Achieving orphan designation for placental insufficiency: annual incidence estimations in Europe.

OBJECTIVE: To determine whether a novel therapy for placental insufficiency could achieve orphan drug status by estimating the annual incidence of placental insufficiency, defined as an estimated fetal weight below the 10th centile in the presence of abnormal umbilical artery Doppler velocimetry, per 10 000 European Union (EU) population as part of an application for European Medicines Agency (EMA) orphan designation. DESIGN: Incidence estimation based on literature review and published national and EU statistics. SETTING AND POPULATION: European Union. METHODS: Data were drawn from published literature, including national and international guidelines, international consensus statements, cohort studies and randomised controlled trials, and published national and EU statistics, including birth rates and stillbirth rates. Rare disease databases were also searched. RESULTS: The proportion of affected pregnancies was estimated as 3.17% (95% CI 2.93-3.43%), using a weighted average of the results from two cohort studies. Using birth rates from 2012 and adjusting for a pregnancy loss rate of 1/100 gave an estimated annual incidence of 3.33 per 10 000 EU population (95% CI 3.07-3.60 per 10 000 EU population). This fell below the EMA threshold of 5 per 10 000 EU population. CONCLUSIONS: Maternal vascular endothelial growth factor gene therapy for placental insufficiency was granted EMA orphan status in 2015 after we demonstrated that it is a rare, life-threatening or chronically debilitating and currently untreatable disease. Developers of other potential obstetric therapies should consider applying for orphan designation, which provides financial and regulatory benefits. TWEETABLE ABSTRACT: Placental insufficiency meets the European Medicines Agency requirements for orphan disease designation.

Successful treatment of severe ABO antibody-mediated rejection using bortezomib: a case report.

BACKGROUND: Our program for ABO-incompatible renal transplantation includes antigen-specific immunoadsorption (extracorporeal columns with the A or B trisaccharides), rituximab, and standard maintenance immunosuppression. Anti-A or -B titers ≤ 8 in the indirect antiglobulin test (IAT) against panel A1 or B RBC are acceptable for transplantation. CASE REPORT: A previously healthy, 15-month-old girl was diagnosed with Wilms' tumor and proteinuria. Denys-Drash syndrome was confirmed. Bilateral nephrectomy was performed. At 3.5 years of age she received an ABO-incompatible renal transplant from her father (A1 to O). The anti-A titers before transplantation were low. She was treated preoperatively with rituximab, immunoadsorption, immunoglobulin and mycophenolate mofetil (MMF). The maintenance immunosuppression protocol included basiliximab, tacrolimus, MMF, and prednisolone. The initial postoperative course was uncomplicated with rapid normalization of serum creatinine. The anti-A titers started to increase on postoperative day 5 (8 NaCl/16 IAT). Despite daily immunoadsorptions the titers rose to 1024 NaCl/1024 IAT on day 9. Renal function deteriorated and hemodialysis was started. A renal biopsy on day 9 showed acute severe antibody-mediated rejection. Additional treatment with bortezomib was given and after 2 doses the titers started to decline, renal allograft function improved and hemodialysis was stopped. On day 21 posttransplant the titers went down, creatinine was 28 µmol/L, and no more immunoadsorptions were performed. CONCLUSION: By using bortezomib, we were able to successfully reverse a severe ABO antibody-mediated rejection.

Gene expression profiling of placentae from women with early- and late-onset pre-eclampsia: down-regulation of the angiogenesis-related genes ACVRL1 and EGFL7 in early-onset disease.

The underlying mechanisms behind the obstetric condition pre-eclampsia (PE) are still unclear. Manifestation of PE is heterogeneous and it has therefore been proposed to be a syndrome with different causes rather than one disease with a specific aetiology. Recently, we showed differences in circulating angiogenic factors between two subgroups-early- and late-onset PE. To further elucidate the differences between the two, we investigated placental gene expression profiles. Whole genome microarray technology and bioinformatic analysis were used to evaluate gene expression profiles in placentae from early- (24-32 gestational weeks, n = 8) and late-onset (36-41 gestational weeks, n = 7) PE. The results were verified by using quantitative real-time (qRT)-PCR. We found significant differences in the expression of 196 genes in early- compared with late-onset PE, 45 of these genes showing a fold change above 2. Bioinformatic analysis revealed alterations in angiogenesis and regulation of cell motility. Two angiogenesis-associated transcripts (Egfl7 and Acvrl1) showed lower expression in early-onset PE versus late-onset PE (P = 0.037 and P = 0.003) and versus gestational age-matched controls (P = 0.007 and P = 0.011). We conclude that angiogenesis-associated genes are regulated in a different manner in the two subgroups, and that the gene expression profiles of early- and late-onset PE diverge, supporting the hypothesis of early- and late-onset PE being at least partly two separate entities.

