Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells.

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.

Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells.

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.

Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis.

The endosomal compartment of polarized epithelial cells is a major crossroads for membrane traffic. Proteins entering this compartment from the cell surface are sorted for transport to one of several destinations: recycling to the original cell surface, targeting to lysosomes for degradation, or transcytosis to the opposite surface. The polymeric immunoglobulin receptor (pIgR), which is normally transcytosed from the basolateral to the apical surface, was used as a model to dissect the signals that mediate this sorting event. When exogenous receptor was expressed in Madin-Darby Canine Kidney (MDCK) cells, it was shown that phosphorylation of pIgR at the serine residue at position 664 is required for efficient transcytosis. Replacement of this serine with alanine generated a receptor that is transcytosed only slowly, and appears to be recycled. Conversely, substitution with aspartic acid (which mimics the negative charge of the phosphate group) results in rapid transcytosis. It was concluded that phosphorylation is the signal that directs the pIgR from the endosome into the transcytotic pathway.

Survival after relapse in children and adolescents with rhabdomyosarcoma: A report from the Intergroup Rhabdomyosarcoma Study Group.

BACKGROUND: Despite advances in therapy, nearly 30% of children with rhabdomyosarcoma experience progressive or relapsed disease, which is often fatal. PATIENTS AND METHODS: To facilitate the development of a retrieval therapy protocol, we studied potential risk factors that were predictive of survival after first relapse in 605 children who were enrolled onto three consecutive Intergroup Rhabdomyosarcoma Study Group protocols. RESULTS: The median survival time from first recurrence was 0.8 years; the estimated percentage of patients who survived 5 years from first recurrence was 17% +/- 2% (mean +/- SD). Univariate analysis showed that tumor histology was an important predictor of 5-year survival (P <.001): the 5-year survival rate was 64% for patients with botryoid tumors (n = 19), 26% for patients with embryonal tumors (n = 313), and 5% for patients with alveolar or undifferentiated sarcoma (n = 273). Further analysis identified prognostic factors within histologic subtypes (P <.001). For patients with embryonal tumors, the estimated 5-year survival rate was 52% for patients who initially presented with stage 1 or group I disease, 20% for those with stage 2/3 or group II/III disease, and 12% for those with group IV disease. For patients with stage 1/group I disease, estimated 5-year survival rates were higher for patients with local (72%) or regional (50%) recurrence than for those with distant (30%) recurrence. Among patients with alveolar or undifferentiated sarcoma, only the disease group predicted outcome: the 5-year survival estimate was 40% for group I versus 3% for groups II through IV. We identified a "favorable risk" group (approximately 20% of patients) whose 5-year estimated survival rate was near 50%; for all other patients, the estimated survival was near 10%. CONCLUSION: This analysis demonstrates that the probability of 5-year survival after relapse for rhabdomyosarcoma is dependent on several factors at the time of initial diagnosis, including histologic subtype, disease group, and stage. These findings will form the basis of a multi-institutional risk-adapted relapse protocol for childhood rhabdomyosarcoma.

Cell biology of the asialoglycoprotein receptor system: a model of receptor-mediated endocytosis.

