An Evaluation of European Countries' Health Systems through Distance Based Analysis.

INTRODUCTION: The issue of evaluating the efficiency of health systems has been elaborated upon frequently. Since "health" is a multi-faceted concept, many variables of different measurement units must be included in its analysis; consequently, this presents a great obstacle for researchers to overcome. MATERIALS AND METHODS: A novel statistical approach for evaluating the efficiency of organizational units is here proposed, which can also be easily applied to the health sector. For these purposes, the health status of the 27 countries belonging to the European Union has been examined by employing a statistical Ivanovic-Jeremic Distance Based Analysis (DBA) on various health indicators. RESULTS: The subsequent outcome of the Distance Based Analysis has shown that Cyprus and Ireland have a most efficient health system sectors. Greece also has exceptional indicators of health service, yet health on the individual level is not comparable. LIMITATIONS: Since it synthesizes many variables into an efficiency score, a DBA can be easily applied to other regions/countries. However, the choice of input and output variables can be considered to be potential limitations since a different choice of variables may cause different efficiency scores for the countries selected. CONCLUSIONS: A DBA approach contributes significantly to the efficiency in the field of research measurement. This analysis can be additionally performed alongside DEA and SFA methods, as a new measure of efficiency.

Detection of zona pellucida proteins during human folliculogenesis.

BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.

Thrombin is a pro-fibrotic factor for rat renal fibroblasts in vitro.

BACKGROUND: Generation of thrombin occurs in response to parenchymal injury. Thrombin not only converts plasma fibrinogen into an insoluble fibrin clot, but also potentially augments inflammation through receptor-mediated activity. This study examines whether thrombin may potentially exacerbate fibrosis by upregulating the function of interstitial fibroblasts in vitro. METHODS: Fibroblasts were isolated by explant outgrowth culture of rat kidneys. Subcultured cells were grown in DMEM+10% FCS supplemented with 0.1-0.5 U/ml thrombin. Functional parameters examined included kinetics (thymidine incorporation and change in cell number), differentiation (Western blotting for alpha-smooth muscle actin; alphaSMA), expression of procollagen alpha1(I) (Northern blotting) and contraction of collagen I lattices. RT-PCR was used to characterise expression of protease-activated receptors (PAR) previously implicated in thrombin's cellular effects. RESULTS: Cell population growth was increased 66 +/- 41 and 47 +/- 41% by 0.1 and 0.5 U/ml thrombin respectively (both p < 0.05 vs. basal). Likewise, 0.5 U/ml thrombin increased corrected procollagen alpha1(I) expression 2.4-fold (p < 0.05 vs. basal) and exacerbated the ability of fibroblasts to contract collagen matrix (p < 0.05 vs. basal). These effects were not associated with any change in expression of the myofibroblast marker alphaSMA. Effects on cell number were inhibited by treatment with (D)-Phe-Pro-Arg-chloromethylketone HCl (PPACK) suggesting that functional effects were mediated by serine protease activity. PAR-1 was the only fully functional known thrombin receptor expressed by these cells. CONCLUSION: Thrombin is a potential unrecognised fibroblast agonist in renal disease. Further studies of thrombin and its receptors may yield valuable insights into the pathogenesis of interstitial fibrosis.

Intracellular cyclic nucleotide analogues inhibit in vitro mitogenesis and activation of fibroblasts derived from obstructed rat kidneys.

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10- 1,000 microM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for alpha-smooth muscle actin; alpha-SMA) and expression of CTGF (Northern blotting), a TGF-beta(1)-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 +/- 4% by 100 microM 8br-cAMP (p < 0.01), 49 +/- 6% by 1,000 microM 8br-cAMP (p < 0.001) and 43 +/- 7% by 1,000 microM 8br-cGMP (p < 0.01). 1,000 microM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 +/- 5 and 60 +/- 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker alpha-SMA by 43 +/- 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 +/- 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.

An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm.

We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.

Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal spermatozoa-ZP binding.

Results of zona pellucida (ZP)-induced acrosome reaction (AR) are reported for 186 normospermic men with unexplained infertility and compared with 34 normal fertile men and 54 patients with disordered ZP-induced AR (DZPIAR) diagnosed after failure of standard IVF. For each ZP-induced AR test, four oocytes that failed to fertilize in IVF were incubated for 2 h with 2x10(6)/ml motile spermatozoa. Spermatozoa tightly bound to the ZP were recovered by aspirating the oocytes with a pipette and the AR assessed using pisum sativum agglutinin labelled with fluorescein. The standard deviation of the difference was 5.2% for repeated tests for ZP-induced AR on different ejaculates from 54 men. The ranges for the ZP-induced AR were 3-98% for normospermic infertile men, 24-95% for fertile men and 0-16% for DZPIAR patients. In the normospermic group, there was a significant correlation between ZP-induced AR and sperm concentration (Spearman r = 0.238, P < 0.001). Using ZP-induced AR < or =16% as the threshold for diagnosis of DZPIAR, the frequency of this condition in normospermic infertile men would be 25%. Thus DZPIAR is common with normospermic idiopathic infertility and this condition should be diagnosed before assisted reproductive technology since it requires intracytoplasmic sperm injection.

