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Biomedical subjects

G Kaplan

Publications and source records attributed to G Kaplan.

At least 217 records · Page 12Linked to original sources
PubMed1989

Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.

Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the level of receptor and within the cell.

Blotting, Northern↗
PubMed1989

[Acute, reversible, interstitial pneumopathy induced by melphalan].

A case of acute interstitial pneumonia with hypoxaemia is described; this occurred after the cessation of cortico steroids in a patient suffering from myeloma treated with melphalan. The absence of any microbes and the lymphocytosis in the bronchoalveolar lavage and the rapid and favourable improvement on cortico steroids led to a diagnosis of melphalan induced pneumonia. This acute form is probably due to a hypersensitivity mechanism and should be distinguished from the majority of cases of sub-acute fibrosing pneumonitis due to melphalan which have been published before. Urgent treatment with glucocorticoids is justified as well as the immediate and final cessation of the medication responsible, because it is this which will affect prognosis.

Acute Disease↗
PubMed1988

Macrophages phagocytose thymic lymphocytes with productively rearranged T cell receptor alpha and beta genes.

The thymus gland is important for the formation of competent T lymphocytes. However, there is long-standing evidence that greater than 95% of newly formed thymocytes do not emigrate to peripheral lymphoid tissues but instead die locally. We have identified a rapid and selective pathway for thymocyte turnover in vitro. The mechanism entails binding, uptake, and digestion by macrophages. The susceptible cells are a subpopulation of double-positive thymocytes. These thymocytes can be enriched by virtue of their high buoyant density in Percoll and prove to have low levels of surface CD3 and little or no surface TCR. However TCR-alpha and -beta genes have undergone rearrangement, and full length alpha and beta transcripts are abundant. Therefore many double-positive cells rearrange and express TCR genes but do not have normal levels of TCR on the cell surface. We propose that thymocytes that undergo high turnover in situ are unable to form receptors that can be selected by MHC molecules in the thymus, and that these cells are recognized and cleared by the macrophage.

Gene Rearrangement, T-Lymphocyte↗
PubMed1988

The nature and kinetics of a delayed immune response to purified protein derivative of tuberculin in the skin of lepromatous leprosy patients.

We have analyzed the nature and kinetics of a delayed, cell-mediated immune response to a purified protein derivative of tuberculin (PPD) in the skin of 154 naturally sensitized patients with lepromatous leprosy. After the intradermal injection of 5 U of PPD, biopsies were taken at 1-21 d and studied for the composition, extent, persistence, and organization of the emigratory cell response by light and electron microscopy. Induration of positive sites occurred promptly, reached a maximum diameter at 4 d, displayed a major extravasatory element, and was evident for as long as 21 d. The cellularity of the site exhibited a biphasic course, reached a maximum at 7 d, involved as much as 70% of the dermis and millions of new cells, and was elevated threefold above preinjection levels at 21 d. The emigratory cells were limited to T cells and circulating monocytes. T cells were more evident as they entered a preexisting lepromatous lesion containing parasitized macrophages and only occasional T cells many of the CD8+ phenotype. The predominant emigratory T cell was CD4+ although CD8+ cells were in evidence. The CD4/CD8 ratio of the lesions started at less than unity and in two distinct steps reached levels as high as 5:1. In most sites CD4+ cells were in the majority at 21 d. A well-defined granulomatous response with epithelioid and giant cells was apparent at 4 d, reached a maximum at 7 d, and involved all PPD sites at this time point. The generation of these differentiated mononuclear phagocytes from newly emigrated monocytes was never observed in the underlying lepromatous lesion but is a constant feature of the tuberculoid leprosy response. Epidermal thickening and keratinocyte proliferation, sequellae of the dermal reaction, reached a maximum at 7 d and gradually resolved by 3 wk. A constant feature of the PPD response was the extensive destruction of preexisting macrophages containing Mycobacterium leprae bacilli or their products. This was associated with the presence of and intimate contact with highly polarized lymphoid cells of unknown phenotype. Cell destruction did not involve other elements of the dermis and spared parasitized Schwann cells. Newly emigrated T cells and monocytes were never seen within the perineural sheath in contact with neural elements. It appears that a single antigenic stimulus leads to a very long-term, defined series of events with distinct temporal patterns. It includes waves of emigratory T cells, the maturation and organization of monocytes, the generation of killer cells, and the extensive destruction of parasitized macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Granuloma↗
PubMed1988

The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.

