Showing papers by "Boston Children's Hospital published in 1989"

Monoclonal antibody-mediated tumor regression by induction of apoptosis

Abstract: To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation of cells bearing APO-1 in vitro in a manner characteristic of a process called programmed cell death or apoptosis. Cell death was preceded by changes in cell morphology and fragmentation of DNA. This process was distinct from antibody- and complement-dependent cell lysis and was mediated by the antibody alone. A single intravenous injection of anti-APO-1 into nu/nu mice carrying a xenotransplant of a human B cell tumor induced regression of this tumor within a few days. Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo. Thus, induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO-1 may be a useful therapeutic approach in treatment of malignancy.

Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics

Abstract: Serial electron microscopy and 3-D reconstructions of dendritic spines from hippocampal area CA 1 dendrites were obtained to evaluate 2 questions about relationships between spine geometry and synaptic efficacy. First, under what biophysical conditions are the spine necks likely to reduce the magnitude of charge transferred from the synapses on the spine heads to the recipient dendrite? Simulation software provided by Charles Wilson (1984) was used to determine that if synaptic conductance is 1 nS or less, only 1% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. If synaptic conductance approaches 5 nS, however, 33% of the hippocampal spine necks are sufficiently thin and long to reduce charge transfer by more than 10%. Second, is spine geometry associated with other anatomical indicators of synaptic efficacy, including the area of the postsynaptic density and the number of vesicles in the presynaptic axon? Reconstructed spines were graphically edited into head and neck compartments, and their dimensions were measured, the areas of the postsynaptic densities (PSD) were measured, and all of the vesicles in the presynaptic axonal varicosities were counted. The dimensions of the spine head were well correlated with the area of PSD and the number of vesicles in the presynaptic axonal varicosity. Spine neck diameter and length were not correlated with PSD area, head volume, or the number of vesicles. These results suggest that the dimensions of the spine head, but not the spine neck, reflect differences in synaptic efficacy. We suggest that the constricted necks of hippocampal dendritic spines might reduce diffusion of activated molecules to neighboring synapses, thereby attributing specificity to activated or potentiated synapses.

Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease.

Abstract: Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix.

Abstract: The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells

Abstract: Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes.

Abstract: Using an in vitro coculture system to mimic the interactions between the cells of the vessel wall, we have previously shown that pericytes and smooth muscle cells (SMC) inhibit the growth of capillary endothelial cells (EC). We have undertaken studies to determine the mechanism of this inhibition. Using conditioned media and affinity-purified antibodies to transforming growth factor beta (TGF-beta), we now demonstrate that activated TGF-beta produced in these cocultures mediates EC growth inhibition. No inhibitory activity was detected when media conditioned by individual cultures of EC, SMC, or pericytes were examined for their effect on EC growth. In contrast, media conditioned by cocultures of EC-SMC and EC-pericytes inhibited EC proliferation to the same degree as the coculture itself. Immunoadsorption of coculture-derived conditioned media with antibodies to TGF-beta eliminated the inhibitory activity. Acid activation of serum-free media conditioned by any of the cells cultured alone yielded inhibitory activity, whereas activation of coculture conditioned media did not increase its inhibitory activity. Addition of anti-TGF-beta neutralizing antibodies to cocultures blocked the pericyte-mediated EC growth inhibition. These results indicate that latent TGF-beta is produced by these cells and it is activated by a mechanism that requires contact between the two cell types.

Treatment of Wilms' tumor. Results of the Third National Wilms' Tumor Study.

Abstract: The Third National Wilms' Tumor Study sought to reduce treatment for low-risk patients and find better chemotherapy for those at high risk for relapse. Eligible patients (1439) were randomized according to stage (I-IV) and histology (favorable [FH] or unfavorable [UH]), and contributed data to survival and relapse-free survival (RFS) analyses. Four-year (postnephrectomy) survival percentages and randomized treatment regimens for low-risk patients were 96.5% for 607 Stage I/FH patients who received dactinomycin (Actinomycin D [AMD], Merck Sharp & Dohme, West Point, PA) and vincristine (VCR) for 10 weeks versus 6 months; 92.2% for 278 Stage II/FH patients; and 86.9% for 275 Stage III/FH patients who received AMD + VCR +/- Adriamycin (ADR, Adria Laboratories, Columbus, OH) for 15 months. Stage II/FH patients also had either zero or 2000 cGy irradiation (RT) postoperatively and Stage III/FH patients either 1000 or 2000 cGy. Four-year survival was 73.0% for 279 high-risk patients (any Stage IV, all UH) who received postoperative radiation therapy (RT) and AMD + VCR + ADR +/- cyclophosphamide (CPM). Statistical analysis of survival and RFS experience shows that the less intensive therapy does not worsen results for low-risk patients and CPM does not benefit those at high risk.

