Evaluation of standard and state of the art analytical technology-bioassays.

The development of biological assays for assessing potency is a critical component for monitoring the quality of therapeutic biologicals. Traditional cell-based bioassays, which are the most widely used, are typically based on a terminal cellular response such as cell proliferation or inhibition. While these assays can be very user-friendly, results often take days and sensitivity is sometimes not sufficient for the needs of the development programme. Recent improvements in analytical technology have led to new approaches in bioassay development. Many of these assays exploit cell signalling pathways far upstream from a terminal cellular response. Bioassays based on a cell signal are much more rapid, sensitive, and indicate stability better than their predecessors. Many of these newer assays are "hybrid" assays which combine the receptor signalling of traditional bioassays with the sensitivity of detection found in immunoassays. One such method, the Kinase Receptor Activation Assay (KIRA), works through the detection of receptor phosphorylation following analyte stimulation. Validations of newer technology assays, such as KIRA, require an individualized strategy due to their unique attributes. A thorough assessment of robustness should be paramount in the validation of these assays. Several examples of new technology platforms for bioassays are also discussed.

Hormonal regulation of astroglial insulin-like growth factor (IGF)-binding protein gene expression by IGFs and insulin.

Primary astroglial cells produce insulin-like growth factor (IGF)-binding proteins (IGFBPs), which modulate the biological activity of IGFs on the developing astroglia. Alterations in the synthesis of astroglial IGFBPs by hormones and growth factors may, therefore, influence the paracrine regulatory actions of IGFs. The objective of this study was to examine the regulation of astroglial IGFBP biosynthesis by IGF-I, IGF-II, and insulin. Primary astroglial cells were cultured from newborn rat cerebral cortices. Conditioned media from astroglial cultures without (control) or with hormonal treatment (10, 100, and 200 ng/ml IGF-I or IGF-II and 0.1, 1.0, and 10 micrograms/ml insulin) were collected and subjected to Western ligand blot analysis. Total RNAs were extracted from the same cultures and subjected to Northern blot analysis. Two IGFBP species of 34K (IGFBP-2) and 40K (IGFBP-3) were identified. After 24 h of treatment, IGF-I, IGF-II, and high concentrations of insulin increased IGFBP-2 and IGFBP-3 levels. At 24 h, IGFBP-2 stable mRNA levels showed an increase corresponding to the protein levels; however, IGFBP-3 stable mRNA levels did not. Time-course studies demonstrated that IGF-I rapidly induced the mRNA levels of both IGFBP-2 and IGFBP-3 within 7.5-12 h. IGFBP-2 mRNA levels showed a biphasic response to IGF-I, a rapid increase at 7.5 h, followed by a decline at 12-18 h, and then another increase at 24 h. In contrast, IGFBP-3 mRNA levels showed a response that peaked at 7.5-12 h and decreased to control levels at 24 h. The stability of IGFBP-2 mRNA at 24 h of IGF-I treatment was not significantly altered from that under control conditions, indicating that the changes in IGFBP-2 mRNA levels were not due to alterations in the stability of the mRNA. In situ hybridization studies demonstrated an increase in the abundance per cell of IGFBP-2 and IGFBP-3 mRNAs in either one or both types of astroglial cells after IGF-I or IGF-II treatment. We conclude that, 1) IGFs differentially modulate the production of astroglial IGFBPs; 2) the increase in IGFBP mRNA levels by IGFs is rapid and biphasic; 3) the effects of IGFs on IGFBP stable mRNAs may occur at the level of gene transcriptions; and 4) the effect of IGFs on IGFBP gene expression may be cell type specific.(ABSTRACT TRUNCATED AT 400 WORDS)

Overexpression of insulin-like growth factor-binding protein-2 in C6 glioma cells results in conditional alteration of cellular growth.

To examine the relationship between the expression of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) and cell growth in a cell type with a defined IGF/IGFBP system, an ovine IGFBP-2 complementary DNA was overexpressed in C6 glioma cells. C6 cells produce IGFBP-3, IGFBP-4, a negligible amount of IGFBP-2, and IGF-I. An ovine IGFBP-2 complementary DNA was transfected into C6 cells, and nine colonies that stably expressed variable levels of IGFBP-2 messenger RNA were selected. Synthesis of corresponding levels of IGFBP-2 was confirmed by ligand blot and immunoblot analyses of conditioned media. Three clones exhibited significantly reduced growth rates, and the remainder showed growth rates similar to those of the wild-type C6 cells. The clones, which overexpressed high levels of IGFBP-2 and IGF-I, had growth rates similar to the wild-type cells, whereas the three clones that overexpressed IGFBP-2 without a concomitant increase in IGF-I had reduced growth rates. In addition, a cell-associated IGFBP was identified in the slow growing clones, but not in the wild-type or the fast growing clones. This cell-associated IGFBP was deduced to be IGFBP-5 based on its molecular size, detection of IGFBP-5 messenger RNA only in slow growing clones, and competition of its binding by heparin. Growth of the slow growing clone, C6BP2-1, could not be overcome by the addition of exogenous IGF-I, suggesting that the cell-associated IGFBP-5 was the dominant regulator of IGF action. These observations suggested that 1) in C6 glioma cells cellular growth is altered by a disturbance in the equilibrium between IGF-I and IGFBPs and/or the functional properties of the IGFBPs; and 2) C6 cells may have a limited capacity to modulate IGF/IGFBP expression in response to changes in endogenous expression of IGFBPs. Endogenous regulation of the balance between IGFs and IGFBPs may be a model of regulation of cellular growth in tumor cells.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

