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Objectives: The developmental and epileptic encephalopathies (DEE) are a heterogeneous group of rare neurodevelopmental disorders, characterised by early-onset seizures that are often intractable, electroencephalographic abnormalities, and developmental delay or regression. There is a paucity of data from sub-Saharan Africa on the genetic basis of DEE. The aim of this study was to investigate the genetic background of DEE using targeted next generation sequencing (NGS) analysis in a tertiary pediatric neurology outpatient department at Tygerberg Hospital, South Africa. In addition, we assessed the value of the genetic results to the parents and managing physicians., Methods: A prospective cohort study of 41 consecutive children with DEE (onset before 3 years of age) that were recruited over a 2-year period (2019-2021). Pre- and post-test genetic counselling were offered to all study participants. The results were categorized as either: positive (pathogenic/likely pathogenic variant identified), inconclusive (variant(s) of unknown significance identified), or negative (no variants identified). Result interpretation and careful matching of the variant to the clinical phenotype was performed. Subsequently, questionnaires were administered to both the physicians and the parents., Results: A genetic underlying cause for DEE was identified in 18 of 41 children (diagnostic yield 43.9%). Variants in SCN1A (n=7), KANSL1 (n=2), KCNQ2 (n=2) and CDKL5 (n=2) were identified in more than one patient. Rarer genes included IQSEC2, SMC1A and STXBP1. All of the identified pathogenic variants fully explained and matched the respective phenotypic description of the patient at the time of clinical diagnosis. In 26% of patients the genetic result facilitated precision medicine management changes to anti-seizure medication. Both parents and physicians expressed benefit of genetic testing in patients with DEE., Conclusion: Targeted NGS analysis proved an efficient diagnostic tool in detection of a genetic cause of DEE in a large proportion of South African children. The 43.9% diagnostic yield is similar to previously reported international pediatric cohorts. Additionally, the genetic findings proved useful for targeted therapeutic decision-making and accurate genetic counseling., Competing Interests: Declaration of Competing Interests None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2022 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Background: Identification of SARS-CoV-2 infected individuals is imperative to prevent hospital transmission, but symptom-based screening may fail to identify asymptomatic/mildly symptomatic infectious children and their caregivers., Methods: A COVID-19 period prevalence study was conducted between 13 and 26 August 2020 at Tygerberg Hospital, testing all children and their accompanying asymptomatic caregivers after initial symptom screening. One nasopharyngeal swab was submitted for SARS-CoV-2 using real-time reverse-transcription polymerase chain reaction (rRT-PCR). An additional Respiratory Viral 16-multiplex rRT-PCR test was simultaneously done in children presenting with symptoms compatible with possible SARS-CoV-2 infection., Results: SARS-Co-V 2 RT-PCR tests from 196 children and 116 caregivers were included in the analysis. The SARS-CoV-2 period prevalence in children was 5.6% (11/196) versus 15.5% (18/116) in asymptomatic caregivers ( p <0.01). Presenting symptoms did not correlate with SARS-CoV-2 test positivity; children without typical symptoms of SARS-CoV-2 were more likely to be positive than those with typical symptoms (10.2% [10/99] vs 1% [1/97]; p <0.01). Children with typical symptoms (97/196; 49.5%) mainly presented with acute respiratory (68/97; 70.1%), fever (17/97; 17.5%), or gastro-intestinal complaints (12/97; 12.4%); Human Rhinovirus (23/81; 28.4%) and Respiratory Syncytial Virus (18/81; 22.2%) were frequently identified in this group. Children-caregiver pairs' SARS-CoV-2 tests were discordant in 83.3%; 15/18 infected caregivers' children tested negative. Symptom-based COVID-19 screening alone would have missed 90% of the positive children and 100% of asymptomatic but positive caregivers., Conclusion: Given the poor correlation between SARS-CoV-2 symptoms and RT-PCR test positivity, universal testing of children and their accompanying caregivers should be considered for emergency and inpatient paediatric admissions during high COVID-19 community transmission periods. Universal PPE and optimising ventilation is likely the most effective way to control transmission of respiratory viral infections, including SARS-CoV-2, where universal testing is not feasible. In these settings, repeated point prevalence studies may be useful to inform local testing and cohorting strategies., Competing Interests: The authors declared no conflict of interest., (© 2022 Published by Elsevier B.V. on behalf of African Federation for Emergency Medicine.)

