Intracellular Bacterial Killing Research Articles

Physicochemical characterization of a polypeptide present in uremic serum that inhibits the biological activity of polymorphonuclear cells.

A granulocyte inhibitory protein was isolated and characterized from uremic serum by using ion-exchange column chromatography, high-performance size-exclusion chromatography, and immunochemical procedures. The purification process concentrated the protein 240-fold and to a purity of greater than 95%. An overall recovery of 45% was achieved; the purified protein had a specific activity of 104 units per mg of protein. The polypeptide had a molecular weight of approximately 28,000 and an isoelectric point of 4.0-4.5. Amino acid sequencing of the NH2 terminus revealed a single sequence (Asp-Ile-Val-Met-Thr-Gln-Ser-Pro-Gly-Thr-Leu-Ser-Val-Ser-Pro-Gly-Glu-Arg-Ala- Thr) that proved to be nonhomologous with other serum proteins that appear during an inflammatory state. The polypeptide inhibited the uptake of deoxyglucose, chemotaxis, oxidative metabolism, and intracellular bacterial killing by polymorphonuclear leukocytes. A specific rabbit polyclonal antibody raised against the protein nullified these inhibitory changes. We contend that the protein is responsible for the leukocyte dysfunction that is commonly seen in patients with uremia.

In vitro Effect of Actinomycin D on Human Neutrophil Function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.

Drug action against intracellularly growingMycobacterium xenopi

The intracellular growth kinetics ofMycobacterium xenopi was studied in the murine J-774 macrophage cell line model. During the initial 4 days of infection, the bacilli divided about every 33 h. Electron microscopy of infected macrophages showed that bacteria inside phagosomes were surrounded by a protective electron-transparent zone (ETZ). This model was used for comparing the extracellular and intracellular activities of the following drugs: pristinamycin (PRISTINA), isoniazid (INH), clofazimine (CLOFA), rifabutin (=ansamycin; ANSA), rifampicine (RIFA), streptomycin (SM), ethambutol (EMB), and five fluoroquinolones, namely, ciprofloxacin (CIPRO), ofloxacin (OFLO), pefloxacin (PEFLO), enoxacin (ENOX) and norfloxacin (NORFLO). All the drugs were tested within their obtainable serum level concentrations in man. Under these conditions, CLOFA, SM, CIPRO, and OFLO were highly active against intracellularly growingM. xenopi, INH and RIFA were moderately active, whereas ANSA, PRISTINA, EMB, PEFLO, ENOX, and NORFLO were only growth inhibiting. The comparison of these data with extracellular activities of the same drugs underlined the discrepancies observed in test-tube drug activity evaluation and its correlation with results of chemotherapy in patients in whom the drug has essentially an intracellular bacterial killing role.

ブリどん食細胞のＰａｓｔｅｕｒｅｌｌａ ｐｉｓｃｉｃｉｄａに対する細胞内殺菌能測定法の検討

Applicability of some methods for determination of intracellular bacterial killing by yellowtail phagocytic cells against Pasteurella piscicida was investigated. The following method was found applicable. A mixture of phagocytic cells prepared from yellowtail kidney, viable Pasteurella piscicida suspension and normal yellowtail serum was prepared, and incubated at 25°C for 30min. Then, 100μg/ml streptomycin and 1, 500units/ml penicillin G were added to the mixture, and incubated again at 25°C for 15min. The phagocytic cells and the remaining extracellular bacteria were separated by centrifuging at 5, 900×g for 5min on Percoll at 1.070 specific gravity. Phagocytic cells were washed three times by modified L-15, and resuspended in modified L-15 at a density of 1×107 cells/ml. After incubation at 25°C for 60min, the phagocytic cells were destroyed by 15% sodium chloride solution containing 0.2% non-ionic detergent, BL-25, at 0°C for 15min. The number of viable bacteria in destroyed phagocytic cells was counted by the pour plate method using BHI agar containing 1.0% sodium chloride.

Pyoderma Gangrenosum

Four patients with pyoderma gangrenosum were treated with hyperbaric oxygen to prepare the wounds for skin grafting. Each wound responded to a course of daily hyperbaric oxygen with reduction of infection and increased capillary angiogenesis. During follow-up periods of 12 to 30 months, all wounds remained healed. Although the exact etiology of pyoderma gangrenosum is unknown, vasculitis with wound ischemia and infection are prominent components. Inspired oxygen partial pressures of 1100 to 1300 mmHg elevate wound oxygen tension despite relative ischemia. The impaired intracellular bacterial killing of hypoxic leukocytes is corrected during each day's 2-hour bolus of hyperbaric oxygen. Daily wound oxygenation increases collagen production by fibroblasts to support capillary angiogenesis.

