Elevated middle cerebral artery peak systolic velocity and risk of death in donor twin affected by twin-twin transfusion syndrome but not twin anemia-polycythemia sequence.

OBJECTIVES: To determine the association between elevated (> 1.5 multiples of the median (MoM)) middle cerebral artery (MCA) peak systolic velocity (PSV) and fetal demise of the donor twin in pregnancies complicated by twin-twin transfusion syndrome (TTTS) in the absence of twin anemia-polycythemia sequence (TAPS). Secondary objectives were to evaluate if donor or recipient MCA-PSV is associated with a risk for their corresponding fetal death, and to compare the proportion of donor fetuses with low MCA pulsatility index (PI) among donor twins with high MCA-PSV and those with normal MCA-PSV to evaluate the contribution of blood-flow redistribution to the fetal brain in donor twins with high MCA-PSV. METHODS: This prospective cohort study included TTTS cases that underwent laser surgery between 2011 and 2022 at a single center. TAPS cases were excluded from the study. Multivariable and Poisson regression analysis were performed to explore the association between isolated elevated donor MCA-PSV and fetal demise, adjusted for TTTS stage, selective fetal growth restriction (sFGR) and other confounders. RESULTS: Of 660 TTTS cases, donor MCA-PSV was not recorded in 48 (7.3%) cases. Of the remaining 612 patients, nine (1.5%) were lost to follow-up and 96 TAPS cases were excluded; thus, 507 cases were included in the study. High donor MCA-PSV was seen in 6.5% (33/507) of cases and was an independent risk factor for donor fetal demise (adjusted relative risk (aRR), 4.52 (95% CI, 2.72-7.50)), after adjusting for confounders. Regression analysis restricted to each Quintero TTTS stage demonstrated that high donor MCA-PSV was an independent risk factor for fetal demise of the donor in Quintero Stage II (aRR, 14.21 (95% CI, 1.09-186.2)) and Quintero Stage III (aRR, 3.41 (95% CI, 1.82-6.41)). Donor MCA-PSV in MoM was associated with fetal demise of the donor (area under the receiver-operating-characteristics curve (AUC), 0.69; P < 0.001), but recipient MCA-PSV in MoM was not associated with fetal demise of the recipient (AUC, 0.54; P = 0.44). A higher proportion of donor twins in the group with high MCA-PSV had a low MCA-PI compared to the group with normal MCA-PSV (33.3% vs 15.5%; P = 0.016). CONCLUSIONS: Elevated donor MCA-PSV without TAPS prior to laser surgery for TTTS is associated with a 4-fold increased risk for donor fetal demise, adjusted for sFGR, TTTS stage and other confounders. Doppler evaluation of donor MCA-PSV prior to laser surgery may help stratify TTTS staging to evaluate the risk of donor fetal demise. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.

Anchoring device to prevent membrane detachment and preterm premature rupture of membranes after fetal intervention.

OBJECTIVE: Anchoring the fetal membrane to the uterine wall via a novel suture delivery system could reduce the risk of preterm premature rupture of membranes (PPROM) after fetoscopic surgery. This study assesses the feasibility of using a novel device designed for minimally invasive suturing to anchor fetal membranes to the uterine wall and to close surgical defects after fetoscopy. METHODS: We tested the suturing device both ex vivo and in vivo. In the ex vivo studies, 12-French trocar defects were created with a fetoscope in five specimens of human uterine tissue with fetal membranes attached. Specimens were examined for integrity of the anchoring stitch. For in vivo studies, trocar defects were created in the two uterine horns of three pregnant ewes, each carrying twins at ~79-90 days gestation. One trocar defect in each ewe was repaired using the suture device, and the other was left unrepaired as a control. The repair sites were examined for membrane anchoring integrity when the defect was created and at delivery. RESULTS: Fetal membranes were successfully anchored to the uterine myometrium using this device in all five trials performed ex vivo. The in vivo trials also revealed successful membrane anchoring compared with controls both at the time of device deployment and five-to-eight weeks after the procedure. CONCLUSIONS: We successfully anchored amniotic membranes to the underlying myometrium via suturing device both ex vivo and in vivo. Further studies are needed to evaluate the efficacy of the device and to determine whether it can successfully anchor fetal membranes percutaneously in human subjects. This article is protected by copyright. All rights reserved.

