Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.     

Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity

Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKCalpha activity, and it can directly interact with the phos- phoinositide PIP2. We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKCalpha beta gamma show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCalpha beta gamma and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKCalpha beta gamma by PIP2, resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP2 and PKCalpha, localize them to forming focal adhesions, and potentiate PKCalpha activity there.     

Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and western blots before immunodetection and sequencing

The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.     

Advanced colorectal cancer is associated with impaired interleukin 12 and enhanced interleukin 10 production

Interleukin 12 (IL-12) is a heterodimeric cytokine that has been demonstrated to have a major role in stimulating a cell-mediated antitumor response. IL-10, a product of T helper 2 lymphocytes, is its most potent inhibitor. The aim of this study was to investigate whether patients with colorectal cancer had an imbalance in production of IL-12 and IL-10 preoperatively, and whether this was associated with advanced disease at surgery. Blood was obtained before surgery from 60 patients with colorectal cancer and from 30 controls. Peripheral blood mononuclear cells were incubated with Staphylococcus aureus Cowan's strain 1 in vitro for 24 h to assess IL-12 expression after stimulation, and serum was used for IL-10 measurement. IL-12 and IL-10 levels were assessed by ELISA. A single pathologist staged the tumors according to the tumor-node-metastasis (TNM) and Dukes' classifications. Patients with colorectal cancer had significantly lower levels of IL-12 (P <0.001) and higher levels of IL-10(P = 0.004) compared to controls. In addition, lower levels of IL-12 were detected in those patients who were node positive (P<0.05), had Dukes' C lesions (P < or = 0.001), and T3 or T4 lesions (P<0.033) when compared to controls. Patients with Dukes' B and C lesions (P<0.01) and T3 and T4 lesions (P<0.05) also had higher levels of IL-10 compared to controls. This study is the first to demonstrate that patients with colorectal cancer have decreased IL-12 production and increased serum IL-10. This suggests an impaired T helper 1 cell-mediated antitumor response and provides some justification for exogenous IL-12 therapy or anti-IL-10 therapy in these patients.     

Joint destruction after glucocorticoids are withdrawn in early rheumatoid arthritis. Arthritis and Rheumatism Council Low Dose Glucocorticoid Study Group

OBJECTIVE: Prednisolone reduced the progression of joint destruction over 2 yr in early, active rheumatoid arthritis. The response to discontinuation of prednisolone under double-blind conditions is now reported. METHODS: A randomized, double-blind, placebo-controlled trial of prednisolone 7.5 mg daily in addition to routine medication over 2 yr in 128 patients with early rheumatoid arthritis, using radiological progression (changes in the Larsen score) and the development of erosions as primary outcome measures. Study medication was blindly discontinued and follow-up maintained for a further year. Other assessments included disability, joint inflammation, pain and the acute-phase response. RESULTS: Similar results were obtained when all available radiographs were included for each year of assessment (maximum 114) and when only patients with radiographs at all time points were included (75 patients). In these 75, the mean progression in the prednisolone group was 0.21 Larsen units in year 1, 0.04 units in year 2 and 1.01 units in year 3 (P = 0.587, 0.913 and 0.039 for change within each year, respectively). The equivalent placebo group means were 2.34, 1.00 and 1.63 Larsen units (P = 0.001, 0.111 and 0.012; difference between groups: 2.13, 0.96 and 0.67 units, P = 0.082, 0.02 and 0.622). The percentage of hands which had erosions at each time point was: prednisolone group: 27.8, 29.2, 34.7 and 39.2; placebo group: 28.2, 48.7, 59.0 and 66.5. There was little evidence for a flare in clinical symptoms after discontinuation of prednisolone. CONCLUSION: Joint destruction resumed after discontinuation of prednisolone. This corroborates the previously reported therapeutic effect and challenges current concepts of disease pathogenesis.     

Hemostatic efficacy of a fibrin sealant dressing in an animal model of kidney injury

Arterial spin tagging techniques have been used to image tissue perfusion in MR without contrast injection or ionizing radiation. Currently, spin tagging studies are performed primarily using single-slice imaging sequences, which are time consuming. This note reports a multislice echo-planar arterial spin tagging technique (Simultaneous Multislice Acquisition with aRterial-flow Tagging, or "SMART"). Multiband RF encoding (Hadamard) is used to provide simultaneous multislice acquisition capability for spin tagging techniques (such as echo planar imaging signal targeting with alternating radio frequency and flow-sensitive alternative inversion recovery). The method is illustrated with a two-slice pulse sequence that was implemented using the FAIR technique to generate two perfusion weighted images simultaneously. Compared with single-slice sequences, this two-slice sequence provided similar image quality, signal-to-noise ratio, and twice the spatial coverage compared with the single-slice technique within the same scan time.     

Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture

The activation of latent transforming growth factor beta (L-TGFbeta) is essential for the action of TGFbeta, which, in turn, is involved in the regulation of expression of some progesterone-responsive genes. One mechanism by which TGFbeta is activated involves thrombospondin (TSP), a protein that binds extracellular proteins. Immunoreactive TSP (irTSP) protein and TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women were localized by immunohistochemistry and in situ hybridization. IrTSP and TSP-1 mRNA were randomly distributed in myometrial smooth muscle cells of some, but not all, tissues of pregnant women at term before labor; but in some areas of most of these tissues, irTSP was intense and commonly localized extracellularly. Intense irTSP and TSP-1 mRNA in myocytes were more common in myometrium during labor. In myometrium from ovulatory women (n = 26), irTSP was localized primarily in vascular smooth muscle cells and was detected occasionally in scattered myocytes. Little TSP-1 mRNA was demonstrable by in situ hybridization in vessels or myocytes of myometrial tissue from ovulatory women (n = 7). By Northern analysis of total RNA, TSP-1 mRNA was detected in myometrial tissue of pregnant women and in human myometrial smooth muscle cells in culture. The levels of TSP-1 mRNA in myometrial tissues of pregnant women during labor (n = 18) were greater than those in myometrium at > 37 wk gestation before labor began (n = 25, p < 0.001). The ratios of TSP-1 to glyceraldehyde 3-phosphate dehydrogenase mRNAs in 3 myometrial tissues during oxytocin-induced labor were not statistically different from those in myometrium during spontaneous labor but were greater than those in myometrium before labor (p < 0.05). The level of TSP-1 mRNA in confluent human myometrial cells in culture was relatively high, was increased by treatment with fetal bovine serum, and was decreased by treatment with platelet-derived growth factor or activators of adenylyl cyclase or protein kinase C. Myometrial cells in culture constitute a useful model for studying the regulation of TSP-1 gene expression in human myometrium.     

Trace element trophic transfer in aquatic organisms: a critique of the kinetic model approach

The bioaccumulation of trace elements in aquatic organisms can be described with a kinetic model that includes linear expressions for uptake and elimination from dissolved and dietary sources. Within this model, trace element trophic transfer is described by four parameters: the weight-specific ingestion rate (IR); the assimilation efficiency (AE); the physiological loss rate constant (ke); and the weight-specific growth rate (g). These four parameters define the trace element trophic transfer potential (TTP = IR.AE/[ke + g]) which is equal to the ratio of the steady-state trace element concentration in a consumer due to trophic accumulation to that in its prey. Recent work devoted to the quantification of AE and ke for a variety of trace elements in aquatic invertebrates has provided the data needed for comparative studies of trace element trophic transfer among different species and trophic levels and, in at least one group of aquatic consumers (marine bivalves), sensitivity analyses and field tests of kinetic bioaccumulation models. Analysis of the trophic transfer potentials of trace elements for which data are available in zooplankton, bivalves, and fish, suggests that slight variations in assimilation efficiency or elimination rate constant may determine whether or not some trace elements (Cd, Se, and Zn) are biomagnified. A linear, single-compartment model may not be appropriate for fish which, unlike many aquatic invertebrates, have a large mass of tissue in which the concentrations of most trace elements are subject to feedback regulation.     

Paravertebral muscles in experimental scoliosis: a light and electron microscopic study

Experimental structural dextroconvex scoliosis was produced in rabbits by costotransversolisis with transversectomy and releasing of paravertebral muscles between TVII and TX on the right side. Two compensatory curves developed on the upper dorsal and lumbar levels. Biopsies of paravertebral muscles in experimental animals included, besides areas of normal tissue, a considerable derangement of the cell contractile apparatus with sarcoplasmic dilation and eventual cell disintegration and necrosis. Histological changes varied along levels, the convexity being more affected. The severity of changes and reduction in body weight and length were correlated with the degree of scoliosis. A selective atrophy of slow-twitch fibers was observed in experimental animals, especially at the level of the main curve, whereas fast-twitch fiber atrophy was more important caudally. Control animal biopsies always appeared normal. Our experimental model shows an overt participation of paravertebral muscles in the establishment of compensatory processes following scoliosis, although the role that paravertebral muscles play in the etiopathogenesis of human idiopathic scoliosis requires further investigation.     

Preferential benefit of implementation of a statewide trauma system in one of two adjacent states

BACKGROUND: Implementation of Oregon's trauma system was associated with a reduction in the risk of death for hospitalized injured patients. An alternative explanation for improved outcome, however, is favorable concurrent temporal trends, e.g., new technologies and treatments. PATIENTS AND METHODS: To control for temporal trends, seriously injured hospitalized patients in Oregon and Washington were compared before either state had a trauma system (1985-1988) and when only the Oregon trauma system had been implemented (1990-1993). The study group consisted of hospitalized injured patients aged 16 to 79 years with one or more index injuries in six body regions, i.e., head, chest, spleen/liver, femur or pelvis fracture, and burns. Hospital discharge claims data were analyzed, converting International Classification of Diseases, Ninth Revision, Clinical Modification, discharge diagnosis codes to Abbreviated Injury Scale scores and Injury Severity Scores using a conversion algorithm. Multivariate logistic regression models were used to estimate the differential risk-adjusted odds of death in Oregon compared with Washington after adjustment for demographics, injury type, and injury severity. RESULTS: Findings indicated no difference in the risk-adjusted odds of death between Oregon and Washington while both states functioned under an ad hoc trauma system (1985-1988). A significant reduction in the risk of death, however, was noted in Oregon for patients with an index injury and an Injury Severity Score > 15 compared with Washington (adjusted odds ratio (OR) = 0.80, 95% confidence interval (CI) = 0.70-0.91) after trauma system implementation in Oregon (1990-1993). Specifically, reductions in the risk of death were demonstrated for patients with head injuries (adjusted OR = 0.70, 95% CI = 0.59-0.82) or liver/spleen injuries (adjusted OR = 0.73, 95% CI = 0.54-0.99). CONCLUSION: Assuming that the two states demonstrated similar concurrent temporal trends, the findings support the conclusion that improved outcomes among injured patients in Oregon may be attributed to the institution of a statewide trauma system.