Cell Adhesion↗
PubMed1988

Keratinocyte growth regulation by the products of immune cells.

We describe a bioassay that allows the in vitro investigation of the stimulatory and suppressive factors derived from immune cells in short-term cultures of human keratinocytes. In agreement with other assays, epidermal growth factor is not mitogenic for human keratinocytes. Supernatant fluid from human PBMC stimulated with Con A, from allo-MLRs, as well as supernatants from nonstimulated PBMC, possess growth-promoting molecules. Our results show that both activated and nonactivated T cells release growth factors. Suppressive molecules are produced preferentially by monocyte cultures. Two T cell products, IFN-gamma and transforming growth factor beta are both inhibitory for keratinocyte proliferation. Two other T cell products, IL-3 and GM-CSF, stimulate keratinocyte proliferation at nanogram concentrations. These results suggest the existence of regulatory circuits between the T cells of a dermal inflammatory infiltrate and the overlying epidermal keratinocytes. This may determine the fine control of epidermal proliferation and turnover leading either to enhanced wound repair or skin pathology.

Cell Differentiation↗
PubMed1988

A combination of levobunolol and dipivefrin for the treatment of glaucoma.

This double-masked prospective study compared the ocular hypotensive efficacy and the safety of 0.5% and 1% levobunolol hydrochloride with 0.5% timolol maleate when each was administered topically twice daily in combination with 0.1% dipivefrin hydrochloride. Forty-three patients whose intraocular pressure was previously controlled by concomitant treatment with timolol and dipivefrin were randomly assigned to receive 0.5% or 1% levobunolol and 0.1% dipivefrin, or to continue to receive 0.5% timolol and 0.1% dipivefrin for three months. In the groups receiving levobunolol and dipivefrin concurrently, continued intraocular pressure control was achieved equal to that attained with timolol and dipivefrin before study entry. We concluded that concomitant treatment with levobunolol and dipivefrin is equal in both efficacy and safety to concomitant treatment with timolol and dipivefrin.

Drug Combinations↗
PubMed1988

Antigen-specific T lymphocytes efficiently cluster with dendritic cells in the human primary mixed-leukocyte reaction.

Experimental conditions have been developed to detect the efficient interaction of antigen-presenting cells and antigen-specific CD4+ T lymphocytes early in the human primary mixed-leukocyte reaction (MLR). When monocytes are depleted from the stimulator population, it is evident that small numbers of allogeneic dendritic cells form multicellular aggregates with responsive T cells. B cells and monocytes in allogeneic stimulator populations do not appear to form aggregates in the first 2 days of the MLR. Upon return to culture, most of the lymphocytes that have clustered with dendritic cells become IL-2 responsive, proliferating lymphoblasts. The nonclustered cells exhibit little growth, while mixtures of clusters and nonclusters proliferate comparably to clusters alone. Cluster-derived lymphocytes respond rapidly to rechallenge with foreign leukocytes from the original donor but are greater than 90% depleted of reactivity to other "third party" donors. Nonclustered lymphocytes, in contrast, are greater than 90% depleted in specific reactivity but respond normally to third party. Therefore antigen-specific (alloreactive) resting CD4+ lymphocytes efficiently and selectively aggregate with dendritic cells. Dendritic-T-cell aggregates represent a stable microenvironment in which the MLR begins and might be useful in the experimental analysis of early events in the sensitization phase of cell-mediated immunity in man.

Antibodies, Monoclonal↗
PubMed1988

The efficacy of a cell-mediated reaction in the disposal of M. leprae in human skin.

