In South Africa and other high prevalence countries, transmission is a significant contributor to... more In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discrimina...
Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, M... more Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular pathogen of macrophages, with alveolar macrophages (AMs) being the primary conduit of infection and disease. The outcome of primary infection is most often a latently infected healthy human host, in whom the bacteria are held in check by the host immune response. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the host immune response fails to control the growth of bacilli, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols coughed out into the environment and inhaled by new hosts. The molecular details of the Mtb-macrophage interaction continue to be elucidated. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. Macrophages demonstrate tremendous phenotypic heterogeneity and functional plasticity which, depending on the site and stage of infection, facilitate the diverse outcomes. Moreover, host responses vary depending on the specific characteristics of the infecting Mtb strain. In this chapter, we describe a contemporary view of the behavior of AMs and their interaction with various Mtb strains in generating unique immunologic lung-specific responses.
Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage... more Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, Nacetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C3 receptor. In vivo stimulation triggered the capacity to internalize up to 45 % of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C3 receptor may be the most useful parameter.
A method is described for the separation of fish leucocytes and the establishment of pure monolay... more A method is described for the separation of fish leucocytes and the establishment of pure monolayers offish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90-99/O of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.
The morphology of mouse peritoneal macrophages and echinoid phagocytes during phagocytosis in vit... more The morphology of mouse peritoneal macrophages and echinoid phagocytes during phagocytosis in vitro was studied. A striking similarity in the function of the foreign .surface receptor is found in the t^vo systems. Glutaraldebydetreated erj'throcytes attached randomly over the entire surface of tht L'ells and were internalized without circumferential attachment between the particles and the phagocyte membrane. The particles seemed to sink directly into the cytoplasm of the cells. Tannin-treated erythrocytes were phagocytosed by the echinoid cells in a similar mode. The camp Semen r-coa ted erythrocytes were attached only in the perinuclear area of the echinoid phagocyte's membrane, but tbe morpbology of their internalization was similar to that mediated by the foreign surt'acf rt-ceptor. A circumferential attachment between the particles and tbe phagocyte membrane did not seem necessaiy. This is also the case for mouse peritoneal macrophages.
Experimental conditions have been developed to detect the efficient interaction of antigenpresent... more Experimental conditions have been developed to detect the efficient interaction of antigenpresenting cells and antigen-specific CD4+ T lymphocytes early in the human primary mixedleukocyte 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 >90% depleted of reactivity to other "third party" donors. Nonclustered lymphocytes, in contrast, are >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.
The thymus is critical for the production of mature, recirculating T cells. Congenitally athymic ... more The thymus is critical for the production of mature, recirculating T cells. Congenitally athymic mammals and neonatally thymectomized mice, have profound deficits in T cell numbers and immunologic functions. Such athymic individuals do not reject skin grafts nor do they resist a variety of infectious agents (1-5), and few lymphocytes are noted in the thymus-dependent areas oflymphoid tissues (5-7). The thymus is also the main site for rearranging TCRa and -0 genes (8-12). The repertoire of a/R receptors is shaped by deleting cells that react directly with self MHC (13) and by selecting cells that recognize foreign antigens in association with self MHC (14, 15). These negative and positive selections are thought to be mediated by bone marrow-derived dendritic cells in the thymus medulla and pharyngeal pouch-derived epithelium in the cortex (16-18).
Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few... more Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few such instances is the biochemical basis of cytotoxicity understood. Recently, we demonstrated that activated macrophages, stimulated by phorbol myristate acetate (PMA), 1 secreted substantial amounts of hydrogen peroxide (1). The release of this oxidant from macrophages correlated with the extracellular lysis of tumor cells
Proceedings of the National Academy of Sciences, 1990
Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed th... more Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.
Proceedings of the National Academy of Sciences, 1979
The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma... more The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma membrane were analyzed. Cells labeled with [ S~methionine were incubated with the nonpermeant reagent trinitrobenzene sulfonic acid to introduce the trinitrophenyl moiety on free amino groups of externally oriented membrane proteins. The cells were then incubated with rabbit anti-dinitrophenyl IgG and the immune complexes formed with the trinitrophenyl-proteins were isolated from detergent lysates of the cells by using fixed Staphylococcus aureus as the immunoadsorbent. Proteins isolated by this method were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The interval between the release of newly synthesized proteins from ribosomes and their appearance at the cell surface, where they became accessible to trinitrobenzene sulfonic acid, was studied in pulsechase experiments. The "transit" time of four major membrane glycoproteins (48,000-310,000 Md) ranged from 36 to 55 min and their appearance on the cell surface occurred in a relatively synchronous fashion. The turnover of most proteins of molecular weight above 50,000 was very slow (ti/2> 80 hr) and was rather synchronous. Two exceptions were the 310,000 Mr protein, which was lost with a t1/2 = 21 hr, and a major glycoprotein (Mr 48,000), which exhibited more complex kinetics. Although the overall turnover of surface proteins was biphasic in nature, the rapid phase of protein loss was largely due to low molecular weight species.