Substantial information about the ASGP-R has accumulated in the 10 years following the initial studies of this receptor by Ashwell and Morell. Many of its biochemical properties, its structure, and its orientation within the plasma membrane are now known. The pathways of ASGP ligand and receptor, with the CURL organelle being a central component, are summarized in Fig. 18. The major pathway of the ligand through the cell, beginning with binding at the cell surface and ending with degradation in lysosomes, has been investigated in detail. Recently, alternate routes of the ligand such as the ligand recycling pathway have been observed. With regard to the itinerary of the receptor, there is now biochemical, kinetic, and morphological evidence to support receptor recycling. The new concept of CURL as an important intracellular organelle has originated from studies of ASGP-R recycling. Its importance in the dissociation and segregation of ligand and receptor as well as in receptor recycling is now evident. In addition, there has been a concurrent investigation of other receptor systems that participate in receptor-mediated endocytosis, providing parallels and contrasts to the ASGP-R of hepatocytes. Many critical issues still exist in the cell biology of the ASGP-R. What are the structural requirements of the receptor for ligand binding and subsequent endocytosis of the receptor-ligand complex? Very little is known about the interactions between the receptor and the lipid bilayer in which it resides. How does the receptor move laterally in the plasma membrane? Are there proteins or glycolipids closely associated with the ASGP-R and, if so, what is their function? What is the mechanism that causes receptor clustering into coated pits? Although the existence of a pathway for ligand recycling has been demonstrated, there are still many issues to be addressed. What signals a particular ligand molecule for recycling? Is it a stochastic process? What is the function of this route of ligand movement? How are the various ligand pathways coordinated and regulated? In addition, there are many unanswered questions regarding the receptor pathway. How does CURL mediate the sorting of ASGP-R from ligand? How are receptors with different destinations (e.g., ASGP-R and IgA receptor) sorted in CURL? What is the mechanism of ASGP-R degradation and how is it regulated? Finally, how does the Golgi function in the ASGP system and what is the relationship between the Golgi and CURL? Future investigation of these issues will require further observations with existing techniques as well as new approaches.(ABSTRACT TRUNCATED AT 400 WORDS)

National estimates of the use of hematopoietic stem-cell transplantation in children with cancer in the United States.

National utilization data for hematopoietic stem-cell transplantation (HSCT) for childhood cancers in the United States have not been reported. We identified cancer encounters for children aged 18 years and younger from 1997 to 2001 in US nonfederal, acute care hospitals. We compared patient, hospital, and resource use characteristics and in-patient mortality associated with HSCT and non-HSCT encounters, estimated the number of HSCT encounters by stem-cell source and cancer type, and examined resource use and mortality in each category. We identified 461,175 cancer encounters, of which 6380 (1.4%) were HSCT encounters. There was wide variation in resource use and mortality by stem-cell source and cancer type. Of note, 17% of HSCT encounters were for patients with acute lymphoblastic leukemia without remission or sarcoma, conditions for which there is little evidence of benefit from HSCT in children. These encounters were associated with high in-patient mortality and long lengths of stay. Also, we observed an increasing use of cord blood over the study period. Future research should examine potentially important sociodemographic differences in patients undergoing HSCT compared to those who do not. Additional analyses incorporating disease stage and severity are needed.

Accurate case finding using linked electronic clinical and administrative data at a children's hospital.

To link hospital administrative data and an electronic medical record at a children's hospital in order to identify children with cancer admitted for fever and neutropenia. Hospital administrative data concerning 13,374 inpatient and outpatient encounters were validated against and linked to clinical data stored in an electronic medical record. Queries of the linked databases identified children with fever and neutropenia. Sensitivity and specificity of the experimental case-finding strategy were determined and compared to a control case-finding strategy utilizing administrative data alone. Linking of the clinical record to the administrative record was achieved in 233 (99%) of the 235 records. Of 1680 data elements reviewed from the administrative data that were also potentially available in the clinical data system, 1679 (99.9%) were verified in the electronic medical record. The experimental strategy for case finding had a sensitivity of 73.1% (95% CI: 58.1, 88.3), specificity 99.6% (95% CI: 99.1, 100). If only administrative data such as diagnosis-related group and hospital service were used for case finding, both the sensitivity (P < 0.01) and specificity (P < 0.01) were significantly lower. Linking a children's hospital administrative data system with clinical data is feasible and can be utilized for specific case finding for a common and costly condition in children.

Transjugular intrahepatic portosystemic shunting (TIPS) for treatment of severe hepatic veno-occlusive disease.