The inability of lepromatous leprosy patients to mount a cellular immune response against Mycobacterium leprae antigens is not understood. The extensive intracellular replication of bacilli in the phagocytes and the relative paucity of T lymphocytes in the lesions suggest that these patients might be incapable of generating normal delayed type hypersensitivity responses in their skin. In order to elucidate this problem we evaluated the patient's response to local antigen administration. Our observations suggest that the majority of lepromatous patients can respond normally to intradermal injections of a soluble antigen such as purified protein derivative of tuberculin. The underlying lepromatous lesions do not inhibit mononuclear cell infiltration or differentiation. Moreover, the generation of a cellular immune response in the lesions appears to modify the lepromatous lesion to a lesion resembling the tuberculoid type. This process involves local T cells recruitment, granuloma formation and a reduction in the bacterial load at the antigen responsive site.

Biopsy↗
PubMed1988

Efficacy of a cell-mediated reaction to the purified protein derivative of tuberculin in the disposal of Mycobacterium leprae from human skin.

The purpose of this study was to evaluate the effects of a delayed-type cell-mediated immune response to Mycobacterium tuberculosis antigen on the Mycobacterium leprae load in the skin of leprosy patients. Twelve patients with the lepromatous form of leprosy have been injected intradermally with 5 units of the purified protein derivative of tuberculin (PPD). Ten individuals responded with areas of induration ranging from 12 to 21 mm in diameter, and two were unresponsive (less than 10 mm). Twenty-one days thereafter, the injected and control sites were biopsied, and the histology, number of acid-fast bacilli, nature and phenotype of the emigrant cells, and ultrastructural characteristics of the lesions were evaluated. Eight of the 10 responding patients showed reductions in the number of acid-fast bacilli by factors ranging from 5 to 10,000. Two responders and both nonresponders exhibited no discernible decline in the number of organisms. The reduction in bacillary load was correlated with an intense mononuclear cell infiltrate, the maintenance of a high CD4+ T-cell/CD8+ T-cell ratio, the formation of granulomata, and the extensive destruction of previously parasitized macrophages.

Antigens, Bacterial↗
PubMed1988

Subjective state of health and survival in elderly adults.

The relationship between subjective state of health and 5-year survival in an elderly cohort was examined. During the 1978 study of the elderly population of Kiryat Ono, a suburban community in the central coastal area of Israel, respondents were asked to evaluate their general health status on a four-level scale, and their vital status was updated until 1983. A multivariate analysis of survival was conducted using the Cox Proportional Hazards Model. Self-rating of health was an independent predictor of survival after controlling for age, sex, continent of origin, number of conditions and medications reported, heart disease and activities of daily living (ADL). The self-rating of health was found to be an important psychosocial parameter in the evaluation of health status, in determining the prognosis of an elderly person, and in analyzing survival.

Aged↗
PubMed1988

Mechanism of dietary modulation of calcitonin levels in Fischer rats.

Aging caused an increase in serum calcitonin that was markedly suppressed by food restriction. Food restriction had no significant effect on gut calcium absorption, urinary calcium excretion, serum 1,25(OH)2 vitamin D, blood ionized calcium, the clearance of calcitonin from plasma, and its rate of inactivation in vitro by the kidney and liver. In contrast, food restriction modulated an increase with age in calcitonin secreting C-cells, thyroidal calcitonin and thyroidal calcitonin mRNA pools. We conclude that the lowering of age-related increase in serum calcitonin levels by food restriction is likely because of lowered thyroid calcitonin pools rather than changes in the metabolism of calcium, which is the primary stimulus for calcitonin secretion. Our findings also suggest that food restriction decreases age-related increase in calcitonin levels, at least in part, by modulating the expression of the calcitonin gene.

Aging↗
PubMed1988

Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.

Codon↗
PubMed1988

VP1 of serotype C foot-and-mouth disease viruses: long-term conservation of sequences.

The nucleotide sequences of the VP1-coding regions of several isolates of serotype C3 foot-and-mouth disease virus (FMDV) were determined. The deduced amino acid sequences were compared with those of serotype C1 FMDV. The results provide evidence for two different lineages of FMDV C3 and document the potential for both long-term conservation and rapid evolution of FMDV.

Amino Acid Sequence↗
PubMed1988

Construction and characterization of poliovirus subgenomic replicons.

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.

Base Sequence↗
PubMed1988

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.

Animals↗
PubMed1988

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.

Base Sequence↗