Pirfenidone reduces in vitro rat renal fibroblast activation and mitogenesis.

BACKGROUND: Fibroblasts have been universally recognised in tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. Recently, pirfenidone (PF) has been shown to both ameliorate progressive fibrosis and reduce established scarring after ureteric obstruction (UUO) in the rat, suggesting that it is a novel anti-fibrotic agent. The objective of this study was therefore to determine if these effects include down-regulation of fibroblast function. METHODS: Cortical fibroblasts were obtained from outgrowth cultures of renal tissue isolated from kidneys 3 days after UUO and constituted 100% of cells studied. Functional studies examined the effects of 20 and 200 microg/ml PF on basal serum stimulated activity. Activation was examined by western blotting for alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Cell proliferation, collagenase activity and collagen production were determined from kinetic studies, zymography for MMP2 and [3H] proline incorporation in collagenous proteins respectively. RESULTS: Proliferation, as measured by [3H] thymidine incorporation, was reduced in dose dependent manner by 20 and 200 microg/ml PF (p<0.05; 200 vs 0 microg/ml). Likewise, 200 microg/ml PF reduced cell population growth over 5 days of culture (p<0.05 vs 0 microg/ml). PF (200 microg/ml) decreased alphaSMA and CTGF protein expression to 66+/-13 and 37+/-26% of basal levels respectively (both p<0.05 vs 0 microg/ml). Synthesis of collagen was unaffected by PF. Maximal dose of PF produced a modest reduction in MMP2 lytic activity (p=0.05). Effects of PF were independent of cell toxicity. CONCLUSIONS: Down-regulation of renal fibroblast activation and proliferation are specific actions of PF.

Pentoxifylline reduces in vitro renal myofibroblast proliferation and collagen secretion.

Interstitial myofibroblasts (MF) are cells with features of both smooth muscle cells and fibroblasts. They have been universally recognized in situations of tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. The objective of this study was to determine if functions of MF relevant to fibrogenesis can be modified in vitro by the phosphodiesterase inhibitor pentoxifylline (PTX). MF were obtained from sub-culture of normal rat kidney explant outgrowths maintained in DMEM + 20% fetal calf serum (FCS), supplemented with antibiotics. Cells were characterized on the basis of growth characteristics and immunohistochemistry. MF constituted >95% of cells at passage 3. Cell culture media was supplemented with the potential antagonist PTX alone (0, 1, 10, 100 microg/ml) and in combination with TGFbeta(1) (5 ng/ml). Population kinetics, proliferation and collagen production were determined from cell growth, [(3)H]thymidine incorporation and [(3)H]proline incorporation in collagenous proteins, respectively. Both serum-stimulated population growth and proliferation were reduced in a linear fashion by 1, 10 and 100 microg/ml PTX (all p < 0.05 versus 0 microg/ml). Effect of PTX on cell population growth was however reversible when PTX was removed. Basal collagen secretion was decreased by PTX at 10 and 100 microg/ml (p < 0.05 versus 0 microg/ml although layer collagen remained unchanged. Collagen production (secreted and cell layer) was augmented by 5 ng/ml TGFbeta(1). These effects on collagen production were partially reduced when 100 microg/ml PTX was added. The authors conclude that myofibroblast function can be altered with agonists/antagonists. Attempts to down-regulate fibrogenic functions of MF may therefore offer a valuable therapeutic strategy.

The human acrosome reaction.

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVF was usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call this condition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.

An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.

The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10-40 micromol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.

The results of "exercise therapy in coronary prone individuals and coronary patients".

The authors followed for at least two years 483 patients (430 coronary prone patients and 53 with proved coronary heart disease), to determine whether physical training could decrease coronary risk factors and improve exercise tolerance in the trained group as compared with the conventionally treated group (way of life, diet, drugs). There was no significant difference among the two groups for blood lipid profile, blood pressure (at rest and during exercise), functional capacity and mortality. In the authors' opinion this could be explained by the inadequate training program (45 minutes twice a week) and by a possible overlapping of the groups.