Regulation of PTC phenotype and function by TGF-β1: implications for transdifferentiation

Introduction There is now increasing evidence that proximal tubular cells (PTCs) contribute to renal interstitial fibrosis by alteration of matrix turnover and by the generation of pro‐fibrotic cytokines such as TGF‐β1. Recent studies suggest that, through a process of transdifferentiation, the PTCs are one source of the interstitial myofibroblasts that directly drive the fibrotic process. The aim of this work was to examine the role and mechanism by which TGF‐β1 may regulate PTC phenotype and function. Methods Experiments were performed using both primary‐cultures of PTC and the human PTC cell line HK2. All experiments were performed on growth‐arrested cells in the absence of serum. Results TGF‐β1 altered cell phenotype, assessed by light microscopy, with cells appearing elongated and spindle‐shaped. This was associated with loss of cell–cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibres and focal adhesions. Disruption of the actin cytoskeleton with cytochalasin‐D prevented phenotypic alterations following addition of TGF‐β1. Transient transfection with Smad‐2/‐4 or Smad‐3/‐4 expression vectors did not alter cell phenotype. Previously, we have demonstrated β‐catenin translocation to PTC nuclei and its association with Smad proteins following addition of TGF‐β1, suggesting the possibility that TGF‐β1 may modulate Wnt signalling. Wnt‐responsive Xtwn‐reporter construct was, however, silent in response to TGF‐β1. Similarly, a second Wnt‐/LEF‐1‐regulated element Toplflash, which does not contain Smad‐binding sites, was insensitive to TGF‐β1 signalling. In contrast, phenotypic changes in response to TGF‐β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell–cell contact and adherens junction disassembly. Removal of TGF‐β1 and addition of 1% FCS, however, reverted cell phenotype to a typical cobblestone epitheliod appearance, suggesting that TGF‐β1 did not result in terminal PTC transdifferentiation. Cells grown on tissue culture dishes coated with either type‐I or type‐III collagen also acquired an elongated fibroblastic phenotype; this effect was exaggerated by the addition of TGF‐β1. In contrast to the cells stimulated with TGF‐β1 alone, following stimulation by both TGF‐β1 and exposure to interstitial collagens, cell phenotype was stable in that it was not reversed upon removal of TGF‐β1 and addition of FCS. Addition of TGF‐β1 to cells grown on type‐IV collagen had no greater effect than TGF‐β1 alone. Addition of TGF‐β1 alone had little effect on the expression of α‐SMA. In contrast, cells grown on either type‐I or type‐III collagen, following addition of TGF‐β1, demonstrated marked increased expression of α‐SMA, which appeared to be incorporated into the cell cytoskeleton. Similarly, the combination of interstitial collagen (either type‐I or type‐III) and TGF‐β1 had synergistic effect on the relocation and down‐regulation of the epithelial markers E‐cadherin and cytokeratin. Finally, the results demonstrated synergistic effects of coating with interstitial collagen (either type‐I or type‐III), on cell ‘fibroblastic’ cell function as assessed by cell migration and by the synthesis of type‐III and type‐IV collagen. Conclusion The results of these in vitro experiments suggest that terminal transdifferentiation of proximal tubular epithelial cells is the result of a combination of the effects of the pro‐fibrotic cytokine TGF‐β1 and exposure of the cells to components of the interstitial extra‐cellular matrix to which the cells are not exposed in the absence of damage to the tubular basement membrane.     

The influence of metamemory on the quality of life of persons with multiple sclerosis.

This study examined the influence of metamemory (i.e., self-report of memory ability and skills), in combination with other factors, on quality of life for people with multiple sclerosis (MS). Participants (482 persons with MS) completed instruments to measure functional limitations, depressive symptoms, metamemory, and perceived quality of life. Participants reported greater satisfaction with their memory performance but less frequent use of memory aids and strategies compared with 115 older adults on whom the instrument was originally tested. Components of metamemory were significantly related to neurological disability, duration of disease, depressive symptoms, age, and quality of life. Depressive symptoms had a higher impact than other variables in the final regression model on quality-of-life prediction.     

