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Baltimore↗
Biomedical subjects
J Kaplan
Publications and source records attributed to J Kaplan.
Inhibition of late endosome-lysosome fusion: studies on the mechanism by which isotonic-K+ buffers alter intracellular ligand movement.
Incubation of alveolar macrophages or hepatocytes in media in which Na+ is replaced by K+ ("isotonic-K buffer") inhibited the movement of internalized ligand from late endosomes to lysosomes (Ward et al.: Journal of Cell Biology 110:1013-1022, 1990). In this study we investigate the mechanism responsible for the isotonic-K+ block in movement of ligand from late endosomes to lysosomes. We observed that iso-K+ inhibition of endosome-lysosome fusion is not unique to alveolar macrophages or hepatocytes but can be seen in a variety of cell types including J774 and Hela cells. The inhibition in intracellular ligand movement was time dependent with the maximum change occurring after 60 minutes. Once established the inhibition resulted in a prolonged and apparently permanent decrease in vesicle movement. Cells were able to recover from the effects of iso-K+ buffers over a time course of 5-10 minutes when placed back in Na(+)-containing media. The effect of iso-K+ buffers was independent of intracellular pH changes and appeared to involve cell swelling. When cells were incubated in iso-K+ buffers under conditions in which cell volume changes were reduced, intracellular ligand movement approached normal levels. Such conditions included replacing Cl- with the less permeant anion gluconate, and by addition of sucrose to isotonic-K+ buffers. Analysis of the mechanism by which changes in cell volume could alter intracellular movement ruled out changes in cyclic nucleotides, Ca2+, or microtubules. These results suggest that changes in cell shape or volume can alter intracellular transport systems by novel routes.
Animals↗
Clinical and genetic heterogeneity in retinitis pigmentosa.
The clinical course of defective vision and blindness has been investigated in relation to different modes of genetic transmission in a large series of 93 families with retinitis pigmentosa (RP). For autosomal dominant RP, two clinical subtypes could be distinguished according to the delay in macular involvement. In the severe form, macular involvement occurred within 10 years, while in the mild form, macular involvement occurred after 20 years. Interestingly, a significant increase of mean paternal age (38.8 years, mean controls in France = 29.1 years, P less than 0.001) was found in this form of RP, a feature which is suggestive of new mutations. For autosomal recessive RP, four significantly different clinical subtypes could be recognized, according to both age of onset and the pattern of development (P less than 0.001), namely cone-rod dystrophy and early-onset severe forms on the one hand (mean age of onset = 7.6 years), late-onset mild forms and senile forms on the other. Similarly, two significantly different clinical subtypes could be recognized in X-linked RP, according to both mode and age of onset, which were either myopia (mean age = 3.5 +/- 0.5 years) or night blindness (mean age = 10.6 +/- 4.1 years. P less than 0.001). By contrast, no difference was noted regarding the clinical course of the disease, which was remarkably severe whatever the clinical subtype (blindness before 25 years). In addition, all obligate carriers in our series were found to have either severe myopia or pigment deposits in their peripheral retina. Finally, sporadic RP represented the majority of cases in our series (42%). There was a considerable heterogeneity in this group, and at least three clinical forms could be recognized, namely cone-rod dystrophy, early onset-severe forms and late onset moderate forms. At the beginning of the disease, the hereditary nature of the sporadic forms was very difficult to ascertain (especially between 7-10 years) and only the clinical course could possibly provide information regarding the mode of inheritance. However, the high level of consanguinity, and the high sex ratio in early onset and severe sporadic forms (including cone-rod dystrophy), was suggestive of an autosomal or X-linked recessive inheritance, while increased paternal age in late onset forms was suggestive of autosomal dominant mutations.
Adolescent↗
Leukocyte immunophenotyping by flow cytometry in a multisite study: standardization, quality control, and normal values in the Transfusion Safety Study. The Transfusion Safety Study Group.
The Transfusion Safety Study (TSS) is a multicenter, cooperative investigation of factors that may determine the occurrence and modify the expression of transfusion-transmitted infections. A flow cytometry laboratory was established in each of the six participating centers in order to avoid alterations in cell phenotypes which may be caused by shipping delays, temperature changes, and handling. As a consequence, in order to assure compatibility of results, stringent standardization, quality control, and proficiency testing procedures were developed. This paper documents (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibodies used in the study; (ii) the effects of variations in light scatter cursor location for certain antibodies; (iii) a quality control program and data management and analysis system, each specifically designed for the study; and (iv) presents extensive data on age- and sex-related reference (normal) ranges for the several individual and paired monoclonal antibodies used in the study. Problems encountered, including obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications.