Mammographically detected duct carcinoma in situ. Frequency of local recurrence following tylectomy and prognostic effect of nuclear grade on local recurrence.

Abstract: Seventy-nine patients with mammographically detected foci of duct carcinoma in situ (DCIS) of histologically confirmed extents of 25 mm or less, were treated by tylectomy without irradiation or axillary dissection. Adequacy of excision was confirmed histologically, by radiographic-pathologic correlation and by postoperative mammographic examination. Eight patients (10.1%) have recurred locally in the immediate vicinity of the biopsy site. Four patients developed recurrent in situ disease identified mammographically, and all were initially treated by reexcision. One of these patients subsequently elected to undergo mastectomy; no residual in situ or invasive disease was detected in the breast or in axillary lymph nodes. Four patients developed recurrent invasive disease; 50% of these recurrences were detected mammographically. All patients were treated by mastectomy with node dissection. Three had confirmed minimal invasive carcinomas and were N0, one patient had a 13-mm invasive lobular carcinoma with a single Group I micrometastasis. All patients, including those treated for a recurrence, are presently free of disease but three patients died of heart disease. Nuclear grade would appear to identify subsets of DCIS more likely to produce local failure after tylectomy alone. Duct carcinoma in situ with high-grade nuclear morphology and comedo-type necrosis was associated with a 19% local recurrence rate after an average interval of 26 months; only one of ten patients with intermediate-grade DCIS developed a local recurrence at 87 months; and none of 33 patients with DCIS of micropapillary/nonnecrotic cribriform type and low-grade nuclear morphology developed local recurrence in the follow-up period.

Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion.

Abstract: Many Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients have a cytogenetic deletion of 15q11q13. While AS and PWS share a similar cytogenetic anomaly, they have very different clinical phenotypes. DNAs from 4 AS patients were examined using 5 chromosome 15q11q13-specific cloned DNA segments. With the present level of resolution, the molecular deletions between AS and those previously reported for PWS did not appear to differ. However, in contrast to the paternal inheritance of the deleted chromosome 15 observed in the majority of PWS patients, maternal inheritance of the deleted chromosome 15 was demonstrated in the AS patients by restriction fragment length polymorphisms (RFLPs).

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues.

Abstract: The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family. We have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse. Expression levels of each mdr gene are dramatically different in various mouse tissues. Specific mdr RNA transcripts of approximately 4.5, 5, and 6 kilobases have been detected. Each of the mdr genes has a specific RNA transcript pattern. These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.

The human mannose-binding protein functions as an opsonin.

Abstract: The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O-polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms.

Dieting Behavior and Eating Attitudes in Children

Abstract: Many studies document the high prevalence of dieting and abnormal eating behaviors in adolescents and young adults. The age of onset of eating disorders remains unclear because there are no published surveys of voluntary dieting and eating attitudes in young children. A total of 318 girls and boys were surveyed from two randomly chosen schools in middle-income neighborhoods. The children were equally distributed among grades 3 through 6. The survey included a children's version of the Eating Attitudes Test and a demographic and dieting questionnaire. Results showed that 45% of the children wanted to be thinner, 37% had already tried to lose weight, and 6.9% scored in the anorexia nervosa range, closely matching the published results concerning teenagers and young adult women. Several questions were isolated by univariate and multiple regression analyses that accurately (P less than .05) predicted children's Eating Attitude Test scores. The fact that young children admit having diet preoccupation and atypical eating attitudes is shown in this study. It may be possible to improve early case identification of children at risk for anorexia nervosa and perhaps improve intervention strategies by focusing on the early development of dieting behavior in young children.

Treatment of pulmonary hemangiomatosis with recombinant interferon alfa-2a.

Abstract: PULMONARY hemangiomatosis is a rare, fatal disorder characterized by diffuse microvascular proliferation within the lung. The disease most often affects children and young adults and usually progresses rapidly, causing death from pulmonary hypertension or bleeding. Most cases have been diagnosed at autopsy. Corticosteroids and cyclophosphamide have been used for this condition, but no successful therapy has been described. We describe a patient with pulmonary hemangiomatosis who presented with digital clubbing and bilateral interstitial pulmonary disease. After a decline in the patient's exercise tolerance, therapy with interferon alfa-2a was initiated and has been continued for 14 months at the time of . . .