Proceedings of the National Academy of Sciences, 1986
The epidermal changes that occur in human cutaneous immune responses have been investigated in th... more The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from lato Ia'. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.
Proceedings of the National Academy of Sciences, 1989
The effect of multiple intradermal injections (four to six) of 10 micrograms of interferon gamma ... more 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.
Proceedings of the National Academy of Sciences, 1989
Experiments were carried out in the skin of patients with leprosy to examine whether suppressor c... more Experiments were carried out in the skin of patients with leprosy to examine whether suppressor cell populations either exist in the skin of multibacillary lepromatous leprosy patients, can be activated with antigen, or are induced to emigrate into a cutaneous site from the circulation. For this purpose, purified protein derivative of tuberculin, a delayed-type antigen that generates a cell-mediated immune response, was introduced into the skin alone or with nonviable tTo whom reprint requests should be addressed at: The Rockefeller University,
Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrop... more Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.
Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to ... more Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to thalidomide therapy were evaluated. Circulating tumor necrosis factor-alpha (TNF alpha) levels were assayed in serum obtained from lepromatous leprosy patients at diagnosis, during multidrug therapy, at the onset of ENL episodes, and during treatment with thalidomide. Patients with systemic ENL demonstrated the highest serum TNF alpha levels, which decreased significantly during thalidomide treatment. Serum TNF alpha in nonreactional patients was associated with mild flu-like symptoms and local inflammatory lesions. Serum interferon-gamma (IFN-gamma) was also elevated in patients with high TNF alpha levels. Thalidomide therapy reduced not only serum TNF alpha levels and the clinical symptoms but also the dermal infiltration of polymorphonuclear leukocytes and T cells. The expression of intercellular adhesion molecule 1 and major histocompatibility complex class II antigens on the epidermal keratinocytes was also down-regulated. These results indicate that the thalidomide-induced alleviation of clinical symptoms of ENL was associated with a reduction of TNF alpha levels.
In South Africa and other high prevalence countries, transmission is a significant contributor to... more In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discrimina...
Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, M... more Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular pathogen of macrophages, with alveolar macrophages (AMs) being the primary conduit of infection and disease. The outcome of primary infection is most often a latently infected healthy human host, in whom the bacteria are held in check by the host immune response. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the host immune response fails to control the growth of bacilli, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols coughed out into the environment and inhaled by new hosts. The molecular details of the Mtb-macrophage interaction continue to be elucidated. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. Macrophages demonstrate tremendous phenotypic heterogeneity and functional plasticity which, depending on the site and stage of infection, facilitate the diverse outcomes. Moreover, host responses vary depending on the specific characteristics of the infecting Mtb strain. In this chapter, we describe a contemporary view of the behavior of AMs and their interaction with various Mtb strains in generating unique immunologic lung-specific responses.
Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage... more Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, Nacetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C3 receptor. In vivo stimulation triggered the capacity to internalize up to 45 % of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C3 receptor may be the most useful parameter.
A method is described for the separation of fish leucocytes and the establishment of pure monolay... more A method is described for the separation of fish leucocytes and the establishment of pure monolayers offish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90-99/O of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.
The morphology of mouse peritoneal macrophages and echinoid phagocytes during phagocytosis in vit... more The morphology of mouse peritoneal macrophages and echinoid phagocytes during phagocytosis in vitro was studied. A striking similarity in the function of the foreign .surface receptor is found in the t^vo systems. Glutaraldebydetreated erj'throcytes attached randomly over the entire surface of tht L'ells and were internalized without circumferential attachment between the particles and the phagocyte membrane. The particles seemed to sink directly into the cytoplasm of the cells. Tannin-treated erythrocytes were phagocytosed by the echinoid cells in a similar mode. The camp Semen r-coa ted erythrocytes were attached only in the perinuclear area of the echinoid phagocyte's membrane, but tbe morpbology of their internalization was similar to that mediated by the foreign surt'acf rt-ceptor. A circumferential attachment between the particles and tbe phagocyte membrane did not seem necessaiy. This is also the case for mouse peritoneal macrophages.
Experimental conditions have been developed to detect the efficient interaction of antigenpresent... more Experimental conditions have been developed to detect the efficient interaction of antigenpresenting cells and antigen-specific CD4+ T lymphocytes early in the human primary mixedleukocyte 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 >90% depleted of reactivity to other "third party" donors. Nonclustered lymphocytes, in contrast, are >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.
