Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line.

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.

Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle-stimulating hormone flare, growth hormone protocol.

OBJECTIVE: To assess the efficacy of a novel protocol-microdose GnRH agonist (GnRH-a), FSH, and GH, for the stimulation of IVF patients who were canceled previously on a standard luteal GnRH-a, FSH, GH protocol. DESIGN: Prospective evaluation using the patient's previous IVF stimulation attempt as historic controls. SETTING: Private practice assisted reproductive technology center. PARTICIPANT(S): Thirty-two patients who had prior ovulation induction cycles canceled using luteal phase GnRH-a suppression followed by exogenous gonadotropins and GH. INTERVENTION(S): Precycle treatment with oral contraceptives followed by follicular phase administration of 40 micrograms leuprolide acetate every 12 hours beginning on cycle day 3 and FSH supplemented with GH beginning on cycle day 5. MAIN OUTCOME MEASURE(S): Paired analysis of E2 day 5, number of follicles, ampules of FSH required, and cancellation rate. The number of oocytes, embryos, embryo quality, implantation rate, and pregnancy rate (PR) were determined for completed cycles on the microdose GnRH-a, FSH, GH protocol. RESULT(S): Controlled ovarian hyperstimulation was superior during microdose GnRH-a, FSH, GH stimulation when compared with the prior luteal GnRH-a cycle. Specifically, there was a higher E2 response, more oocytes, fewer cycle cancellations, and no premature LH surge or luteinization. The microdose GnRH-a, FSH, GH protocol produced an average of 10 oocytes and a 50% ongoing PR. CONCLUSION(S): The microdose GnRH-a, FSH, GH protocol is superior to standard protocols for the treatment of patients with decreased ovarian reserve undergoing controlled ovarian hyperstimulation for IVF.

A model for the incorporation of intracytoplasmic sperm injection into a private practice in vitro fertilization program.

OBJECTIVE: To describe a method for the training of personnel and the implementation of intracytoplasmic sperm injection (ICSI) into an IVF program. The results of the first 75 cycles are reviewed. DESIGN: Retrospective review of the first 75 consecutive ICSI procedures. SETTING: Private, community-based IVF program. MAIN OUTCOME MEASURES: The fertilization rate, damage rate, ongoing pregnancy rate (PR), and implantation rate were measured. RESULTS: Nine percent of the injected oocytes were damaged. The fertilization rate was 60%, and the cleavage rate was 98%. Fifty-nine percent of the cycles resulted in an ongoing pregnancy, and the implantation rate per embryo was 26%. CONCLUSIONS: A high initial PR can be obtained with ICSI using a systematic training regimen.

Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate.

The relationship of the circulating level of progesterone (P) on the day of human chorionic gonadotropin (hCG) injection to occurrence of clinical pregnancy was examined in 133 leuprolide acetate human menopausal gonadotropin (hMG) in vitro fertilization cycles in women having at least three embryos transferred. Progesterone concentrations greater than 0.5 ng/mL were associated with a significantly lower rate of pregnancy (12/59, 20%) compared with less than 0.5 ng/mL (40/74, 54%, P less than 0.005). The higher P cycles were associated with greater patient age and hMG dose, although these relationships appeared to be indirect. Luteinizing hormone (LH) concentrations remained suppressed. Ovarian stimulation may cause excessive luteinization and an adverse cycle outcome even in the presence of low LH levels. Prospective use of P levels may be helpful to determine optimal hCG timing.

Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.

OBJECTIVE: To determine the relationship between blastocyst score and pregnancy outcome. DESIGN: Retrospective review of blastocyst transfer in an IVF clinic. SETTING: Private assisted reproductive technology unit. PATIENT(S): 107 patients undergoing blastocyst culture and transfer of two embryos. INTERVENTION(S): Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5), followed by transfer of two blastocysts. MAIN OUTCOME MEASURE(S): Implantation rates, pregnancy rates, and twinning were analyzed. RESULT(S): When a patient received two top-scoring blastocysts (64% of patients), implantation and pregnancy rates were 70% and 87%, respectively. The twinning rate in this group was 61%. When only one top-quality blastocyst was available for transfer (21% of patients), the implantation and pregnancy rates were 50% and 70%. The twinning rate for this group was 50%. In contrast, when only low-scoring blastocysts were available for transfer (15% of patients), implantation and pregnancy rates were 28% and 44%, and the twinning rate was 29%. No monozygotic twins were observed in this group of patients. CONCLUSION(S): The ability to transfer one high-scoring blastocyst should lead to pregnancy rates greater than 60%, without the complication of twins.

Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates.

OBJECTIVE: To access the effect of augmenting IVF with assisted hatching in the treatment of poor-prognosis patients. DESIGN: Thirty-three poor-prognosis IVF patients were treated with assisted hatching and were compared with 43 control subjects without assisted hatching. SETTING: Center for Reproductive Medicine, Swedish Medical Center, Englewood, Colorado. PARTICIPANTS: Seventy-six women undergoing IVF with a poor prognosis for pregnancy. Poor prognosis was defined as Elevated day 3 FSH level; age > or = 39 years; and multiple prior IVF failures. MAIN OUTCOME MEASURES: Pregnancy and implantation rates per embryo. RESULTS: The incidence of ongoing pregnancy in the assisted hatching group was 64% compared with 19% in the control group. Implantation rate per embryo transferred was 33% in the assisted hatching group versus 6.5% in the control group. CONCLUSIONS: These results demonstrate that assisted hatching, when applied to poor-prognosis patients, improves embryonic implantation and pregnancy rates.

Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers.

OBJECTIVE: To determine whether the transfer of blastocysts on day 5, developed in sequential culture media, resulted in an increase in implantation rate compared with embryos transferred on day 3. DESIGN: Comparative study of embryo culture regimes. SETTING: Private practice assisted reproductive technology center. PATIENT(S): Twenty-three patients undergoing routine IVF cycles. INTERVENTION(S): Culture of embryos to day 3 in either standard culture conditions or a serum-free chemically defined medium. One hundred one embryos were subsequently cultured from day 3 to day 5 in a second serum-free medium specifically designed to support development of the blastocyst. MAIN OUTCOME MEASURE(S): Embryo cell number and quality on day 3. Blastocyst development on day 5. Implantation rate (determined by fetal heart) and ongoing pregnancy rate (PR). RESULT(S): Implantation rates for embryos transferred at the blastocyst stage of development were twice that observed for embryos transferred on day 3, around the eight-cell stage. Significantly more embryos were required for transfer on day 3, compared with day 5, to establish similar PRs. CONCLUSION(S): Viable human blastocysts can be obtained in sequential culture media in the absence of coculture and serum. Transfer of blastocysts in IVF will facilitate high PRs while limiting the number of embryos transferred and therefore minimizes the risk of multiple gestation.

Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs.

OBJECTIVE: To determine whether previously described advanced blastocyst development and high implantation rates are confirmed in an expanded multicenter trial. DESIGN: Retrospective review. SETTING: Two private assisted reproductive technology units. PATIENT(S): One hundred seventy-four patients who underwent blastocyst culture and transfer. INTERVENTION(S): Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5) followed by ET. MAIN OUTCOME MEASURE(S): The number and percentage of blastocysts developed, implantation rates, pregnancy rates, and parameters that affected outcome were analyzed. RESULT(S): Only 3 of 174 patients failed to achieve blastocyst-stage ET. The mean blastocyst development rate was 48%. The ongoing pregnancy rate was 66.3% per oocyte retrieval, with a mean (+/-SE) of 2.2 +/- 0.05 blastocysts transferred and an implantation rate of 48% per blastocyst transferred. CONCLUSION(S): Blastocyst culture and transfer is an effective means of treating patients who respond well to gonadotropins. High pregnancy rates can be accomplished with low numbers of embryos transferred. Patients who failed to achieve ET were rare.

Lead poisoning in children. The ramifications and the road to prevention.

Environmental lead poisoning is easier to prevent than to treat, and it can be destructive or deadly if ignored. Dr Schlenker, medical director of the Milwaukee Health Department, shares his approach to screening young children for potentially dangerous blood lead levels, educating the community about prevention of lead poisoning, and eradicating environmental sources of lead.

Primary prevention of childhood lead poisoning through community outreach.