Severe veno-occlusive disease (VOD) of the liver is a frequent cause of morbidity and mortality in patients undergoing transplantation. While surgical portosystemic shunts have been reported to be useful in the treatment of severe hepatic VOD with intractable ascites, few of these patients are surgical candidates. We report a case of severe VOD after autologous peripheral blood progenitor cell transplantation treated with transjugular intrahepatic portosystemic shunting (TIPS). This procedure resulted in marked improvement in the patient's ascites, coagulation status and urinary output. The safety and efficacy of this non-surgical approach for the treatment of patients with severe VOD requires prospective studies.

A case controlled comparison of open and laparoscopic splenectomy in children.

BACKGROUND: This case controlled study compares the efficacy, safety, and cost of laparoscopic splenectomy (LS) and open splenectomy (OS) for hematologic disorders in children. METHODS: The records of 82 consecutive children and adolescents undergoing splenectomy for hematologic disorders between August 1994 and September 1997 were reviewed retrospectively. RESULTS: Fifty patients underwent LS by a lateral approach and 32 underwent OS through a left subcostal incision. Mean age was 7.76 years for LS and 6.9 years for OS. Patient weights were similar: (LS, mean 30.5 kg; OS, mean 27.6 kg). Hematologic indications included hereditary spherocytosis in 43 children (LS 26, OS 17), sickle cell anemia with sequestration in 13 (LS 7, OS 6), immune thrombocytopenic purpura in 14 (LS 8, OS 6), and 12 with other disorders (LS 9, OS 3). Concomitant cholecystectomy was performed in 10 of 50 LS and 6 of 32 OS cases. Accessory spleens were identified in 8 of 32 (25%) OS and 9 of 50 (18%) LS cases (P = .578). No LS procedures required conversion to OS. The mean estimated blood loss was 54.4 mL for LS and 49.0 mL for OS (P = .233). LS required a longer operative time (115 vs 83 minutes, P = .002), less need for postoperative intravenous narcotic (51% vs 100%, P < .0001), lower total narcotic doses (0.239 vs 0.480 mg/kg morphine, P = .006), shorter length of hospital stay (1.4 +/- 0.97 vs 2.5 +/- 1.43 days, P = .0001), and lower average total hospital charges ($5713 vs $6564) than OS. There were no deaths or major complications in either group. CONCLUSIONS: Laparoscopic splenectomy is a safe and effective procedure in children with hematologic disorders resulting in longer operative times, less narcotic administration, shorter length of stay, and lower total hospital charge.

Pilot study of a point-of-use decision support tool for cancer clinical trials eligibility.

Many adults with cancer are not enrolled in clinical trials because caregivers do not have the time to match the patient's clinical findings with varying eligibility criteria associated with multiple trials for which the patient might be eligible. The authors developed a point-of-use portable decision support tool (DS-TRIEL) to automate this matching process. The support tool consists of a hand-held computer with a programmable relational database. A two-level hierarchic decision framework was used for the identification of eligible subjects for two open breast cancer clinical trials. The hand-held computer also provides protocol consent forms and schemas to further help the busy oncologist. This decision support tool and the decision framework on which it is based could be used for multiple trials and different cancer sites.

Dicentric (9;20)(p11;q11) identified by fluorescence in situ hybridization in four pediatric acute lymphoblastic leukemia patients.

Four children with acute lymphocytic leukemia (ALL) and a dic(9;20) are described. All four patients were diagnosed with pre-B-cell All, and the three for whom information was available were CD10+. Age at diagnosis ranged from 23 months to 12 years. All patients achieved remission, with two in continuous remission for 2 years 6 months and 3 years, one patient relapsed, dying 3 years 2 months after diagnosis, and one patient was lost to follow-up. These four patients were initially diagnosed as having a deletion of 9p and loss of one chromosome 20. Re-examination of the karyotypes indicated a possible dic(9;20). The dicentric chromosome was verified using dual-color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 20 on interphase nuclei. Three of the four patients had multiple chromosomal abnormalities in addition to the translocation; one was hypodiploid, one was pseudodiploid, and two were hyperdiploid. This dicentric chromosome was recently described in four adult and nine pediatric patients with ALL [8, 9]. All reported patients had CD10+ pre-B-cell All, and achieved remission, as was the case for our four pediatric dic(9;20) patients. Two of our three patients for whom follow-up is available are in continuous remission as were two adults and five pediatric patients in the previous reports. These studies confirm the dic(9;20) as a recurring abnormality in ALL. Due to the subtle nature of the translocation, FISH is very useful in confirming the chromosomal abnormality.

