Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons.

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day-70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos.

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos.

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

Expression of the uterine Mx protein in cyclic and pregnant cows, gilts, and mares.

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.

Chronic subcutaneous octreotide decreases gastrointestinal blood loss in blue rubber-bleb nevus syndrome.

BACKGROUND: A patient affected by blue rubber-bleb nevus syndrome had chronic gastrointestinal bleeding requiring weekly blood transfusions. Despite multiple surgical and endoscopic procedures to treat the venous malformations, the patient continued to bleed primarily from lesions in the small bowel. Therefore, this patient was treated with octreotide, a somatostatin analog known to decrease splanchnic blood flow and that is used for acute and chronic gastrointestinal bleeding. METHODS: Octreotide therapy, 5.7 microg/kg subcutaneously twice daily, was initiated, and the patient was followed up clinically. Complete blood counts, blood glucose concentration, pancreatic enzyme concentration, liver function tests, and growth hormone concentration were monitored during treatments. RESULTS: During the 4 weeks after initiation of octreotide therapy, hemoglobin concentration was maintained without the need for transfusions. Octreotide decreased the patient's monthly need for blood transfusion from 52 +/- 7 mL. kg-1. mo-1 of packed red blood cells to 23 +/- 7 mL. kg-1. mo-1. She had no detectable side effects or growth inhibition. Other medical interventions including -epsilonaminocaproic acid, nadolol, and total parenteral nutrition with bowel rest were not as effective as octreotide alone. CONCLUSION: Octreotide decreased the patient's need for blood transfusions. Possible mechanisms include altering blood flow to the gastrointestinal tract and direct effects on the venous malformations.

Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes.

Interferon-tau (IFN tau) acts locally on the endometrium to suppress estrogen and oxytocin receptor expression and block luteolysis in ruminants. Systemic administration of conceptus homogenates or recombinant ovine IFN tau does not block luteolysis or enhance pregnancy rates in sheep or cattle, respectively. However, IFN tau up-regulates expression of the antiviral protein Mx throughout the entire uterine wall during early pregnancy. These studies determined if conceptus-derived IFN tau also up-regulates Mx expression in components of the circulating immune system that migrate through the endometrial wall. In experiment one, peripheral blood mononuclear cells (PBMC) were isolated from ewes at D26 post-artificial insemination (AI) and Mx mRNA levels examined by Northern and slot-blot hybridization. Pregnancy resulted in a two-fold increase in Mx mRNA levels compared to bred, non-pregnant ewes at D26. In experiment two, PBMC were isolated from ewes at AI, and every three days from D9 to D30. Results showed a four-fold increase in Mx mRNA levels in PBMC from pregnant versus bred, non-pregnant ewes at D15. Increased Mx mRNA, which remained elevated through D30, was accompanied by increased levels of Mx protein. These results show that pregnancy recognition signaling rapidly induces Mx gene expression in PBMC, and are the first to suggest that IFN tau activates gene expression in components of the circulating immune system.

Early operation in children with pancreas transection.

BACKGROUND/PURPOSE: The preferred treatment for children with major pancreatic ductal injury remains controversial. This report compares our results using early operation with previously reported series using both operative and nonoperative management. METHODS: This is a retrospective analysis of all children with pancreatic transection identified at Children's Medical Center of Dallas, Texas, from 1995 through 1999. RESULTS: There were 11 children with pancreatic transection. There was a delay of 2.3 days before presentation to a hospital in 6. Transection was diagnosed within 12 hours of presentation in 5 children. In the other 6 there was a mean delay of 36 hours. Nine children underwent operation within 72 hours of injury. Two late presenters initially were treated nonoperatively, and both developed a pseudocyst. The length of hospital stay in patients undergoing early operation averaged 11 days. Early postoperative morbidity occurred in 4 children and late morbidity in 1. CONCLUSIONS: Major pancreatic injuries are uncommon in children, and the diagnosis often is delayed. A high index of suspicion and repeated computed tomography scans should lead to earlier diagnosis. When compared with nonoperative management, early pancreatic resection more expeditiously returns the child to good health and lessens the inconvenience and emotional stress associated with prolonged hospitalization.

Management of parapneumonic collections in infants and children.