Introduction: The aim was to analyse prognosticators in acinic cell carcinoma (AciCC) in two head and neck referral centers in Amsterdam, the Netherlands., Materials and Methods: Eighty- nine cases of AciCC treated between 1979 and 2016 were retrospectively reviewed. Five, - 10 -and 20- year estimates of survival were executed as well as univariate analysis of prognosticators., Results: The majority of AciCC were T1-T2; 89%. Two percent had nodal disease (2%). The most affected organ was the parotid gland (84%) with a female preponderance (67%). Mean age was 52 years with most cases diagnosed in the fourth to sixth decade. The majority of patients received adjuvant radiotherapy. Elective neck dissection (END) in the N0 neck showed no metastases. High grade transformation (HGT) was found in 21% of cases. Median follow up was 101.9 months. Median time to recurrence was 26 months. Nine patients developed distant metastases (DM) of whom 6 had HGT-AciCC. Median survival with DM was 7 months. Five,- ten -and twenty- year estimates were 84%, 81% and 81% for recurrence free survival respectively. Negative clinical features were advanced stage disease and tumour size > 2.6 cm. Negative histological features were a high mitotic rate, HGT, close and involved surgical margins and necrosis., Conclusion: AciCC- HGT excluded- of the head and neck has an excellent prognosis and shows acceptable long term results. END can be considered as part of the standard treatment due to the relative high incidence of HGT- AciCC and low accuracy of cytology., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The introduction of adjuvant systemic treatment has significantly improved recurrence-free survival in patients with resectable high-risk melanoma. Adjuvant treatment with immune checkpoint inhibitors and targeted therapy, however, substantially impacts health care budgets, while the number of patients with melanoma who are treated in the adjuvant setting is still increasing. To evaluate the socioeconomic impact of the three adjuvant treatments, a cost-effectiveness analysis (CEA) was carried out., Materials and Methods: Data were obtained from the three pivotal registration phase III clinical trials on the adjuvant treatment of patients with resected high-risk stage III in melanoma (KEYNOTE-054, CheckMate 238, and COMBI-AD). For this CEA, a Markov model with three health states (no evidence of disease, recurrent/progressive disease, and death) was applied. From a societal perspective, different adjuvant strategies were compared according to total costs, life years (LYs), quality-adjusted life years (QALYs), and incremental cost-effectiveness ratios. To evaluate model uncertainty, sensitivity analyses (deterministic and probabilistic) were carried out., Results: In the adjuvant setting, total costs (per patient) were €168 826 for nivolumab, €194 529 for pembrolizumab, and €211 110 for dabrafenib-trametinib. These costs were mainly determined by drug acquisition costs, whereas routine surveillance costs varied from €126 096 to €134 945. Compared with routine surveillance, LYs improved by approximately 1.41 for all therapies and QALYs improved by 2.02 for immune checkpoint inhibitors and 2.03 for targeted therapy. This resulted in incremental cost-effectiveness ratios of €21 153 (nivolumab), €33 878 (pembrolizumab), and €37 520 (dabrafenib-trametinib) per QALY gained., Conclusions: This CEA compared the three EMA-approved adjuvant systemic therapies for resected stage III melanoma. Adjuvant treatment with nivolumab was the most cost-effective, followed by pembrolizumab. Combination therapy with dabrafenib-trametinib was the least cost-effective. With the increasing number of patients with high-risk melanoma who will be treated with adjuvant treatment, there is an urgent need to reduce drug costs while developing better prognostic and predictive tools to identify patients who will benefit from adjuvant treatment., Competing Interests: Disclosure SS is chair of the Centre for Personalized Cancer Treatment (CPCT), member of the SkylineDx Supervisory board (paid to institute), and declares patents on assays detecting CECs, EpCAM-neg CTCs, and CTC-ARv7 detection and outcome to cabazitaxel. AV is advisory board member for Bristol-Myers Squibb, Ipsen, Merck Sharp & Dohme, Novartis, Pierre Fabre, Pfizer, Roche, Eisai, and Sanofi. All other authors have declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin-containing tubes, were collected from patients with non-small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin-containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Young infants with a possible serious bacterial infection (SBI) are a very common presentation to emergency centres (ECs). It is often difficult to distinguish clinically between self-limiting viral infections and an SBI. Available evaluation algorithms to assist clinicians are mostly from high-income countries. Data to inform clinical practice in low- and middle-income countries are lacking., Objectives: To determine the period prevalence of SBI and invasive bacterial infection (IBI) and describe current practice in the assessment and management of young infants aged 21-90 days presenting with a possible SBI to a Paediatric Emergency centre (PEC) in Cape Town, South Africa., Methods: A retrospective cross-sectional review of infants 21-90 days old presenting to the Tygerberg Hospital PED between 1 January 2016 and 31 May 2016., Results: A total of 248 infants 21-90 days were included in the study. Sixty-two patients (25%, 95% CI 20-30) had an SBI and 13 (5.2%, 95% CI 3-8) had an IBI. One hundred and sixty-five infants had a possible SBI based on WHO IMCI criteria. The sensitivity of the WHO IMCI criteria in detecting SBI was 82.3% (95% CI 70.5-90.8) and the specificity 38.7% (95% CI 31.7-46.1). More than half (51.2%) of the infants received antibiotics within the 48 h prior to presentation, of which 33.5% included intramuscular injection of Ceftriaxone. Only 20 (8.0%) patients in this age group were discharged home after initial evaluation. A significant relationship was noted between fever and the risk of SBI (p-value 0.010) and IBI (p-value 0.009). There also appeared to be a significant relationship between nutritional status and IBI (p-value 0.013)., Conclusion: Period prevalence of SBI and IBI was higher compared to that published in the literature. Validated evaluation algorithms to stratify risk of SBI are needed to assist clinicians in diagnosing and managing infants appropriately in low- and middle-income settings., Competing Interests: The authors declared no conflict of interest., (© 2020 African Federation for Emergency Medicine. Publishing services provided by Elsevier.)