Nutrition and somatomedin. XVI: Somatomedins and somatomedin inhibitors in fasted and refed rats

Nutritional deprivation is associated with poor growth and decreased levels of net circulating somatomedin activity, as measured by bioassay. Since somatomedin activity reflects the contributions both of somatomedins (which stimulate cartilage) and of somatomedin inhibitors (which antagonize the ability of the somatomedins to stimulate cartilage), we asked if changes in net somatomedin activity could involve progressive underlying alterations in levels of both somatomedins and somatomedin inhibitors. Groups of rats were killed during three days of fasting and 24 hours of refeeding. Fasting was associated with a rise in serum beta-hydroxybutyrate from 1.6 to 12.6 mmol/L after one day, followed by a decline to 4 mmol/L at three days. Somatomedins (low-MW) were separated from somatomedin inhibitors (high-MW) by gel permeation chromatography at acid pH on Sephadex G-50 and TSK-2000 HPLC. Somatomedins fell 35% after one day of fasting, and decreased to 59% below control levels after three days (P less than .05). Somatomedins did not change with six hours of refeeding, but then rose rapidly, reaching control levels after 24 hours. Somatomedins were correlated with change in weight (r = .41, P less than .05), but not with glucose or beta-hydroxybutyrate. Inhibitors rose to 195% above control-levels after one day of fasting, and continued to rise to 375% above control after three days (P less than .01). In contrast to the delayed change in somatomedins with refeeding, there was an abrupt fall in inhibitors (41% below three-day fasted levels after six hours), returning to control levels after 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sonographic appearances in postpartum thyroiditis.

During the postpartum period about 50% of women with circulating thyroid autoantibodies develop a transient autoimmune thyroiditis. To determine the sonographic appearances in postpartum thyroiditis (PPT), serial ultrasound (US) scans of the thyroid were performed in 135 postpartum women who were divided into three clinical groups: Group 1, 37 antibody positive subjects who developed PPT; Group 2, 28 antibody positive subjects in whom thyroid function remained normal; Group 3, 70 antibody negative controls. Thyroid hypoechogenicity was observed in 14/31 patients (45%) who were scanned between 4 and 8 weeks postpartum and who subsequently developed PPT (Group 1) compared with 4/24 patients (17%) in Group 2 (P less than 0.05) and 1/65 patients (1.5%) in Group 3 (P less than 0.001). In antibody positive patients, the positive predictive value of an abnormal scan during this period was 78%. Between 15 and 25 weeks postpartum thyroid hypoechogenicity was present in 32/37 patients (86%) in Group 1 compared with 11/28 patients (39%) in Group 2 (P less than 0.001) and 2/70 patients (3%) in Group 3 (P less than 0.001). Sonographic abnormality persisted beyond 32 weeks postpartum in 36/41 antibody positive patients (87%) who had exhibited thyroid hypoechogenicity earlier during the study and who had late scans. The characteristic US appearance in PPT is thyroid hypoechogenicity. The role of sonography in the prediction, diagnosis and follow up of patients with PPT is discussed.     

Sinusoidal lining cell damage: the critical injury in cold preservation of liver allografts in the rat

We have previously defined viability limits in a rat transplantation model. All liver allografts stored in a simple preservation solution (NaCl 0.9%, CaCl2 2 mM) at 4 degrees C for 4 hr or at 37 degrees C for 1 hr were viable upon transplantation, but all those stored at 4 degrees C for 8 hr or at 37 degrees C for 2 hr were nonviable. Only cold-preserved, nonviable livers showed increased vascular resistance, platelet trapping and an initially low, but then high, rise in aspartate transaminase (AST) upon reperfusion, all suggesting injury to the microcirculation, with secondary injury to the hepatocyte. In the present study, we investigated the morphological changes that occur in livers stored for the defined critical times, using light and electron microscopy after perfusion-fixation. Accurate and reproducible identification of specimens as belonging to viable or nonviable and warm- or cold-preserved could be made in this way. Preservation in the cold first resulted in reversible changes consisting of cellular swelling, alterations of intracellular organelles, and partial denudation of the sinusoidal lining (cold-preserved viable group). Later, under conditions of nonviable cold preservation, detachment of cell bodies of sinusoidal lining cells with nuclear changes and almost complete denudation of the sinusoidal lining was observed. Endothelial cells of larger vessels were only injured mildly. In contrast, under conditions of warm preservation, changes involving mitochondria and later nuclei were found in hepatocytes, and blebbing was more extensive. Endothelial cells were spared relatively. We also examined livers stored in isotonic citrate solution at 4 degrees C for 8 hr and 16 hr, the critical times determined for this solution in another model of rat liver transplantation. The findings were very similar to storage in saline with respect to the changes in the sinusoidal lining cells after cold preservation for the two critical times. The results provide convincing evidence of a qualitative difference between warm and cold preservation injury, with relatively selective damage to hepatocytes or sinusoidal lining cells, respectively. Endothelial damage represents the primary event, resulting in the loss of organ viability following hypothermic storage. Thus morphology may serve as a useful viability marker after preservation.     