Review: Biochemical markers to predict preeclampsia.

Worldwide the prevalence of preeclampsia (PE) ranges from 3 to 8% of pregnancies. 8.5 million cases are reported yearly, but this is probably an underestimate due to the lack of proper diagnosis. PE is the most common cause of fetal and maternal death and yet no specific treatment is available. Reliable biochemical markers for prediction and diagnosis of PE would have a great impact on maternal health and several have been suggested. This review describes PE biochemical markers in general and first trimester PE biochemical markers specifically. The main categories described are angiogenic/anti-angiogenic factors, placental proteins, free fetal hemoglobin (HbF), kidney markers, ultrasound and maternal risk factors. The specific biochemical markers discussed are: PAPP-A, s-Flt-1/PlGF, s-Endoglin, PP13, cystatin-C, HbF, and α1-microglobulin (A1M). PAPP-A and HbF both show potential as predictive biochemical markers in the first trimester with 70% sensitivity at 95% specificity. However, PAPP-A is not PE-specific and needs to be combined with Doppler ultrasound to obtain the same sensitivity as HbF/A1M. Soluble Flt -1 and PlGF are promising biochemical markers that together show high sensitivity from the mid-second trimester. PlGF is somewhat useful from the end of the first trimester. Screening pregnant women with biochemical markers for PE can reduce unnecessary suffering and health care costs by early detection of mothers at increased risk for PE, thus avoiding unnecessary hospitalization of pregnant women with suspect or mild PE and enabling monitoring of the progression of the disease thereby optimizing time for delivery and hopefully reducing the number of premature births.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERß mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Increased cystatin C expression in the pre-eclamptic placenta.

Trophoblast invasion is regulated by proteinases and their inhibitors. Cystatin C inhibits cysteine proteinases. The serum concentration of cystatin C is increased in late pregnancy and pre-eclampsia. We aimed to investigate whether the expression of cystatin C is increased in the pre-eclamptic placenta and to investigate the expression pattern of cystatin C mRNA and protein in placental tissue. Tissue samples from the central part of the placenta from 13 normal and 22 pre-eclamptic pregnancies were included. We used real-time polymerase chain reaction (RT-PCR) and in situ hybridization for mRNA expression analysis and immunohistochemistry and Western blotting for protein expression analysis. RT-PCR showed a significantly higher expression of cystatin C mRNA in pre-eclampsia than in normal pregnancy, with the highest expression in cases with severe pre-eclampsia. In situ hybridization revealed a distinct pattern of high expression in the extravillous trophoblast cells of the basal plate and low expression in the syncytiotrophoblast covering villi. The cystatin C protein distribution matched the mRNA expression pattern. Western blot analysis revealed an increased protein expression in cases with severe pre-eclampsia and confirmed the presence of cystatin C in amniotic fluid samples. The high expression of cystatin C mRNA in the extravillous trophoblast cells of the basal plate suggests a role for cystatin C in the regulation of proteases in placentation. Placental expression and secretion of cystatin C could contribute to the elevated maternal plasma levels seen in pre-eclampsia.

Early recurrence of nephrotic syndrome (immunoglobulin m nephropathy) after renal transplantation successfully treated with combinations of plasma exchanges, immunoglobulin, and rituximab.

In contrast to focal segmental glomerulosclerosis, which is well known to recur early in a renal graft, there are only few cases described with recurrence of immunoglobulin M (IgM) nephropathy after transplantation. We herein describe a patient with early recurrence of IgM nephropathy. A 15-year-old boy with nephrotic syndrome (IgM nephropathy) proceeding to end-stage renal disease was on dialysis before living related renal transplantation. Native kidneys were not removed. Standard immunosuppression including steroids, tacrolimus, and mycophenolate mofetil yielded initially good graft function with the s-creatinine falling to 73 micromol/L. Proteinuria was present on day 1, increasing to 20 g/L after 3 days. S-creatinine increased to 158 micromol/L and urine production diminished. A graft biopsy showed no rejection or glomerulopathy but protein vacuoles were seen within tubular cells indicating massive proteinuria. Treatment with plasma exchanges, immunoglobulin, and steroids was started. Hemodialysis was necessary. Proteinuria improved to 3.5 g/L, but s-creatinine continued to rise and a second graft biopsy showed vascular rejection (Banff type IIA). The patient was treated with antithymocyte globulin and further plasma exchanges. A single dose of rituximab was given. Five months after transplantation the s-creatinine was 67 micromol/L and there was no proteinuria. In this case early recurrence of nephrotic syndrome occurred on the first posttransplant day in combination with later occurring vascular rejection. Successful treatment included a combination of plasma exchanges, rituximab, immunoglobulin, and antithymocyte globulin.

Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients.

BACKGROUND: The aim of this study was to analyse the effects of an estradiol (E(2))-progesterone substitution protocol on the endometrial expression of estrogen-sensitive genes during the peri-implantation period. METHODS: Peripheral blood and endometrial biopsies were obtained from 13 infertile women both in a natural cycle (NC), on days 5 and 7 after ovulation (NC5, NC7), and in an artificial (substituted) cycle (AC), on days 5 and 7 of progesterone addition (AC5, AC7). Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were assayed by immunohistochemistry. Matrix metalloproteinase-26 (MMP-26) mRNA and tissue inhibitor of metalloproteinase-4 (TIMP-4) mRNA were semiquantitatively assessed in tissue sections using in situ hybridization (ISH) and quantified in tissue extracts using real-time PCR. RESULTS: Levels of both E(2) and progesterone were higher in the peripheral blood in AC than in NC. Also on day AC5, expressions of ERalpha, PR and MMP-26 mRNA (focally) were increased in the epithelium and TIMP-4 mRNA in the stroma. Expression levels of these genes dropped significantly between AC5 and AC7, but not between NC5 and NC7. Abnormally high levels in AC5 samples suggest overstimulation with E(2), and the rapid decrease between AC5 and AC7 suggests overstimulation with progesterone. CONCLUSIONS: In ACs, increased levels of E(2) in the blood exaggerate the endometrial expression of estrogen-sensitive genes, whereas higher levels of progesterone in the blood in the secretory phase exaggerate the drop in expression of these genes. Dramatic variations in the gene expression may not be optimal for the implantation process.

Endometrial TIMP-4 mRNA is expressed in the stroma, while TIMP-4 protein accumulates in the epithelium and is released to the uterine fluid.

We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.

Gene expression profiling of human placentas from preeclamptic and normotensive pregnancies.

The aim of this study was to investigate patterns of gene expression in placental samples from patients with preeclampsia (PE), persistent bilateral uterine artery notching (without PE), and normal controls. This study included placental tissue from nine women with PE, seven with uncomplicated pregnancies and five with bilateral uterine artery notching in Doppler velocimetry tracings. Human cDNA microarrays with 6500 transcripts/genes were used and the results verified with real-time PCR and in-situ hybridization. Multidimensional scaling method and random permutation technique demonstrated significant differences among the three groups examined. Within the 6.5K arrays, 6198 elements were unique cDNA clones representing 5952 unique UniGenes and 5695 unique LocusLinks. Multidimensional scaling plots showed 5000 genes that met our quality criteria; among these, 366 genes were significantly different in at least one comparison. Differences in three genes of interest were confirmed with real-time PCR and in-situ hybridization; acid phosphatase 5 was shown to be overexpressed in PE samples and calmodulin 2 and v-rel reticuloendotheliosis viral oncogene homolog A (RELA) were downregulated in PE and uterine artery notch placentas. In conclusion downregulation of RELA and calmodulin 2 might represent an attempt by the placenta to compensate for elevations in intracellular calcium, possibly caused by hypoxia and/or apoptosis, in both pregnancies with uterine artery notching and preeclampsia.

Extended antipaternalism.

Extended antipaternalism means the use of antipaternalist arguments to defend activities that harm (consenting) others. As an example, a smoker's right to smoke is often invoked in defence of the activities of tobacco companies. It can, however, be shown that antipaternalism in the proper sense does not imply such extended antipaternalism. We may therefore approve of Mill's antipaternalist principle (namely, that the only reason to interfere with someone's behaviour is to protect others from harm) without accepting activities that harm (consenting) others. This has immediate consequences for the ethics of public health. An antipaternalist need not refrain from interfering with activities such as the marketing of tobacco or heroin, boxing promotion, driving with unbelted passengers, or buying sex from "voluntary" prostitutes.

Matrix metalloproteinase-26 (matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer.