Fetal cardiovascular changes during open and fetoscopic in-utero spina bifida closure.

OBJECTIVE: Laparotomy-assisted fetoscopic closure of spina bifida utilizing heated-humidified carbon dioxide gas has been associated with less maternal morbidity than open in-utero spina bifida closure. Fetal cardiovascular changes during these surgical interventions are not well defined. Our objective was to compare fetal bradycardia (defined as fetal heart rate (FHR)<110 bpm over 10 minutes) and changes in umbilical artery Doppler parameters throughout open in-utero closure with those observed during laparotomy-assisted fetoscopic closure. METHODS: We conducted a prospective cohort study of 22 open and 46 fetoscopic consecutive in-utero closures between 2019 and 2023. Both cohorts had similar preoperative counseling and clinical management. FHR and umbilical artery velocimetry were systematically obtained during preoperative assessment, every 5 minutes during the intraoperative period, and in the postoperative assessment. FHR, pulsatility indexes and end-diastolic flows were segmented into hourly periods during surgery, and the lowest values were averaged for analysis. Umbilical vein maximum velocities were measured in the fetoscopic cohort. Each fetal heart rate recording time point was correlated to maternal parameters, including heart rate, systolic and diastolic blood pressures. RESULTS: Fetal bradycardia occurred in 4/22 cases (18.2%) of open in-utero closure and in 21/46 cases (45.7%) of fetoscopic closure. FHR gradually decreased in both cohorts after general anesthesia and decreased further during surgery. FHR were significantly lower after two hours of surgery in the fetoscopic closure than in the open in-utero closure group. In addition, the FHR (BPM) change in the final stages of the fetal surgery from the baseline FHR was significantly lower in the fetoscopic cohort (-32.3 (-35.7, -29.1)) compared to the open cohort (-23.5 (-28.1, -18.8)) (p=0.002). Abnormal end-diastolic flow (defined as absent or reversed end-diastolic flow) in the umbilical artery Doppler velocity occurred in 3/22 (13.6%) of the open closure cohort and in 23/46 (50%) of the fetoscopic closure cohort (p=0.004). There were no differences in umbilical artery end-diastolic flow and pulsatility index between closure techniques during the various stages of assessment. CONCLUSIONS: We observed a decrease in the FHR and abnormalities in umbilical artery Doppler parameters in both open in-utero and fetoscopic closure groups. Fetal bradycardia was more prominent during fetoscopic closure following heated-humidified carbon dioxide insufflation, but the FHR recovered after cessation of the heated-humidified carbon dioxide. Changes in FHR and umbilical artery Doppler parameters during in-utero spina bifida closure were observed to be transient, no cases required emergency delivery and no fetoscopic closure were converted to open closure. These observations should inform algorithms for perioperative management of fetal bradycardia associated with in-utero spina bifida closure. This article is protected by copyright. All rights reserved.

Role of gestational age at time of placental laser surgery in outcomes of twin-to-twin transfusion syndrome.

