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Biomedical subjects

L Harvath

Publications and source records attributed to L Harvath.

53 records · Page 3Linked to original sources
PubMed1985

Studies of human C5a as a mediator of inflammation in normal human skin.

C5a is an 11,000-D fragment of the fifth component of complement (C5) with potent anaphylatoxic and leukocyte chemotactic activities. C5a is believed to play an important role in the pathophysiology of certain skin disorders and systemic diseases with cutaneous manifestations. However, there is very little known about the in vivo reactivity of C5a in man. In this study we examined the effects of intradermal injections of human C5a in 17 normal volunteers. C5a was prepared by interacting highly purified human C5 with zymosan bound alternative pathway C5 convertase under conditions resulting in consumption of approximately 90% of the C5 substrate. C5a produced in this manner was chemotactic for human neutrophils and monocytes (0.5 X 10(-7) to 10(-9) M) and caused neutrophil aggregation and myeloperoxidase release (concentrations greater than or equal to 10(-10) M) in vitro. In vivo, C5a produced immediate wheal and flare reactions in all volunteers, and was active in doses as low as 1 ng (10(-13) mol). Intradermal testing with 20 ng of C5a in eight volunteers produced a maximal mean wheal of 11.75 mm (+/- 0.80 mm SEM) 20 min after anaphylatoxin injection, and a maximal mean erythema of 62.50 mm (+/- 3.27 mm SEM) 10 min after C5a administration. Reactions at C5a test sites were dose-related, associated with marked pruritus in some subjects, resolved without lesion formation, and were not associated with late phase reactions. In vivo testing revealed that human C5a was a more potent mediator of wheal and flare reactions than histamine, 48/80, human C3a, or morphine sulfate. Skin biopsies from eight volunteers 20 min after intradermal injection of 20 ng of C5a revealed a neutrophil-predominant perivascular infiltrate, endothelial cell edema, and sites of leukocytoclasis. Mast cell degranulation was observed on both light and electron microscopy of biopsies from C5a test sites. Although erythema at C5a injection sites was reduced by pretreating volunteers with hydroxyzine, whealing reactions and cellular infiltrates in biopsies were unaffected by this H1-antihistamine. Moderate doses of systemic corticosteroids did not alter clinical or histologic reactions at C5a injection sites in two volunteers. This study, using doses within the potential physiologic range of the anaphylatoxin, provides a comprehensive assessment of the effect of human C5a on normal human skin.

Adult↗
PubMed1984

Oxidized N-formylmethionyl-leucyl-phenylalanine: effect on the activation of human monocyte and neutrophil chemotaxis and superoxide production.

F-met-leu-phe, a potent neutrophil and monocyte chemoattractant, can be oxidized to F-met-leu-phe sulfoxide by metabolically activated neutrophils. We investigated the interaction of human neutrophils and monocytes with chemically prepared oxidized derivatives of F-met-leu-phe: F-met-leu-phe sulfoxide and F-met-leu-phe sulfone. We compared the derivatives with nonoxidized parent F-met-leu-phe for specific binding to neutrophils and monocytes, and for effectiveness as chemoattractants and stimuli of superoxide production. We observed that neutrophils did not migrate to either derivative over the concentration range of 10(-9) to 10(-3) M. In marked contrast, monocytes migrated to both oxidized peptides, with optimal chemotaxis occurring at derivative concentrations 10- to 100-fold higher than the optimal parent F-met-leu-phe concentration. F-met-leu-phe sulfoxide and sulfone stimulated neutrophil and monocyte superoxide production and specifically bound to both neutrophils and monocytes at optimal concentrations that were 10- to 100-fold greater than the optimal parent F-met-leu-phe concentration. These results suggest that the higher concentrations of oxidized derivatives required for monocyte migration and for neutrophil and monocyte superoxide production are due to a reduced affinity of the oxidized derivatives for the F-met-leu-phe receptor. The finding that oxidized F-met-leu-phe binds to the neutrophil receptor without eliciting a chemotactic response suggests that the F-met-leu-phe receptor complex or chemotaxis transduction mechanism is different in human neutrophils and monocytes.

Chemotaxis, Leukocyte↗
PubMed1983

Abnormal polymorphonuclear leukocyte motility in dermatologic diseases.