Background: Children born preterm are at risk of visuospatial attention orienting and processing dysfunctions, which can be quantified early in life using visually-guided eye movement responses., Aims: To identify the prevalence and perinatal risk factors for visuospatial attention orienting and processing dysfunctions in children born preterm of 1 year of corrected age (CA)., Study Design: 123 children born between 26 and 33 weeks of gestation underwent a nonverbal visuospatial test at 1y CA, using an eye tracking-based paradigm. For the detected high-salient (cartoon and contrast), intermediate-salient (form and motion) and low-salient (color) stimuli, we quantified the reaction time to fixation (RTF). RTFs were compared to normative references from an age-matched control group (N = 38). The prevalence of perinatal risk factors (gestational age and weight, indices of neurological damage, overal sickness, respiratory failure, and retinopathy) was compared between the groups with normal and delayed RTFs., Results: At 1y CA, the preterm group had 7-20% less detected stimuli than the control group, particularly for intermediate and low-salient stimuli. Compared to normative RTFs, modest delays were found for high-salient cartoon (in 19% of preterm children) and contrast (8%), intermediate-salient motion (23%) and form (21%), and low-salient color stimuli (8%). These children had a significantly higher prevalence of perinatal risk factors for respiratory failure and intraventricular hemorrhages., Conclusions: Children born between 26 and 32 weeks have a modest risk (8-23%) of visuospatial attention and processing dysfunction. This warrants early monitoring and support of general visual development in preterm children at risk of respiratory distress and disrupted cerebral blood flow., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: In epidemiological and clinical studies, whole blood assay (WBA) has been used as a measure to characterize inter-individual differences in the cytokine response of individuals exposed to inflammatory agents, such as endotoxins. Several short-time repeatability studies have shown stable cytokine levels in individuals over periods of days, weeks or months, but little is known about the long-term stability of cytokine reactivity., Methods: We studied cytokine response levels in LPS-stimulated whole blood in a cohort of 193 farmers and agricultural industry workers at two time points with a five-year interval., Results: IL-10 and IL-1β responses measured with a five-year time interval showed a weak positive correlation (r = 0.22 and 0.27, respectively), whereas no correlation was observed for TNFα (r = 0.06). Cytokine reactivity measured repeatedly at the same time point showed high correlations (IL-10 r = 0.80, IL-1β r = 0.53 and TNFα r = 0.74), suggesting that the observed weak correlations over time are reflective of actual variations in cytokine reactivity over time., Conclusions: Repeatability of ex vivo cytokine reactivity showed to be differential for the measured cytokines, being more stable for IL-10 and IL-1β than for TNFα. However, in general, repeatability of ex vivo cytokine reactivity was weak, reflecting that cytokine reactivity can mostly be explained by (short term) intra-individual (immunological) or time varying environmental factors and less by genetic or other time-invariant factors. Therefore, WBA should be regarded as a viable tool to study relationships with current health status and exposure, and only partially as a predictor for a future response., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: In 2010, a new subtype of salivary gland cancer (SGC), (mammary analogue) secretory carcinoma (SC), was defined, characterized by the ETV6-NTRK3 fusion gene. As clinical behavior and outcome data of this histological subtype tumor are still sparse, we aimed to describe the clinicopathological course and outcome of a series of translocation positive SC patients., Patient and Methods: We re-evaluated the pathological diagnosis of a subset of SGCs, diagnosed in 4 of 8 Dutch head and neck centers. Subsequently, tumors with a morphological resemblance to SC were tested for the ETV6-NTRK3 fusion gene using RT-PCR. Furthermore, patients prospectively diagnosed with SC were included. The clinical characteristics and outcomes were retrieved from the patient files., Results: Thirty-one patients with ETV6-NTRK3 fusion gene positive SC were included. The median age was 49 years, 17 patients (55%) were male. Eighteen tumors (58%) arose in the parotid gland. One patient presented with lymph node metastasis. All patients underwent tumor resection and 4 patients had a neck dissection. Four patients had re-resection and 15 patients (48%) received postoperative radiotherapy. One patient developed a local recurrence, no regional recurrences or distant metastases were observed. After a median follow-up of 49 months the 5- and 10-year overall survival were 95%, the 5- and 10-year disease free survival were 89%., Conclusion: The clinical course of SC is favorable with a low rate of locoregional recurrence and excellent survival. Given the low incidence of nodal metastases, elective neck treatment, i.e. surgery and/or radiotherapy, does not seem to be indicated., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: In the Netherlands there is an ongoing debate regarding environmental health risks of livestock farming for neighbouring residents. This explorative study aims to determine the prevalence of carriage of extended-spectrum β-lactamase and/or plasmid-mediated AmpC-producing Enterobacteriaceae (ESBL/pAmpC-E) in the general population living in a livestock-dense area, and to study associations between determinants, including exposure through contact with animals and the environment, and human carriage of ESBL/pAmpC-E., Methods: A cross-sectional study was performed among 2432 adults (aged 20-72 years) in 12 temporary research centres in the south of the Netherlands, consisting of a questionnaire and analysis of a faecal sample to assess carriage of ESBL/pAmpC-E. Risk factors were analysed using logistic regression., Results: The prevalence for carriage of ESBL/pAmpC-E was 4.5% (109/2432; 95% CI 3.7-5.4) ranging from 1.4% to 10.9% among the research centres. ESBL/pAmpC resistance genes were detected in Escherichia coli and Klebsiella pneumoniae isolates obtained from these 109 persons and the most common ESBL-resistance genes were bla CTX-M-15 , bla CTX-M-14/17 and bla CTX-M-1 , originating from 76 participants. Travel in the previous 12 months to Africa, Asia or Latin America (OR 2.82; 95% CI 1.71-4.63), having kept cows for a hobby in the previous 5 years (OR 3.77; 95% CI 1.22-11.64), usage of proton-pump inhibitors (OR 1.84; 95% CI 1.05-3.23), and living within 1000 m of a mink farm (OR 2.26; 95% CI 1.28-3.98) were identified as risk factors. Exposure to poultry was not identified as a risk factor., Conclusions: Overall, living in close proximity to livestock animals and farms does not seem to be a risk factor for carriage of ESBL/pAmpC-E., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients., (© 2016 by The American Society of Hematology.)

