Introducing a post-registration interprofessional learning programme for healthcare teams.

INTRODUCTION: Few studies have evaluated interprofessional learning (IPL) and teamworking in active clinical teams. The aim of this study was to evaluate an IPL programme offered to established clinical teams by assessing team climate before, during and after the intervention. METHODS: A previously validated questionnaire, that explored team members' views of team climate, was administered before the IPL programme, at four months following facilitated meetings, and again at eight months. Responses were analysed using one-sample and independent samples t-tests. RESULTS: Nine teams, made up of 79 individuals, agreed to join the IPL programme. After four months, during which time the teams were supported by an educational facilitator, the overall team climate increased by 8.0% of the maximum possible score of the questionnaire (95% confidence interval = 7.4% to 8.6%). This difference was highly statistically significant (p-value <0.001) and similar increases in scores were seen in each section of the questionnaire. This significant change was sustained after a further four months when the programme continued without the support of an educational facilitator. CONCLUSION: An IPL programme, such as the one described in this paper, can improve team climate and raise awareness of professional roles within established clinical teams.

An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity.

The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.

The association of human adipose angiotensinogen gene expression with abdominal fat distribution in obesity.

OBJECTIVE: To investigate in obese subjects the relationship between angiotensinogen gene expression in the abdominal omental and subcutaneous adipose tissue on the one hand and body fat distribution as measured by waist-to-hip ratio (WHR) on the other hand and to compare angiotensinogen gene expression between the two adipose tissue regions. SUBJECTS: Twenty obese subjects undergoing weight reduction surgery with adjustable gastric banding (12 men, eight women; WHR 0.89-1.09; body mass index (BMI) 29-51 kg/m2, age 26-54 y). MEASUREMENTS: Omental and subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured by competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Angiotensinogen mRNA levels were one-third higher in the omental than in the subcutaneous adipose tissue region (P=0.02). The 18S rRNA levels did not differ significantly between the two adipose tissue regions. WHR correlated positively and significantly with angiotensinogen mRNA in both the subcutaneous and the omental adipose tissue (r=0.5). This relationship was independent of age and BMI. However, WHR did not correlate with 18S rRNA in any of the adipose tissue regions. CONCLUSION: The angiotensinogen gene in adipose tissue might be involved in the development of upper-body obesity.

Acetylcholine-induced calcium signaling along the rat colonic crypt axis.

BACKGROUND & AIMS: Acetylcholine-induced calcium signaling dynamics have been described in cell monolayers derived from colonic mucosa, but not in intact colonic crypts. The aim of this study was to characterize the spatiotemporal characteristics of calcium signaling induced by acetylcholine in isolated intact rat colonic crypts and to identify the muscarinic receptor subtype coupled to this signaling pathway. METHODS: Isolated crypts from the distal colon of male Wistar rats were loaded with the calcium-sensitive dye Fura-2 and imaged with a charge-coupled device video camera. RESULTS: Acetylcholine mobilized intracellular calcium with an EC50 of 3.9 micromol/L. The response was initiated at the base of the crypt and progressed toward the surface. The velocity of propagation was dose dependent. Addition of muscarinic antagonists inhibited the response (pKb values calculated for pirenzepine and 4-DAMP, 6.08 and 8.65, respectively). Microperfusion of acetylcholine initiated a calcium signal throughout the lower half of the crypt. Microinjection of inositol 1,4,5-triphosphate induced a propagation of a calcium signal along the crypt axis. Heptanol inhibited the velocity of acetylcholine-induced wave propagation by 33%. CONCLUSIONS: M3 muscarinic receptors are coupled to the mobilization of calcium from intracellular stores of intact, isolated rat colonic crypts. Intercellular communication potentiates the propagation of the acetylcholine-induced calcium signal along the crypt axis.

Ethene oxide and bone induction. Controversy remains.

There is controversy as to whether ethene oxide ("ethylene oxide", EO) sterilization destroys the bone-inducing capacity of demineralized bone matrix (DBM) or not. Correctly performed studies seem to support both opinions. Bone conductive properties of fresh frozen, defatted bone grafts are greatly impaired by EO sterilization, whereas purified inductive proteins resist EO. Studies showing destruction of osteoinductive capacity used nonpulverized DBM, whereas the others used powder. This could be the key to resolving the controversy, because if EO treatment reduces the cells' ability to penetrate a cortical graft and to reach inductive proteins inside it, it may appear noninductive after EO sterilization, even though BMP molecules may be intact. On the other hand, cells could easily penetrate the powder implants. We compared the effect of EO sterilization on the inductive capacity of demineralized cortical bone with that of DBM powder, using allogeneic material in rats. Cortical pieces lost all inductive capacity by EO sterilization, whereas the powder yielded a calcium content which was at best one fourth of the unsterilized. The concentrations of residual EO, ethene chlorohydrin and ethene glucol at implantation were far below approved levels. Another difference between studies is the humidity during EO treatment. In our hands, humidification reduced bone yield by half. In conclusion, EO sterilization may impair the biological performance of bone inductive implants by reducing cell penetration into bulk material. However, DBM powder, when correctly sterilized, also yielded scanty amounts of bone.

Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection.

The identity of cells responsible for transmission of human cytomegalovirus (HCMV) in blood products or bone marrow transplants is unknown. We have tested the capacity of HCMV to in vitro infect human peripheral blood mononuclear cells (PBMC) from healthy donors and found that certain PBMC are permissive to HCMV infection. In vitro-infected viable cells were double stained for surface expression of different HMCV proteins and for cell-type-specific antigens to allow the identification of sensitive cells. All analysis were performed on viable cells, using HCMV-specific monoclonal antibodies and automated flow cytofluorimetry. PBMC were infected either with the laboratory-adapted HCMV strain AD169 or with a virus isolate obtained from a viremic patient. Up to 25% of all PBMC could express the major immediate-early antigen as well as the pp65 antigen, known at the lower matrix protein. Infected cells were mainly CD14+ monocytes, but also a small population of large CD8+ cells were susceptible to HCMV infection. CD19+ B lymphocytes were resistant to HCMV infection. Different populations of infected cells were enriched by using Dynabeads coated with cell-type-specific antibodies, and the presence of infectious virus was demonstrated by incubating the selected and sonicated cell material on human fibroblasts. Only material from infected monocytes and from CD3+ CD8+ cells gave rise to HCMV-specific plaques. The presence of HCMV mRNA as a sign of active viral transcription of the major immediate-early and late pp150 genes in infected cells was demonstrated by using nested reversed polymerase chain reaction. A common denominator was found for all cells that could be infected with HCMV. The CD13 antigen, a 130- to 150-kDa integral membrane protein identical to the enzyme aminopeptidase N, was expressed on all HCMV-permissive cells.

Identification of blood mononuclear cells permissive of cytomegalovirus infection in vitro.

We conclude that both the experimental strain Ad169 and a CMV isolate obtained from a patient could infect PBMCs in vitro. The identity of infected cells was established as CD14+ monocytes and a small population of CD3/CD8+ large granular lymphocytes. No evidence of sensitivity to infection was obtained in small lymphocytes, in either the B or T cell population. Furthermore, flow cytometric analysis of cells expressing CMV-encoded antigens is a sensitive assay, as is the demonstration of viral RNA using reversed PCR and nested primer pairs. Since three different types of analyses all indicated active production of structural CMV antigens in infected cells, we conclude that monocytes and some CD8+ large lymphocytes might serve as reservoirs for latent CMV infection and might be responsible for the transfer of CMV infection. Presently, it cannot be determined whether PBMCs are indeed sensitive to a primary infection. Direct sequencing of virus isolates from in vitro-infected cells will have to be carried out to settle this issue. However, we favor the explanation that CMV causes a primary in vitro infection, since, as discussed above, various activating substances can induce the expression of CMV-encoded antigens in blood cells from seropositive donors. Finally, we found that the presence of the CD13 marker was a common denominator of all cells sensitive to CMV infection. We are presently attempting to elucidate whether the CD13 molecule is instrumental in the infection of cells by CMV.

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae.

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.

Interleukin 4 instructs uncommitted B lymphocytes to switch to IgG1 and IgE.

Mouse interleukin 4 (IL 4) is a T cell-produced lymphokine with multiple effects on different cells types of the hematopoietic lineages. IL 4 has pronounced effects on B lymphocytes, where it induces high levels of IgG1 and IgE secretion in lipopolysaccharide-stimulated cultures that would otherwise secrete predominantly IgG3 and IgG2b (of the non-IgM isotypes). An important question is how IL 4 exerts its effect. Two main possibilities exist: (a) IL 4 instructs uncommitted B lymphocytes to IgG1 and IgE production; (b) IL 4 selects and expands an already precommitted B cell. In this study we show, by the use of limiting dilution analysis, that IL 4 dramatically increases the precursor frequency of IgG1 and IgE-secreting cells with no significant effect on the clone size, clearly suggesting that IL 4 instructs uncommitted B cells to switch to IgG1 and IgE. The fraction of total Ig precursors that can switch to the two isotypes is furthermore high. The high precursor frequency for IgE obtained in the presence of IL 4 further demonstrates that IL 4 is an important modulator of IgE responses.