Blood Transfusion↗
Obstructive sleep apnea syndrome.
Obstructive sleep apnea syndrome is the most common cause of hypersomnolence in patients referred to sleep disorders centers. This type of sleep apnea is characterized by loud snoring, nocturnal oxyhemoglobin desaturation, and disrupted sleep that leads to daytime hypersomnolence. The anatomic configuration of the pharynx and the physiologic responses to occlusion of the upper airway play a major role in the pathogenesis of this disorder. Polysomnography can accurately identify obstructive sleep apnea, and the multiple sleep latency test allows an objective measurement of daytime alertness. Weight loss and training the patient to sleep in a lateral position are frequently used to alleviate mild cases. Nasally applied continuous positive airway pressure is an extremely effective modality for treating moderate and severe obstructive sleep apnea. Surgical correction of obvious anatomic defects has a role in diminishing obstructive sleep apnea, but the exact role of surgical intervention in patients without obvious anatomic defects remains unknown. The choice of therapy should be tailored to the individual patient with sleep apnea, and careful follow-up is essential to ensure a positive response to therapy.
Humans↗
Fusion of sequentially internalized vesicles in alveolar macrophages.
Previously we reported that internalized ligand-receptor complexes are transported within the alveolar macrophage at a rate that is independent of the ligand and/or receptor but is dependent on the endocytic apparatus (Ward, D. M., R. S. Ajioka, and J. Kaplan. 1989. J. Biol. Chem. 264:8164-8170). To probe the mechanism of intracellular vesicle transport, we examined the ability of vesicles internalized at different times to fuse. The mixing of ligands internalized at different times was studied using the 3,3'-diaminobenzidine/horseradish peroxidase density shift technique. The ability of internalized vesicles to fuse was dependent upon their location in the endocytic pathway. When ligands were administered as tandem pulses a significant amount of mixing (20-40%) of vesicular contents was observed. The pattern of mixing was independent of the ligands employed (transferrin, mannosylated BSA, or alpha macroglobulin), the order of ligand addition, and temperature (37 degrees C or 28 degrees C). Fusion was restricted to a brief period immediately after internalization. The amount of fusion in early endosomes did not increase when cells, given tandem pulses, were chased such that the ligands further traversed the early endocytic pathway. Little fusion, also, was seen when a chase was interposed between the two ligand pulses. The temporal segregation of vesicle contents seen in early endosomes was lost within late endosomes. Extensive mixing of vesicle contents was observed in the later portion of the endocytic pathway. This portion of the pathway is defined by the absence of internalized transferrin and is composed of ligands en route to lysosomes. Incubation of cells in iso-osmotic medium in which Na+ was replaced by K+ inhibited movement of internalized ligands to the lysosome, resulting in ligand accumulation within the late endocytic pathway. The accumulation of ligand was correlated with extensive mixing of sequentially internalized ligands. Although significant amounts of ligand degradation were observed, this compartment was devoid of conventional lysosomal markers such as acid glycosidases. These results indicate changing patterns of vesicle fusion within the endocytic pathway, with a complete loss of temporal ligand segregation in a prelysosomal compartment.
Animals↗
Studies on Pneumococcus vaccine alone or mixed with DTP and on Pneumococcus type 6B and Haemophilus influenzae type b capsular polysaccharide-tetanus toxoid conjugates in two- to five-year-old children with sickle cell anemia.