For hatcheries, not only is it important to have a high level of hatchability, but the quality of the chicks provided also has to be good, because broiler farmers are looking for chicks with a high growth potential, resulting in a greater slaughter yield at the end of the rearing period. However, chick quality has proven to be a difficult and subjective matter to define. Therefore, the aim of this study was to investigate the predictive value of different chick quality measurements for BW at slaughter age. Body weight, chick length, shank length, and toe length measurements as well as Tona score determination were performed on 1-d-old chicks and were linked to posthatch performance parameters. Different breeder lines (Cobb and Ross) and breeder ages (39, 42, and 53 wk of age) were used to investigate line and age effects. In addition, variability between people and repeatability in time of these quality measurements were determined. Body weight at 7 d of age appeared to be the best predictor of BW at slaughter age among all the quality measurements performed. Body weight at 1 d of age had the second greatest predictive value, closely followed by the ratio between BW at 1 d of age and chick length squared. Chick length and shank length both had low to no predictive value whatsoever for posthatch performance. The lack of significant correlations between the Tona score and posthatch performance could be explained by the absence of day-old chicks with anomalies (and thus a suboptimal Tona score) because a distinction had already been made, as is done in practice, between top-grade and lower grade chicks.

Despite thorough selection during the last decade, the incidence of ascites is still high in modern broiler strains. Although ascites occurs mostly at the end of the rearing period, there are indications that the etiology of this problem may have started during embryonic development. Recent studies have shown that the post-hatch performance of the broiler chick might be influenced by changing the environmental conditions in the incubator during embryonic development. This study investigated the effect of increasing incubator CO(2) concentration up to 0.7%, by nonventilation during the first 10 d of incubation, on the embryonic development of 2 commercial broiler strains (Cobb and SAS) differing in their susceptibility for ascites syndrome. The Cobb strain is suspected to be less susceptible than the SAS strain. Overall, the chick embryos of the Cobb strain had a faster development than those of the SAS strain as expressed by their higher BW from embryonic day (ED)10 until ED18. Nonventilation stimulated embryonic development resulting in higher embryonic BW, early hatch, and narrower spread of hatch in both strains. In the SAS strain, nonventilation improved hatchability by more than 10%. Gas composition of the air cell in the egg of the nonventilation groups (both Cobb and SAS) had higher partial pressure of CO(2) and lower partial pressure of O(2) from ED11 until ED14 compared with the ventilation groups. During the entire incubation period, partial pressure of CO(2) was higher in eggs of the Cobb strain compared with the SAS strain. Plasma triiodothyronine, thyroxine, and corticosterone levels were different at the end of the incubation period and during hatching due to nonventilation at the beginning of incubation. It is concluded that nonventilation during the first 10 d of incubation had a stimulatory effect on embryonic development of the 2 broiler strains with no effect of heart weights but with effects on hormone levels, air cell pressures, and hatching parameters.

This study investigated the effect of high CO2 (4%) from embryonic day (ED)10 until ED16 on the acid-base balance and related parameters in the chicken embryo. From ED10 to ED16, blood was taken from a vein from the chorioallantois membrane and was analyzed for pH, partial pressure of CO2, partial pressure of O2 (pO2), [HCO3(-)], [K+], and [Ca2+]. Allantoic fluid was taken for measurement of pH, NH3-N, phosphate, and calcium concentration. The right tibia was ashed, and calcium was measured with atomic absorption spectroscopy. Embryos exposed to high CO2 showed a consistent higher blood pH than control embryos. Notwithstanding this alkalosis, bicarbonate concentration was significantly higher in the CO2 group from ED12 until ED16. Potassium concentration in the blood was significantly higher in the CO2 group from ED11 until ED16. The pH of the allantois was significantly higher on ED14 and ED15. Ammonia N concentration was significantly higher in the CO2-incubated embryos on ED12 and ED13, whereas phosphate did not differ between groups. Calcium per tibia dry weight did not differ between incubation conditions. We can conclude that embryos adapt to high CO2 during the second half of incubation by increasing blood HCO3(-). It appears that this increase in HCO3(-)is mainly the result of the stimulated intracellular exchange of H+ with K+, although temporary reabsorption of HCO3(-)by the kidney cannot be excluded.

