Possible role of nucleus-membrane interaction in capping of surface membrane receptors.

Interaction of multivalent ligands and cell surface receptors can induce redistribution of these receptors to form patches and caps. In this study, we have investigated the role of nucleus-membrane interaction in the capping of membrane components. Mouse L cells and leukemia EL4 cells were enucleated with the aid of cytochalasin B, yielding cytoplasts and karyoplasts. Capping of surface receptors was induced by allo- and hetero-immune sera followed by fluorescein-conjugated antiglobulin serum, or by the plant lectin concanavalin A. Capping could easily be induced in intact cells, but virtually no capping was detected in the nucleus-free cytoplasts. Interestingly, karyoplasts, which posses cell-membrane components but very little cytoplasm, could be easily induced to cap their surface antigens. Hence, cap formation of membrane components seems not to be an autonomous membrane process. The data suggest that interaction of surface membranes and inner cell components associated with the nucleus is involved in the movement of surface membrane receptors.     

Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vitro.

Cytotoxic hybridomas were generated by polyethylene glycol-induced fusion of cytotoxic T lymphocytes (CTL) and BW5147 lymphoma cells. The CTL populations used for fusion were obtained from BALB/c (H-2d) mice primed with leukemia EL4 of C57BL/6 (H-2b) and restimulated either in vivo or in vitro. To circumvent possible CTL-mediated nonspecific lysis of BW5147 cells during fusion, the CTL were transiently inactivated by trypsin prior to fusion. Four cytolytically active hybridomas were obtained, cloned, and subcloned. Hybrid clones lysed all H-2b leukemic target cells tested but not lipopolysaccharide- or concavanalin A-stimulated C57BL/6 lymphoblasts or non-H2b target tumor cells. The mechanism of hybridoma-mediated killing of target cells in vitro appears to be similar to that of parental CTL, although some differences have been observed. The hybridomas appear to possess neither natural killing nor antibody-dependent cytolytic activity. Clones of hybrids propagated in culture for over 6 months without the addition of known external stimulus (i.e., independent of cell growth factor and antigen) exhibit specific lytic activity against H-2b tumor cells. Such autonomous hybridomas will provide a tool for studying the mechanism of CTL-mediated lysis and the nature of the CTL receptors.     

Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.     

Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient "compromised" bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.     

DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH) OF THE SPINE: A CAUSE OF BACK PAIN? A CONTROLLED STUDY

This is the first controlled study of the frequency of backpain in a European caucasian population with diffuse idiopathicskeletal hyperostosis (DISH). Elderly patients admitted to hospital for reasons other thanback pain were assessed for the presence of spinal DISH usingthe routine lateral chest radiograph films. A total of 106 probands(82 males, 24 females) with a mean age of 70 years fulfilledthe criteria for DISH as defined previously. One hundred andseventyeight patients (117 males, 61 females) not meeting thesecriteria were used as controls. The prevalence of back painwas assessed by a blinded interviewer using a structured questionnaire.Our primary hymthesis was that spinal DISH positive probandshad not had back pain more often than controls. The controlledstudy showed no statistically significant difference in painfrequency between spinal DISH positive probands and controlsat any spinal level. We conclude that back pain does not occur more often in radiographicallydefined DISH positive probands than in controls. The radiologicalfinding of spinal DISH, as far as it does not lead to stenosisof the spinal canal or dysphagia, thus seems to be a findingwithout clinical relevance. KEY WORDS: Spine, Radiographs, Pain, Osteoarthritis, Forestier's disease, Ankylosing vertebral hyperostosis     

Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozen-thaw transfers

In-vitro fertilization patients (n = 15) at risk of ovarian hyperstimulation syndrome (OHSS) (oestradiol > or =4500 pg/ml on the day of human chorionic gonadotrophin administration and 25 or more follicles of intermediate or large size) underwent aspiration of all follicles and cryopreservation of all fertilized oocytes at the pronuclear stage. Patients were monitored for up to 2 weeks post- retrieval. Subsequent transfer of cryopreserved-thawed embryos was performed in programmed cycles using exogenous oestrogen and progesterone for endometrial preparation. Two patients (13%) developed OHSS necessitating hospitalization and vaginal aspiration of ascitic fluid. Two other patients (13%) developed moderate OHSS requiring ascitic fluid vaginal aspiration in the office setting, with dramatic improvement of the condition. Subsequent transfer of cryopreserved- thawed embryos yielded a clinical pregnancy rate of 58% per transfer and ongoing or delivery rates of 42 and 67% per transfer and per patient respectively. By eliminating pregnancy potential with cryopreservation of all prezygotes and examining the pregnancy potential with subsequent cryopreserved-thawed transfers, it is concluded that OHSS is reduced, but not eliminated for patients at risk. Subsequent transfer of cryopreserved-thawed prezygotes in a programmed cycle with exogenous steroids yields an excellent pregnancy rate.      

Diabetic ketoacidosis

Diabetic ketoacidosis (DKA) is a true pediatric and medical emergency. Diagnosis should be entertained and confirmed within 30 min of presentation. Any delay in making the diagnosis or instituting fluid and electrolyte correction is likely to increase morbidity and mortality. Slow and careful monitoring and correction of water, sodium and potassium levels should decrease DKA-associated problems with either continuous intravenous low-dose insulin or intramuscular insulin protocols designed to slowly bring the hyperglycemic and hyperosmotic state towards normal homeostasis. Special attention should be paid to potassium replenishment. Most patients do not require bicarbonate replacement. Cerebral edema, when it occurs, is associated with an approximately 50% morbidity and mortality; therefore, all attempts should be made at early recognition and prevention since treatment is less than ideal. Recurrent ketoacidosis is often related to omitted insulin and major psychosocial turmoil in the family, such as depression substance abuse, physical and/or sexual abuse. Prevention of recurrent DKA remains a major challenge for diabetologists and involves detailed assessment of family psychodynamics plus responsibility for home monitoring and insulin administration by a mature adult. Sick day guidelines should be taught and reviewed frequently in an effort to decrease ketoacidosis and metabolic decompensation during episodes of intercurrent illness. □ Cerebral edema, diabetic ketoacidosis, diabetes mellitus, DKA, sick day guidelines, sick day management, type 1 diabetes mellitus     

A STD/HIV prevention trial among adolescents in managed care

OBJECTIVE: To determine if sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) infection, risk assessment, and education tools provided as part of office-based primary care reduce adolescent risky sexual behaviors. DESIGN: A randomized intervention trial with 3- and 9-month follow-up. SETTING: Five staff-model managed care sites in Washington, DC (n = 19 pediatricians). PATIENTS: Consecutive 12- to 15-year-olds receiving a general health examination; 81% minority. Participation rate = 215/432 (50%). Nine-month follow-up rate = 197/215 (92%). INTERVENTION: Audiotaped STD risk assessment and education about staying safe (safer = condoms, safest = abstinence). MAIN OUTCOME MEASURES: Adolescent-reported sexual intercourse and condom use. RESULTS: More intervention adolescents reported pediatrician discussion on 11/13 sexual topics. Although more vaginal intercourse (odds ratio [OR] = 2.46, 95% confidence interval [CI] = 1.04-5.84) was reported in the intervention group at 3 months, this was not true of overall sexual intercourse (OR = 1.55, 95% CI =.73-3.32). More sexually active adolescents reported condom use in the intervention group at 3 months (OR = 18.05, 95% CI = 1.27-256.03). At 9 months, there were no group differences in sexual behaviors; however, more signs of STD were reported by the control (7/103) than the intervention group (0/94). CONCLUSIONS: STD risk assessment and education tools administered in a single office visit facilitated STD/HIV prevention education. Any impact on sexual activity and condom use was short-lived. Further research is needed to develop brief, office-based sexual risk reduction for young adolescents.