Pneumococcus vaccine, injected alone or mixed with diphtheria-tetanus toxoid-pertussis, did not elicit significant concentrations of pneumococcus type 6 antibodies in 2- to 5-year-old sickle cell anemia patients (n = 22). Reinjection 5 months later failed to elicit a booster response to pneumococcus type 6. We then injected conjugates of pneumococcus type 6B and of Haemophilus influenzae type b (Hib), each bound to tetanus toxoid (TT), alternatively at monthly intervals into sickle cell anemia patients of the same age group (n = 25); most received 3 injections of each vaccine. Pneumococcus vaccine was administered to 19 patients and Hib to 1 at approximately 1 year of age. Blood samples were taken before each and approximately 6 months after the last injection. Infrequent and minimal local reactions and only 6 episodes of fever (3%) occurred after injection of the conjugates. Pneumococcus type 6B-TT elicited a rise in the geometric mean concentration of pneumococcus type 6 antibodies (Ab) from 104 ng of antibody nitrogen (AbN)/ml in preimmunization sera to 385 ng of AbN/ml after the first injection (P less than 0.01). There were further increases after the 2 subsequent injections; 6 months after the third injection, the mean concentration was 940 ng of AbN/ml and 15 of 16 (94%) had greater than 300 ng of AbN/ml. Hib-TT elicited a 160-fold increase of Hib antibodies to a geometric mean concentration of 39.0 micrograms of Ab/ml after the first injection. These levels rose approximately 2-fold following 2 additional injections to 71.7 micrograms/ml and declined to 10.7 micrograms/ml at the 6-month sampling.(ABSTRACT TRUNCATED AT 250 WORDS)
Anemia, Sickle Cell↗
Potential of radio-frequency balloon angioplasty: weld strengths, dose-response relationship, and correlative histology.
Previous studies have established the feasibility of combining tissue heat generated by radio-frequency (RF) current and mechanical pressure to manage problems of percutaneous transluminal angioplasty (PTA) that are thought to cause postangioplasty restenosis (PARS). In the current in vitro study of normal and atherosclerotic human aortic layers (intima-media and media-adventitia) separated artificially, the purposes were to identify a dose-response relationship between the total RF energy delivered and the resultant weld strength between vascular tissues and to study the histologic correlates. Twenty-eight control and 100 experimental specimens were evaluated. The mean weld strength for all specimens with no RF current (at 0 J) was 4.1 g +/- 2.0; at 100 J, 5.9 g +/- 2.8; at 200 J, 28.5 g +/- 3.3; at 300 J, 50.0 g +/- 5.5; and at 500 J, 82.0 g +/- 8.2. Inspection of treated specimens revealed vascular molding (depression in the surface corresponding in dimensions to those of the electrode gap in the treatment chamber). Histologic examination revealed necrosis in the region underlying the electrode gap, a hypocellular fusion zone, indistinct boundaries between welded specimen parts, and transition zones to normal histologic characteristics at either end of each specimen. RF energy combined with mechanical pressure produces dose-dependent thermal welds in artificially dissected vascular tissues, molding of vascular tissue, and cellular destruction in the media. These findings may have clinical application in the management or prevention of all forms of PARS in humans and in the treatment of spontaneous aortic dissection.
Angioplasty, Balloon↗
Movement of receptors and ligands through the endocytic apparatus in alveolar macrophages.
The ability of alveolar macrophages to remove potentially injurious agents is essential for normal lung function. One mechanism by which macrophages affect this function is by the internalization and clearance of noxious agents from lung fluid. Extracellular macromolecules may be removed as a result of nonspecific uptake via fluid-phase pinocytosis or as a result of binding to surface receptors followed by a highly efficient internalization of the receptor-ligand complex. The ability of macrophages to bind, internalize, and thus sequester extracellular macromolecules is facilitated by the fact that membrane components involved in this process may be reutilized. That is, once internalized, membrane components are not invariably degraded but can be recycled for further use. In this commentary we examine some of the features known about the recycling process in both macrophages and other cell types. We demonstrate that although the rate of internalization of a membrane component is unique to that component, the rate of movement of internalized components within cells is a function of the endocytic apparatus and is independent of the ligands or receptors contained in the internalized vesicle. We also demonstrate how alterations in the rates of recycling may result in changes in the distribution of membrane proteins expressed on the cell surface and affect the ability of a cell to respond to the environment.
Animals↗
A phase I trial of recombinant interleukin-2 combined with recombinant interferon-gamma in patients with cancer.
