Fibrinogen assembly, secretion, and deposition into extracellular matrix by MCF-7 human breast carcinoma cells.

A hallmark of breast carcinoma is the deposition of fibrinogen (FBG) without subsequent conversion to fibrin in the tumor stroma. In this study, the ability of the MCF-7 human breast cancer epithelial cell line to synthesize, secrete, and deposit FBG into the extracellular matrix (ECM) was examined. Whereas MCF-7 cells produced low levels of intact FBG, abundant levels of FBG intermediate complexes or degraded Aalpha, Bbeta, and gamma chain polypeptides were observed. Most of the Bbeta chain was degraded and missing an NH2-terminal peptide fragment. Reverse transcription-PCR analysis indicated that only gamma chain mRNA was present in detectable steady-state levels, although Southern hybridization revealed that the FBG Aalpha, Bbeta, and gamma chain genes were intact in MCF-7 cells. Immunostaining showed that extracellular FBG was bound to the surface of MCF-7 cells in a punctate pattern, reminiscent of receptor binding, rather than a fibrillar pattern characteristic of mature ECM. A similar punctate pattern of staining was observed when MCF-7 FBG was added to fibroblasts that normally assemble exogenous FBG into an extensive, fibrillar ECM, suggesting that MCF-7 cells are defective in assembly of a fibrillar ECM. The loss of FBG Bbeta chain NH2-terminal peptides may contribute to the lack of intact FBG assembly in MCF-7 cells, which may further affect its ability to assemble FBG into a fibrillar ECM. Taken together, the data suggest that endogenous synthesis and secretion of FBG is, at least in part, the source of FBG deposition in the ECM of breast cell carcinomas.

Calcium modulates plasmin cleavage of the fibrinogen D fragment gamma chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the gamma chain.

Plasmin sensitive sites are found on the A alpha, B beta and gamma chains of fibrinogen at regions joining the two C-terminal D fragments with the central E fragment. We have developed a monoclonal antibody (MoAb) reactive with this plasmin sensitive region on the human fibrinogen gamma chain and mapped its epitope. MoAb J88B reacts with gamma chains of both native as well as with reduced and denatured fibrinogen and fibrin, the CNBr fragment of the fibrinogen central domain, plasmin cleaved fragments D, gamma-gamma dimers, but not with plasmic fragments E. These data indicate that J88B maps to the plasmin sensitive domain localized to gamma 63-78. MoAb J88B failed to react with synthetic peptide gamma 70-78, which suggests that the epitope includes the newly exposed N-terminal residues gamma 63-70 of the early plasmic fragment D1A. As calcium has a marked influence on plasmin cleavage of C-terminal sites on the gamma chain, the effects of calcium on modulating plasmin cleavage of D1A to D1 were assessed in the absence or presence of J88B. The results indicated that calcium delays and J88B (+/- calcium) protects the gamma chain from plasmin cleavage at the N-terminus of D1A, suggesting that this enzymatically labile site is calcium-sensitive. Thus, MoAb J88B should prove useful in studies examining the structure of plasmin cleaved fibrinogen and fibrin.

Characterization of a monoclonal antibody, D73H, that maps to a highly conserved region on fibrinogen Bbeta chain.

The primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bbeta chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bbeta chain of plasmin fragment D, localizing its epitope to Bbeta134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bbeta243-253, further localizing the epitope to Bbeta243-305. In silico analysis indicated that Bbeta243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bbeta252-258, Bbeta262-269, and Bbeta279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bbeta262-269 (FGRKWDPY) is predominantly alpha-helical and located on the surface of the molecule adjacent to a bend imposed in the beta chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bbeta261-272 competitively inhibited the binding of D73H to the Bbeta chain of denatured intact fibrinogen and reduced and denatured Bbeta chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bbeta chain residues Bbeta261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

Tumors and fibrinogen. The role of fibrinogen as an extracellular matrix protein.

The progression of a tumor from benign and localized to invasive and metastatic growth is the major cause of poor clinical outcome in cancer patients. Much like in a healing wound, the deposition of fibrin(ogen), along with other adhesive glycoproteins, into the extracellular matrix (ECM) serves as a scaffold to support binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during angiogenesis and tumor cell growth. Inappropriate synthesis and deposition of ECM constituents is linked to altered regulation of cell proliferation, leading to tumor cell growth and malignant transformation. Fibrin deposition occurs within the stroma of a majority of tumor types. In contrast, abundant FBG, not fibrin, is present within the stroma of breast cancers. It is thought to originate from exudation of plasma FBG and subsequent deposition into the tumor stroma and not endogenous synthesis and secretion of FBG by breast tumor cells. However, we show that MCF-7 human breast cancer cells synthesize and secrete FBG polypeptides, suggesting that the origin of FBG in the stroma of breast carcinoma may be due to endogenous synthesis and deposition. Moreover, FBG assembles into ECM as conformationally altered FBG, not as fibrin. Studies in our laboratory demonstrate that FBG alters the ability of breast cancer cells to migrate. Together, the results of studies from our laboratory, as well as the laboratories of others, indicate that the presence of fibrin(ogen) within the tumor stroma likely affects the progression of tumor cell growth and metastasis. This review focuses on FBG within tumors and its relationship with other tumor constituents, ultimately focusing on the role of FBG in breast cancer.