BACKGROUND/PURPOSE: Video-assisted thoracoscopic surgery (VATS) has a recognized role in treatment of empyema thoracis. The purpose of this report is to show the value of initial VATS as the primary treatment of parapneumonic collections. METHODS: A retrospective review was done of 139 children who required surgical consultation for parapneumonic collections between January 1992 and July 1998. Management options were (M1) thoracentesis, chest tube drainage, or fibrinolytic therapy and delayed thoracotomy for unresolved collections; (M2) thoracentesis, chest tube drainage, fibrinolytic therapy with delayed VATS if the child remained ill; or (M3) primary VATS. Comparative data included age, duration of prehospital illness, oxygen requirements, white blood cell count, bacterial culture results, number of procedures performed per patient, duration of chest tube drainage, complications, and length of stay. Kruskal-Wallis 1-way analysis was used, with significance at P less than .05. RESULTS: A total of 60 children were treated by M1, 38 by M2, and 41 by M3. Age, duration of prehospital illness, oxygen requirements, white blood cell count, bacterial culture results, and complication rates were comparable. The median length of stay was 12 days for M1, 11 days for M2, and 7 days for M3, with M3 significantly shorter at P<.001. The number of procedures was a median of 2 in M1, 2 in M2, and 1 in M3, with M3 significantly fewer at P<.001. Duration of chest tube drainage was a median 5 days for M1 and 3 days for M2 and M3, with M1 significantly longer at P<.001. There were 9 thoracotomies in the M1 group, 3 in the M2 group, and none in the M3 group. One child in M3 required a second VATS. CONCLUSIONS: Primary VATS has significantly decreased the number of procedures, duration of chest tube drainage and length of stay for children with parapneumonic effusions. Primary VATS appears to be of value in management of bacterial pneumonia with effusion.

Comparison of laparoscopic and open splenectomy in children with hematologic disorders.

OBJECTIVE: To compare laparoscopic and traditional open splenectomy in children with nonmalignant hematologic disorders. STUDY DESIGN: Retrospective review of 36 consecutive nonrandomized splenectomies (16 laparoscopic and 20 open) performed for hematologic disorders at a single pediatric institution during the past 3 years. The two-sided Mann-Whitney U test for non-parametric variables was used for statistical analysis. RESULTS: An open procedure was performed on 20 patients (mean age, 9.7 years), five of whom had a concomitant cholecystectomy. A laparoscopic splenectomy was performed on 16 children (mean age, 10.3 years), seven of whom had a concomitant cholecystectomy. The mean anesthesia and operative times were longer in the laparoscopic than in the open group (p < 0.001). However, the mean number of hours of postoperative analgesia was less in the laparoscopic group (p < 0.005). Patients who had laparoscopic splenectomy were also discharged home earlier (p < 0.01) and resumed a regular diet sooner. Mean operating room charges were higher in the laparoscopic group (p < 0.001), but total hospitalization costs were not significantly different. Postoperative complication rates were similar. The hematologic response was comparable. CONCLUSIONS: laparoscopic splenectomy is feasible and safe in children with hematologic disorders. Although it currently requires more operative time than the open approach, it is superior with regard to duration of postoperative analgesia, duration of hospital stay, and recovery of bowel function.

Laryngeal atresia in the newborn: surgical implications.

Congenital atresia of the larynx is a rare, life-threatening anomaly in which early recognition and appropriate perinatal management are essential for survival. Few long-term survivors have been reported in the literature, with most documented cases studied at necropsy. The authors present a case of a full-term male newborn with Smith and Bain Type I laryngeal atresia, who has survived and developed normally during the 10-months since birth. Rapidly progressive cyanosis following umbilical cord occlusion, lack of phonation, and no air movement with respiratory efforts are typically present and should alert the clinician to the possibility of laryngeal atresia. Positive pressure ventilatory assistance may be possible through a patent pharyngoglottic duct or tracheoesophageal fistula until a surgical airway is established. If this is not possible, emergent tracheostomy may be a lifesaving procedure in the first minutes of life.

Laparoscopic Nissen fundoplication in the infant.

Twenty-five children weighing less than 8 kg underwent laparoscopic Nissen fundoplication for treatment of refractory gastroesophageal reflux (GER) between January 1993 and April 1995. Mean patient age was 6 months and weight was 5.1 kg. Mean operative time was 114 min, including gastrostomy in 20 patients. Nineteen patients are alive without evidence of GER with a mean follow-up of 359 days. Perioperative complications were unusual and mild. There were 6 late deaths due to progression of underlying disease and not attributable to the procedure. Laparoscopic Nissen fundoplication provides effective antireflux protection to very small children with acceptable morbidity and mortality through short-term follow-up.

