Interaction of transforming growth factor β1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator

The effect of transforming growth factor- (TGF-) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF- treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF- favors the development of sclerosis.Abbreviations FN Fibronectin - GEC Glomerular epithelial cells - TGF- Transforming growth factor - uPA Urokinase-type plasminogen activator     

The molecular basis for inherited bullous diseases

In the past 5 years enormous progress have been made in our understanding of the molecular basis for a number of inherited skin diseases characterized by easy blistering of the skin and the mucous membranes after minor physical trauma. This increased fragility of the skin or its appendages is due to molecular defects in genes coding for different intra- and extracellular structural proteins which are responsible for mechanical strength at their sites of expression. These diseases encompass the group of epidermolysis bullosa and disorders of cornification such as bullous forms of ichthyosis, palmoplantar keratoderma, and pachyonychia congenita. On the basis of clinical, morphological, and ultrastructural observations the epidermolysis bullosa group has been divided into three major categories. In epidermolysis bullosa simplex blister formation appears within the basal cell layer of the epidermis, and many mutations have been found in the genes of keratin 5 and 14 which are both expressed in basal keratinocytes. Epidermolytic hyperkeratosis leads to an epidermal separation in the suprabasal cell layers. In these patients numerous point mutations have now been described in the suprabasally expressed genes of keratin 1 and 10. In ichthyosis bullosa of Siemens blisters occur in the more upper suprabasal epidermis coincidental with the expression of keratin 2e, and mutations have been detected in the corresponding gene. In epidermolytic palmoplantar hyperkeratosis the suprabasal epidermal splitting is restricted to palms and soles of the patient. In keratin 9, which reveals such an exclusive expression pattern, molecular defects have indeed been recognized. Most recently in two different clinical subtypes of pachyonychia congenita, which is characterized by defective nails and focal palmoplantar hyperkeratosis, point mutations have been found in the genes coding for keratins 6, 16, and 17. In junctional epidermolysis bullosa the separation takes place within the dermal-epidermal basement membrane at the level of the lamina lucida, and mutations have been found in three genes coding for different laminin chains, in the ₄ gene of ₆₄ integrin, and in the gene of collagen XVII. In dystrophic epidermolysis bullosa the tissue separation occurs beneath the basement membrane within the papillary dermis at the level of the anchoring fibrils, and several mutations have been identified in the collagen VII gene. The rapid unraveling of molecular defects in these disabling or even lethal inherited skin diseases makes possible a more precise and earlier prenatal diagnosis, creates new options for suitable therapeutic regimens, and even offers the hope of curing these diseases by means of somatic cell gene therapy.Abbreviations BM Basement membrane - BPAg Bullous pemphigoid antigen - DEB Dystrophic epidermolysis bullosa - EB Epidermolysis bullosa - EBS Epidermolysis bullosa simplex - EHK Epidermolytic hyperkeratosis - EPPK Epidermolytic palmoplantar keratoderma - IBS Ichthyosis bullosa of Siemens - JEB Junctional epidermolysis bullosa - KIF Keratin intermediate filaments - NC Noncollagenous domain - NEPPK Nonepidermolytic palmoplantar keratoderma - PC Pachyonychia congenita     

A volume-activated anion conductance in insulin-secreting cells

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K⁺-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5-triphosphate (ATP) in the pipette solution but was Ca²⁺ independent. Na⁺ and Cl^– substitution studies suggested that the swelling-activated current is Cl^– selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

Reconstitution of chemokine-induced actin polymerization in undifferentiated human leukemia cells (HL-60) by heterologous expression of interleukin-8 receptors

The chemokines interleukin-8 (IL-8) and GRO bind in neutrophils to the interleukin-8 receptor and (IL-8R and ) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R and to pertussis toxin-insensitive or . To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing , and were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R and was confirmed by binding studies with radiolabeled ligands. IL-8R bound IL-8 with high affinity (Kd1 nM) and GRO with low affinity (Kd 1 M), whereas IL-8R bound both IL-8 and GRO with high affinity (Kd1 nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of or , but not in this response.accepted by M.J. Parnham     

