[Cell-based therapy to treat stress urinary incontinence: which cell type at what cost?]. / Zellbasierte Therapie der Belastungsinkontinenz: Welche Zelle zu welchen Kosten?

In Germany, 6-8 million woman and men suffer urinary incontinence, which represents 12.5 % of the population. It is estimated that by the middle of this century, it will increase to almost 30 %. The primary reason will be primarily related to the aging population but also to patient awareness and seeking a solution. In addition to the cost which is covered by the health insurance, the patient will spend more than half a billion euro/year out-of-pocket, not to mention the social stigma associated with urinary incontinence. The current common treatment options are symptomatic but do not restore functionality. One option might be tissue engineering or stem cell therapy. This article describes the likelihood that this therapy will change the approach in treating stress urinary incontinence. Boundaries and legal aspects are highlighted as well as approximated cost. These treatment costs might be currently higher than the standard treatment options, but the investment to reduce these costs are paid indirectly by society.

A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men.

OBJECTIVE: To determine whether the two major isoflavones in red clover differ in their effect on low-density lipoprotein cholesterol (LDL-C). DESIGN: A randomised, placebo-controlled, double-blind trial; two parallel groups taking one of the two isoflavones within which treatment and placebo were administered in a crossover design. SETTING: Free-living volunteers. SUBJECTS: A total of 46 middle-aged men and 34 postmenopausal women. INTERVENTION: Two mixtures of red clover isoflavones enriched in either biochanin (n=40) or formononetin (n=40) were compared. Placebo and active treatment (40 mg/day) were administered for 6 weeks each in a crossover design within the two parallel groups. MAIN OUTCOME MEASURES: Plasma lipids were measured twice at the end of each period. RESULTS: Baseline LDL-C concentrations did not differ significantly between men (n=46) and women (n=34), nor between those randomised to biochanin or formononetin. Interaction between time and treatments, biochanin, formononetin and corresponding placebos (two-way ANOVA) on LDL-C showed a significant effect of biochanin treatment alone. The biochanin effect was confined to men; median LDL-C was 3.61 (3.05-4.14) mmol/l with biochanin and 3.99 (3.16-4.29) mmol/l with the corresponding placebo (RM ANOVA with Dunnett's adjustment P<0.05). The difference between placebo and biochanin effects on LDL-C was 9.5%. No other lipid was affected and women failed to respond significantly to treatment. CONCLUSION: Isolated isoflavones from red clover enriched in biochanin (genistein precursor) but not in formononetin (daidzein precursor), lowered LDL-C in men. This may partly explain the previous failure to demonstrate cholesterol-lowering effects with mixed isoflavones studied predominantly in women. SPONSORSHIP: Novogen Ltd, North Ryde NSW, Australia, provided partial support including provision of tablets and outside monitoring.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Cryopreservation of reduced cytochrome C for determination of N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion production in human whole blood.

Various methods are available for measuring the production of reactive oxygen species by phagocytes, but they are limited in their use by the need for their immediate application, cell isolation and of cell-activation by unphysiological stimuli. In addition, after measurement of reactive oxygen metabolites using oxidizing agents, the reduced compounds formed have to be determined during or immediately after their formation. In the present study, an improved cytochrome C assay was investigated which allowed measurements of superoxide anions in whole blood samples following activation of phagocytes by physiological stimuli such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The fMLP-stimulated production of superoxide anion (O(2)(-)) showed a sigmoidal-shaped fMLP dose-response curve, and constant O(2)(-) production rates (nmol.1(-1)x10(6) granulocytes) could be determined reliably up to a blood granulocyte concentration of 1 x 10(4) x microl(-1). To allow the determination of reduced cytochrome C later after its formation, the effect of long-term storage at -20 degrees C on the stability of reduced cytochrome C was tested up to 16 weeks. The results obtained show that the determination of reduced cytochrome C in whole blood represents a simple and reliable method. Most importantly, O(2)(-)-reduced cytochrome C can be frozen and stored without any alterations, at least up to 2 weeks. Thus the method seems to be superior to other methods of detection, especially when the experimental conditions do not allow immediate spectrophotometry (e.g. mountain medicine, space medicine). Under such conditions the present assay would allow reliable measurement of reduced cytochrome C, even after weeks of cryopreservation.