OBJECTIVE: There are conflicting data on whether fetoscopic laser photocoagulation of placental anastomoses (FLP) for treating twin-to-twin transfusion syndrome (TTTS) is associated with lower rates of overall survival. The objective of this study is to characterize survival and other associated morbidity after FLP across gestational ages of FLP. METHODS: This is a secondary analysis of prospectively collected data on patients with monochorionic-diamniotic twins that had FLP for TTTS at two centers between 2011 and 2022. Patients were divided into gestational age epochs for FLP before 18 wks, 18 0/7 - 19 6/7 wks, 20 0/7 - 21 6/7 wks, 22 0/7 - 23 6/7 wks, 24 0/7 - 25 6/7 wks and after 26 wks. Demographic characteristics, sonographic characteristics of TTTS and operative characteristics were compared across the gestational age epochs. Outcomes including overall survival, preterm delivery, preterm prelabor rupture of membranes (PPROM), intrauterine fetal demise (IUFD) and neonatal demise (NND) were also compared across gestational age epochs. Multivariate analysis was performed by fitting logistic regression models for these outcomes. Kaplan-Mejer curves were constructed to compare the interval from PPROM to delivery for each gestational age epoch. RESULTS: There were 768 patients that met inclusion criteria. The dual survival rate was 61.3% for FLP performed prior to 18 weeks compared to 78.0% - 86.7% across later gestational age epochs. This appears to be related to increased rates of donor IUFD following FLP performed before, versus after 18 weeks (28.0% vs. 9.3% - 14.1%). Rates of recipient IUFD/NND and donor NND were similar regardless of gestational age of FLP. Rates of PPROM were higher for earlier FLP, ranging from 45.6% for FLP before 18 weeks to 11.9% for FLP at 24 - 26 weeks gestational age. However, the gestational age of delivery was similar across gestational age epochs with a median of 31.7 weeks. In multivariate analysis, donor loss was independently associated with FLP before 18 weeks after adjusting for selective fetal growth restriction, Quintero stage and other covariates. PPROM and PTD were also associated with FLP before 18 weeks after adjusting for cervical length, placental location, trocar size, laser energy and amnioinfusion. CONCLUSION: FLP performed at earlier gestational ages is associated with lower overall survival, which is driven by higher risk of donor IUFD, as opposed to differences in PPROM or PTD. Counseling regarding survival should account for gestational age of presentation. This article is protected by copyright. All rights reserved.

Management of large placental chorioangioma: two-port laser approach for fetal intervention.

OBJECTIVES: There is a paucity of literature providing evidence-based guidelines for the management of large placental chorioangioma (≥ 4 cm in diameter). The objectives of this study were to compare outcomes between patients managed expectantly and those undergoing in-utero intervention and to describe the different in-utero techniques used for cessation of blood flow to the tumor and the associated outcome. METHODS: This was a retrospective cohort study of 34 patients referred for the management of large placental chorioangioma in a single center between January 2011 and December 2022, who were managed expectantly or underwent in-utero intervention. In-utero intervention was performed when the fetus developed any signs of impending compromise, including high combined cardiac output (CCO), worsening polyhydramnios or abnormal fetal Doppler velocimetry findings. Interventions included radiofrequency ablation (RFA), interstitial laser ablation (ILA) and single-port or two-port fetoscopic laser photocoagulation (FLP). Treatment selection was dependent on the proximity of the tumor to the umbilical cord insertion (UCI) and placental location. The two-port technique was performed in patients with a chorioangioma with large feeding vessels (≥ 3 mm) located in the posterior placenta, in which one port was used for occlusion using bipolar forceps and the other port was used for laser photocoagulation of the feeding vessels downstream. The single-port technique was used for chorioangioma with small feeding vessels (< 3 mm) located in the posterior placenta. ILA or RFA was performed in cases with an anterior placenta. Supportive treatments, including amnioreduction and intrauterine transfusion (IUT), were performed for worsening polyhydramnios and suspected fetal anemia based on middle cerebral artery Doppler flow studies, respectively. Comparative statistical analysis between cases undergoing expectant management vs in-utero intervention was performed. Descriptive details were provided for patients who underwent in-utero intervention. RESULTS: Thirty-four cases of large chorioangioma were evaluated, of which 25 (73.5%) were managed expectantly and nine (26.5%) underwent intervention. The frequency of polyhydramnios was significantly higher in the intervention group compared with the expectant-management group (66.7% vs 8.0%, P < 0.001). The live-birth rate among expectantly managed cases with large chorioangioma was significantly higher compared with that in cases that underwent in-utero intervention (96.0% vs 62.5%, P = 0.01). In the intervention group, preoperative CCO was elevated in all cases with available information and preoperative hydrops was present in 33.3% (3/9) of cases. One patient experienced fetal demise following IUT prior to planned FLP. Among the remaining eight patients, four underwent two-port FLP, two underwent single-port FLP, one underwent ILA and one underwent both ILA and RFA. All three cases in which hydrops was present at the time of intervention resulted in fetal demise. CONCLUSIONS: In-utero interventions aimed at cessation of blood flow in the feeding vessels are a therapeutic option for the management of cases with large chorioangioma. The two-port percutaneous technique appears to improve the efficiency of FLP when a large chorioangioma with large feeding vessels is located in the posterior placenta. We propose that in-utero interventions for large chorioangioma should be initiated prior to the development of fetal hydrops. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Primary vs patch-based skin closure for in-utero spina bifida repair.