Pediatric germ cell tumors.

Malignant germ cell tumors are relatively uncommon, accounting for approximately 3% of all childhood malignancies. Occurring with an incidence of approximately 4 per million among children less than 15 years of age, they account for approximately 225 new cases per year in the United States. Germ cell tumors occur in both gonadal and extragonadal sites, with extragonadal and testicular tumors predominating in children less than 3 years of age and with the gonads being the main location of tumors during and after puberty. They occur more frequently in girls than boys. Germ cell tumors are interesting for several reasons: (1) abnormal migration of primordial germ cells account for many of the childhood germ cell tumors; (2) markers exist to allow evaluation of the extent of resection and the development of recurrence for many of the tumors; and (3) the introduction of platinum-based chemotherapy has markedly improved the survival rate for germ cell tumors, as well as the salvage rate for recurrent or metastatic disease.

Functional hyposplenism due to a primary epithelioid hemangioendothelioma of the spleen.

We present a unique case of functional hyposplenism due to massive involvement of the spleen by a rare tumor, an epithelioid hemangioendothelioma, in a 9-year-old girl. To our knowledge, three previous cases of this disorder involving the spleen have been reported, but this is the first associated with functional hyposplenism.

Favorable outcome in children with Beckwith-Wiedemann syndrome and intraabdominal malignant tumors.

Children with Beckwith-Wiedemann syndrome (BWS) have an increased risk of developing Wilms' tumors, hepatoblastomas, and adrenal tumors. This study evaluates disease-free survival in children with BWS and intraabdominal tumors. Sixteen tumors occurred in 13 children with BWS (8 boys, 5 girls). Diagnoses included Wilms' tumor (10) (2 bilateral, 20%), hepatoblastoma (2), bladder rhabdomyosarcoma (1), and adrenal cortical tumor (1). In the 10 children with Wilms' tumor, the average age at diagnosis was 3.5 years (range, 7 months to 5 years). Nine of 10 had initial tumor resection, chemotherapy, and radiation therapy (when indicated). One child with bilateral disease had tumor biopsy, chemotherapy, and partial nephrectomy. Tumors were classified as stage I (5), stage II (2), stage IV (1) and stage V (2), all with favorable histology. Disease-free survival rate was 100% with median follow-up of 9 years (range, 4 to 22 years). One patient had a left adrenal tumor detected during screening sonography 11 years after Wilms' tumor resection. Two infants with advanced-stage hepatoblastoma responded to chemotherapy, allowing subsequent complete hepatic resection. Both tumors had unfavorable histology. Both completed postoperative chemotherapy and have no evidence of disease (NED) with normal alpha-fetoprotein levels at 21 and 12 months, respectively, after tumor detection. One patient with stage III (group 3) bladder rhabdomyosarcoma underwent partial cystectomy following chemoradiation and is alive (NED) after 20 months. Children with BWS should be screened at regular intervals (every 3 to 6 months) for renal, adrenal, and hepatic tumors. The exact duration of screening is not yet determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-free synthesis and co-translational processing of the human asialoglycoprotein receptor.