BACKGROUND: The prevalence of childhood lead poisoning has substantially decreased in Milwaukee, Wisconsin over the past decade. Nevertheless, by the mid 1990s, 30% of children were still found to have elevated blood levels. OBJECTIVE: To extend the reach of the Milwaukee Health Department (MHD) to previously underserved families in 1 inner city neighborhood with extremely old housing, the Sixteenth Street Community Health Center (SSCHC) implemented, in 1995, the Community Lead Outreach Project (CLOP). Going door-to-door, CLOP attempted to identify children 6 months to 6 years old with elevated blood lead levels (BLL), referring those > or = 20 micrograms/dL to MHD and enrolling those with BLLs 10-19 micrograms/dL in a program of prevention education and environmental clean-up with the specific aim of preventing BLLs increasing to 20 micrograms/dL and above. METHODS: A team of community outreach workers led by a nurse-coordinator visited, over a 4-year period, families in their homes in 13 census tracts surrounding the SSCHC. During the home visits, capillary blood samples for BLLs were drawn, environmental assessments and scoring were conducted, lead poisoning prevention education provided and repair and cleaning of household lead hazards demonstrated. For control and comparison, BLL data for the entire city by ZIP Code and provider were obtained from the Milwaukee Health Department. Odds ratios for changes in the proportions of children screened > or = 10 micrograms/dL were calculated and compared for the years 1996 through 1999. The odds ratios of changes for various populations were compared for significant differences using tests of homogeneity. To control for age confounding, proportions of elevated BBLs for all groups and for all years were age-standardized, using the direct method. RESULTS: Over the entire study, 20.9% of the children screened had BLLs > or = 10 micrograms/dL and 3.0% were > or = 20 micrograms/dL. For 395 children with BLLs 10-19 micrograms/dL enrolled in the CLOP follow-up program, the mean BLL was 12.9 micrograms/dL. Mean levels at the first, second and third follow-up visits were 10.8, 10.3 and 9.8 micrograms/dL respectively, showing an overall decline of 3.1 micrograms/dL or 24%. At the first follow-up visit, 97% of the children tested were < 20 micrograms/dL while 76% were < 10 micrograms/dL. By the second follow-up visit, 100% were < 20 micrograms/dL. Initial environmental scores averaged 24.7, declining to 19.0 at first, 17.8 at second and 14.8 at third follow-up visits. For the entire CLOP population, the proportion of children testing > 10 micrograms/dL declined each year from 46.3% in 1996 to 22.5% in 1999. The geographic area in which CLOP operated recorded the highest screening penetration rate in the city: 61%. The odds ratio for CLOP clients to have elevated blood lead levels at the end of the study period, in contrast with the beginning, was 0.34 compared to 0.55 for the entire city and 0.75 for private physicians serving the same general population. Comparison of odds ratios showed the CLOP target population enjoyed a decrease in rate of elevated BLL 1.6 times that of the city-wide average, p-value = 0.016 and more than double that of the patients of area private providers. CONCLUSIONS: We conclude that the Sixteenth Street Community Health Center Childhood Lead Outreach Project has successfully accessed populations of children with high rates of lead poisoning who had escaped more traditional screening venues and effectively intervened to reduce their BLLs to < 10 micrograms/dL. Moreover, CLOP produced impressive and unanticipated primary prevention benefits in the community at large. The demonstrated ability of community outreach workers to access high-risk populations and reduce exposure to lead hazards suggests the potential of this strategy for extension to other geographic areas, to the patients of private physicians and to address other prevalent, urban health problems like asthma, injuries and violence.

The effects of lead in Milwaukee's water.

Lead concentrations in household drinking water were matched with blood lead levels in 46 women of childbearing age (37 of whom were pregnant) who reside within the city of Milwaukee. The women represented a geographically diverse sample. Water lead concentrations ranged from less than 10 micrograms/L (65% of all homes studied) to 160 micrograms/L. None o the women tested had blood lead levels within the range most recently designated by the Centers for Disease Control (CDC) as "at risk" for the developing fetus (greater than or equal to 10 micrograms/dL). Within the limits of this study, there was no biologically significant correlation between blood lead and drinking water lead levels, suggesting that household water in Milwaukee with first-draw lead concentrations above EPA standards, either proposed or current, may have little or no biological effect.

Collaborating with private sector physicians: the example of childhood lead poisoning prevention.

Collaboration between public and private health sectors to achieve public health goals can be difficult. The experience of childhood lead poisoning prevention programs in two cities where private practitioners dramatically increased blood lead screenings is used as an example of how successful collaborative relationships can be constructed. Seven action steps toward effective collaboration are offered: establish a rationale, document the problem, attend to logistics, clarify reimbursement, make known available support, build demand, and demonstrate leadership.

Measles in Milwaukee.

