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Biomedical subjects

Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 37 records · Page 2Linked to original sources
PubMed1989

The generation of antigen-specific, major histocompatibility complex-restricted cytotoxic T lymphocytes of the CD4+ phenotype. Enhancement by the cutaneous administration of interleukin 2.

We have examined an in vitro system in which PBMC from purified protein derivative (PPD)-sensitized patients generate CTL after in vitro activation with antigen. These cells selectively destroy mycobacterial antigen PPD-pulsed monocyte targets. These CTL are of the CD4+ phenotype and exhibit MHC class II restriction. After exposure to antigen these cells require 5-7 d for maximal development, whereas, a separate antigen-independent population is generated within 3-4 d. CD8+ cells are poorly, if not at all, cytotoxic under similar conditions. Cells with properties of the NK and LAK lineage are also present in these cultures and kill other specific targets. Human rIL-2 was injected into the skin of lepromatous patients at 10-micrograms doses, given at 48-h intervals, for three doses. Peripheral blood cells obtained 8-14 d after the initiation of IL-2 injection demonstrated enhanced antigen-dependent destruction of monocyte targets. The efficacy of antigen-dependent and -independent populations and their amplification by IL-2-dependent mechanisms is discussed in terms of the local destruction of parasitized macrophages and the subsequent disposal of M. leprae.

Antigens, Bacterial↗
PubMed1989

Endocytic and secretory repertoire of the lipid-loaded macrophage.

We have studied the effects of intracellular lipid storage on macrophage function. Mouse peritoneal macrophages were lipid loaded by three regimens modeling loading through the scavenger receptor [acetylated low density lipoprotein (Ac-LDL) cells], by extracellular matrix-bound LDL [low density lipoprotein complexed with dextran sulfate (DS-LDL) cells], and by conditions of reduced cholesterol acceptors in the medium [low serum, oleic acid, Ac-LDL (LS/OI) cells]. Significantly increased cholesterol levels in all three regimens were measured by cholesterol determination and Oil Red O staining of fixed cells. Lipid-laden cells were equal to control macrophages in binding and ingesting immunoglobulin-coated sheep erythrocytes, reflecting Fc-mediated endocytosis. The lipid-laden cells were compared to control cells for secretory functions of macrophages that could be important in the atherosclerotic artery. They were still capable of producing all secretory products examined, but the quantities of H2O2 and arachidonic acid metabolites were reduced in some cases, and fibrinolytic activity appeared to be increased. Western blot analysis showed a five-fold increase in the release of tumor necrosis factor by DS-LDL-loaded cells. We suggest that the location of intracellular lipid pools as well as the type of lipids (and/or lipid complexes) ingested may determine the extent of functional changes.

Animals↗
PubMed1989

Mononuclear phagocytes of blood and bone marrow: comparative roles as viral reservoirs in human immunodeficiency virus type 1 infections.

We examined human immunodeficiency virus (HIV) production in cultured mononuclear cells from 36 seropositive homosexual males. Production was detected by using an HIV p24 antigen ELISA assay in blood mononuclear cells in 54% of asymptomatic, 71% of acquired immunodeficiency syndrome (AIDS)-related complex, and 100% of AIDS patients. When the peripheral blood mononuclear cells were separated into monocytes and CD4+ T cells, we found that the CD4+ T-cell fraction was preferentially infected in the three clinical stages. The ability to isolate HIV from blood monocyte-derived macrophages was similar in the three stages (24-33%) and required coculture with phytohemagglutinin-stimulated lymphoblasts. Bone marrow and blood mononuclear cells cultured simultaneously yielded virus from both sources in 13 individuals. Again the prime source of virus was the nonadherent bone marrow mononuclear cells, which contained CD4+ T cells, and not the adherent monocyte-enriched fraction. We conclude that blood mononuclear cells yield productive virus more readily as disease progresses and that infection is detected in association with CD4+ T-cell-enriched fractions. In our large sample of patients, monocyte infection was detected in only a small fraction, suggesting that this cell type is neither a primary nor an exclusive reservoir of HIV infection.

AIDS-Related Complex↗
PubMed1989

Effect of multiple interferon gamma injections on the disposal of Mycobacterium leprae.