Rhabdoid Tumor of Kidney: A Report of 111 Cases from the National Wilms' Tumor Study Pathology Center

Abstract: We review 111 cases of rhabdoid tumor of kidney (RTK), including 79 entered on the National Wilms' Tumor Study (NWTS). Median age at diagnosis was 11 months, with a range from 0 to 106 months. The male:female ratio was 1.5:1. Gross features included a characteristic involvement of perihilar renal parenchyma. A wide histological spectrum was encountered, including nine major morphological patterns (classical, epithelioid, sclerosing, lymphomatoid, histiocytoid, etc.). These appearances invite confusion with other renal neoplasms. Ultrastructural studies were performed in 20 cases; immunocytochemical studies were performed in 11. Vimentin was demonstrated in all tumors; epithelial membrane antigen was seen in 7. Nonspecific decoration of cytoplasmic inclusions by a variety of immunostains was found in several cases. Several findings suggested that RTK might arise from primitive cells involved in formation of the renal medulla. There was no evidence of a histogenetic relationship to Wilms' tumor, although RTK may overlap with mesoblastic nephroma and clear cell sarcoma. Of the 70 NWTS patients with adequate follow-up, 56 (80%) have died. Every patient presenting with distant metastases died, whereas 10 of 20 with negative nodes survived. Survival rates were higher for girls (56.3% versus 11.1%). None of the histological variables had independent prognostic significance.

Mortality in Children and Adolescents With Sickle Cell Disease

Abstract: A study of the natural history of sickle hemoglobinopathies was begun in March 1979. By August 1987, a total of 2824 patients less than 20 years of age were enrolled. There have been 14,670 person-years of follow-up. Seventy-three deaths have occurred. Most of the deaths were in patients with hemoglobin SS. The peak incidence of death was between 1 and 3 years of age, and the major cause in these young patients was infection. Cerebrovascular accidents and traumatic events exceeded infections as a cause of death in patients greater than 10 years of age. There was limited success in identifying risk factors for death. Comparison of this study's overall mortality of 2.6% (0.5 deaths per 100 person-years) with previous reports indicates improvement of survival in US patients less than 20 years of age with sickle hemoglobinopathies. This improvement is most likely due to parental education and counseling about the illness and the early institution of antibiotics in suspected infections.

Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.

Abstract: Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.

Development of a modified treatment evaluation inventory

Abstract: Kazdin's (1980a) Treatment Evaluation Inventory (TEI) is the major instrument used to assess parents' acceptance of procedures for behavior problem children. The length of the TEI, however, as well as problems with its scaling and wording limits its value as a clinical research instrument. In the present study, three experiments were conducted to develop a modified TEI. In Experiment 1, 153 parents completed the TEI to evaluate a behavioral treatment for noncompliant and oppositional children. A factor analysis of the data was used to obtain a reliable factor structure for the TEI and to construct a 9-item TEI-Short Form (TEI-SF) with a 5-point scale, consistent anchors on the scale, and simplified text and instructions. Experiment 2 evaluated the psychometric characteristics of the TEI and the TEI-SF. These data indicated the TEI-SF is a sound alternative to the original TEI. Experiment 3 compared the readability and completion time of the two instruments.

Induction of human IgE synthesis requires interleukin 4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens.

Abstract: The induction of IgE synthesis by IL-4 requires T cells and monocytes, as well as T cell- and monocyte-derived cytokines. Optimal cytokine combinations, however, fail to induce highly purified B cells to secrete IgE, indicating that additional signals are required. We show herein that the induction of human IgE synthesis by rIL-4 requires cognate interaction between the T cell receptor/CD3 complex on T cells and MHC class II antigens on B cells: mAbs directed against these molecules completely blocked IL-4-dependent IgE induction. mAbs against cell adhesion molecules (CD2, CD4, LFA-1) also inhibited IgE synthesis induced by IL-4, confirming that cell-cell contact is necessary for IgE induction. The requirement for cognate T/B cell interaction was further shown by comparing the IgE-inducing ability of two human IL-4-producing alloreactive T cell clones: F6, which recognizes MHC class II antigens on both B cells and monocytes, and A1, which recognizes an HLA-DP- associated epitope expressed on monocytes, but not on B cells. When incubated with B cells and monocytes from a normal donor bearing the appropriate alloantigen, clone F6, but not clone A1, induced vigorous IgE synthesis, although both clones proliferated and secreted IL-4. Taken together, our results suggest that at least two, possibly synergizing, signals are required for the T cell-dependent induction of IgE synthesis by B cells: one signal is delivered by cognate T/B cell interaction, the other by T cell-derived IL-4.