This study investigated the effect of non-ventilation of the incubator during the first 10 days of incubation and its combination with dexamethasone administration at day 16 or 18 of incubation on hatching parameters and embryo and post-hatch chick juvenile physiology. A total of 2400 hatching eggs produced by Cobb broiler breeders were used for the study. Blood samples were collected at day 18 of incubation, at internal pipping stage (IP), at the end of hatch (day-old chick) and at 7-day-post-hatch for T(3), T(4) and corticosterone levels determination. From 448 to 506 h of incubation, the eggs were checked individually in the hatcher every 2h for pipping and hatching. The results indicate that non-ventilation during the first 10-day shortened incubation duration up to IP, external pipping (EP) and hatch, had no effect on hatchability and led to higher T(3) levels at IP but lower corticosterone levels at 7-day-post-hatch. The injection of dexamethasone at days 16 and 18 of incubation affected hatching and blood parameters in both the ventilated and non-ventilated embryos differentially and the effect was dependent on the age of the embryo. Dexamethasone increased T(3) levels and T(3)/T(4) ratios but the effect was greater with early non-ventilation of eggs. Dexamethasone decreased hatchability but the effect was greater when injected at day 16 and especially in ventilated embryos. The effects of incubation protocols and dexamethasone treatments during incubation were still apparent in the hatched chicks until 7 days of age. The changes in T(3), T(4) and corticosterone levels observed in response to the early incubation conditions and late dexamethasone treatments in this study suggest that incubator ventilation or non-ventilation may influence the hypothalamic-pituitary-adrenal axis (HPA) regulation of stress levels (in terms of plasma corticosterone levels) and thyroid function in the embryo with impact on incubation duration, hatching events and early post-hatch life of the chick. Our results also suggest that some stages of development are more sensitive to dexamethasone administration as effects can be influenced by early incubation protocols.

In this study, broiler embryos were exposed during the second half of incubation [embryonic day (ED) 10 until ED18] to 4% CO(2). The CO(2) was set to reach 2% on ED11 and 4% from ED12 onward. Two experiments were conducted with the same setup. Embryo weight was measured and partial pressure of CO(2) and O(2) in the air cell was analyzed at several embryonic ages. Times of internal pipping, external pipping, and hatching were recorded. Chicks were raised until d 7 posthatch. Plasma corticosterone, triiodothyronine, and thyroxine concentrations were determined. Embryonic growth was not retarded and hatchability did not decrease in the CO(2)-incubated group, demonstrating that chicken embryos can tolerate high (4%) concentrations of CO(2) between ED10 and ED18. In the first experiment, partial pressure of CO(2) in the air cell was significantly higher in the CO(2) group on ED11, ED12, ED13, and ED14, but disappeared thereafter. This difference was not observed in the second experiment. A change in the hatching process of the CO(2) group was seen. Relative growths of newly hatched chicks until d 7 posthatch were equal in the CO(2) group and the control group. However, corticosterone and thyroxine concentrations were significantly higher in the CO(2)-incubated chicks on d 7 posthatch.

Ascites is a metabolic disorder that accounts for over 25% of overall mortality in the broiler industry. This disorder is manifested between wk 5 and 6 posthatch, but there are previous indications that predisposition may be identified during embryonic development. In this current study, we determined embryonic physiological and metabolic parameters that may be associated with ascites predisposition. For this purpose, we used broiler eggs from 3 lines that differed in ascites sensitivity. These included an ascites-sensitive dam line (DAS), an ascites-resistant dam line (DAR), and an ascites-sensitive sire line (SASL). Eggs were incubated for 21 d under standard conditions. The following parameters were measured during incubation: egg weights at setting, egg weight losses at 18 d, embryo body weights and embryo heart weights throughout development, air cell partial gas pressures (pCO2 and pO2) levels at d 18 and at internal pipping (IP); plasma triiodothyronine, thyroxine, and corticosterone levels at d 18, IP, and hatch; heat production from d 17 until hatch, hematocrit values at hatch, and posthatch growth rate to 7 d along with hematocrit values. The data obtained revealed that selection for ascites sensitivity or rapid growth rate had no consistent influence on some of these parameters such that they could be wholly associated with ascites sensitivity for predictive purposes. Whereas differences in embryonic developmental patterns were apparent throughout embryonic development, these differences in physiological and metabolic parameters may be due partly to genetic differences unrelated to ascites sensitivity.

Ascites is a prevalent cardiovascular disease among modern broilers with negative impacts on production and animal welfare. The peak of mortality due to ascites occurs at the end of the growing period, but the etiology of this problem may start during embryonic development. A few recent reports have demonstrated that the signs of ascites susceptibility are manifested during the late stages of incubation. In the current study, we used a nondestructive method based on egg acoustic resonance parameters [resonant frequency (RF) and damping] to establish a relationship between embryo physiological events during early development in broiler eggs and susceptibility to ascites. The hatching eggs of 3 broiler lines differing in ascites susceptibility were used for this study: ascites-resistant dam line (DAR), ascites-sensitive dam line (DAS), and ascites-sensitive sire line (SASL). These lines were selected on the basis of fast growth, high breast meat yield, and ascites induction at low temperatures such that the order of ascites susceptibility in terms of mortality was SASL >> DAS > DAR. Eggs were incubated under standard conditions in forced-draft incubators. We measured egg weights at setting, albumen pH, Haugh units (HU) at setting, and embryo weights at d 11 and 18, at internal pipping (IP), and at hatch. The durations of IP, external pipping (EP), and hatching were also determined. At 2 hourly periods during incubation, egg RF and damping were also measured. There were differences in egg weights between DAR and SASL vs. DAS, but albumen HU, albumen pH, and the ratio of yolk weight to egg weight were similar. There were differences in RF, damping, embryonic growth rates, and hatching events. Changes in resonant frequency and damping, which certainly suggest eggshell differences among lines, were not totally related to variations in physiological events during early and late embryonic development. A comparison between DAR and DAS, between DAS and SASL, or DAR and SASL indicates that sensitivity to ascites and selection for rapid growth rate in ascites-sensitive lines have different effects on embryonic parameters. We concluded that the sensitivity of broiler breeders to ascites does not influence egg internal quality, but the occurrence of ascites sensitivity in broilers could not be reliably predicted by early in ovo acoustic resonance parameters and hatching events.