Mystery of the toxic flea dip: an interactive approach to teaching aerobic cellular respiration.

We designed an interrupted case study to teach aerobic cellular respiration to major and nonmajor biology students. The case is based loosely on a real-life incident of rotenone poisoning. It places students in the role of a coroner who must determine the cause of death of the victim. The case is presented to the students in four parts. Each part is followed by discussion questions that the students answer in small groups prior to a classwide discussion. Successive parts of the case provide additional clues to the mystery and help the students focus on the physiological processes involved in aerobic respiration. Students learn the information required to solve the mystery by reading the course textbook prior to class, listening to short lectures interspersed throughout the case, and discussing the case in small groups. The case ends with small group discussions in which the students are given the names and specific molecular targets of other poisons of aerobic respiration and asked to determine which process (i.e., glycolysis, citric acid cycle, or the electron transport chain) the toxin disrupts.

Age and forgetting rate with pictorial stimuli.

The rate of forgetting standardized line drawings of common objects was assessed in groups of young (M age = 22 years) and older (M age = 70.5 years) subjects. The two groups forgot equal quantities of pictorial stimuli over successive intervals of 10 min, 2 hr, and 48 hr, after being matched for original learning. In contrast, the older subjects showed the expected age decrement in reproduction of geometric designs from memory. These findings indicate that aging does not affect retention of pictures when differences in learning and retrieval abilities are controlled.

Reminiscence interviews as stress management interventions for older patients undergoing surgery.

A standard reminiscence interview and one that focused on successfully met challenges reduced state anxiety and enhanced coping self-efficacy when measured against both attention-placebo and no-intervention control groups in a sample of 104 elderly male patients facing surgery. Age-peer interviewers did not elicit significantly greater overall reductions in state anxiety or increases in coping self-efficacy scores than younger interviewers (nonpeers), but did produce significantly higher coping self-efficacy scores than nonpeers when administering the challenge reminiscence interview.

Comparing mind-body wellness interventions for older adults with chronic illness: classroom versus home instruction.

Two versions of a multicomponent mind-body wellness intervention were tested. One hundred seventy-eight physician-referred HMO patients (mean age = 64.5) were randomly assigned to a classroom intervention, a home study intervention, or a wait-list control group. Both interventions provided instruction on mind-body relationships, relaxation training, cognitive restructuring, problem solving, communication, behavioral treatment for insomnia, nutrition, and exercise. The home version was delivered by class videotapes and readings. Compared with the control condition, both interventions led to significant decreases in self-reports of pain, sleep difficulties, and symptoms of depression and anxiety. The home course also led to a significant decrease in self-reported frequency of medical symptoms, and the classroom program resulted in a significant decrease in "chance" health locus of control beliefs. No effects were obtained for health behaviors, life satisfaction, HMO satisfaction, and other health locus of control beliefs. A lower cost, more accessible home study version of a mind-body wellness program can be an effective alternative to classroom instruction.

A classroom mind/body wellness intervention for older adults with chronic illness: comparing immediate and 1-year benefits.

The authors tested the efficacy of a mind/body wellness intervention for older adults with chronic illness. They randomly assigned 243 physician-referred patients from an urban HMO to a classroom intervention or a wait-list control group. The intervention provided instruction on mind/body relationships; relaxation training; cognitive restructuring; problem-solving; communication; and behavioral treatment for insomnia, nutrition, and exercise. At posttreatment, the intervention group had significant decreases in self-reported sleep difficulties, pain, anxiety, and depression symptoms compared with controls. The intervention also led to a significant decrease in "chance" and "powerful others" health locus of control beliefs. At 1-year follow-up, the intervention group maintained benefits in sleep and health locus of control and also reported a significant increase in health behaviors compared with controls. Pain, anxiety, and depression benefits were not maintained. This type of classroom intervention appears to have some lasting effects on health behaviors and beliefs.

A multifactorial study of age bias among rehabilitation professionals.

