Mechanisms of postprandial protein accretion in human skeletal muscle. Insight from leucine and phenylalanine forearm kinetics.

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Abstract

The relative role of protein synthesis and degradation in determining postprandial net protein deposition in human muscle is not known. To this aim, we studied forearm leucine and phenylalanine turnover by combining the arteriovenous catheterization with tracer infusions, before and following a 4 h administration of a mixed meal in normal volunteers. Forearm amino acid kinetics were assessed in both whole blood and plasma. Fasting forearm protein degradation exceeded synthesis (P < 0.01) using either tracer, indicating net muscle protein loss. The net negative forearm protein balance was quantitatively similar in whole blood and in plasma. After the meal, forearm proteolysis was suppressed (P < 0.05- < 0.03), while forearm protein synthesis was stimulated (P < 0.05- < 0.01). However, stimulation of protein synthesis was greater (P < 0.05- < 0.01) in whole blood (leucine data: +50.4 +/- 7.8 nmol/min x 100 ml of forearm; phenylalanine data: +30.4 +/- 11.6) than in plasma (leucine data: +17.8 +/- 5.6 nmol/min x 100 ml of forearm; phenylalanine data: +5.7 +/- 2.1). Consequently, the increment of net amino acid balance was approximately two to fourfold greater (P < 0.04- < 0.03) in whole blood than in plasma. In conclusion, meal ingestion stimulates forearm protein deposition through both enhanced protein synthesis and inhibited proteolysis. Plasma data underestimate net postprandial forearm protein synthesis, suggesting a key role of red blood cells and/or of blood mass in mediating mealenhanced protein accretion.

Authors

P Tessari, M Zanetti, R Barazzoni, M Vettore, F Michielan

Heterogeneity and clinical significance of glomerular-binding antibodies in systemic lupus erythematosus.

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Abstract

We used an ELISA employing extracts of human glomerular basement membrane (GBM) to detect, characterize, and evaluate the clinical significance of glomerular-binding IgG in patients with SLE nephritis. Most patients with SLE nephritis exhibited GBM-binding IgG, although many patients with active nonrenal SLE or symptomatic, drug-induced lupus had similar reactivity, albeit at lower levels. IgG binding to GBM in SLE nephritis patients was decreased by DNase pretreatment of GBM, restored after DNase with nuclear antigens (most notably with nucleosomes), inhibited by exogenous nuclear antigens (particularly nucleosomes), but unaffected by exposure of serum to DNase/high ionic strength. The characteristics of IgG binding to GBM largely paralleled the patients' underlying autoimmune response, which was dominated either by antibodies to DNA/nucleosomes or to nucleosomes alone. Binding of lupus sera to nonrenal extracellular matrix (even with nucleosomes) was not equivalent to GBM. Collagenase pretreatment of GBM variably decreased IgG binding, depending on the level and type of binding. SLE nephritis patients with high levels of GBM-binding IgG exhibited more severe disease clinically, but the same renal histopathology, as patients with lower levels. The level of GBM-binding IgG at presentation did not predict the therapeutic response, but decreased in responders to therapy. In sum, glomerular-binding IgG in lupus nephritis binds to epitopes on chromatin, which adheres to GBM in part via collagen. These autoantibodies appear necessary, but not sufficient, for the development of nephritis, and correlate with clinical rather than histopathologic parameters of disease activity.

Authors

J B Lefkowith, M Kiehl, J Rubenstein, R DiValerio, K Bernstein, L Kahl, R L Rubin, M Gourley

Endothelin antagonism in experimental hepatic fibrosis. Implications for endothelin in the pathogenesis of wound healing.