OBJECTIVE: During in-utero spina bifida (SB) repair, closure of large defects is often challenging, requiring tissue graft for watertight skin closure. No prior studies have compared primary skin closure vs patch-based repair. Our objective was to compare neonatal and 1-year outcomes associated with these two types of skin closure for in-utero SB repair. METHODS: This was a prospective cohort study of 102 patients undergoing open prenatal SB repair from September 2011 to August 2021 at a single institution. All patients met the inclusion criteria of the Management of Myelomeningocele Study (MOMS), and the surgical procedure for in-utero SB repair was similar to that described in the MOMS trial. During the surgery, if primary skin approximation was not feasible due to the large size of the defect, the decision was at the discretion of the pediatric neurosurgeon to utilize a patch for closure. Neonatal outcomes at birth and 1-year outcomes were compared between the primary skin and patch-based closure groups. RESULTS: Of 102 patients included in the study, 70 (68.6%) underwent primary skin closure and 32 (31.4%) patch-based closure. The patch type included acellular bovine skin matrix (Durepair®; n = 31) and human acellular dermal matrix (Alloderm®; n = 1). Fetuses with myeloschisis were more likely to require patch-based repair than those with myelomeningocele. The median time of fetal repair was 4 min longer for patch-based compared with primary skin closure (37 vs 33 min; P = 0.001). Following patch-based repair, neonates had a longer length of stay in the neonatal intensive care unit (NICU) by 24 days (adjusted risk ratio, 2.40 (95% CI, 1.41-4.29)) compared to those that underwent primary skin closure. There was no difference between the two groups in the other neonatal outcomes, including the need for ventriculoperitoneal shunt placement and cerebrospinal fluid leakage. Outcome at 1 year of age was available for 90 infants. Need for wound revision within their first year after birth was more common in infants who underwent patch-based vs those with primary skin closure (19.4% vs 5.1%; P = 0.05). There was no difference between the two groups in other 1-year outcomes, including the need for ventriculoperitoneal shunt placement by 1 year of age and surgery for tethered cord. CONCLUSIONS: Patch-based closure during SB repair is often needed in fetuses with myeloschisis and is associated with prolonged fetal surgery time, long NICU stay and need for wound revision within the first year after birth. Further studies are required to identify optimal patches for SB repair or alternative methods to improve outcome. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Interventions to prevent preterm delivery in women with short cervix before fetoscopic laser surgery for twin-twin transfusion syndrome.

OBJECTIVE: Preoperative short cervical length (CL) remains a major risk factor for preterm birth after laser surgery for twin-twin transfusion syndrome (TTTS), but the optimal intervention to prolong pregnancy remains elusive. The objective of this study was to compare secondary methods for the prevention of preterm birth in twin pregnancies with TTTS undergoing fetoscopic laser photocoagulation (FLP), in the setting of a short cervix at the time of FLP, in five North American Fetal Treatment Network (NAFTNet) centers. METHODS: This was a secondary analysis of data collected prospectively at five NAFTNet centers, conducted from January 2013 to March 2020. Inclusion criteria were a monochorionic diamniotic twin pregnancy complicated by TTTS, undergoing FLP, with preoperative CL < 30 mm. Management options for a short cervix included expectant management, vaginal progesterone, pessary (Arabin, incontinence or Bioteque cup), cervical cerclage or a combination of two or more treatments. Patients were not included if the intervention was initiated solely on the basis of having a twin gestation rather than at the diagnosis of a short cervix. Demographics, ultrasound characteristics, operative data and outcomes were compared. The primary outcome was FLP-to-delivery interval. Propensity-score matching was performed, with each treatment group matched (1:1) to the expectant-management group for CL, in order to estimate the effect of each treatment on the FLP-to-delivery interval. RESULTS: A total of 255 women with a twin pregnancy complicated by TTTS and a short cervix undergoing FLP were included in the study. Of these, 151 (59%) were managed expectantly, 32 (13%) had vaginal progesterone only, 21 (8%) had pessary only, 21 (8%) had cervical cerclage only and 30 (12%) had a combination of treatments. A greater proportion of patients in the combined-treatment group had had a prior preterm birth compared with those in the expectant-management group (33% vs 9%; P = 0.01). Mean preoperative CL was shorter in the pessary, cervical-cerclage and combined-treatment groups (14-16 mm) than in the expectant-management and vaginal-progesterone groups (22 mm for both) (P < 0.001). There was no significant difference in FLP-to-delivery interval between the groups, nor in gestational age at delivery or the rate of live birth or neonatal survival. Vaginal progesterone was associated with a decrease in the risk of delivery before 28 weeks' gestation compared with cervical cerclage and combined treatment (P = 0.03). Using propensity-score matching for CL, cervical cerclage was associated with a reduction in FLP-to-delivery interval of 13 days, as compared with expectant management. CONCLUSIONS: A large proportion of pregnancies with TTTS and a short maternal cervix undergoing FLP were managed expectantly for a short cervix, establishing a high (62%) risk of delivery before 32 weeks in this condition. No treatment that significantly improved outcome was identified; however, there were significant differences in potential confounders and there were also likely to be unmeasured confounders. Cervical cerclage should not be offered as a secondary prevention for preterm birth in twin pregnancies with TTTS and a short cervix undergoing FLP. A large randomized controlled trial is urgently needed to determine the effects of treatments for the prevention of preterm birth in these pregnancies. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Effect of intra-amniotic fluid pressure from polyhydramnios on cervical length in patients with twin-twin transfusion syndrome undergoing fetoscopic laser surgery.