The effect of multiple intradermal injections (four to six) of 10 micrograms of interferon gamma on the number of Mycobacterium leprae in the skin of patients with polar lepromatous leprosy and borderline lepromatous leprosy was evaluated. To achieve a maximum zone of induration and cell emigration a preparatory dose of the lymphokine was required. A second group of three injections, given 3-4 days after the initial series, resulted in lesser degrees of induration and was more in keeping with a partial local hyporesponsive state. A marked emigration of T cells and monocytes into the dermis resulted from injections of interferon gamma and persisted for greater than 21 days. A preponderance of CD4+ cells in the infiltrate was seen within a few days and CD4/CD8 ratios remained elevated for greater than 5 weeks. The bacillary load of injected sites evaluated 21 days after lymphokine administration was reduced in 14/17 patients by factors ranging from 5- to 1000-fold. This occurred predominantly within diffuse lesions and occurred rarely in nodular sites. Biopsy samples of injected sites taken 6 months later demonstrated progressive 10-fold reductions in bacilli and the continued presence of a granulomatous response.

Administration, Topical↗
PubMed1989

Most CD4+ T cells from human immunodeficiency virus-1 infected patients can undergo prolonged clonal expansion.

We have addressed the capacity of HIV-1 infection to alter the growth of primary CD4+ T cells, but at the clonal level. Single T cells were expanded in the presence of PHA, IL-2, and small numbers of accessory dendritic cells. We report two new findings. First, T cells from seropositive individuals, even those with AIDS and markedly reduced CD4+ counts, exhibit a normal cloning efficiency, and proliferative capacity. This result is in contrast to two prior reports of a low cloning efficiency in CD4+ T cells from HIV-1-infected patients. Second, when we added high doses of exogenous HIV-1 to T cell clones from control subjects, we observed infection but not cytotoxicity or loss of CD4+ cells, following addition of virus stocks at days 0, 3, and/or 7 of clonal growth. The same HIV-1 isolates markedly reduced CD4+ T cells in bulk mononuclear cultures. When tested at day 11, HIV-1 mRNA was expressed in some cells of exogenously infected clones by in situ hybridization; when tested at day 18, several clones could transactivate a TAT-sensitive cell line. These findings suggest that the loss of CD4+ T cells in infected individuals is not the inevitable result of the activation of latent infection, or spread of a productive infection, during clonal growth.

Acquired Immunodeficiency Syndrome↗
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

Properties of isolated red pulp macrophages from mouse spleen.

Dense monolayers of large, adherent macrophages were prepared from the red pulp of mouse spleen. These sinus-lining phagocytes resembled liver Kupffer cells in morphology, as well as expression of F4/80 and class II MHC antigens and receptors for IgG. C3-coated red cells attached at low levels to spleen macrophages, but attachment and endocytosis were enhanced on fibronectin-coated surfaces. The ionophore A23187 induced spleen macrophages to synthesize prostaglandin E2, but like Kupffer cells, spleen macrophages did not synthesize leukotrienes and made relatively small amounts of HETE and 12-hydroxyheptadecanoic acid. Resident spleen macrophages did not produce H2O2, but splenic inflammatory cells, induced by infection of animals with Listeria monocytogenes, actively released H2O2. We conclude that the functional properties of resident, sinusoidal-lining macrophages in liver and spleen are similar to one another but distinct from other pools of phagocytes.

Animals↗
PubMed1988

The dynamics of granuloma formation in experimental visceral leishmaniasis.

We have examined the temporal sequence of events leading to the formation of hepatic granulomas after the intravenous injection of L. donovani amastigotes into BALB/c mice. Parasite ingestion by permissive Kupffer cells (KC) occurred promptly, and local KC aggregations were the foci about which granulomas were subsequently formed. Infected KC were recognized by the uptake of colloidal carbon and the expression of the macrophage-specific antigen recognized by F4/80 mAb. Peroxidase-positive granulocytes migrated rapidly and were followed by monocytes and L3T4+ T cells that enclosed the infected KC. Thereafter, Ly-2+ T cells were prominent members of the granulomatous lymphoid population. Parasites multiplied until 4 wk, and then a prompt reduction in infected cells occurred. This was associated with a sharp decline in the L3T4+ T cells of the granulomas and the maintenance of the Ly-2+ subset. In comparison, athymic nu/nu mice developed smaller, more slowly appearing granulomas that contained granulocytes and monocytes and exhibited progressive parasite replication. Upon rechallenge, the entire process was completed in 2 wk, and infected KC in the euthymic mice were never observed. We hypothesize that the effectiveness of the granulomatous response requires the destruction of parasitized host cells (KC), in a lymphokine rich environment. We further suggest that the Ly-2+ T cell serves as an important effector cell in this process, either by direct cytotoxicity or by supporting the cytotoxic potential of other cell types in the granuloma.

Animals↗
PubMed1988

Stimulus-dependent myristoylation of a major substrate for protein kinase C.