The thymus is critical for the production of mature, recirculating T cells. Congenitally athymic ... more The thymus is critical for the production of mature, recirculating T cells. Congenitally athymic mammals and neonatally thymectomized mice, have profound deficits in T cell numbers and immunologic functions. Such athymic individuals do not reject skin grafts nor do they resist a variety of infectious agents (1-5), and few lymphocytes are noted in the thymus-dependent areas oflymphoid tissues (5-7). The thymus is also the main site for rearranging TCRa and -0 genes (8-12). The repertoire of a/R receptors is shaped by deleting cells that react directly with self MHC (13) and by selecting cells that recognize foreign antigens in association with self MHC (14, 15). These negative and positive selections are thought to be mediated by bone marrow-derived dendritic cells in the thymus medulla and pharyngeal pouch-derived epithelium in the cortex (16-18).
Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few... more Mononuclear leukocytes are able to destroy tumor cells under a variety of circum- stances. In few such instances is the biochemical basis of cytotoxicity understood. Recently, we demonstrated that activated macrophages, stimulated by phorbol myristate acetate (PMA), 1 secreted substantial amounts of hydrogen peroxide (1). The release of this oxidant from macrophages correlated with the extracellular lysis of tumor cells
Proceedings of the National Academy of Sciences, 1990
Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed th... more Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.
Proceedings of the National Academy of Sciences, 1979
The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma... more The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma membrane were analyzed. Cells labeled with [ S~methionine were incubated with the nonpermeant reagent trinitrobenzene sulfonic acid to introduce the trinitrophenyl moiety on free amino groups of externally oriented membrane proteins. The cells were then incubated with rabbit anti-dinitrophenyl IgG and the immune complexes formed with the trinitrophenyl-proteins were isolated from detergent lysates of the cells by using fixed Staphylococcus aureus as the immunoadsorbent. Proteins isolated by this method were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The interval between the release of newly synthesized proteins from ribosomes and their appearance at the cell surface, where they became accessible to trinitrobenzene sulfonic acid, was studied in pulsechase experiments. The "transit" time of four major membrane glycoproteins (48,000-310,000 Md) ranged from 36 to 55 min and their appearance on the cell surface occurred in a relatively synchronous fashion. The turnover of most proteins of molecular weight above 50,000 was very slow (ti/2> 80 hr) and was rather synchronous. Two exceptions were the 310,000 Mr protein, which was lost with a t1/2 = 21 hr, and a major glycoprotein (Mr 48,000), which exhibited more complex kinetics. Although the overall turnover of surface proteins was biphasic in nature, the rapid phase of protein loss was largely due to low molecular weight species.
Proceedings of the National Academy of Sciences, 1986
The epidermal changes that occur in human cutaneous immune responses have been investigated in th... more The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from lato Ia'. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.
Proceedings of the National Academy of Sciences, 1989
The effect of multiple intradermal injections (four to six) of 10 micrograms of interferon gamma ... more 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.
Proceedings of the National Academy of Sciences, 1989
Experiments were carried out in the skin of patients with leprosy to examine whether suppressor c... more Experiments were carried out in the skin of patients with leprosy to examine whether suppressor cell populations either exist in the skin of multibacillary lepromatous leprosy patients, can be activated with antigen, or are induced to emigrate into a cutaneous site from the circulation. For this purpose, purified protein derivative of tuberculin, a delayed-type antigen that generates a cell-mediated immune response, was introduced into the skin alone or with nonviable tTo whom reprint requests should be addressed at: The Rockefeller University,
Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrop... more Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.
Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to ... more Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to thalidomide therapy were evaluated. Circulating tumor necrosis factor-alpha (TNF alpha) levels were assayed in serum obtained from lepromatous leprosy patients at diagnosis, during multidrug therapy, at the onset of ENL episodes, and during treatment with thalidomide. Patients with systemic ENL demonstrated the highest serum TNF alpha levels, which decreased significantly during thalidomide treatment. Serum TNF alpha in nonreactional patients was associated with mild flu-like symptoms and local inflammatory lesions. Serum interferon-gamma (IFN-gamma) was also elevated in patients with high TNF alpha levels. Thalidomide therapy reduced not only serum TNF alpha levels and the clinical symptoms but also the dermal infiltration of polymorphonuclear leukocytes and T cells. The expression of intercellular adhesion molecule 1 and major histocompatibility complex class II antigens on the epidermal keratinocytes was also down-regulated. These results indicate that the thalidomide-induced alleviation of clinical symptoms of ENL was associated with a reduction of TNF alpha levels.
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