OBJECTIVES: To determine the relationship between intra-amniotic pressure and cervical length (CL) in patients with twin-twin transfusion syndrome (TTTS) undergoing fetoscopic laser photocoagulation (FLP), and to identify pre- or intraoperative factors associated with increased intra-amniotic pressure in this population. METHODS: This was a prospective cohort study of patients undergoing FLP for TTTS. Exclusion criteria were triplet or higher-order gestation and prior cervical cerclage, amnioreduction or FLP procedure. CL was assessed using preprocedure transvaginal ultrasound. Intra-amniotic pressure measurements were obtained on initial placement of the trocar into the amniotic cavity, using a direct hydrostatic pressure gauge. The relationship between intra-amniotic pressure and CL was assessed using multivariate linear regression analysis, including relevant preoperative and intraoperative variables. RESULTS: In total, 283 pregnancies met the inclusion criteria. Quintero stage of TTTS was I in 33 pregnancies, II in 88, III in 150 and IV in 12. Mean gestational age (GA) at FLP was 20.7 ± 3 weeks. Mean intra-amniotic pressure was 23.1 ± 9 mmHg. On unadjusted linear regression analysis, there was no significant association between intra-amniotic pressure and preoperative CL (P = 0.24) or GA at delivery (P = 0.22). On multivariate analysis, the factors associated significantly with intra-amniotic pressure were: number of prior term deliveries (P = 0.03), recipient maximum vertical pocket (P < 0.0001), Quintero stage IV (P = 0.01) and type of anesthesia (sedation vs general anesthesia; P = 0.01). CONCLUSION: In pregnancies with TTTS, intra-amniotic pressure is not associated with CL or GA at delivery. This novel finding suggests that cervical shortening in this population is not mechanically driven. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Match of psychosocial risk and psychosocial care in families of a child with cancer.

OBJECTIVE: The Psychosocial Assessment Tool (PAT) was developed to screen for psychosocial risk, aimed to be supportive in directing psychosocial care to families of a child with cancer. This study aimed to determine (i) the match between PAT risk score and provided psychosocial care with healthcare professionals blind to outcome of PAT assessment, and (ii) the match between PAT risk score and team risk estimation. METHODS: Eighty-three families of children with cancer from four pediatric oncology centers in the Netherlands participated (59% response rate). The PAT and team risk estimation was assessed at diagnosis (M = 40.2 days, SD = 14.1 days), and the content of provided psychosocial care in the 5-month period thereafter resulting in basic or specialized care. RESULTS: According to the PAT, 65% of families were defined as having low (universal), 30% medium (targeted), and 5% high (clinical) risk for developing psychosocial problems. Thirty percent of patients from universal group got basic psychosocial care, 63% got specialized care, and 7% did not get any care. Fourteen percent of the families at risk got basic care, 86% got specialized care. Team risk estimations and PAT risk scores matched with 58% of the families. CONCLUSIONS: This study showed that families at risk, based on standardized risk assessment with the PAT, received more specialized care than families without risk. However, still 14% of the families with high risks only received basic care, and 63% of the families with standard risk got specialized care. Standardized risk assessment can be used as part of comprehensive care delivery, complementing the team.