Spontaneous formation of germinal centers in autoimmune mice.

The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1-2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220(+), PNA(+), and GL-7(+) B cells, and FDC-M1(+) follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.

Health providers' opinions on provider-client relations: results of a multi-country study to test Health Workers for Change.

A multi-centre study in four African countries was undertaken to test the acceptability and effectiveness of Health Workers for Change, a methodology to explore provider-client relations within a gender-sensitive context. This intervention addresses the interpersonal component of quality of care. The methodology, consisting of six workshops, was implemented by research teams in Zambia, Senegal, Mozambique and Uganda. It was found to be acceptable within in a range of cultural and primary health care settings. The workshops allowed difficult issues such as prejudice and bribery to be discussed openly, fostered problem solving and the development of practical plans to address problems that could strengthen district health systems.

Secretory bulk flow of soluble proteins is efficient and COPII dependent.

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley alpha-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.

Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin.

We present evidence that Bacillus anthracis lethal toxin (LT) suppresses rather than induces proinflammatory cytokine production in macrophages. Suppression is observed with extremely low levels of LT and involves inhibition of transcription of cytokine messenger RNA. Thus, LT may contribute to anthrax pathogenesis by suppressing the inflammatory response.

Imaging of the oral cavity using optical coherence tomography.

Optical coherence tomography is a new method for noninvasively imaging internal tooth and soft tissue microstructure. The intensity of backscattered light is measured as a function of depth in the tissue. Low coherence interferometry is used to selectively remove the component of backscattered signal that has undergone multiple scattering events, resulting in very high resolution images (< 15 microns). Lateral scanning of the probe beam across the biological tissue is then used to generate a 2-D intensity plot, similar to ultrasound images. This imaging method provides information that is currently unobtainable by any other means, making possible such diverse applications as diagnosis of periodontal disease, caries detection, and evaluation of restoration integrity. This chapter presents an overview of this exciting new imaging technique and its current application to dental diagnosis.

Selective disruption of interleukin 4 autocrine-regulated loop by a tyrosine kinase inhibitor restricts activity of T-helper 2 cells.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4-mediated proliferation of both D10 and the IL-4-dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor alpha activation of NF-kappaB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4. (Blood. 2000;95:3816-3822)

Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma co-association with transcription factor NFAT.

T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARgamma involvement in the regulation of T cell function. Here we show that PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14) prostaglandin J(2), but not PPARalpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARgamma2-expressing, not the PPARgamma-lacking, subpopulation of transfected Jurkat cells. The activated PPARgamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARgamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARgamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.

Molecular cloning of FKHRL1P2, a member of the developmentally regulated fork head domain transcription factor family.

Here we report the expression of a fork head domain protein in human T helper cells. We cloned and characterized a fork head cDNA from human T helper cell mRNA using differential display RT-PCR. The cDNA contains a 546-nucleotide (nt) open reading frame (ORF) that codes for the carboxyl-terminal 180 amino acids (aa) of the recently identified fkhrl1 gene. This ORF does not contain the characteristic DNA-binding domain found in members of the forkhead protein family. In-vitro transcription/translation of this cDNA expressed a protein of approximately 20 kDa. We have generated antibodies that specifically immunoprecipitated the in-vitro-translated 20-kDa protein. This antibody also recognizes in human T lymphocytes a 70-kDa protein corresponding in size to that predicted for the fkhrl1 gene product. The mRNA levels for fkhrl1 is elevated in T helper-induced lymphocytes in comparison to PHA-stimulated T lymphocytes. Further characterization of FKHRL1 and its related family members should shed light on the transcriptional mechanisms of this fork head gene subfamily and their role in T helper cell differentiation and regulation of cell growth.