Alterations in the synthesis of insulin-like growth factor binding proteins and insulin-like growth factors in rat C6 glioma cells transfected with a gap junction connexin43 cDNA.

When C6 glioma cells are stably transfected with a connexin43 cDNA and gap junctions are increased, the rate of cellular proliferation is decreased. To determine if this phenomenon is related to alterations in IGFBP and IGF synthesis, we have compared IGFBPs and IGFs in the conditioned media from primary rat astroglia, C6, and transfected C6 clones Cx43-13 (high expresser), and Cx43-12 and Cx43-14 (intermediate expressers). Primary astroglia produced IGFBP-2 (34 kDa) and IGFBP-3 (40-45 kDa). C6 cells synthesized high levels of IGFBP-3 and low levels of IGFBP-2, and a 24 kDa IGFBP (IGFBP-4). Cx43-13 cells did not synthesize IGFBP-3, but produced low levels of IGFBP-2 and high levels of IGFBP-4. Cx43-12 and Cx43-14 secreted IGFBP profiles similar to the parent C6 line, but with reduced levels of IGFBP-2. The lack of IGFBP-3 in Cx43-13 cells was not due to the presence of proteases. Northern analysis showed IGFBP-2 mRNA to be readily detectable only in the primary astroglia. IGFBP-3 mRNA was detected in the primary astroglia, C6, Cx43-12 and Cx43-14, but not in Cx43-13. In contrast, IGFBP-4 mRNA was readily detected only in the Cx43-13. IGF-II concentrations in the media were low to undetectable for both C6 and transfected cells. IGF-I concentrations were significantly lower in the media from transfected cells compared to the C6 cells. Stable mRNA levels for IGF-I were lower in transfected cells, with the lowest levels observed in the Cx43-13 cells. Although C6 cells did not respond mitogenically to exogenous IGF-I or IGF-II, Cx43-13 cells responded to IGF-I or IGF-II in a dose dependent manner. Conditioned media from Cx43-13 cells decreased the DNA synthesis of C6 cells, and this effect could be reversed by the addition of IGF-II. The decreased synthesis of the autocrine/paracrine growth factor IGF-I together with decreased levels of a positive modulator IGFBP-3, and the increased levels of a negative modulator IGFBP-4 in the extracellular milieu, may be responsible for the reduced proliferative capacity in cells expressing abundant connexin43.

Visual pattern integration as a function of antidepressant medication in depressed patients.

On the basis of an earlier study that reported depressed patients to be superior to normal control subjects in visual iconic integration, the present investigation sought to determine if this finding was replicable and if medication produced it. Newly admitted depressed patients entering drug treatment, depressed patients already in drug treatment, and healthy control subjects were twice tested, with a 5-week interval between sessions. Fully medicated depressed patients, whether newly admitted or already in drug treatment, were superior to normal control subjects in visual integration, and healthy control subjects were superior to depressed patients just entering treatment. At the first testing, the number of days of medication treatment was significantly correlated with the adequacy of visual integration.

Problems in a treatment program for substance misuse. The process of reorganizing into assessment teams and modules.

The previous problems of a treatment program for substance misuse at a V.A. hospital are discussed, along with the implementation of a reorganization of this program into assessment teams and treatment modules, and the effects of this reorganization on program, staff and patients.

The love of war: Vietnam and the traumatized veteran.

Many veterans treated for posttraumatic stress disorder (PTSD) keep alive their war experiences because of their significance and meaning. For these veterans, combat was a positive as well as a negative experience. The authors suggest that many veterans suffer from PTSD because they are continuing to live out their war experiences and to hold onto the meaning of these experiences. Effective treatment requires these veterans to develop a competent peacetime self that incorporates the positive features of the warrior identity.

Combat and personality change.