[Are the long-term results of venous aortocoronary bypass procedures really so bad? A prospective 13-year-long study of 428 patients]. / Zijn de lange-termijnresultaten van veneuze aortocoronaire overbruggingsoperaties wel zo slecht? Een prospectief onderzoek over 13 jaar bij 428 patiënten.

OBJECTIVE: To assess the long-term results of coronary surgery using venous bypasses. DESIGN: Prospective study. SETTING: St. Antonius Hospital, Nieuwegein, the Netherlands. METHOD: In 446 successive patients subjected to coronary surgery between 1 April 1976 and 1 April 1977, a complete long-term follow-up study was carried out after an average of 11.5 years (1.5 months-14.3 years). RESULTS: The peroperative mortality was 3%, a peroperative myocardial infarction occurred in 6.3% and complete revascularization was achieved in 77.6% of the patients. In 90 of 100 patients selected at random, cardiac catheterization was performed 13.5 months on average after the operation. The patency of the single grafts was 89.8%, that of the side-to-side and end-to-side anastomoses of the sequential grafts was 90.4% and 83%, respectively. Mortality, myocardial infarction, necessity of a second operation and angina pectoris occurred with low frequency during the first five years after the operation. Subsequently, complications increasingly occurred. Ten years after the operation 18.8% of the patients had died, 11.9% had suffered a myocardial infarction, 13.4% had been subjected to a second operation and 6.4% to balloon angioplasty, while 46.4% had once more developed angina pectoris. Thirteen years after the operation 29% of the patients had remained completely free from cardiac problems. CONCLUSION: Although arterial bypasses have been increasingly used in recent years, an indication for the use of venous transplants will continue to exist in the future, also, for certain groups of patients (those younger than 50 or older than 75 years). Our study clearly shows that with venous bypasses, also, satisfactory long-term results can be obtained.

[Chronic fatigue syndrome in young persons]. / Het chronische-vermoeidheidsyndroom bij jongeren.

The prevalence of chronic fatigue syndrome (CFS) in teenagers is 10-20 per 100,000 inhabitants in the Netherlands. The natural course of the disorder is not favourable according to the literature. Proposed criteria for the diagnosis 'CFS' in adolescence are: absence of a physical explanation for the complaints, a disabling fatigue for at least six months and prolonged school absenteeism or severe motor and social disabilities. Exclusion criterion should be a psychiatric disorder. Factors that attribute to the persistence of fatigue are somatic attributions, illness enhancing cognitions and behaviour of parents as well as physical inactivity. The role of the physician and the role of parents can enhance the problems. The treatment should focus on decreasing the somatic attributions, on reinforcement by the parents of healthy adolescent behaviour, on the gradual increase of physical activity and on decreasing attention (including medical attention) for the somatic complaints.

Ovine brucellosis: repeated vaccination with Rev 1 vaccine and the prevalence of the disease in the Winburg district.

The effect of repeated vaccination of ram lambs with Brucella melitensis Rev 1 vaccine was studied in relation to complement fixation titres. The presence of Brucella organisms in semen and changes in neutrophil densities of the semen were also studied. A survey of the incidence of ovine brucellosis in the Winburg district on the basis of a clinical examination of rams and the complement fixation test is also reported on. Repeated vaccination of ram lambs with Rev 1 vaccine resulted in high complement fixation titres for Brucella abortus in all cases and slight Brucella ovis titres in some cases. Neither Brucella organisms, nor increased numbers of neutrophils could be found in semen samples collected from the experimental animals. The incidence of ovine brucellosis in commercial and stud flocks in the Winburg district was in the order of 2%. As a fairly high incidence of brucellosis occurred in 2 stud Döhne merino flocks where ram lambs are vaccinated with Rev 1 at weaning, the administration thereof in this breed should take place at an earlier age. A second dosage at weaning should not have any disadvantageous effects. In order to protect newly vaccinated ram lambs from contracting brucellosis from older, infected cases, the separation of age groups is essential. The incidence of clinically detectable testicular lesions in rams from the Winburg district was in the order of 2%.