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Abstract

The liver's response to chronic injury is fibrosis, which is analogous to wound healing in other organs. Hepatic wounding is characterized by the "activation" of resident stellate cells (lipocytes, Ito cells) to myofibroblast-like cells that produce increased amounts of smooth muscle alpha-actin and extracellular matrix. Stellate cells possess abundant endothelin (ET) receptors (ETA/B) and, therefore, are subject to the effects of ET-1 and 3. In this study, we investigated whether ETs contribute to the activation of stellate cells and consequently, fibrogenesis. In cultured stellate cells, ET-1 and sarafotoxin S6C (a potent ETB receptor agonist) stimulated stellate cell activation, as assessed by expression of smooth muscle alpha-actin. Furthermore, the mixed ETA/B receptor antagonist, bosentan, blocked this process. Next, we administered bosentan during the induction of liver injury in two mechanistically distinct forms of hepatic wounding. Bosentan reduced levels of type I collagen and cellular fibronectin mRNAs in whole-liver tissue extracts in both models. In freshly isolated stellate cells from injured livers, bosentan reduced expression of activation markers, including smooth muscle alpha-actin and extracellular matrix mRNAs. We further demonstrate that endothelin antagonism after establishment of fibrosing injury reduced stellate cell activation and matrix production. These data indicate that ET contributes to stellate cell activation and fibrogenesis. Because ET is upregulated in diverse forms of parenchymal injury, we speculate that ET may play an important role in the wound-healing response.

Authors

D C Rockey, J J Chung

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

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Abstract

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

Authors

R Pfundt, F van Ruissen, I M van Vlijmen-Willems, H A Alkemade, P L Zeeuwen, P H Jap, H Dijkman, J Fransen, H Croes, P E van Erp, J Schalkwijk

Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor.

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Abstract

Brain tumor-associated cerebral edema arises because tumor capillaries lack normal blood-brain barrier function; vascular permeability factor (VPF, also known as vascular endothelial growth factor, VEGF) is a likely mediator of this phenomenon. Clinically, dexamethasone reduces brain tumor-associated vascular permeability through poorly understood mechanisms. Our goals were to determine if suppression of permeability by dexamethasone might involve inhibition of VPF action or expression, and if dexamethasone effects in this setting are mediated by the glucocorticoid receptor (GR). In two rat models of permeability (peripheral vascular permeability induced by intradermal injection of 9L glioma cell-conditioned medium or purified VPF, and intracerebral vascular permeability induced by implanted 9L glioma), dexamethasone suppressed permeability in a dose-dependent manner. Since 80% of the permeability-inducing activity in 9L-conditioned medium was removed by anti-VPF antibodies, we examined dexamethasone effects of VPF expression in 9L cells. Dexamethasone inhibited FCS- and PDGF-dependent induction of VPF expression. At all levels (intradermal, intracranial, and cell culture), dexamethasone effects were reversed by the GR antagonist mifepristone (RU486). Dexamethasone may decrease brain tumor-associated vascular permeability by two GR-dependent mechanisms: reduction of the response of the vasculature to tumor-derived permeability factors (including VPF), and reduction of VPF expression by tumor cells.

Authors

J D Heiss, E Papavassiliou, M J Merrill, L Nieman, J J Knightly, S Walbridge, N A Edwards, E H Oldfield

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

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Abstract

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Authors

M Costas, T Trapp, M P Pereda, J Sauer, R Rupprecht, V E Nahmod, J M Reul, F Holsboer, E Arzt

Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression.

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Abstract

Porcine pancreatic islets were microencapsulated in alginate-polylysine-alginate capsules and transplanted intraperitoneally into nine spontaneously diabetic monkeys. After one, two, or three transplants of 3-7 x 10(4) islets per recipient, seven of the monkeys became insulin independent for periods ranging from 120 to 804 d with fasting blood glucose levels in the normoglycemic range. Glucose clearance rates in the transplant recipients were significantly higher than before the graft administration and the insulin secretion during glucose tolerance tests was significantly higher compared with pretransplant tests. Porcine C-peptide was detected in all transplant recipients throughout their period of normoglycemia while none was found before the graft administration. Hemoglobin A1C levels dropped significantly within 2 mo after transplantation. While ketones were detected in the urine of all recipients before the graft administration, all experimental animals became ketone free 2 wk after transplantation. Capsules recovered from two recipients 3 mo after the restoration of normoglycemia were found physically intact with enclosed islets clearly visible. The capsules were free of cellular overgrowth. Examination of internal organs of two of the animals involved in our transplantation studies for the duration of 2 yr revealed no untoward effect of the extended presence of the microcapsules.

Authors

Y Sun, X Ma, D Zhou, I Vacek, A M Sun

A novel missense mutation in the amino-terminal domain of the human androgen receptor gene in a family with partial androgen insensitivity syndrome causes reduced efficiency of protein translation.