Further studies on the variation among cows, bulls and calves in the ability of their blood polymorphonuclear leucocytes to kill Staphylococcus aureus

Bovine neutrophils (PMN) were isolated from anticoagulated blood stored for up to 6 h at room temperature. PMN were incubated with Staphylococcus aureus and 0·5% heated bovine serum for 2 h at 37 °C with constant rolling. An average of 90% of the bacteria were killed but there were large variations in the bactericidal activity of PMN from bulls and calves. The bacterial survival after 2 h incubation with PMN from 32 bulls and 16 calves ranged from 2 to 40%, and from 4 to 33% respectively. The rate of bacterial kill was log-linear with time for incubation periods of up to 2 h for PMN of low activity but bacterial phagocytosis was initially much more rapid for PMN of high activity with half the Staph. aureus killed within 10 min. Three healthy cows with a known history of susceptibility to E. coli mastitis gave average values in this test. There was a tendency for PMN with low overall bactericidal activity to show higher numbers of PMN-associated bacterial survivors—possibly due to reduced intracellular bacterial killing in addition to a low rate of phagocytosis.

Combined neutrophil and T-cell deficiency: Initial report of a kindred with features of the hyper-IgE syndrome and chronic granulomatous disease

A six year old female presented with a recent history of pyoderma gangrenosum involving her legs and arms associated with an episode of Mycoplasma-like pneumonia. This was followed by Aspergillus osteomyelitis involving her left ulna and right femur. Both the skin lesions and the osteomyelitis responded to prolonged treatment with antifungal and antibiotic agents. Investigation of this patient revealed (1) an elevated serum IgE (4,800 units/ml), (2) a defect in neutrophil chemotaxis that appeared to be due to immune complexes, (3) an abnormal nitroblue tetrazolium (NBT) result (0 percent stimulated and unstimulated), and (4) depressed mitogen responses to concanavalin A, phytohemagglutinin, and pokeweed mitogen, negative results of intradermal skin tests, and negative dinitrochlorobenzene (DNCB) serialization. The patient's clinically unaffected sibling had similar findings except for a positive DNCB response. In both children, intracellular bacterial killing of catalase-positive and negative organisms was normal. Kindred studies revealed widespread T-cell abnormalities consistent with autosomal dominant inheritance. Tissue typing studies showed that affected siblings shared the A1, B8, OR3 haplotype. This kindred is unique in that both the proband and the sibling have abnormalities of both the hyper-IgE syndrome and chronic granulomatous disease.

Immunocompetence in obesity.

Thirty-eight % of obese children and adolescents showed a variable impairment of cell-mediated immune responses in vivo and in vitro and reduction of intracellular bacterial killing by polymorphonuclear leukocytes. The obese group had a higher incidence of iron deficiency and moderately lower serum zinc concentrations. Levels of serum immunoglobulins, complement components C3 and C4, and numbers of T and B lymphocytes were comparable in the two groups. Serum triglycerides, cholesterol and lipoproteins were normal in all subjects. Immunologic changes correlated with the presence of subclinical deficiencies of iron and zinc. Therapy with these micronutrients for 4 weeks resulted in improvement in immunologic responses.

Host Defense in Infantile Osteopetrosis

Since infants with malignant osteopetrosis often die from infection at an early age, we studied several aspects of host defense in five such infants. No consistent abnormality was found in cellular or humoral immunity. Monocyte cellular chemotaxis and phagocytosis were normal in four tested infants. However, all four of these infants had decreased intracellular bacterial killing by monocytes. Neutrophil function tests in five infants showed that two had defective bacterial phagocytosis and four had reduced cellular chemotaxis, decreased nitroblue tetrazolium reduction, and decreased intracellular bacterial killing. The severity of the decreased bactericidal capacity of granulocytes did not correlate with the number of circulating immature granulocytes. Our data suggest that abnormal function of circulating monocytes and granulocytes may contribute to impaired host resistance to infection. We postulate that this defect may reflect a more generalized inherited abnormality of phagocytic cells and perhaps osteoclasts that plays a role in the pathogenesis of infantile osteopetrosis.

691 IMMUNOCOMPETENCE IN OBESITY

Obese individuals are susceptible to respiratory and cutaneous infections and post-operative wound sepsis. In a group of 21 children and adolescents diagnosed to be obese on the basis of weight-for-height and skin-fold thickness, a comprehensive assessment of immunity function showed serum immunoglobulins within the normal range, slight elevation of serum IgE and adequate antibody response. Subpopulations of circulating lymphocytes were comparable in proportion and number in the obese and the non-obese. Cutaneous delayed hypersensitivity and mitogeninduced lymphocyte DNA synthesis were reduced, the latter particularly when autologous plasma was used in cell cultures. Serum levels of complement components, opsonization and phagocytosis were normal, but intracellular bacterial killing was impaired. In the obese group, the mean serum lipid concentration was higher, and transferrin saturation and zinc level lower than in the control group. It is suggested that alterations in the immunocompetence of obese individuals may in part be the result of changed nutritional milieu and that such changes may contribute to increased susceptibility to infection.