Instruments for measuring pain-related problems in adolescents with chronic pain are sparse, especially those based on the personal experiences of these adolescents. This study aimed to develop and test such an instrument, the pain-related problem list for adolescents (PPL). A sample of 129 adolescents with chronic pain without documented physiological etiology completed the 57-item problem list, which was based on interviews with a similar group of adolescents with chronic pain. Principal components analysis yielded four domains: problems related to (1) concentration; (2) mobility; (3) adaptability; and (4) mood. The questionnaire was shortened to 18 items and has good reliability (total alpha = 0.82; concentration alpha = 0.86; mobility alpha = 0.77; adaptability alpha = 0.71; and mood alpha = 0.78); the validity also proved to be adequate, especially in the general population sample. The PPL provides a tool to assess the impact of chronic pain in adolescents. Future research should focus on further validation of the PPL in a large clinical population and establishing its test-retest reliability.

A 42-year-old, previously healthy cattle inspector presented with a 7-day history of fever, a painful left knee, malaise and muscular pain. He did not suffer from an underlying disease, nor was he immunocompromised. After 12 days of hospitalization, a unilocular abscess in the left psoas muscle was diagnosed. Nocardia farcinica was isolated from the aspirate. No connection with his work could be demonstrated. The patient was successfully treated with trimethoprim-sulfamethoxazole for 11 months.

In parts of the developing world, pesticide poisoning causes more deaths than infectious diseases. Use of pesticides is poorly regulated and often dangerous; their easy availability also makes them a popular method of self-harm. In 1985, the UN Food and Agriculture Organisation (FAO) produced a voluntary code of conduct for the pesticide industry in an attempt to limit the harmful effects of pesticides. Unfortunately, a lack of adequate government resources in the developing world makes this code ineffective, and thousands of deaths continue today. WHO has recommended that access to highly toxic pesticides be restricted--where this has been done, suicide rates have fallen. Since an Essential Drugs List was established in 1977, use of a few essential drugs has rationalised drug use in many regions. An analogous Minimum Pesticides List would identify a restricted number of less dangerous pesticides to do specific tasks within an integrated pest management system. Use of safer pesticides should result in fewer deaths, just as the change from barbiturates to benzodiazepines has reduced the number of deaths from pharmaceutical self-poisoning.

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.

Overexpression of cell surface glycoproteins of the CD44 family is an early event in the colorectal adenoma-carcinoma sequence. This suggests a link with disruption of APC tumor suppressor protein-mediated regulation of beta-catenin/Tcf-4 signaling, which is crucial in initiating tumorigenesis. To explore this hypothesis, we analyzed CD44 expression in the intestinal mucosa of mice and humans with genetic defects in either APC or Tcf-4, leading to constitutive activation or blockade of the beta-catenin/Tcf-4 pathway, respectively. We show that CD44 expression in the non-neoplastic intestinal mucosa of Apc mutant mice is confined to the crypt epithelium but that CD44 is strongly overexpressed in adenomas as well as in invasive carcinomas. This overexpression includes the standard part of the CD44 (CD44s) as well as variant exons (CD44v). Interestingly, deregulated CD44 expression is already present in aberrant crypt foci with dysplasia (ACFs), the earliest detectable lesions of colorectal neoplasia. Like ACFs of Apc-mutant mice, ACFs of familial adenomatous polyposis (FAP) patients also overexpress CD44. In sharp contrast, Tcf-4 mutant mice show a complete absence of CD44 in the epithelium of the small intestine. This loss of CD44 concurs with loss of stem cell characteristics, shared with adenoma cells. Our results indicate that CD44 expression is part of a genetic program controlled by the beta-catenin/Tcf-4 signaling pathway and suggest a role for CD44 in the generation and turnover of epithelial cells.

Background: Inhalation therapy with sodium cromoglycate is recommended as the first-line prophylactic treatment for moderate asthma in children. The availability of spacer devices with face-masks has extended the applicability of metered-dose inhalers to younger children. We studied the feasibility and effects of this therapy compared with placebo in children aged 1-4 years., Methods: 218 children aged 1-4 years with moderate asthma were recruited through 151 general practitioners between March, 1995, and March, 1996. They were randomly assigned sodium cromoglycate (10 mg three times daily) or placebo, given by inhaler with spacer device and face-mask for 5 months. Rescue medication (ipratropium plus fenoterol aerosol) was available during the baseline period of 1 month and the intervention period. Parents completed a daily symptom-score list. The primary outcome measure was the proportion of symptom-free days in months 2 to 5. Analysis was by both intention to treat and on treatment., Findings: 167 (77%) children completed the trial. 131 (78%) of these children used at least 80% of the recommended dose. Of the 51 children who stopped prematurely, 23 had difficulties with inhaled treatment. The mean proportion of symptom-free days for both groups was greater for the treatment period than for the baseline period (95% CI for mean difference 5.1 to 17.5 cromoglycate, 11.9 to 23.3 placebo). However there were no differences between the sodium cromoglycate and placebo groups in the proportion of symptom-free days (mean 65.7 [SD 25.3] vs 64.3 [24.5]%; 95% CI for difference -8.46 to 5.70) or in any other outcome measure., Interpretation: Our study in a general practice setting shows that inhalation therapy with a spacer device and face-mask is feasible in a majority of children below the age of 4 years. However, long-term prophylactic therapy with inhaled sodium cromoglycate is not more effective than placebo in this age-group.