In response to combat, some soldiers develop a feeling of satisfaction in killing. The authors label this reaction the "heart of darkness experience," after the story by Joseph Conrad (1903/1982). They describe their clinical experience of seeing this response as part of a spectrum of reactions ranging from no personality change to rather gross personality change. After exploring psychological factors involved in this change, they suggest relevant treatment considerations.

Patterns of utilization of medical care and perceptions of the relationship between doctor and patient with chronic illness including chronic fatigue syndrome.

To what extent do personal constructs affect the relationship between doctor and patient when the ill patient does not readily recover with treatment? Questionnaires were returned anonymously by 609 patients with a self-reported diagnosis of chronic fatigue syndrome, who were considered chronically ill. Findings were compared with those of an earlier study of a population of 397 general medical patients. The chronically ill patients lost an average of 65 days of work per year due to illness compared to general medical patients who missed six or fewer days per year because they were ill. The chronically ill patients also reported a 66% higher frequency of iatrogenic illness, spent more money on health care, took more medication, saw more specialists, and were more litigious than the general medical population. Research suggested several patterns of relationships between doctors and patients, and attitudes to health and illness, which may alert doctors to patients' perceptions, beliefs, encoded constructs, and patterns of relating that affect responses to treatment. More attention by doctors to patients who are experiencing the stress of chronic illness is indicated.

Some interpersonal and attitudinal factors characterizing patients satisfied with medical care.

This research investigated the extent to which interpersonal and attitudinal factors affect the doctor-patient relationship. A questionnaire survey of people living in northeastern Kansas who had experience with medical care was conducted. Dependent measures were over-all patient satisfaction, patient's attitude toward death of self or others, causality orientation, attitude toward health and disease, and perception of the doctor-patient relationship. The majority of test subjects (92%) reported satisfaction with their medical care. A significant correlation between fear of death of significant others and scores on causality scales reflects feelings of not being in control of one's life. Other associations indicate that people who do not feel in control of their lives depend on traditional and folk remedies. Scores showing low fear of own death correlated significantly with the rated greater sense of responsibility for own health and treatment. Patients who rated themselves as more needy were unsatisfied with their care and those whose doctors called them by first name tended to be more content. Even in populations satisfied with their medical care, we suggest that the quality of care could be improved by attention to interpersonal and attitudinal factors.

Use of first and last names and milieu characteristics.

As a part of a cooperative study of 79 psychiatric wards in 18 Veterans Administration hospitals with 12,000 resident patients, data were obtained on how staff members and patients address each other. It was found that first name usage by staff members and patients correlates with certain characteristics of psychiatric wards. Predominant first name usage tended to occur on wards with a high degree of patient autonomy, patient participation in discharge planning, nursing personnel wearing street clothes, etc. These characteristics are consonant with a particular ward model, namely that of a therapeutic community.

Insulin-like growth factor binding protein-4 gene expression is induced by transfection of gap junction connexin43 gene in a C6 glioma cell line.

When C6 glioma cells are stably transfected with a connexin43 cDNA and the gene overexpressed, the rate of cellular proliferation is decreased. To determine if this phenomenon is related to alterations in IGFBP synthesis, we have compared the conditioned media of primary rat astroglia, C6, clones Cx43-13 (high expresser of the transfected connexin43 gene), and Cx43-12 and Cx43-14 (intermediate expressers). Primary astroglia produced IGFBP-2 (M(r) 34 K) and IGFBP-3 (40-45 K). C6 cells synthesized high levels of IGFBP-3 and low levels of IGFBP-2, and a 24 K IGFBP (IGFBP-4). Cx43-13 cells did not synthesize IGFBP-3, but produced low levels of IGFBP-2 and high levels of IGFBP-4. Cx43-12 and Cx43-14 secreted IGFBP profiles similar to the parent C6 line, but with reduced levels of IGFBP-2. Northern analysis showed the changes in IGFBPs in the conditioned media to be correlated with alterations in stable mRNA levels. IGFBP-4, a inhibitor of IGF biological action, was produced in greater quantities by the slowly proliferating Cx43-13 cells. Alterations in IGFBP-4 synthesis may be responsible, at least in part, for the reduced proliferative capacity in cells with abundant connexin43.

Expression of IGF system genes during T-antigen driven pituitary tumorigenesis.

Expression of IGF-I, IGF-II, the Type-I IGF receptor and six IGF binding proteins were examined in three different T-ag-driven mouse tumors. Unlike the widespread expression of IGF-II in pancreatic beta-cell tumors, IGF-II was not widely expressed in the two different pituitary tumors examined indicating that a mechanism independent of focal IGF-II expression can also drive T-antigen tumorigenesis. In addition, multiple IGF binding proteins were expressed in all three tumor types. This expression, however, was generally heterogeneous with no specific changes to indicate a required role for any IGF binding protein in T-antigen tumorigenesis.