Mechanisms of cytokine signal transduction: IL-2, IL-4 and prolactin as hematopoietin receptor models.

Cytokines, hormones and hematopoietic growth factors transduce biological signals across the cell membrane via a highly conserved family of single membrane-spanning receptors. The intracellular signal transducing machinery responsible for mediating these responses has remained largely unknown. However, recent identification of a homologous class of tyrosine kinases, Janus Kinases (JAKs), and a related family of transcription factors, signal transducers and activators of transcription (STATs), has shed new light on the molecular mechanisms responsible for mediating hematopoietin signaling and immune response. Current research efforts within the field of cytokine signaling have now shifted to understanding how these molecules are activated by hematopoietic receptors, positively and negatively regulated by kinases and phosphatases, and how they impact on gene transcription to ultimately coordinate cell homeostasis, proliferation and differentiation. This article will review some of our results identifying the involvement of JAKs, STATs, and secondary effector molecules activated following engagement of hematopoietic receptors for IL-2, IL-4, and prolactin. Here, we provide evidence for the ingenious ability of cytokine receptors to selectively recruit and activate these proteins among a repertoire of possible alternative biochemical messengers as a means to affect unique and general cell responses.

Dental OCT.

We present here the first in vivo optical coherence tomography (OCT) images of human dental tissue. A novel dental optical coherence tomography system has been developed. This system incorporates the interferometer sample arm and transverse scanning optics into a handpiece that can be used intraorally to image human dental tissues. The average imaging depth of this system varied from 3 mm in hard tissues to 1.5 mm in soft tissues. We discuss the application of this imaging system for dentistry and illustrate the potential of our dental OCT system for diagnosis of periodontal disease, detection of caries, and evaluation of dental restorations.

Immunocompromised tumor-bearing mice show a selective loss of STAT5a/b expression in T and B lymphocytes.

Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.

Two discrete regions of interleukin-2 (IL2) receptor beta independently mediate IL2 activation of a PD98059/rapamycin/wortmannin-insensitive Stat5a/b serine kinase.

Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1-5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of Stat5b with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor beta (IL2Rbeta) that could independently restore the ability of a truncated IL2Rbeta mutant to mediate Stat5a/b phosphorylation and DNA binding to the gamma-activated site of the beta-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rbeta region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation.

Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580.

The present study of prolactin (PRL) receptor-mediated recruitment of signal transducers and activators of transcription (STATs) demonstrates that PRL activates STAT3, in addition to STAT1 and STAT5 as previously reported, and that STAT1, STAT3 and STAT5 are mediators of PRL effects in cells whether of lymphoid, myeloid or mammary epithelial origin. Furthermore, receptor mutants M240 and T280 that do not mediate PRL-induced JAK2 activation and cell proliferation, are also unable to mediate STAT activation, supporting the proposed model of JAK2 as the initial effector protein used by PRL receptors. On the other hand, tyrosine phosphorylation analysis and electrophoretic mobility shift assays showed that receptor mutant G328, which lacks four of the five conserved cytoplasmic tyrosine residues of PRL receptors, retained the ability to activate JAK2 and STAT1, STAT3 and STAT5. These results support the notion that phosphotyrosyl residues other than those of the receptor, i.e., JAK2, are involved in recruiting STAT proteins to the activated PRL receptor complex.

Growth signaling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors.

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

Measurement of Laser-Plasma Electron Density with a Soft X-ray Laser Deflectometer.

A soft x-ray laser (wavelength lambda = 15.5 nanometers) was used to create a moiré deflectogram of a high-density, laser-produced plasma. The use of deflectometry at this short wavelength permits measurement of the density spatial profile in a long-scalelength (3 millimeters), high-density plasma. A peak density of 3.2 x 10(21) per cubic centimeter was recorded.