CD44 is a major cell-surface receptor for hyaluronate (HA). By alternative RNA-splicing a large number of CD44 variants are generated. To explore the role of CD44 splicing in the regulation of cell binding to HA, three different isoforms of CD44 were transfected in the CD44 negative B-cell lymphoma line Namalwa and in the fibroblastoid cell line COS7. We observed that whereas the standard form of CD44 (CD44s) mediated adhesion of Namalwa to HA, Namalwa transfected with CD44v3-10 or CD44v8-10 was unable to bind to either immobilized or soluble HA. After stimulation of CD44 with an activating anti-CD44 mAb or with phorbol ester, the binding of CD44s to HA was 5- to 10-fold higher than that of the other two isoforms. By contrast, COS7 cells transfected with CD44s, CD44v8-v10, or CD44v3-v10 bound equally effectively to HA. Hence, in addition to alternative splicing, cell type determines CD44 binding to HA.

The SCL/tal-1 gene on chromosome 1 is disrupted in up to 30% of immature T-cell malignancies, thus representing the most commonly recognized chromosomal abnormality in this disorder. Abnormalities of the gene occur rarely by chromosomal translocation into the T-cell receptor (TCR) delta locus and commonly by a site-specific 95-kb deletion, SIL-SCL (tald). Analysis of the SIL-SCL deletion by Southern blotting and polymerase chain reaction (PCR) in a series of 52 immature T-cell malignancies showed a type A deletion in 21% of cases, but no type B deletions. The type A deletion correlated with malignancies of the TCR alpha beta lineage, either on the basis of TCR alpha beta expression or bilateral TCR delta deletion. Fifty percent (5 of 10) of TCR alpha beta-expressing cells demonstrated the abnormality, whereas 0% (0 of 11) of TCR gamma delta-expressing cells did so. Six of eight SIL-SCL type A cases had undergone bilateral delta deletion, whereas only one of 31 cases with an apparently normal SCL gene had done so. These data demonstrate an association between SCL disruption and TCR alpha beta lineage differentiation and suggest that the SIL-SCL deletion occurs at the same stage of ontogeny as TCR delta deletion.

A simple high-performance liquid chromatographic method is presented for the determination of trace amounts of sulphadimidine (SDD), its hydroxylated metabolites and N4-acetyl-SDD in blood plasma, urine, hepatocyte culture media and microsomal incubations. The synthesis of 5-hydroxy-SDD and an improved method for the isolation of 4-methylhydroxy-SDD from urine are described and their respective specific absorption coefficients at 265 nm are calculated by on-line radiochemical and ultraviolet detection. The limit of detection of the analytical method is 0.05 micrograms/ml for SDD and its hydroxy metabolites and 0.2 micrograms/ml for N4-acetyl-SDD. Linear calibration graphs for SDD and its metabolites were constructed from 0.2 to 50 micrograms/ml. The method has been applied to biotransformation studies in vivo and in vitro.

Paraffin-embedded squamous cell carcinomas of the uterine cervix selected for the presence of human papillomavirus (HPV) genotype 16 (n = 19) by polymerase chain reaction, were studied for transcription of the early open reading frame E7 (ORF E7). Nonradioactive RNA in situ hybridization (RISH) was performed using in vitro generated biotinylated probes. Hybrids were visualized by streptavidin gold and silver enhancement staining in combination with confocal laser scan microscopy. Quality of mRNA was verified by detection of beta-actin gene transcripts before E7 expression was studied. In all carcinomas containing HPV 16 DNA and showing beta-actin mRNA signals (n = 13), clear E7 ORF transcription could be found. Additional RNA-PCR on purified cytoplasmic RNA of snapfrozen tissue of identical carcinomas (n = 7) showed E6-E7 specific transcripts in all E7 RISH positive samples. These results indicate continuous expression of E7 ORF in all cervical carcinomas containing HPV 16 DNA and support an active role of the E7 ORF in the pathogenesis of cervical cancer.

We carried out a light and electron microscopic study on end plates and related structures in external intercostal muscles from subjects aged between 4 and 77 years. Light microscopically end plates maintained the same size, did not increase in number and showed no sprouting of terminal axons, all indicating that major compensatory histological changes to maintain adequate neuromuscular transmission with age were not required. At the ultrastructural level end plates became more complex mainly at the postsynaptic side. The latter included increased length and branching of the postsynaptic membrane with enlargement of the postsynaptic area, and degeneration of junctional folds. However, some neuromuscular junctions (NMJs) showed little branching of the postsynaptic membrane, even in old age. At the presynaptic side nerve terminals with an irregular shape were noted in the aged. Schwann cell processes were seen to intrude into the primary synaptic cleft. From these data we suggest that degeneration of the postsynaptic membrane with consequent focal denervation of NMJs is a primary event in the age-related changes of end plates. The muscle fibres showed a minor degree of type grouping in old age doubtless due to loss of motor neurons with age.