Laparoscopic splenectomy in childhood hematologic disorders.

Laparoscopic splenectomy is a new technique that is being utilized in patients with a variety of mostly hematologic disorders. Its application in children has not been extensively documented. Between January 1994 and February 1995, 11 children less than 15 years of age underwent elective laparoscopic splenectomy. Data collected from this treatment group were compared to that from the ten most recent open splenectomy patients with comparable hematologic disorders. All procedures in both groups were successful in relief of symptoms, increase in platelet count, and/or increase in hematocrit. Operative times averaged 147 mm in the laparoscopic group, compared to 112 mm in the open group. Estimated blood loss was 32 ml in the laparoscopic group and 86 ml in the open group. Days to laparoscopic patient discharge were 3.6, compared to 5.3 days in the open group. There were no wound complications or need for perioperative platelet transfusions in the laparoscopic patients. Patient response has been uniformly positive in the laparoscopic group. Reusable access trocars are utilized for two of the four working ports. Stapling devices and special tissue morselizers are not required. There are no additional operating room or surgeons fees incurred in the laparoscopic procedures. This series demonstrates that laparoscopic splenectomy is a safe, cost-efficient alternative to open splenectomy in children with a variety of hematologic disorders.

Distinction between peroxisomal bifunctional enzyme and acyl-CoA oxidase deficiencies.

The clinical distinction between patients with a disorder of peroxisome assembly (e.g., Zellweger syndrome) and those with a defect in a peroxisomal fatty acid beta-oxidation enzyme can be difficult. We studied 29 patients suspected of belonging to the latter group. Using complementation analysis, 24 were found to be deficient in enoylcoenzyme A hydratase/3-hydroxyacylcoenzyme A dehydrogenase bifunctional enzyme and 5 were deficient in acyl-CoA oxidase. Elevated plasma very long-chain fatty acids (VLCFA), impaired fibroblast VLCFA beta-oxidation, decreased fibroblast phytanic acid oxidation, normal plasmalogen synthesis, normal plasma L-pipecolic acid level, and normal subcellular catalase distribution were characteristic findings in both disorders. The elevation in plasma VLCFA levels and impairment in fibroblast VLCFA beta-oxidation were more severe in bifunctional-deficient than in oxidase-deficient patients. The clinical course in bifunctional deficiency (profound hypotonia, neonatal seizures, dysmorphic features, age at death approximately 9 months) was more severe than in oxidase deficiency (moderate hypotonia without dysmorphic features, development of a leukodystrophy, age at death approximately 4 yr). Based on these findings, accurate early diagnosis of these deficiencies of peroxisomal beta-oxidation enzymes is possible.

Complex colon duplication mimicking an obstructed, non-functioning kidney in a newborn with imperforate anus and spinal dysraphism.

Gastrointestinal (GI) duplications contain tissue resembling several portions of the GI tract and are associated with vertebral and genitourinary (GU) abnormalities [1-4]. We report a newborn with low, imperforate anus and lumbosacral dysraphism, who presented with a large cystic mass in the left renal fossa and pelvis. The flank mass (felt initially to be a dysplastic kidney and ureter) proved to be a complex GI duplication with histologic evidence of gastric, small bowel, and colonic mucosa, as well as respiratory epithelium and pancreatic tissue.

The jaundiced newborn.

Evaluation of the newborn with hyperbilirubinemia is an important and commonly encountered problem in all clinical pediatric-based practices. Cholestatic jaundice is always pathologic and warrants prompt assessment. The timely evaluation and directed treatment of any jaundiced newborn are essential to prevent serious metabolic consequences and irreversible hepatic damage.

Bladder management in children with genitourinary sarcoma.