OBJECTIVE: Matrix metalloproteinases (MMPs) are key players in the degradation of extracellular matrix and basement membranes, and are thus important in tumor invasion. Recently, MMP-26 (endometase), a novel matrilysin-type member of the MMP family, was cloned from an endometrial tumor. This study examines the expression of MMP-26 mRNA in hyperplastic, premalignant and malignant endometrial samples, and compares with normal endometrial tissue. METHODS: Endometrial carcinoma samples (19) were histologically classified as well, intermediately and poorly differentiated. Samples with hyperplasia (n = 15) were classified as simple, complex, or complex with atypia. Normal endometrial specimens (n = 39) were classified according to an ideal 28-day menstrual cycle and subsequently grouped in the early, middle, and late parts of the cycle. All samples were analyzed using in situ hybridization and real time PCR. The probes used for in situ hybridization and real time PCR recognized non-overlapping sequences. MMP-26 protein was localized by immunohistochemistry. RESULTS: MMP-26 mRNA was exclusively localized in the epithelial component of normal, hyperplastic, premalignant, as well as malignant samples. It was not found in the stroma of any tissue category. Quantifications with real time PCR as well as semi-quantifications of the in situ hybridization signal revealed maximal levels in normal tissue at midcycle and in endometrial hyperplasia both with and without atypia. The amount of MMP-26 mRNA decreased progressively with loss of histological differentiation in malignant samples. Immunostaining localized MMP-26 in epithelial glandular and luminal cells, in vessel walls, and in tumor cells. Since the pattern of MMP-26 expression mimicked that of ER-alpha, we searched the MMP-26 promoter region for a potential estrogen response element (ERE). A sequence at position -130 to -116 had high homology to the consensus sequence of an ERE. Based on these observations, we suggest that ER-alpha is involved in regulation of the MMP-26 gene. CONCLUSIONS: MMP-26 mRNA is selectively localized in the epithelial compartment of normal, hyperplastic, and malignant endometrial tissue. Expression is high in normal and hyperplastic endometria, but is downregulated in the late part of the cycle and in malignant tumors. The expression pattern of MMP-26 mRNA mimics that of ER-alpha, and the promoter region of the MMP-26 gene has a potential ERE.

[Novel matrix metalloproteinases in cycling endometrium]. / Nové matrix metaloproteinázy v cyklickém endometriu.

OBJECTIVE: To examine the expression pattern of some novel matrix metalloproteinases (MMPs) in cycling endometrium. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden. METHODS: We studied MMP-12, -16, -17, -19 and -26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. Cycle variation was examined in frozen samples using in situ hybridization. RESULTS: Three distinct pattern of MMP mRNA expression were detected in cycling endometrium. MMP-12 was expressed predominantly in perimenstrual period, MMP-16, -17 and 19 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period. CONCLUSION: Different endometrial expression patterns of novel MMPs during menstrual cycle may indicate their specific roles for menstruation, endometrial growth and remodelling and implantation.

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle.

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.

Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours. Coordinated expression with MMP-26.

We have previously reported that endometrial expression of matrix metalloproteinase (MMP)-26 mRNA comes to a maximum in the early secretory phase. Since tissue inhibitor of metalloproteinase (TIMP)-4 is a potent inhibitor of MMP-26, the objective of this study was to identify the pattern of TIMP-4 mRNA expression in the normal endometrial cycle. We also evaluated hyperplastic, pre-malignant (atypical hyperplasia) and malignant endometrial tissue. Endometrial TIMP-4 mRNA was localized in tissue sections using in situ hybridization, and quantified in tissue extracts using real-time PCR. Estrogen receptor alpha (ERalpha) was assayed in the same set of samples using immunohistochemistry. In situ hybridization demonstrated TIMP-4 mRNA in the stroma of both normal and pathological tissues. TIMP-4 mRNA increased in the proliferative phase to a maximum in the early secretory phase, and then decreased in the late part of the cycle. Expression was comparable in normal and hyperplastic (including atypical) endometrial samples, whereas lower levels were detected in malignant tumours. Since this general pattern of expression suggests estrogen dependence, we evaluated ERalpha in our samples. Tissue sections from the normal proliferative phase, hyperplasia and pre-malignant atypical hyperplasia tissue stained strongly for ERalpha, whereas weak staining was seen in the secretory phase and in malignant tumours. Thus, low level of ERalpha was accompanied by down-regulated TIMP-4 mRNA, supporting the hypothesis that ERalpha contributes to regulation of the TIMP-4 gene. In addition, we identified a putative estrogen response element (ERE) in the promoter region of the TIMP-4 gene at position -930 to -916. Similarities in the cyclic patterns of TIMP-4 mRNA and MMP-26 mRNA, together with the fact that TIMP-4 is a potent inhibitor of MMP-26, suggest a functional relationship, and furthermore a role in human implantation.

Management of children with dilating vesico-ureteric reflux in Sweden.