OBJECTIVES: To assess different dimensions of age bias among rehabilitation professionals and to determine the effect of patient gender and behavior on these biases. DESIGN: Between-subjects questionnaire study. SETTING: Randomly selected institutions accredited with the Commission on the Accreditation of Rehabilitation Facilities (CARF). PARTICIPANTS: Nine hundred seventy-four rehabilitation professionals from 9 disciplines at 23 CARF-accredited facilities. INTERVENTIONS: Each professional received a vignette case history of an amputation patient who varied in age (36yr, 76yr), gender (male, female), and patient behavior (ideal, depressed, noncompliant). The vignette was followed by the Professional Bias Questionnaire (PBQ), which included 25 questions assessing the professionals' responses to the patient. MAIN OUTCOME MEASURES: Main effects and interaction effects for age, gender, and behavior for the PBQ. RESULTS: Factor analysis of the PBQ questionnaire yielded 4 reliable scales: the professional's judgment of the patient's psychologic neediness, postdischarge potential, worthiness for rehabilitation, and their personal reaction to working with the patient. Analyses of variance resulted in significant age by behavior interaction effects for 3 of the 4 scales. Older patients were viewed more negatively than equivalent younger patients when they were noncompliant. Professionals also had a more negative personal reaction to depressed older patients compared with equivalent younger patients. In contrast, gender bias was only found in 1 specific situation and did not interact with age bias. CONCLUSION: Several forms of bias toward older adult patients were found among rehabilitation professionals, but they were present only when patient behavior was less than ideal. Age bias continues to be a critical issue in the equity of rehabilitation services and should be addressed with training and policy changes.

Applying cognitive-behavioral psychotherapy to the chronically ill elderly: treatment issues and case illustration.

A large percentage of older adults must endure at least one chronic medical illness. Clinically significant depression and anxiety are common among these patients. Specific psychotherapy approaches as well as adaptations required to address the unique issues of this population have not been delineated in the literature. We outline a cognitive-behavioral therapy approach and discuss five treatment issues we have found to be important for this population. These issues include: (1) resolving practical barriers to participation; (2) accepting depression as a separate and reversible problem; (3) limiting excess disability; (4) counteracting the loss of important social roles and autonomy; and (5) challenging the perception of being a "burden." A case study of a chronic obstructive pulmonary disease (COPD) patient with depression is presented and recommendations for future research are suggested.

Social discomfort and depression in a sample of adults with leg amputations.

We examined the relationship between social discomfort and depression in a sample of 89 adults with leg amputations at two outpatient clinics. It was hypothesized that individuals who reported being uncomfortable with social contacts involving acknowledgement of their amputation or prosthesis were more prone to depression than other patients. A set of questions addressing different aspects of social discomfort demonstrated internal consistency and were used as a scale. Social discomfort was significantly correlated with scores on the Center for Epidemiologic Studies-Depression Scale depression scale (r = .41). Multiple regression analysis showed that social discomfort was a significant independent predictor of depression, after holding constant the effects of age, gender, social support, time since amputation, reason for amputation, and perceived health. These data indicate that health care professionals should view the expression of social discomfort by amputee patients as a possible "marker" for depression. Further studies are planned to develop and validate an expanded social discomfort scale.

Rehabilitation staff perceptions of characteristics of geriatric rehabilitation patients.

OBJECTIVE: To determine if professionals treating older rehabilitation patients regarded them as having different characteristics than younger rehabilitation patients, to derive factors from these perceptions, and to examine the impact of the discipline of the professional and other factors on these perceptions. DESIGN: Rehabilitation professionals at a random sample of facilities accredited by the Commission on Accreditation of Rehabilitation Facilities were surveyed to determine their level of agreement with 60 items addressing characteristics of older rehabilitation patients. The items were derived from focus groups with rehabilitation staff members. PARTICIPANTS: One thousand sixty-three rehabilitation professionals from nine disciplines (nursing, occupational therapy, physiatry, physical therapy, psychology, social work, speech pathology, therapeutic recreation, and vocational counseling) responded to the questionnaire and were included in the study. RESULTS: There was a wide range of agreement levels across the 60 items (range of median agreement, 12.7% to 93.5%). Factor analysis resulted in six categories of perceptions regarding older rehabilitation patients: (1) physical limitations, (2) motivational deficits, (3) psychological distress/need for support, (4) maturity and positive coping skills, (5) need for privacy/decreased adaptability, and (6) discharge complications. Significant differences across disciplines were found for five of six factors. Nurses agreed more strongly with the negative psychological factors (2, 3, and 5) compared to physical therapists, psychologists, and social workers. Physicians scored significantly higher than two other disciplines on the physical limitations factor. These differences may be related to the distinct role each discipline plays in the rehabilitation process. Older professionals also scored higher on four factors, likely because of personal rather than professional experience with aging. CONCLUSIONS: Treating professionals recognize differences between younger and older rehabilitation patients. Many of these perceived differences can be viewed as variables that require more effort and skill on the part of the treating professional. The training of rehabilitation professionals needs to better prepare individuals from all disciplines to adapt to age-specific differences.