Twenty-six patients with metastatic cancer were entered into a phase I trial of concurrent recombinant interleukin-2 (IL-2) and recombinant interferon-gamma (IFN-gamma). IL-2 was administered as a continuous intravenous infusion for 5 days. IFN-gamma was administered by a daily intramuscular (IM) injection during the 5 days of IL-2 administration. Treatment was repeated twice after 9-day rest periods. After a 2-week rest, patients without evidence of tumor progression were retreated. Natural killer (NK)- and lymphokine-activated killer (LAK)-cell activity were assayed in each patient before treatment, on day 1, and on day 5 of each cycle. Constitutional symptoms occurred in most patients but were not dose-limiting. Other toxicities included hypotension responsive to fluids, transient elevations in liver function tests, erythema/pruritus, eosinophilia, and transient leukopenia/thrombocytopenia. The maximum-tolerated dose (MTD) of the combination was 1 x 10(6) U/m2/d of IL-2 combined with 0.50 mg/m2/d of IFN-gamma. The dose-limiting toxicity was pulmonary manifesting as rales and shortness of breath. The dose of the combination that resulted in the optimal generation of in vivo LAK-cell activity was a dose of at least 0.25 mg/m2/d of IFN-gamma combined with 1 x 10(6) U/m2/d of IL-2. Objective clinical responses were seen in five of 26 patients. These included a partial response of 2 months duration in a patient with non-Hodgkin's lymphoma (NHL), mixed responses in a patient with NHL and two patients with renal cell carcinoma (RCC), and an ongoing assessable response in a patient with bone metastases from RCC. The recommended dose for phase II trials of this combination is 0.50 mg/m2 of IFN-gamma and 1 x 10(6) U of IL-2.
Adolescent↗
Usher syndrome type I is not linked to D1S81 (pTHH 33): evidence for genetic heterogeneity.
Usher syndrome is an autosomal recessive disease associating congenital sensorineural deafness and retinitis pigmentosa. Two clinical forms have been recognized, namely a) congenital and severe (type I) and b) later and moderate (type II). A linkage of the D1S81 probe (THH 33) with the gene for type II has been recently demonstrated by Kimberling et al. 1990. Here, a panel of 29 individuals from 6 kindreds with Usher syndrome type I has been tested for possible allelism at the D1S81 locus. A negative lod-score was found with this probe and close linkage to this region could be excluded. These different results support the view that the clinical heterogeneity in Usher syndrome is accounted for by an obvious genetic heterogeneity.
Blotting, Southern↗
Evidence against linkage of the gene for Usher's syndrome type 1 with group specific component (GC) on chromosome 4.
A linkage of the gene for Usher's syndrome with group specific component (GC) on the long arm of chromosome 4 has been suggested by Pelias et al., in 1988. A panel of 38 individuals from 7 kindreds with Usher's syndrome type 1 has been established to test this hypothesis. A negative lodscore was found and close linkage was excluded.
Abnormalities, Multiple↗
Exclusion of linkage between D3S47 (C17) and ADRPII gene in two large families of moderate autosomal dominant retinitis pigmentosa: evidence for genetic heterogeneity.
Autosomal dominant forms of retinitis pigmentosa appear among the most frequent types of retinal degenerations. Two clinical subtypes have been recognized, namely the early onset, severe form (type I) and the late onset, moderate form (type II). A linkage between the D3S47 probe (C17) with the gene of the type I has been recently demonstrated by Humphries et al., 1989. Here, two families with type II of the disease have been tested for possible allelism at the D3S47 locus. A negative lod-score was found with this probe and a linkage with this region could be excluded. These results support the hypothesis of a genetic heterogeneity in autosomal dominant forms of retinitis pigmentosa.
Genes, Dominant↗
[An example of detection of heterozygotes and antenatal diagnosis in four families with anhidrotic ectodermal dysplasia].
Anhidrotic ectodermal dysplasia is an X-linked inherited skin disorder; only affected males exhibit the complete syndrome, whereas females may have a few mild features. The gene involved in this disease is located in the proximal area of the long arm of the X chromosome, in the q13 position. Molecular analysis is very helpful for calculating the risk of transmission in sisters with normal phenotypes and affected individuals (family 1 provides an example), but cannot solve all problems (example of family 4). The best results are obtained when there are two informative markers, each located on either side of and very close to the mutant gene. Molecular analysis can also be applied to chorionic villi sampled at the tenth week of gestation in order to achieve antenatal diagnosis in male fetuses in high risk families. Until recently, antenatal diagnosis could be performed only at the twentieth week of gestation by the demonstration of inadequate development of skin glands in skin biopsy specimens sampled under fetoscopy. Family 2 provides an example of antenatal diagnosis and highlights the risk of error that always exists in molecular analysis studies.
Ectodermal Dysplasia↗
[Molecular biology--its proper use in genetics].
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Blotting, Southern↗
Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease.