AIM: To evaluate the management and outcome of children with dilating vesico-ureteric reflux diagnosed before 2 y of age. METHODS: This retrospective, multicentre study was part of a programme for quality assurance in Sweden. A total of 2309 unselected children, aged 0-2 y, were investigated after the first urinary tract infection. Voiding cystourethrography was performed in a total of 1953 children, of whom 584 had reflux. Of these children, 303 (119 boys and 184 girls) had reflux with dilatation (grade 3-5). RESULTS: Follow-up after 4-6 y was reported in 272 of the 303 children. Spontaneous regression of dilating reflux occurred in more than half of the patients and was significantly more frequent in boys than in girls (p = 0.047). In children with grade 3 reflux and grade 4-5 reflux, there were pyelonephritic recurrences in 18% and 45% of the boys and 28% and 70% of the girls, respectively (p < 0.001). One hundred and eighty-one children (65%) were managed conservatively, 58 (21%) were treated with subureteric injection and 33 (12%) with ureteric reimplantation. There were considerable differences in treatment strategies between centres. CONCLUSION: This study of an unselected cohort of children with urinary tract infection and dilating reflux showed spontaneous resolution of dilating reflux in more than half of the subjects and more often in boys than in girls. Pyelonephritic recurrences were more common in girls than in boys, and more frequent in grade 4-5 reflux than in grade 3. The results indicate important differences between the sexes and that boys and girls should be assessed separately when treatment strategies are studied.

[MMP-26 expression in endometrial explants treated with estradiol and progesterone]. / Exprese MMP-26 v endometriálních explantech pod vlivem estradiolu a progesteronu.

OBJECTIVE: To examine possible estrogen dependent endometrial expression of MMP-26 in vitro. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden. METHODS: We studied MMP-26 mRNA in 14 normal endometrial samples obtained from the proliferative phase of the menstrual cycle. Samples were cultured for five days either with estradiol alone or in combination with progesterone. Samples cultured with ethanol represented control groups. MMP-26 mRNA expression was examined in frozen samples using in situ hybridization. Immunohistochemistry was used to study the presence of estrogen and progesterone receptors in endometrial explants. RESULTS: MMP-26 mRNA expression was highest in fresh (non cultured) samples. Signal intensity decreased during the first two days of culture and was negligable in the following days. Nuclear intensity for estrogen and progesterone receptor was high after five days of culture. CONCLUSION: We did not find MMP-26 mRNA in vitro expression to be directly estrogen dependent.

Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium.

The human endometrium is a dynamic tissue, which undergoes extensive tissue remodelling during the menstrual cycle. Due to their involvement in such processes, several well-characterized matrix metalloproteinases (MMP) have previously been studied in the endometrium. MMP-26 is a newly described matrilysin. We studied MMP-26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Tissue distribution and cycle variation was examined using in-situ hybridization, Northern blot analyis and real time PCR. The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. MMP-26 was localized exclusively in epithelial cells of both glands and the luminal surface. Expression increased during the proliferative phase to a maximum at mid-cycle, then decreased to non-detectable levels in the late secretory and menstrual phases. Expression of MMP-26 mRNA in endometrial tissue explants in vitro required stimulation with both estradiol and progesterone. The tissue content of c-jun mRNA was assayed, since c-jun, as part of the enhancer complex AP-1, may be involved in regulation of MMP-26 gene transcription. The pattern of c-jun expression over the menstrual cycle was similar to that of MMP-26. Epithelial expression in the peri- and post-ovulatory stages of the menstrual cycle suggests the involvement of MMP-26 in reproductive processes.

Antibiotic treatment for five days is effective in children with acute cystitis.

UNLABELLED: Short courses of antibiotics are often recommended to treat children with acute cystitis despite lack of firm evidence to support such management. The aim of this study therefore was to analyse the short-term outcome of such treatment. The retrospective analysis included 300 children (252F, 48M) fulfilling the criteria of first-time acute cystitis and managed according to a protocol recommending 5 d treatment. In 214 (71%) the treatment was given according to the protocol and in the others for 7 or 10 d. Nitrofurantoin was used in 150 (50%) and trimethoprim without or with sulfonamide in 129 (43%). The short-term results were excellent with 96% of the children being free from symptoms at the first follow-up visit after a median of 6 d. Only 2 girls had persisting bacteriuria and thus the frequency of bacteriological treatment failure was 1%. Recurrence within 30 d occurred in 4 girls (2%). CONCLUSION: A 5 d treatment with antibiotics is adequate in children with acute cystitis. Routine follow-up visits after a first acute cystitis may not be necessary, providing that the bacteria causing the infection are sensitive to the prescribed antibiotic and that there is no history of defective bladder or bowel emptying.