All measles cases in Milwaukee for 1989 and the first quarter of 1990 are analyzed according to epidemiological and clinical parameters. Two major outbreaks are described with special attention to the demographics of the populations involved and the variety and severity of clinical outcomes. Health care and health insurance as they relate to the treatment and prevention of measles during this time are also discussed.

The impact of health care financing on childhood immunization practices.

OBJECTIVE: To examine the impact of patient insurance status and third-party payment methods on physician immunization practices. DESIGN: Family practice physicians and pediatricians were surveyed to determine whether differences existed in office immunization practices for five childhood vaccines across insurance and payment classes. SETTING: Milwaukee, Wis. PARTICIPANTS: Of 202 Milwaukee area physicians who administer immunizations routinely, 161 (79.7%) returned the questionnaire. RESULTS: Physicians reported immunizing uninsured patients in their offices less often than patients with insurance. When insurance does not pay for immunizations, most physicians (81.6%) said that they left the decision of whether to pay for private immunizations or seek free immunizations from the city health department to the family. Physicians estimated that approximately half of their uninsured patients decline private immunizations. Some physicians (20%) who treat patients receiving Medicaid reported that they immunize patients with Title 19 coverage less often than patients with other types of insurance. No significant differences in frequency of immunization were reported for patients insured by capitated-payment health maintenance organizations, fee-for-service health maintenance organizations, or traditional insurance covering immunizations. CONCLUSIONS: Physicians reported that they do not immunize uninsured and underinsured children as frequently as insured children. Further research is recommended to evaluate the impact of Medicaid enrollment on access to immunization and to develop innovative financing arrangements to ensure that no children leave their physicians' offices without being immunized.

Ca(2+)-activated C1- channels in a human biliary cell line: regulation by Ca2+/calmodulin-dependent protein kinase.

Biliary epithelial cells contribute to bile formation through absorption and secretion of fluid and electrolytes. Recent studies indicate that membrane Cl- permeability is regulated in part by increases in intracellular Ca2+ concentration. The purpose of these studies was to evaluate the effects of intracellular Ca2+ on channel activity, using the human Mz-ChA-1 cholangiocarcinoma cell line as a model, and to assess the possible roles of Ca(2+)-dependent kinases in channel regulation. Exposure to ionomycin (1 microM) activated ion channels in the cell-attached configuration in 63 of 74 attempts, increasing open probability (NPo) from 0 to 0.26 +/- 0.15 (n = 17). Multiple channels were present in each patch, and the effects of ionomycin were reversed by subsequent addition of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (2 mM) to the bath. With Cl(-)-containing solutions, channels had a slope conductance of 14 +/- 4 pS (n = 11), and the mean open time was estimated to be 5.3 +/- 1.8 ms. These channels were anion selective, and currents were carried by efflux of Cl- at the resting potential. Exposure to the Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist calmidazolium (100 microM) decreased NPo in ionomycin-stimulated cells to 0.02 +/- 0.06 (n = 19). The protein kinase C antagonist chelerythrine (50 microM) was without effect. In parallel studies in subconfluent cell monolayers, CaMKII antagonists were also potent inhibitors of ionomycin-stimulated 125I efflux. These findings indicate that Ca(2+)-dependent increases in membrane Cl- permeability are related in part to opening of 14.pS anion channels through a mechanism that depends on both Ca2+ and CaMKII. These channels represent a potential target for pharmacological modulation of biliary cell transport and function.

Emergency department vaccination of preschool-age children during a measles outbreak.

STUDY OBJECTIVE: To determine the effectiveness of an emergency department vaccination program for preschool-age children during a measles outbreak. DESIGN: Cross-section study. SETTING: Urban pediatric ED with an annual census of 24,000. PARTICIPANTS: Children, 12 to 59 months old, who presented to our ED between April 1 and April 30, 1994. INTERVENTION: Staff trained in rationale for and protocol of ED vaccination offered measles-mumps-rubella (MMR) vaccine, free of charge, to all eligible children. RESULTS: Of the 541 children seen, 7% lacked measles vaccination; MMR vaccination status could not be determined in 10%. From history it was determined that all the others had been vaccinated. Of the vaccination-eligible children, 25% were vaccinated in the ED. Of the eligible children who were not vaccinated, parents declined in half of the cases and physicians did not offer vaccination in the other half. Eligible children with physical injury were more likely to be vaccinated, and those with upper respiratory tract infections were less likely to be vaccinated than were eligible children with other diagnoses (P < .05). CONCLUSION: During a measles outbreak, few children receiving care at a busy pediatric ED were definitively identified as vaccination eligible. Only a few children identified as eligible for vaccination were vaccinated. Significant logistic barriers to effective ED vaccination exist.