Renal function in relation to three candidate genes in a Chinese population

We recently found in a white population that the genes encoding angiotensin-converting enzyme (ACE, I/D polymorphism), -adducin (Gly460Trp), and aldosterone synthase (–344C/T) jointly influence renal function. We therefore investigated in a Chinese population the associations between the serum concentrations of creatinine and uric acid and these three genetic polymorphisms. We genotyped 471 ethnic Han Chinese subjects from 125 nuclear families recruited in northern China via random population sampling (75%) and at specialized hypertension clinics (25%). We performed population-based and family-based association analyses using generalized estimating equations (GEE) and quantitative transmission disequilibrium test (QTDT), respectively, while controlling for covariables. The participants were 39.7 years old and included 235 women (49.9%). The blood pressure measured at the subjects homes averaged 126/80 mmHg. Mean values were 71 µmol/l for serum creatinine, 111 ml min^–1 1.73 m^–2 for calculated creatinine clearance, and 236 µmol/l for serum uric acid. With adjustment for covariables, GEE analyses of single genes demonstrated that serum uric acid, but not serum creatinine, was positively associated with the ACE D allele. Serum uric acid concentrations were 15.8 µmol/l (95% confidence interval 3.3–28.2) and 25.7 µmol/l (11.1–40.2) higher in DD homozygotes than in ID and II subjects, respectively. Further GEE analyses of the three genes combined showed that the association between serum uric acid and the ACE polymorphism was confined to carriers of the -adducin Gly and/or aldosterone synthase C alleles. Sensitivity analyses in parents and offspring separately as well as QTDT analyses were confirmatory. Among 114 informative offspring carrying the -adducin Gly allele serum uric acid was significantly and positively associated with the transmission of the ACE D allele (=20.7 µmol/l). In conclusion, the present study extends our previous findings on the combined effects of the three candidate genes and supports the concept that these genetic polymorphisms jointly influence renal function.     

“Proprioceptive signature” of cursive writing in humans: a multi-population coding

The goal of the present study was to investigate the firing behavior of populations of muscle spindle afferents in all the muscles acting on the ankle while this joint was being subjected to writing-like movements. First it was proposed to determine whether the ensemble of muscle spindles give rise to a unique, specific, and reproducible feedback information characterizing each letter, number or short word. Secondly, we analyzed how the proprioceptive feedback on the whole encodes the spatial and temporal characteristics of writing movements using the vector population model. The unitary activity of 51 primary and secondary muscle spindle afferents was recorded in the tibial and common peroneal nerves at the level of the popliteal fossea, using the microneurographic method. The units recorded from belonged to the tibialis anterior, the extensor digitorum longus, the extensor hallucis longus, the peroneus lateralis, the gastrocnemius-soleus and the tibialis posterior muscles. The writing-like movements were randomly imposed at a natural velocity via a computer-controlled machine in a two-dimensional space. In general, muscle spindle afferents from any of the six muscles responded according to the tuning properties of the parent muscle, i.e. increasing their discharge rate during the phases where the direction of movement was within the preferred sensory sector of the parent muscle. The whole trajectory of the writing movements was coded in turn by the activity of Ia afferents arising from all the muscles acting on the joint. Both single afferent responses and population responses were found to be highly specific and reproducible with each graphic sign. The complex multi-muscle afferent pattern involved, with its timing and distribution in the muscle space, seems to constitute a true proprioceptive signature for each graphic symbol. The ensemble of muscle spindle afferents were therefore found to encode the instantaneous direction and velocity of writing movements remarkably accurately. It was concluded that the proprioceptive feedback from all the muscles with which the moving joint is equipped provides the CNS with highly specific information that might contribute to a graphic sign identification process.This work was supported by grants from the Ministère de la Recherche, ACI Cognitique     

Shoulder muscle activity in Parkinson’s disease during multijoint arm movements across a range of speeds

Bradykinesia is one of the primary symptoms of Parkinson disease and leads to significant functional limitations for patients. Single joint movement studies, that have investigated the mechanism of bradykinesia, suggest that several features of muscle activity are disrupted, including modulation of burst amplitude and duration, and the number of bursts. It has been proposed that it is the blending of these different burst deficits that collectively defines bradykinesia. This study adds two new approaches to the study of bradykinesia. First, we examined the features of shoulder muscle activities during multijoint arm movement in bradykinetic and control subjects, such that previously reported single joint hypotheses could be tested for generalized arm movement. Second, we directly manipulated speed while keeping distance constant for a large range of speeds. In this manner, we could compare individual trials of muscle activity between controls and subjects with Parkinsons disease (PD) for movements matched for both speed and movement duration. Our results showed that while a multiple burst pattern of shoulder muscles was a common strategy for all subjects (young, elderly controls and PD), subjects with PD showed several burst abnormalities, including deficits in initial agonist burst amplitude and duration at both fast and slow speeds. Subjects with PD also (1) failed to produce a one-burst pattern at fast speeds and, instead, produced a predominance of multiple burst patterns and (2) showed a relationship between the number of burst deficits and the severity of disease. These results extend the findings of single joint studies to multi-joint and similarly indicate that a combination of burst modulation abnormalities correlate with bradykinesia and disease severity.     

Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddles syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na⁺. We tested the effect of extracellular Na⁺ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na⁺ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddles syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na⁺ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na⁺ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddles syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na⁺ concentration [Na⁺]_i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na⁺]_i-app. We conclude that the inhibitory effect of extracellular Na⁺ removal on rundown is due to an inhibition of endocytic ENaC retrieval.