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Abstract

The role of the androgen receptor (AR) in male sexual differentiation is revealed in part by the analysis of naturally occurring mutations in families with androgen insensitivity syndrome (AIS). We have investigated a family with partial AIS affecting three generations and have identified a G to A substitution in the AR gene at the fourth position 3' from the A of the ATG initiation codon changing the second amino acid residue from glutamic acid to lysine (EK2). Transient expression of the mutant EK2-pCMVhAR expression vector in COS cells revealed decreased translation with a 20-50% reduction in mutant protein relative to wild type AR by immunoblot analysis. The rate of dissociation of [3H]methyltrienolone from the EK2 mutant (half-time [t1/2] = 1.7 +/- 0.08 SE h) was increased compared with wild type AR (t1/2 = 2.4 +/- 0.11 h). Cotransfection studies using an androgen responsive luciferase reporter vector demonstrated a 50% reduction in transcriptional activation by EK2. These functional alterations are consistent with the partial AIS phenotype in affected males, corroborate the AR amino-terminal domain effect on kinetics of androgen binding, and provide physiological evidence for earlier translation experiments identifying the nucleotide sequence for optimal translation initiation.

Authors

C S Choong, C A Quigley, F S French, E M Wilson

Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection.

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Abstract

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are present in the peripheral blood and liver of chronically infected patients. The current study was performed to study the relationship between the strength of the CTL response, liver disease severity, and viral load. The results may be summarized as follows: first, using CTL precursor frequency (CTLpf) analysis to quantitate the peripheral blood CTL response, chronically infected patients were less strongly sensitized to a panel of well-defined HCV epitopes than they were to an epitope within the influenza matrix protein. Second, HCV-specific CTLpf did not correlate with disease activity or viral load in the majority of patients on a cross-sectional basis, although it did increase in three patients concomitant with sharp increases in liver disease. Finally, interferon therapy did not enhance the CTLpf against the HCV epitopes studied in these patients, indicating that its antiviral effect is independent of the CTL response. Since the HCV-specific CTLpf in the blood is actually quite low, the CTL may contribute to ongoing liver disease in these patients while being quantitatively inadequate to destroy all of the infected hepatocytes, thereby facilitating HCV persistence and contributing to chronic liver disease.

Authors

B Rehermann, K M Chang, J G McHutchison, R Kokka, M Houghton, F V Chisari

Endogenous interleukin 6 production in multiple myeloma patients treated with chimeric monoclonal anti-IL6 antibodies indicates the existence of a positive feed-back loop.

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Abstract

In vitro as well as in vivo observations have shown that IL6 plays a key role in the pathogenesis of multiple myeloma. Therefore we started a phase I/II dose escalating study with chimeric monoclonal anti-IL6 antibodies (cMab) in multiple myeloma (MM) patients resistant to second-line chemotherapy. Here we describe the pharmacological data as well as a new method for calculating the endogenous IL6 production. The cMab (CLB IL6/8; Kd: 6.25 x 10(-12) M) was given in two cycles of 14 daily infusions, starting on day 1 and day 28. Daily dose: 5 mg in patients 1-3, 10 mg in patients 4-6, and 20 mg in patients 7-9 (total dose 140, 280, and 560 mg of anti-IL6, respectively). Using the pharmacokinetic data of free IL6 and the binding characteristics of the cMab, the endogenous IL6 production could be calculated from day to day using a one-compartment open model. The median half-life time of this antibody was 17.6 d. No human antichimeric antibodies were induced. Pre-treatment median endogenous IL6 production in the MM patients was 60 micrograms/d (range 13.8-230; normal controls < 7 micrograms/d). During treatment with anti-IL6 cMabs, the endogenous IL6 production immediately decreased in all patients to below 3 micrograms/d and never reached the pre-treatment value during the treatment period, except in two patients who developed an active infection, resulting in an IL6 production of 128 and 1,208 micrograms/d, respectively. We concluded that in MM patients endogenous IL6 production is 2-30 times higher than in healthy individuals. The anti-IL6 cMab strongly suppress this endogenous IL6 production, probably by blocking a positive feed-back loop, but this cMab does not prevent infection-induced IL6 production. The chimeric anti-IL6 Mabs have a long half-life time, a low immunogenicity, and are able to block IL6-dependent processes in vivo.

Authors

H C van Zaanen, R P Koopmans, L A Aarden, H J Rensink, J M Stouthard, S O Warnaar, H M Lokhorst, M H van Oers