In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery.
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Macrophages are normal targets for Salmonella during natural infections, and it has been demonstrated that attenuated bacteria can deliver nucleic acid vaccine constructs. Therefore, we assessed if attenuated Salmonella can be used for the in vivo delivery of transgenes to their natural cellular target, in an attempt to correct genetic defects associated with monocytes/macrophages. This system would offer the distinct advantage of achieving a specific targeting of defective cells in a non-invasive form. Using a reporter gene, we demonstrated that attenuated Salmonella could be used as an effective in vitro delivery system to transfer genetic material into nondividing cells like murine macrophages. In vivo, the oral administration of attenuated Salmonella allows targeted delivery of transgenes to macrophages and subsequently expression of transgenes at a systemic level. IFNgamma-deficient mice (GKO) were thus selected as a model for the in vivo validation of the Salmonella-based delivery approach. Attenuated Salmonella, used as the carrier for a eukaryotic expression vector encoding the murine IFNgamma gene, was able to restore the production of this cytokine in GKO macrophages. Their oral administration to IFNgamma-deficient mice also re-established, in these immunocompromised animals, the natural resistance to bacterial infections. These results demonstrate, for the first time, that attenuated Salmonella can be successfully used in vivo as a DNA delivery system for the correction of a genetic defect associated with monocyte/macrophages. (+info)
Acquired disorders of phagocyte function complicating medical and surgical illnesses.
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There is evidence that acquired dysfunction of neutrophils, monocytes, or macrophages is an important cause of infection in patients with diabetes mellitus, renal or hepatic failure, alcoholism, autoimmune diseases, influenza or human immunodeficiency virus infection, burns, and trauma. Distinguishable mechanisms of acquired phagocyte dysfunction include inhibitory effects of metabolic disturbances (e.g., hyperglycemia, uremia), chemical toxins (e.g., ethanol), viral proteins on phagocyte activation, and pathologic activation of phagocytes in the circulation (e.g., after hemodialysis, burns, or cardiopulmonary bypass). Although the burden of morbidity and mortality resulting from acquired phagocyte dysfunction appears to be vast, research in this area has been hampered by the complexity of the underlying illnesses and by limitations of laboratory assays and clinical study methodology. Given the advent of improved assays of phagocyte functions and treatments that can enhance these functions, there is a pressing need for more prospective studies of acquired phagocyte dysfunction. (+info)
Association of Pseudomonas cepacia with chronic granulomatous disease.
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Pseudomonas cepacia was recovered from a number of infected sites in three patients with chronic granulomatous disease of childhood. The organisms were identified on the basis of their oxidative utilization of a variety of carbohydrates and their positive beta-galactosidase and oxidase activities. They were resistant to most antimicrobial agents and moderately susceptible to chloramphenicol. Peripheral blood leukocytes isolated from two siblings with chronic granulomatous disease, including one of the patients in this series, failed to kill P. cepacia in vitro. Prolonged prophylactic and antimicrobial therapy may well have played a significant role in the colonization and infection of these patients with P. cepacia. (+info)
Complement-induced impairment of innate immunity during sepsis.
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This study defines the molecular basis for defects in innate immunity involving neutrophils during cecal ligation/puncture (CLP)-induced sepsis in rats. Blood neutrophils from CLP rats demonstrated defective phagocytosis and defective assembly of NADPH oxidase, the latter being due to the inability of p47(phox) to translocate from the cytosol to the cell membrane of neutrophils after cell stimulation by phorbol ester (PMA). The appearance of these defects was prevented by in vivo blockade of C5a in CLP rats. In vitro exposure of neutrophils to C5a led to reduced surface expression of C5aR and defective assembly of NADPH oxidase, as defined by failure in phosphorylation of p47(phox) and its translocation to the cell membrane, together with failure in phosphorylation of p42/p44 mitogen-activated protein kinases. These data identify a molecular basis for defective innate immunity involving neutrophils during sepsis. (+info)
Urokinase-deficient and urokinase receptor-deficient mice have impaired neutrophil antimicrobial activation in vitro.
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Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). We have shown that neutrophil recruitment to the lung during P. aeruginosa pneumonia is impaired in uPAR-deficient (uPAR-/-) mice but is normal in uPA-/- mice. However, both uPA-/- mice and uPAR-/- mice have impaired lung clearance of P. aeruginosa compared with wild-type (WT) mice. To determine the role of uPA and uPAR in antibacterial host defense, we compared neutrophil bacterial-phagocytosis, respiratory burst, and degranulation among uPA-/-, uPAR-/-, and WT mice. Neutrophil phagocytosis was significantly diminished comparing uPA-/- and uPAR-/- mice with WT mice at all time points. The generation of superoxide by both uPA-/- and uPAR-/- neutrophils was about half of that seen in WT neutrophils. Degranulation of azurophilic granules was significantly diminished in uPA-/- neutrophils compared with either uPAR-/- or WT neutrophils. By contrast, agonist-stimulated release of specific granules was not diminished in either uPA-/- or uPAR-/- mice compared with WT. We conclude that the uPA/uPAR system modulates several of the crucial steps in neutrophil activation that result in bacterial killing and effective innate host defense. (+info)
Ultrastructural studies of parallel tubular arrays in human lymphocytes.
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Parallel tubular inclusions were found in peripheral blood lymphocytes from 18 patients with various hematologic disorders, primarily lymphoproliferative processes, and 1 apparently healthy individual. The inclusions varied in size from 1000 to 6000 A and were usually membrane bounded. The microtubule-like structures comprising the inclusions ranged in size from 150 to 300 A and were packed in wall-to-wall contact with each other. Dense amorphous material and small dark crystalloids were frequently noted in the inclusions. There appeared to be a spatial and structural relationship of the inclusions with the centriole. The highest percent of lymphocytes with inclusionss (greater than 90%) were found in a patient with a lympho-proliferative disorder in whom 95% of the peripheral blood lymphocytes typed as T cells by spontaneous rosette formation with sheep red blood cells. (Am J Pathol 78:59-70, 1975) (+info)
The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes.
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The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined. (+info)
Congenital sensory neuropathy as a differential diagnosis for phagocytic immunodeficiency.
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There are few reports about congenital indifference to pain or Hereditary and Sensory Autonomic Neuropathy (HSAN). Several investigations for pathophysiology of this syndrome have been performed and different classifications about it. In this report we present a case of HSAN type II with general absence of pain and self amputations and leprosy-like damage of extremities which was suspected to be phagocytic immunodeficiency due to past history of repeated ulcer and abscess formation. (+info)