Bacterial lipopolysaccharide (LPS), the major surface component of gram-negative bacteria, exerts a profound effect on the immune system by enhancing the release of proteins and arachidonic acid metabolites from macrophages (for review see ref. 1). The molecular mechanism(s) by which LPS induces these various secretory responses is unknown. We previously reported that LPS promotes the myristoylation of several macrophage proteins including one with a relative molecular mass (Mr) of 68K2. We have now found that by several criteria the 68K myristoylated protein is similar or identical to the 80/87K protein, a major specific substrate for protein kinase C (PKC) found in brain and fibroblasts (for review see refs 7,8). We have also found that the myristoylated PKC substrate is quantitatively associated with the membrane fraction. Myristoylation of the PKC substrate may target it to the membrane and constitute a transduction pathway for stimulus-response coupling.

Animals↗
PubMed1988

Calcium ionophore synergizes with bacterial lipopolysaccharides in activating macrophage arachidonic acid metabolism.

LPS, a major component of Gram-negative bacterial cell walls, prime macrophages for greatly enhanced arachidonic acid [20:4] metabolism when the cells are subsequently stimulated. The LPS-primed macrophage has been used as a model system in which to study the role of Ca2+ in the regulation of 20:4 metabolism. The Ca2+ ionophore A23187 (0.1 microM) triggered the rapid release of 20:4 metabolites from LPS-primed macrophages but not from cells not previously exposed to LPS. Macrophages required exposure to LPS for at least 40 min before A23187 became effective as a trigger. A23187 (0.1 microM) also synergized with PMA in activating macrophage 20:4 metabolism. The PMA effect could be distinguished from that of LPS since no preincubation with PMA was required. A23187 greatly increased the amount of lipoxygenase products secreted from LPS-primed macrophages, leukotriene C4 synthesis being increased 150-fold. LPS-primed macrophages, partially permeabilized to Ca2+ with A23187, were used to titrate the Ca2+ concentration dependence of the cyclooxygenase and lipoxygenase pathways. Cyclooxygenase metabolites were detected at an order of magnitude lower Ca2+ concentration than were lipoxygenase products. The data suggest that Ca2+ regulates macrophage 20:4 metabolism at two distinct steps: an increase in intracellular Ca2+ regulates the triggering signal and relatively higher Ca2+ concentrations are required for 5-lipoxygenase activity.

Animals↗
PubMed1988

How killer cells kill.

Killer lymphocytes, the commandos of the immune system, attack tumor cells and cells infected by viruses. They kill by secreting protein molecules that link to form pores in target cells; the cells promptly leak to death. Study of the process may make it possible to improve the killers' efficiency in fighting cancer and such viral infections as AIDS.

Animals↗
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

Interferon gamma induces the myristoylation of a 48-kDa protein in macrophages.

The lymphokine interferon gamma (IFN-gamma) induces the selective myristoylation of a macrophage protein with an apparent molecular mass of 48 kDa. The myristic acid-protein bond is resistant to treatment with hydroxylamine, suggesting that the fatty acid moiety is in an amide linkage. As little as 1 unit of IFN-gamma per ml induces the myristoylation of the 48-kDa protein, with half-maximal myristoylation being observed with 4 units/ml. The effect is observed within 1 hr after exposure to IFN-gamma and is maximal by 3-4 hr, after which it declines. IFN-alpha does not induce the myristoylation of the 48-kDa protein, and IFN-beta does so very poorly. Neither IFN-alpha nor IFN-beta has any effect on IFN-gamma-induced myristoylation of the 48-kDa protein. The 48-kDa protein is constitutively myristoylated in murine macrophages that have been activated in vivo by intraperitoneal injection of Corynebacterium parvum, suggesting that it may be an early intermediate in the activation of macrophages.

Acylation↗
PubMed1988

Perforin-dependent and -independent pathways of cytotoxicity mediated by lymphocytes.

There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

A calcium- and perforin-independent pathway of killing mediated by murine cytolytic lymphocytes.

Cytotoxic T lymphocytes have been thought to lyse cellular targets in the past by a calcium-dependent pathway. This notion was recently supported by the identification and purification of a pore-forming protein (perforin) from the granules of these cell types. Here, we show that perforin is absent from a number of cell lines that nevertheless display vigorous cytolytic activity toward target cells. The cytotoxic activity of eight murine CTL lines is completely or partially retained in the absence of calcium. The calcium-independent lytic activity is associated with two subcellular fraction peaks isolated by Percoll gradient centrifugation, e.g., a heavy density band migrating with granule markers and a lighter band corresponding to free cytosolic material. These results suggest a complex picture of lymphocyte-mediated killing involving probably multiple mechanisms and mediators that may operate in concert or independently in the delivery of the lethal hit.

Animals↗