Mechanism of calcification: Role of collagen fibrils and collagen‐phosphoprotein complexes in vitro and in vivo

Abstract: Samples of decalcified chicken bone together with varying concentrations of phosphoproteins from bone or egg yolk (phosvitin) were used in vitro as heterogenous nucleators for the induction of Ca-P apatite crystals. The lag time between exposure of the collagen-phosphoprotein complexes and the time nucleation of crystals occurred decreased as the concentration of Ser(P) and Thr(P) increased. Enzymatic cleavage of the phosphate groups by wheat germ and phosphatase reversed this effort, indicating that the phosphate group per se principally facilitated the nucleation of Ca-P crystals by the phosphoprotein complex and collagen.

The human mannose-binding protein gene. Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10.

Abstract: The human mannose-binding protein (MBP) plays a role in first line host defense against certain pathogens. It is an acute phase protein that exists in serum as a multimer of a 32-kD subunit. The NH2 terminus is rich in cysteines that mediate interchain disulphide bonds and stabilize the second collagen-like region. This is followed by a short intervening region, and the carbohydrate recognition domain is found in the COOH-terminal region. Analysis of the human MBP gene reveals that the coding region is interrupted by three introns, and all four exons appear to encode a distinct domain of the protein. It appears that the human MBP gene has evolved by recombination of an ancestral nonfibrillar collagen gene with a gene that encodes carbohydrate recognition, and is therefore similar to the human surfactant SP-A gene and the rat MBP gene. The gene for MBP is located on the long arm of chromosome 10 at 10q11.2-q21, a region that is included in the assignment for the gene for multiple endocrine neoplasia type 2A.

Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1)

Abstract: The human erythrocyte anion-exchange protein (band 3 or AE1) was cloned from a fetal liver cDNA library. Three overlapping clones, encompassing 3637 nucleotides, were analyzed in detail. These encode a 911-amino acid protein (Mr 101,791) and detect a single 4.7-kilobase species in human reticulocyte RNA. The corresponding gene is located on chromosome 17. The protein is similar in structure to other anion exchangers and is divided into three regions: a hydrophilic, cytoplasmic domain that interacts with a variety of membrane and cytoplasmic proteins (residues 1-403); a hydrophobic, transmembrane domain that forms the anion antiporter (residues 404-882); and an acidic, C-terminal domain of unknown function (residues 883-911). The N-terminal domain contains several conserved sections (e.g., residues 57-86, 102-164, 219-347, and 375-403), some of which may contribute to binding sites for ankyrin, protein 4.1, or protein 4.2. The membrane domain is highly conserved with the exception of a single segment (residues 543-567) that contains several sites for cleavage of the protein by extracellular proteases. Based on hydropathy analyses and the wealth of available topographical and functional data, a model is proposed in which the protein crosses the membrane 14 times.

Spinal cord injury without radiographic abnormality in children--the SCIWORA syndrome.

Abstract: Spinal cord injury in children frequently occurs without fracture or dislocation. The clinical profiles of 55 children with spinal cord injury without radiographic abnormalities (SCIWORA) are reported in detail to illustrate features of this syndrome. No patient had vertebral fracture or dislocation on plain films and tomographies. There were ten upper cervical (C1-C4), 33 lower cervical (C5-C8), and 12 thoracic cord injuries; of these, 22 were complete or severe lesions and 33 were mild lesions. The mechanism of the neural injury probably relates to the inherent elasticity of the juvenile spine, which permits self-reducing but significant intersegmental displacements when subjected to flexion, extension, and distraction forces. The spinal cord is therefore vulnerable to injury even though the vertebral column is spared from disruption, and this vulnerability is most evident in children younger than 8 years. All but one of the 22 children with profound neurologic injuries were younger than 8 years (p less than 0.000001), whereas 24 of 33 children with mild injuries were older. Younger children were also more likely to have severe upper cervical lesions (p less than 0.05); lower cervical lesions were distributed evenly through the ages of 6 months to 16 years. Thoracic injuries most commonly resulted from distraction or crushing. Distraction invariably involved violent forces, and crush injuries were usually caused by children being run over while lying prone, when the spinal column was acutely bowed towards the spongy abdominal and thoracic cavities. Fifteen children had delayed onset of neurologic deficits; nine of these had transient warning symptoms of paresthesia, subjective paralysis, and Lhermitte's phenomenon 30 minutes to 4 days before the onset of deterioration. Eight other children suffered a second SCIWORA 3 days to 10 weeks after the initial SCIWORA. The spines in these children were presumably rendered incipiently unstable by the initial injury and thus were susceptible to additional, often more severe, neurologic trauma. The long-term neurologic outcome in children with SCIWORA is solely determined by their admission neurologic status. Realistically, the outcome can thus only be improved by: 1) ruling out occult fractures and subluxation which will require surgical fusion; 2) identifying patients likely to have delayed deterioration; and 3) preventing recurrent SCIWORA. Our experience and recommendations in these regards are discussed.