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86.     

Myc stabilization in response to estrogen and phospholipase D in MCF-7 breast cancer cells

Estrogen, which has been strongly implicated in breast cancer, suppresses apoptosis in estrogen receptor (ER) positive MCF-7 breast cancer cells. Phospholipase D (PLD), which is commonly elevated in ER negative breast cancer cells, also suppresses apoptosis. Survival signals generated by both estrogen and PLD are dependent upon elevated Myc expression. We report here that estrogen- and PLD-induced increases in Myc expression are due to reduced turnover of Myc protein. Estrogen and PLD suppressed phosphorylation of Myc at Thr58 - a site that targets Myc for degradation by the proteasome. The data provide a mechanism for elevated Myc expression in hormone-dependent and hormone-independent breast cancer.     

DAX-1, as an androgen-target gene,inhibits aromatase expression: a novel mechanism blocking estrogen-dependent breast cancer cell proliferation

Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. In situ estrogen production by aromatase is a critical determinant for breast cancer growth and progression. On the contrary, clinical and in vitro studies indicate that androgens have a protective role in mammary carcinogenesis. Here, we demonstrated, in hormone-dependent breast cancer cells, the existence of a functional interplay between the androgen receptor (AR), the orphan nuclear receptor DAX-1 and the aromatase enzyme involved in the inhibition of the estrogen-dependent breast cancer cell proliferation exerted by androgen signaling. Indeed, our results revealed, in MCF-7 cells, that ligand-activated AR induces the expression of the orphan nuclear receptor DAX-1 by direct binding to a newly identified androgen-response-element within the DAX-1 proximal promoter. In turn, androgen-induced DAX-1 is recruited, in association with the corepressor N-CoR, within the SF-1/LRH-1 containing region of the aromatase promoter, thereby repressing aromatase expression and activity. In elucidating a novel mechanism by which androgens, through DAX-1, inhibit aromatase expression in breast cancer cell lines, these findings reinforce the theory of androgen- opposing estrogen-action, opening new avenues for therapeutic intervention in estrogen-dependent breast tumors.     

Estrogen withdrawal-induced human breast cancer tumour regression in nude mice is prevented by Bcl-2

We recently showed that estrogen induces expression of the anti-apoptotic protein, Bcl-2 in MCF-7 human breast cancer cells. Since estrogen-dependent breast tumours can regress following estrogen withdrawal, we hypothesized that stable Bcl-2 expression would prevent estrogen-withdrawal induced regression of MCF-7 tumours. We therefore established tumours in ovariectomized female nude mice implanted with an estrogen-release pellet using untransfected MCF-7 cells or MCF-7 cells stably transfected with a Bcl-2 cDNA sense or antisense expression vector. All tumours grew at similar rates indicating that Bcl-2 levels have no effect on tumour formation. After removal of the estrogen pellet, Bcl-2 antisense tumours and untransfected MCF-7 tumours regressed means of 49% and 52%, respectively, after estrogen pellet removal whereas Bcl-2 sense tumours were significantly stabilized. Regressing tumours displayed characteristics of apoptotic cells. These results show that Bcl-2 can prevent hormone-dependent breast tumour regression and are consistent with the notion that decreased Bcl-2 levels following estrogen withdrawal renders hormone-dependent breast tumour cells sensitive to apoptotic regression.     

Systematic mapping of posttranslational modifications in human estrogen receptor-alpha with emphasis on novel phosphorylation sites

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.     

Design and synthesis of estrogen receptor ligands with a 4-heterocycle-4-phenylheptane skeleton

The estrogen receptor (ER), a member of the nuclear receptor (NR) family, is involved in the regulation of physiological effects such as reproduction and bone homeostasis. Approximately 70% of human breast cancers are hormone-dependent and ERα-positive, and, thus, ER antagonists are broadly used in breast cancer therapy. We herein designed and synthesized a set of ER antagonists with a 4-heterocycle-4-phenylheptane skeleton.     

The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata,an exercise in comparative metabolism for food and wine science students

Comparative cellular metabolism can be a difficult area of biochemistry to teach in the undergraduate laboratory class. Student practicals involving animal tissues generally require approval from animal ethic committees, and the relevance for students whose primary interest in biochemistry is in the area of food and wine sciences, is often questioned. In this report, we present an undergraduate practical exercise in which glucose catabolism via the pentose phosphate pathway is compared in two types of yeast with direct relevance to the wine and food industries, Saccharomyces cerevisiae and Kloeckera apiculata. The exercise is carried out as a demonstration to second year undergraduate students, studying metabolic biochemistry. It is of some value in that it illustrates comparative cellular metabolism in wine yeasts and introduces the students to the safe use of radioisotopes.