Prompted by our recent finding that lymphokine-activated killer (LAK) cells mediate both veto and natural suppression, we tested the ability of adoptively transferred LAK cells to block two in vivo alloreactions which complicate bone marrow transplantation: resistance to transplanted allogeneic bone marrow cells, and lethal graft-vs-host disease. Adoptive transfer of either donor type B6D2 or recipient-type B6 lymphokine-activated bone marrow cells, cells found to have strong LAK activity, abrogated or inhibited the resistance of irradiated B6 mice to both B6D2 marrow and third party-unrelated C3H marrow as measured by CFU in spleen on day 7. The ability of lymphokine-activated bone marrow cells to abrogate allogeneic resistance was eliminated by C lysis depletion of cells expressing asialo-GM1, NK1.1, and, to a variable degree, Thy-1, but not by depletion of cells expressing Lyt-2, indicating that the responsible cells had a LAK cell phenotype. Similar findings were obtained by using splenic LAK cells generated by 3 to 7 days of culture with rIL-2. Demonstration that allogeneic resistance could be blocked by a cloned LAK cell line provided direct evidence that LAK cells inhibit allogeneic resistance. In addition to inhibiting allogeneic resistance, adoptively transferred recipient-type LAK cells prevented lethal graft-vs-host disease, and permitted long term engraftment of allogeneic marrow. Irradiation prevented LAK cell inhibition of both allogeneic resistance and lethal graft-vs-host disease. These findings suggest that adoptive immunotherapy with LAK cells may prove useful in preventing graft rejection and graft-versus-host disease in human bone marrow transplant recipients.
Animals↗
Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages.
The rate of movement of different receptors and ligands through the intracellular endocytic apparatus was studied in alveolar macrophages. Cells were exposed to iodinated alpha-macroglobulin-protease complexes, mannose terminal glycoproteins, diferric transferrin, and maleylated proteins. By use of the diaminobenzidine density shift procedure, we demonstrated that these ligands were internalized into the same endocytic vesicle. We then compared the rates of transfer to the lysosome or recycling to the cell surface of different ligands/receptors contained in the same endosome. We found that although the rate constant for degradation was ligand specific, the lag time prior to the initiation of degradation was the same for all three ligands. We also found that molecules taken up nonspecifically by fluid-phase pinocytosis had the same lag time prior to degradation as ligands internalized via receptor-mediated endocytosis. These data suggest that different molecules within the same endocytic compartment are transferred to the lysosome (or degradative compartment) at the same rate. We measured the rate of return of receptors to the cell surface by either inactivating surface receptors by protease treatment at 0 degrees C, or by incubating cells with saturating amounts of nonradioactive ligand at 37 degrees C. We then measured the rate of appearance of "new" receptors on the cell surface. Using these approaches, we found that three different receptors were transferred from internal pools to the cell surface at the same rate. The rate of transfer was independent of whether receptors were initially occupied or unoccupied. Our observations indicate that receptor/ligands, once inside alveolar macrophages, are transported by vesicles which transfer their contents as a cohort from one compartment to another. The rate of movement of these receptors is determined by the movement of vesicles and is independent of their content.
3,3'-Diaminobenzidine↗
Effect of volume and pH on surface receptor number in macrophages.
Incubation of rabbit alveolar macrophages in hypo-osmotic solutions transiently increases cell volume and inhibits membrane internalization, resulting in an increase in surface receptor number. Since recent reports suggest that hypo-osmotic treatment decreases intracellular pH, and that reduced pH inhibits receptor internalization, pH was measured in hypo-osmotically treated macrophages. We found that cells incubated in iso-osmotic solutions of pH less than 7.2 exhibited a decrease in intracellular pH upon exposure to hypo-osmotic solutions, while cells in iso-osmotic solutions of pH greater than 7.2 had an increase in pH upon exposure to hypo-osmotic solutions. The relative increase in surface receptor number was unaffected by the initial pH or by the direction of change in pH. Incubation of cells in high K+/low Na+ hypotonic buffers induced a persistent increase in cell volume and surface receptor number. Cell volume and surface receptor number fell to baseline values after restoration of isotonicity by the addition of hypertonic sucrose. These manipulations had little effect on intracellular pH. We conclude that the inhibition of membrane internalization observed in cells exposed to hypo-osmotic solutions is independent of changes in intracellular pH. The inhibition of internalization observed in this system may be due directly to forces produced as a consequence of cell swelling.
Animals↗