Absence of a late-phase response or increase in histamine responsiveness after bronchial provocation with adenosine 5'-monophosphate in atopic and non-atopic asthma

1. Adenosine 5'-monophosphate (AMP) causes bronchoconstriction in atopic and non-atopic asthma by a mechanism believed to involve histamine release from airway mast cells. To determine whether preformed mast cell mediators, principally histamine, can initiate a late-phase bronchoconstriction we have investigated the effect on the airways over a 24 h period of a single bronchial challenge with AMP. 2. Six atopic asthmatic subjects (all late responders to inhaled allergen) and six non-atopic asthmatic subjects were studied on two occasions for a 24 h period after inhalation of the provocation concentration of AMP required to produce a 20% fall in forced expiratory volume in 1 s (FEV1) from baseline (PC20) and 0.9% (w/v) sodium chloride placebo, respectively. The atopic asthmatic subjects were studied on a further occasion after challenge with the PC20 allergen. 3. Inhalation of the PC20 AMP resulted in an immediate fall in FEV1 to a mean maximum 25.5% below baseline without resulting in any late decrease in airway calibre. No significant increase in non-specific bronchial responsiveness as determined by measuring the PC20 histamine before, and at 3, 9 and 24 h after, AMP challenge, occurred. Inhalation of the PC20 allergen caused a reproducible late-phase bronchoconstriction and increase in non-specific bronchial responsiveness in all the atopic asthmatic subjects studied. 4. These results suggest that preformed mast cell mediators, principally histamine, play no role in the initiation of the late-phase reaction in allergen-provoked asthma, although they may contribute to the inflammatory changes involved.     

Altered gastric emptying in the head-injured patient: relationship to feeding intolerance

Most patients with moderate to severe head injury initially do not tolerate enteral feedings postinjury. This intolerance is more prolonged than that found in patients suffering other types of trauma. The authors prospectively evaluated 12 patients with moderate to severe head injury (Glasgow Coma Scale score between 4 and 10) throughout their hospitalization for liquid gastric emptying as a possible mechanism for intolerance to enteral feeding. During Week 1, the majority of patients displayed a delay in gastric emptying. Patients also displayed an abnormal biphasic response (gastric emptying faster than normal during the early stage but prolonged later). By Week 2, many patients still had delayed and abnormal biphasic responses to gastric emptying. By Week 3, an improvement was observed with the majority of patients exhibiting rapid gastric emptying, but delays and abnormal biphasic responses were still seen. Patients who initially had rapid or normal gastric emptying tolerated full-strength full-rate feedings significantly earlier compared with those who experienced delayed gastric emptying (8.5 +/- 0.5 days vs. 13.7 +/- 3.2 days, p less than 0.001). All patients tolerated full-strength full-rate feedings by Day 16 postinjury (range 7 to 16 days) except the two patients who displayed delayed gastric emptying for prolonged periods of time (mean 25 days). This is the first study to longitudinally evaluate gastric emptying following head injury. The authors suggest that patients with moderate to severe head injury often experience alterations in gastric emptying which may affect their ability to tolerate enteral feedings.     

Guidelines for the collection, processing and storage of human bone marrow and peripheral stem cells for transplantation

SUMMARY. There are no current U.K. or international guidelines or regulations covering the production, processing and storage of haemopoietic cells such as to allow their engraftment following myeloablative therapy. This paper seeks to provide such guidelines. It enumerates how quality control and assurance can be applied to this area of transfusion medicine; procedural steps relating to bone marrow harvest on peripheral blood stem cell collection are outlined and recommended doses of nucleated cells suggested for both procedures. General specifications for identification, storage and transportation of bone marrow and peripheral blood stem cells are included and specific laboratory procedures related to the provision of haemopoietic cells for engraftment are outlined. Umbilical cord blood transplants and long-term bone marrow culture are alluded to but these are still in a research phase.