Iron status, immune response and susceptibility to infection.

Nutritional factors can modulate immune responses. The concentration of iron, amongst other nutrients, influences host defence mechanisms. In experimentally induced iron deficiency in animals, morbidity and mortality on bacterial challenge are increased several-fold. Cell-mediated immunity and intra-cellular bacterial killing by polymorphonuclear leucocytes are impaired in iron-deficient individuals. This impairment is likely to be mediated by the effect of iron lack on cell proliferation, DNA synthesis and activity of iron-containing and iron-dependent enzymes involved in killing and elimination of microbes. Conversely, the availability of the free iron is a critical determinant for bacterial multiplication. It is not surprising then that epidemiological and clinical data on the frequency of infections--bacterial, fungal and others--in iron-deficient, iron-overloaded and healthy groups differ so widely. Vulnerability to infection based on the individual's iron status must be the net result of the effect of iron, or the lack of it, on microbial growth on the one hand and on immunocompetence of the host on the other. The key to keeping these interactions within physiological bounds is 'optimal iron nutrition'.

Impaired immunocompetence associated with iron deficiency

Summary Immunocompetence of 20 children with iron deficiency was evaluated. Serum immunoglobulin levels were normal; in the few with concurrent infection these values were elevated. Serum concentration of complement C3 was normal; in three out of four children with infection, it was reduced. Serum antibody responses to tetanus toxoid and S. typhi were adequate. Cutaneous hypersensitivity was reduced, and the in vitro lymphocyte transformation response to phytohemaggultinin stimulation was impaired. The proportion of T lymphocytes forming spontaneous rosettes with sheep red blood cells was slightly reduced. Opsonic activity of plasma and phagocytosis by polymorphonu-clear leukocytes was normal. Intracellular bacterial killing and reduction of nitroblue tetrazolium were distinctly impaired; they correlated with the severity of iron deficiency. Treatment of iron deficiency by oral or parenteral administration of iron corrected the immunologic abnormalities. It is suggested that iron deficiency is associated with secondary impairment of immunocompetence which is reversible.

Intraleukocytic bactericidal activity in patients receiving corticosteroid and radiation therapy, and in patients with diabetes mellitus.

Bactericidal activities of peripheral white blood cells obtained from patients and from healthy persons were examined in vitro. The results obtained are summarized as follows. 1. Peripheral white blood cells from patients receiving corticosteroid and radiation therapy showed decreased levels of intracellular bactericidal activities against Staphylococcus aureus. The leukocytes from almost all patients examined displayed intense activities of intracellular bacterial killing against Streptococcus pyogenes. 2. Only polymorphonuclear leukocytes (PMNs) and macrophages obtained from patients in severe stages of diabetes mellitus exhibited decreased levels of intracellular bactericidal activities against S. aureus. 3. The leukocytes from all patients examined exhibited the same levels of intracellular bactericidal effects against S. pyogenes as leukocytes from healthy persons. 4. Pseudomonas aeruginosa, which was phagocytized by PMNs obtained from healthy persons, demonstrated a remarkable degree of resistance to any intracellular bactericidal effect.

Effect of Alcohol and Various Diseases on Leukocyte Mobilization, Phagocytosis and Intracellular Bacterial Killing

Abstract Alcoholics are more susceptible than the general population to pneumonia, apparently in some cases because of nutritional deficiencies. Ethyl alcohol produced a profound depression in the rate of leukocyte mobilization into traumatized skin of nutritionally normal people, and was comparable to that observed in terminal phases of medical shock. Diabetic patients showed a less marked decrease in mobilization. No significant depression of polymorphonuclear-leukocyte mobilization was found in patients with cirrhosis or uremia, in coma or undergoing prolonged general Anesthesia for major surgery. Human polymorphonuclear leukocytes, obtained after alcohol was given, or leukocytes exposed to alcohol in vitro showed no decrease in ability to ingest or kill ingested bacteria. Diminished leukocyte mobilization may contribute substantially to the increased susceptibility to infection in the lung parenchyma noted in alcoholics and in diabetic patients or those with severe prolonged shock.

Intracellular killing of bacteria.

DRAMATIC morphologic changes occur in polymorphonuclear leukocytes during bacterial phagocytosis. Cytoplasmic granules disappear, and the granular membranes fuse with phagocytic vacuoles. These granules (lysosomes) containing hydrolytic enzymes and cationic proteins with specific antimicrobial properties are solubilized during phagocytosis and surround the bacteria in phagocytic vacuoles. Despite identification of bactericidal factors (for example, muramidase and phagocytin) and knowledge of their shift to the phagocytic vacuole, the functional mechanism of intracellular bacterial killing is still uncertain. Peritoneal mononuclear leukocytes without muramidase or phagocytin are able to kill bacteria rapidly, and in a reverse situation, polymorphonuclear leukocytes from patients with chronic granulomatous disease . . .