The human asialoglycoprotein receptor is a 46-kDa membrane glycoprotein. It is initially synthesized as a 40-kDa precursor species possessing two N-linked high-mannose oligosaccharides which is subsequently converted to the 46-kDa mature product upon modification of its oligosaccharides of the complex form [Schwartz, A. L. & Rup, D. (1983) J. Biol. Chem. 258, 11 249-11 255]. To investigate further the biosynthesis of the human asialoglycoprotein receptor, we have utilized a cell-free wheat germ translation system supplemented with dog pancreatic microsomal membranes and programmed with HepG2 and human liver RNA. The primary translation product of the human receptor is a single 34-kDa species and this species is expressed throughout human fetal and adult development. The primary translation product possesses no cleavable signal peptide and is cotranslationally glycosylated to form the 40-kDa precursor species. In addition, the human asialoglycoprotein receptor is co-translationally inserted into microsomal membranes such that a 4-kDa cytoplasmic tail is susceptible to trypsin digestion.

Influence of the N-linked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor.

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein of 46,000 Da which possesses two N-linked oligosaccharide chains (Schwartz, A. L., and Rup, D. (1983) J. Biol. Chem. 258, 11249-11255). In order to examine the role of N-linked oligosaccharides in the biosynthesis, intracellular routing, and function of the ASGP-R, we have used Hep G2 cells, which have a large number of ASGP-R, and two inhibitors of glycosylation, swainsonine and tunicamycin. In the presence of swainsonine, newly synthesized ASGP-R is a 43,000-Da species which is endoglycosidase H-sensitive, appears on the Hep G2 cell surface, and specifically binds 125I-asialoorosomucoid (ASOR). In the presence of tunicamycin newly synthesized ASGP-R is a 34,000-Da nonglycosylated species which appears on the Hep G2 cell surface where it specifically binds 125I-ASOR. There is no major effect on subsequent uptake and degradation of 125I-ASOR in cells whose ASGP-R was synthesized in the presence of tunicamycin. The turnover of ASGP-R synthesized in the presence of either swainsonine or tunicamycin is not significantly altered from that found for the normal 46,000-Da species. Thus, it appears that the two N-linked oligosaccharide chains of the human ASGP-R do not play a major role in the intracellular routing, turnover, or function of ASGP-R.

Melanotic neuroectodermal tumor of infancy: an important mimicker of paratesticular rhabdomyosarcoma.

We report a case of a paratesticular tumor in a 6-month-old infant. This tumor was originally believed to represent a poorly differentiated sarcoma of the cord but upon further pathological consultation it was recognized as a rare melanotic neuroectodermal tumor of infancy involving the epididymis. Since the former is a highly aggressive lesion and the latter an indolent tumor, treatment of these 2 entities differs markedly. Therefore, although the histological distinction between these lesions is often difficult, it is of critical importance.

A case-control retrospective study of the efficacy of granulocyte-colony-stimulating factor in children with neuroblastoma.

PURPOSE: We conducted a retrospective case-control study to examine the effect of granulocyte-colony-stimulating factor (G-CSF) on the duration of the neutrophil nadir and other clinical parameters in children with neuroblastoma. PATIENTS AND METHODS: We retrospectively reviewed 85 courses of the same chemotherapy in 16 consecutive neuroblastoma patients. The first nine patients received no growth factor and the following seven patients received G-CSF. Data obtained included days of neutropenia, fever rate and duration, hospitalization rate and duration, antibiotic duration, and infection rate. RESULTS: Patients who received G-CSF had a significant decrease in the period of neutropenia (mean 5.4 +/- 2.6 days per course vs. 11.4 +/- 4.1 days per course in the control group; p < 0.001). There were no statistically significant differences in episodes of fever per course, rate of hospitalization per course, duration of hospitalization, or duration of antibiotic therapy. Control patients had documented infections during 16% (nine of 56) of their chemotherapy courses, whereas the patients receiving G-CSF had infections during 7% (two of 29) of their courses, but this difference was not statistically significant (p = 0.318). We calculated that a study of 220 courses in each group would be needed to have adequate power to confirm that this difference is statistically significant. CONCLUSIONS: The administration of G-CSF in this patient population did result in fewer days of neutropenia, a finding that has been reported previously in several adult studies. However, we conclude that the clinical benefit of more rapid hematologic recovery in children remains uncertain and deserves further investigation in a large, prospective multicenter trial.