Induction of the gap-pgk operon encoding glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase of Xanthobacter flavus requires the LysR-type transcriptional activator CbbR.

In a previous study, a gene (pgk) encoding phosphoglycerate kinase was isolated from a genomic library of Xanthobacter flavus. Although this gene is essential for autotrophic growth, it is not located within the cbb operon encoding other Calvin cycle enzymes. An analysis of the nucleotide sequence upstream from pgk showed the presence of a gene encoding glyceraldehyde-3-phosphate dehydrogenase and the 3' end of an open reading frame encoding a protein which is 50% identical to transketolase encoded by cbbT of X. flavus. Gene fusions between pgk and lacZ demonstrated that the gap and pgk genes are organized in an operon. Induction of the Calvin cycle in heterotrophically growing cells resulted in a sixfold increase in phosphoglycerate kinase activity in parallel with the appearance of ribulosebisphosphate carboxylase activity. This superinduction of phosphoglycerate kinase did not occur in an X. flavus strain in which cbbR, encoding the transcriptional activator of the cbb operon, was disrupted. The failure to superinduce the gap-pgk operon is not caused by the absence of a functional Calvin cycle, since the expression of this operon in an X. flavus strain with a defective ribulosebisphosphate carboxylase enzyme was the same as the expression in the wild type. It is therefore concluded that the expression of both the cbb and gap-pgk operons is controlled by CbbR.

Characterization of fluorescein transport in isolated proximal tubular cells of the rat: evidence for mitochondrial accumulation.

The uptake and accumulation of the organic anion fluorescein-sodium (Flu-Na) was investigated in freshly isolated proximal tubular cells (PTC) of the rat kidney. Furthermore, the influence of other organic anions on Flu-Na uptake was studied in order to characterize Flu-Na transport in PTC. Flu-Na showed concentration-dependent, saturable and probenecid-sensitive transport. Comparing the transport parameters with para-aminohippurate (PAH), Flu-Na exhibited a higher affinity, but lower capacity to the organic anion transport system. The apparent Km for Flu-Na transport was 59 +/- 15 microM with a Vmax of 186 +/- 26 pmol/mg of protein/min, and for PAH 207 +/- 11 microM and 740 +/- 46 pmol/mg of protein/min, respectively. Dose-dependent inhibition of Flu-Na uptake with PAH resulted in an apparent inhibition constant Ki of 249 microM. This is in good agreement with the apparent Km of PAH, indicating that Flu-Na uptake is regulated by the PAH transport system. It is suggested that cellular uptake of both organic anions is mediated by a carrier at the basolateral membrane. However, after incubating cells with different concentrations of phenol red the percentage of maximum inhibition was 84%, which was significantly different from the 32% with PAH, suggesting that another transport system may be involved in Flu-Na uptake. Experiments with confocal laser scanning microscopy showed cellular uptake of Flu-Na and accumulation in subcellular structures. After superfusion of PTC with rhodamine 123 these structures were identified as mitochondria.

CbbR, a LysR-type transcriptional activator, is required for expression of the autotrophic CO2 fixation enzymes of Xanthobacter flavus.

Xanthobacter flavus is able to grow autotrophically with the enzymes of the Calvin cycle for the fixation of CO2, which are specified by the cbbLSXFP gene cluster. Previously, the 5' end of an open reading frame (cbbR), displaying a high sequence similarity to the LysR family of regulatory proteins and transcribed divergently from cbbLSXFP, was identified (W. G. Meijer, A. C. Arnberg, H. G. Enequist, P. Terpstra, M. E. Lidstrom, and L. Dijkhuizen, Mol. Gen. Genet. 225:320-330, 1991). This paper reports the complete nucleotide sequence of cbbR and a functional characterization of the gene. The cbbR gene of X. flavus specifies a 333-amino-acid polypeptide, with a molecular weight of 35,971. Downstream from cbbR, the 3' end of an open reading frame displaying a high similarity to ORF60K from Pseudomonas putida and ORF261 from Bacillus subtilis was identified. ORF60K and ORF261 are located at the replication origin of the bacterial chromosome. Inactivation of cbbR, via the insertion of an antibiotic resistance gene, rendered X. flavus unable to grow autotrophically. This was caused not by an inability to oxidize autotrophic substrates (e.g., formate) but by a complete lack of expression of the cbb genes. The expression of the CbbR protein in Escherichia coli was achieved by placing cbbR behind a strong promoter and optimization of the translational signals of cbbR. CbbR binds specifically to two binding sites in the cbbR-cbbL intergenic region.