Between 1977 and 1991, 14 patients were treated for genitourinary sarcoma (mean age, 7.4 years). The primary site was bladder in six patients, prostate in five, and vagina in three. Histological study showed embryonal rhabdomyosarcoma in 12 and leiomyosarcoma in 2 children. Initial therapy included biopsy followed by chemotherapy in all patients. Subsequently, five patients had anterior exenteration, four underwent partial cystectomy, and one patient had a radical prostatectomy; four patients were treated with chemotherapy and external beam pelvic irradiation (4,000 to 6,000 rads) alone. Overall survival for the group is 100% (follow-up 6 to 168 months). Two of four patients undergoing partial cystectomy had bladder augmentation at the time of surgery. All patients having partial cystectomy had negative surgical margins, are tumor free, and have volitional voiding. Two of four patients (50%) initially treated with chemotherapy and radiotherapy alone have had significant bladder deterioration requiring bladder reconstruction. There has been an evolution toward less radical, initial surgical intervention in pediatric genitourinary sarcoma; however, surgical resection continues to be the primary curative modality. Partial cystectomy with or without primary reconstruction may be preferable to exenteration for selected patients. Primary reconstruction at the time of partial cystectomy leaves a functional bladder and excellent long-term results. Children treated with chemotherapy and radiotherapy protocols alone must be monitored for late bladder deterioration.

Transplantation of beta cells from transgenic mice into nude athymic diabetic rats restores glucose regulation.

We have shown that elevated plasma D-glucose levels in experimentally-induced diabetic nude athymic rats can be reduced by intraperitoneal transplantation of microcarrier-attached insulin producing beta cells from the mouse pancreatic beta cell line, beta TC-1. The reduction in the level of hyperglycemia was observed as early as two days following cell transplantation and was associated with a concomitant increase in plasma insulin levels. beta TC-1 cell transplanted diabetic rats had plasma D-glucose levels similar to those found in non-diabetic control animals and remained normoglycemic throughout the 39 day experimental period. The beta TC-1 cell transplanted diabetic rats also had near normalization of body weight, food and water intake and of urine output when compared to control diabetic and non-diabetic rats. Similarly, they exhibited improved blood glucose clearance following intravenous D-glucose administration. These results suggest that beta TC-1 cells regulate D-glucose homeostasis following transplantation into diabetic rat recipients in a manner similar to that of endogenous pancreatic beta cells.

Alcohol and the adolescent trauma population.

Trauma is the leading killer of children and adolescents between 1 and 21 years of age. Alcohol-impaired driving represents the single greatest cause of mortality and morbidity of children over the age of 6. We retrospectively reviewed 878 consecutive adolescent (age range, 16 to 20 years) trauma admissions for blood alcohol concentration (BAC). Four hundred sixty-seven patients had BAC drawn, 258 were BAC-negative (group I), 209 (48%) were BAC-positive (group II). The adolescent drinkers were then compared with a group of 748 adult drinkers (group III). Groups I and II differ in sex, age, time of day of the accident, Injury Severity Score, Glasgow Coma Score, and Revised Trauma Score, whereas group II and III differ by type of accident, type of injury, socioeconomic factors (bad debt), time of day of the injury, and BAC. There were no significant differences in TRISS predicted survival, actual survival, nor mean length of stay. We conclude that (1) alcohol is a significant contributor to injury during adolescence, and (2) adolescent drinkers differ from adult drinkers in their habits, demographics, and socioeconomic status. These socioeconomic differences have implications for the access to and cost-effectiveness of interventions.

Impaired liver regeneration in the analbuminemic rat.

Albumin has been implicated as both an inhibitor and a stimulator of liver regeneration. We examined the rate of liver regeneration following partial hepatectomy in rats which are genetically analbuminemic. Adult male analbuminemic and Sprague-Dawley control rats underwent either a 70% partial hepatectomy or a sham operation. Twenty-four hours postoperatively, rats were injected iv with tritiated thymidine and the liver remnants were resected 1 hr later. Liver weight, protein and DNA contents, and the rate of tritiated thymidine incorporation into DNA were determined. The rate of DNA synthesis in control sham-operated rats was greater (P less than 0.05) than that of analbuminemic rats. Following partial hepatectomy, there was a statistically significant (P less than 0.01) increase in DNA synthesis in control rats; no significant increase was noted in the analbuminemic rats. No significant differences in liver remnant weight or protein and DNA contents were noted between control and analbuminemic sham-operated or partially hepatectomized rats. Our data suggest that albumin plays a role in the regulation of DNA synthesis in both the resting and the regenerating liver and it may be essential in maintaining a normal rate of liver regeneration.