[Ulcers of the colon in association with nonsteroidal anti-inflammatory drugs (NSAID)--a rare cause of gastrointestinal bleeding? Report of 3 cases]. / Kolonulzera nach der Einnahme nichtsteroidaler Antiphlogistika--eine seltene Ursache gastrointestinaler Blutungen? Bericht von 3 Fällen.

Although adverse side effects of nonsteroidal anti-inflammatory drugs (NSAID) can affect the whole gastrointestinal tract, most reports refer to upper gastrointestinal tract complications. We report on 3 patients with lower gastrointestinal bleeding (patient 1 and 2) respectively detectable fecal blood loss (patient 3) after the use of NSAID. Patient 1 and 3 were taking NSAID over at least 6 months for the treatment of rheumatic diseases while patient 2 reported a single use of 2 g acetylsalicylic acid. Colonoscopy showed a single ulcer of the colon in patients 1 and 2. Due to acute bleeding patient 1 required interventional endoscopic treatment. Colonoscopy of patient 3 revealed multiple colonic ulcerations. Gastroduodenoscopy also detected adverse NSAID-effects on the upper gastrointestinal tract in patient 1 and 3 (ulcers of the stomach, erosive duodenitis). NSAID-medication was discontinued in all patients and, additionally, mesalazine was administered to patient 3. Consecutively, symptoms and lesions disappeared. Our cases stress the clinical importance of NSAID-toxicity distal to the small intestine which may exist concomitantly to lesions of the upper gastrointestinal tract and is not obligatory dose-dependent.

Measles herd immunity. The association of attack rates with immunization rates in preschool children.

OBJECTIVE: To examine the association between incidence of measles and immunization coverage among preschool-age children. DESIGN: An ecological study in which measles incidence was compared with immunization coverage among census tracts. The independent effects of race and population density were controlled for. SETTING: A recent measles outbreak in Milwaukee, Wis. Immunization coverage data were estimated from a retrospective, school-based survey of Milwaukee grade school students. PATIENTS: One thousand eleven persons (less than or equal to 17 years) who had confirmed measles from September 1989 through June 1990. MAIN OUTCOME MEASURES: Confirmed measles cases grouped by census tract, corresponding census tract preoutbreak immunization coverage, racial breakdown, and population density. RESULTS: Census tracts stratified into four levels, with mean immunization rates of 50.4%, 60.2%, 69.9%, and 81.0%, had respective median attack rates of 11.6, 5.0, 1.7, and 0.0 cases per 1000 persons (P less than .01). The association between immunization coverage and measles attack rate remained significant even after controlling for race and population density. CONCLUSIONS: Modest improvements in low levels of immunization coverage among 2-year-olds confer substantial protection against measles outbreaks. Coverage of 80% or less may be sufficient to prevent sustained measles outbreaks in an urban community.

[Antineutrophil cytoplasmic antibodies in chronic inflammatory bowel diseases]. / Antineutrophile zytoplasmatische Antikörper bei chronisch-entzündlichen Darmerkrankungen.

Serum samples from 83 patients (42 women, 41 men, mean age 41 [19-85] years) with chronic inflammatory bowel diseases (ulcerative colitis: n = 41, Crohn's disease: n = 42) of differing degrees of activity were tested for antineutrophil cytoplasmic antibodies (ANCA) by immunofluorescence microscopy and various ELISA techniques. Seven patients with ulcerative colitis and one with Crohn's disease were suffering from associated primary sclerosing cholangitis. ANCA were detected in 18 sera, 13 from patients with ulcerative colitis (31.7%) and five from patients with Crohn's disease (11.9%). Six of the eight patients with primary sclerosing cholangitis were ANCA-positive. Nine sera showed a cytoplasmic (c-ANCA-) pattern and 9 others showed a partially atypical perinuclear (p-ANCA-) pattern. Among the ANCA-positive sera, ELISA techniques showed that two had antibodies against serine proteinase 3, two against lactoferrin, two against elastase and one against myeloperoxidase. There was no correlation between the anatomical pattern or activity of the disease and the presence of ANCA. The antineutrophil cytoplasm antibodies demonstrable in chronic inflammatory bowel disease appear to be directed against so far unknown antigens. They are particularly frequent in patients with associated primary sclerosing cholangitis.