Outcome in three groups of patients with typhoid fever in Indonesia between 1948 and 1990.

The outcome in three groups of patients with bacteriologically confirmed typhoid fever caused by Salmonella typhi, treated during three episodes between 1948 and 1990 in Java, Indonesia, was compared by retrospective analysis of hospital records. The study population consisted of three groups of patients. Group I (n = 50) was treated in Batavia (the present Jakarta) from 1948 to 1950, Group II (n = 61) in Yogyakarta from 1952 to 1956, Group III (n = 105) in Semarang from 1989 to 1990. Main outcome measures were days until defervescence, early relapses during hospitalization, duration of hospital stay, complications and mortality. Group I received supportive treatment only, Group II low doses of chloramphenicol (total 12.5 g) and Group III full doses of chloramphenicol (total 27 g); occasionally other antibiotics were used. In Group I, II and III the mean number of days until defervescence was 16, 8 and 6 and the mean number of days in hospital 43, 47 and 15, respectively. Mortality was 26%, 10% and 5% and complications occurred in 38%, 18% and 13%, respectively. Between Group I and Group II the differences in mortality and complications were statistically significant (P < 0.05). Compared to Group I the proportion of early relapses was higher in Group II, but was zero in Group III. There were significantly fewer gastrointestinal complications in Group II than in Group I (P < 0.01) and even fewer in Group III. When no antibiotic against S. typhi was available, typhoid fever had a protracted course, and only 74% of patients survived. Even with low dosages of chloramphenicol, defervescence was earlier and mortality and complications decreased dramatically, but early relapses were frequent. Full doses of chloramphenicol for a sufficient period of time only slightly reduced mortality and complications further, but eliminated early relapses completely.

The role of the centre of projection in the estimation of slant from texture of planar surfaces.

Displays were presented consisting of a perspective projection of a regular square grid, made up of vertical and horizontal equally spaced white lines, that was slanted in depth. The surface was viewed monocularly, through a circular aperture. A range of slants was shown (0 degree, 10 degrees, 20 degrees, 30 degrees, 40 degrees, 50 degrees, or 60 degrees) and the observers' task was to match the slant by means of a mouse-driven probe. The viewing distance (50, 75, or 100 cm) as well as the focal distance (25, 50, 75, 100, or 125 cm) were varied. We expected the estimation error to be smallest when the viewing distance and the focal distance coincided. This was not the case. Instead, subjects seemed to use the perspective deformation of the texture elements in the stimulus display to make a slant estimation, regardless of the specific combination of viewing distance and focal distance.

Fructosebisphosphatase isoenzymes of the chemoautotroph Xanthobacter flavus.

Xanthobacter flavus employs two fructosebisphosphatase (FBPase)-sedoheptulosebisphosphatase (SBPase) enzymes. One of these is constitutively expressed and has a high FBPase-to-SBPase ratio. The alternative enzyme, which is encoded by cbbF, is induced during autotrophic growth. The cbbF gene was expressed in Escherichia coli, and the FBPase was purified to homogeneity. The purified enzyme has a specific FBPase activity of 114 mumol/min/mg of protein, a Michaelis constant for fructosebisphosphate of 3 microM, and a low FBPase-to-SBPase ratio. CbbF was activated by ATP and inhibited by Ca2+.

The LysR-type transcriptional regulator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor.

Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 microM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparent K(dNADPH) of CbbR was determined to be 75 microM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64 degree bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9 degree. From the results of these in vitro experiments, we conclude that CbbR responds to NADPH. The in vivo regulation of the cbb and gap-pgk operons may therefore be regulated by the intracellular concentration of NADPH.