IgG1 induction factor: a single molecular entity with multiple biological functions.

A cDNA clone coding for the murine IgG1 induction factor has been isolated. The translation products directed by this clone were analyzed in different biological assays. The data obtained show that the IgG1 induction factor: Is involved in the regulation of IgG responses, by increasing IgG1 and decreasing IgG3 and IgG2b secretion; Induces hyper-Ia expression on resting B lymphocytes; Synergizes with anti-Ig in inducing DNA synthesis in resting B lymphocytes; Synergizes with DxS in inducing DNA synthesis by B lymphocytes; It induces DNA synthesis by either the T cell line CTL-L or Con-A blasts. Thus, this lymphokine in addition to IgG1 inducing activity has also BSF-1, BCGF-II and TCGF like activities. The fact that a single molecule can perform all the above listed functions has implications for our view of lymphocyte activation. It indicates that considering the B cell response as an ordered series of independently controlled events, is an oversimplified view of the dynamic process through which B cells are activated and also indicate the functional interconnection of the different elements of the immune system.

IgG1 induction factor.

The IgG1 induction factor elevates the IgG1 response and reduces the IgG2b and IgG3 response in LPS-stimulated spleen cells. The factor is a lymphokine produced by T cells. The precursor frequency for cells secreting the IgG1 induction factor is at least tenfold lower as compared to those secreting interleukin 2. Some biochemical properties of the lymphokine are listed. The effects of gamma interferon in B cell-stimulated cultures are shown. Isolation of a cDNA clone coding for the IgG1 induction factor has been achieved and results of these studies are reviewed. Evidence is given that this lymphokine is the same as B cell stimulating factor 1, and we propose that it be renamed interleukin 4. Finally, the possible mechanisms of interleukin 4 are discussed.

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II.

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.

Jaw morphology in edentulous individuals: a radiographic cephalometric study.

The jaw morphology in the mandible and maxilla was studied by cephalometric analysis in two groups of edentulous subjects and in two groups of individuals in possession of all teeth. The predominating facial type in these groups was the orthognathic type. The alveolar bone height differed significantly between men and women in the edentulous groups but not in the subjects in possession of all teeth. Thus, there seemed to be a sex difference in alveolar bone diminution after extraction of all teeth. The gonial angle was significantly greater in both men and women in the edentulous groups than in either sex in the groups in possession of all teeth. Thus, the basal bone morphology in the mandible seemed to be changed in the same manner in both sexes in the edentulous groups after extraction of all teeth, in contrast to the alveolar bone diminution. Indications were found that the gonial angle might be age related. The chin angle as such was found to be less than 60 degrees in the majority of edentulous individuals which makes this value seem to be of minor importance when selecting edentulous individuals for surgical deepening of the vestibular sulcus.

Secretion of IgG1 induction factor by T cell clones and hybridomas.

IgG1 induction factor elevates the IgG1 response induced by lipopolysaccharide and suppresses the lipopolysaccharide-induced IgG3 and IgG2b responses in cultures of mouse spleen cells. We have developed new T cell lines secreting this factor by cloning mixed lymphocyte culture populations. Using supernatants of one of these T cell lines it was found that the assay is quantitative, reproducible and accurate, both when induction of IgG1 as well as reduction of IgG3 and IgG2b were measured. Using this analysis, different conditions to induce maximal production of the factor were tested. The cell line was thereafter used as fusion partner with a T cell lymphoma. The hybrids were selected in the presence of T cell growth factor and all of them secreted IgG1 induction factor.

Partial biochemical characterization of IgG1-inducing factor.

IgG1 induction factor elevates the IgG1 and suppresses the IgG3 and IgG2b responses in lipopolysaccharide-stimulated murine spleen cell cultures. By the use of a quantitative assay, it was found that the three activities, induction of IgG1 and reduction of IgG3 and IgG2b synthesis, were found in the same fractions after different chromatographic procedures, suggesting that the same molecule was responsible for the effects. The factor was precipitated by 60-90% saturation of ammonium sulfate and was sensitive to proteolytic cleavage and to treatment with a buffer of pH 10. It had an apparent molecular mass of 20 kDa as judged by gel filtration chromatography and was separated into two peaks after isoelectric focusing, pI 7.4-7.2 and 6.4-6.2, respectively. Finally it was weakly hydrophobic and negatively charged at pH 7.55. These characteristics indicate that the factor is different from many previously characterized lymphokines and similar or identical to the B cell stimulating factor-1 (BSF-p1). The relevance of these findings to the mechanism of the immunoglobulin class switch is discussed.