Bilateral Symmetrical Calcifications (B.S.C.) in cerebro represent calcium deposits usually found in the basal ganglia and/or dentate nucleus. They can be the result of diverse disorders, but can also present themselves without any underlying disease. Most often they are asymptomatic, but if the calcifications are extensive, extrapyramidal and cerebellar signs may arise. The following case concerns a patient with pseudohypoparathyroidism. Besides the usual signs and symptoms found in this disease, the patient also showed extensive B.S.C. The etiology and clinical symptoms of B.S.C. will be discussed. Furthermore, attention will be given to the syndrome of the pseudohypoparathyroidism and the effects of hypocalcemia.

Antibodies elicited by HIV-1 strains, and which neutralize such strains in vitro, bind to synthetic peptides of 5-8 amino acids in length. These amino acids, although variable, have a fixed location between two cysteines in the carboxyl terminus of the HIV-1 external envelope. Nine peptides of 9 amino acids corresponding to the gp120 domains of European and American (LAV-1, NY5, CDC4, SF2), Haitian (RF) and African (ELI, MAL, Z3, Z6) HIV-1 strains, were synthesized using LAV-1 and RF neutralization epitopes as models. Serum of chimpanzees infected with LAV-1, HTLV-IIIB or HTLV-IIIRF reacted predominantly with the homologous peptide, although cross-reactivity with heterologous peptides occurred: 8 out of 11 human sera with HTLV-IIIB-neutralizing activity bound the LAV-1/HTLV-IIIB peptide, and 6 out of 7 sera with HTLV-IIIRF-neutralizing activity bound the RF peptide. African sera reacted most frequently with the Z3 peptide (78%) while only 35% (p = 0.0001) of European and 20% (p less than 0.0001) of American sera recognized it. Recognition patterns of children from the USA and Europe were different. Although multiple reactivities were observed, blocking experiments favoured cross-reactivity as the explanation. Based on the antibody profiles of nonapeptide recognition, peptides LAV-1, RF and SF2 were clustered, as were NY5 and CDC4, and so were Z6, MAL and ELI. This antigenic relatedness of HIV-1 strains could not entirely be explained by the physico-chemical characteristics of the nonamers per se. Resemblance was observed with the clustering of HIV-1 strains based on the divergence of the nucleotide sequence of entire HIV-1 envelopes. This implies a role of peripheral envelope residues, in the context of infectious particles or infected cells, in determining the specificity of antibodies reactive to the V3 domain. Therefore, the neutralization domain in this variable region may be considered part of a conformational structure involving several envelope regions which appear distinct from each other in the primary sequence.

A 9-year-old girl suffering from progressive fluctuating dystonia is reported. Some problems of diagnosis are discussed. The differential diagnosis is described and a comparison is made with cases from the literature.

Chimpanzees infected with the HIV-1 strains HTLV-IIIB or LAV-1 in primary, secondary or tertiary passages developed neutralizing antibodies binding to variable domain V3 in the carboxyl terminal half of the external envelope (amino acids 309-317). Nonapeptide antigens reflecting either the HTLV-IIIB/LAV-1 neutralization epitope (IQRGPGRAF, designated 3B) or peptide analogues (ITKGPGRVI, designated RF; IQRGPGRVI, designated 3B/RF; ITKGPGRAF, designated RF/3B) were previously shown to be able to distinguish antibody populations in a polyclonal response of rabbits to these peptides. Sera from chimpanzees infected with the HIV-1 strains HTLV-IIIB and LAV-1 were tested for the presence of antibodies reactive to these nonapeptides. Sera from 3 chimpanzees infected with a primary LAV-1 or HTLV-IIIB passage, 2 chimpanzees infected with blood from the primary infected chimpanzees and from 1 chimpanzee infected with blood from a secondary passage animal, all bound peptides 3B and 3B/RF, sharing the sequence IQRGPGR, in equally high tires. In 2 primary passage animals and in 1 secondary passage animal, the capacity to bind to peptides 3B and 3B/RF was equally high, indicating clonality of these B-cell responses. Contrasting results were obtained with the sera from 1 primary, 1 secondary and 1 tertiary passage animal, showing stronger binding to the 3B/RF peptide than to the 3B peptide. The animals with antibodies binding strongly to the 3B peptide had an early HTLV-IIIB-induced cell-fusion-inhibiting (CFI) antibody response, while the animals with antibodies binding strongly to the 3B/RF peptide had a late HTLV-IIIB-induced CFI antibody response. This difference in binding to the 3B peptide might result from antigenic variation in the neutralization domain of the inoculum virus(es). The conservation of the antibody specificity for the neutralization epitope of the inoculum strain might open the way to type circulating virus strains by antibody specificity for a panel of peptide analogues derived from the V3 domain of the external envelope of distinct HIV-1 strains.

We report the pedigree of a family in which a mother and her two children, a boy and a girl, all suffer from a similar, though variably expressed cerebral disorder, seen on CT as uni- or bilateral cavities within the supratentorial white matter in communication with the ventricular system. Additional white matter hypodensity around the lateral ventricles without ventricular widening provides preliminary evidence of a primary disease of myelination, in the absence of histopathological confirmation. This is probably the first report of "porencephaly" which shows a pattern of autosomal dominant inheritance.