A variety of skin diseases have been reported to have associated abnormalities in PMN chemotactic motility; however, the relationship of abnormal leukocyte motility to the pathogenesis of these diseases is poorly understood. A common feature in skin diseases associated with depressed chemotaxis has been the frequency of recurrent pyogenic infections. Since the chemotaxins and methodologies for evaluating leukocyte chemotaxis differ among laboratories, little is known about the specificity of these chemotactic defects. Further information regarding PMN motility defects may be obtained from detailed studies of migration to various doses of chemotaxins. The commercial availability of potent, structurally defined chemotaxins, such a N-formylated peptides, provides laboratories the opportunity to evaluate migration to "common" chemotaxins. In addition, the development of the multiple microwell chemotaxis chamber has made possible the opportunity to collect substantial leukocyte motility data from relatively small blood samples. Further investigations of leukocyte motility in various dermatologic diseases are clearly needed. A more systematic approach, i.e. (1) use of several different chemotaxins at various doses in chemotaxis assays and (2) use of standard, structurally defined chemotaxins, would facilitate comparative analyses of clinical studies among laboratories. It is likely that this approach would provide more specific information about the relevance of leukocyte motility defects in these diseases.

Acrodermatitis↗
PubMed1982

Two neutrophil populations in human blood with different chemotactic activities: separation and chemoattractant binding.

Normal human blood contains a population of neutrophils that migrates to various chemoattractants and a population that fails to migrate. The percentage of neutrophils migrating to optimal concentrations of chemoattractants was quantified: 20 to 40% migrated to N-formylmethionyl-leucyl-phenylalanine, 30 to 50% migrated to human C5a, 25 to 35% migrated to human leukocyte-derived chemotactic factors, 20 to 30% migrated to casein, 15 to 20% migrated to pepstatin, and 1 to 5% migrated to medium alone. Neutrophil migration to the most active chemoattractant was not increased when other chemoattractants were added, indicating that the population of neutrophils migrating to the most active attractant was the same population that was migrating to the other attractants. The percentage of neutrophils migrating to a chemoattractant was not altered by prolonging the assay incubation period or by replacing the attractant with new chemoattractant during the assay, and the percentage was independent of the neutrophil concentration added to the chemotaxis chamber. Nonmigrating neutrophils were isolated with a chemotaxis collection chamber, and they were examined for radiolabeled chemotactic peptide binding. The binding of radiolabeled N-formylmethionyl-leucyl-phenylalanine by nonmigrating and migrating neutrophils was identical.

Cell Separation↗
PubMed1982

Only the chemotactic subpopulation of human blood monocytes expresses receptors for the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine.

Human peripheral blood monocytes comprise a subpopulation of 20 to 40% that is capable of responding to chemoattractants and a remaining subpopulation that cannot respond. We were able to obtain 99%-pure attractant-responsive monocytes by using a newly constructed separation chamber. The binding of the radioactive chemotactic peptide N-formylmethionyl-leucyl-[3H]phenylalanine to migrating and nonmigrating populations was then studied. The binding was saturable at room temperature in the presence of azide. Saturation occurred at 5 x 10(-8) M, and 50% of the maximal binding was obtained at 10(-8) M, the concentration that induced optimal chemotaxis. The nonmigrating monocytes did not bind the peptide under the same conditions, which shows that at least one reason for a nonresponsiveness to chemotaxin is apparently a lack of receptors. By Scatchard analysis we calculated an equilibrium dissociation constant ranging from 23 to 37 nM; the number of binding sites per cell ranged from 64,000 to 77,000. The binding was very rapid. Fifty percent of the optimal binding occurred at 3.5 min, and equilibrium was reached after 20 to 30 min. Chemotactic deactivation of the monocytes reduced the number of available binding sites by 60%.

Azides↗
PubMed1980

Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly.

A neutrophil chemotaxis assay was developd which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 microns thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20-50% fall off polyvinylpyrrolidone-coated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm x 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 microliters of attractant per assay well. Neutrophil chemotaxis was complete within 10-20 min at 37 degrees C, with 20-30% of the cells migrating to N-formyl-methionyl-leucyl-phenylalanine and 40-50% migrating to complement derived C5a.

Carbonates↗
PubMed1979

Defective initiation of oxidative metabolism in polymorphonuclear leukocytes.

The polymorphonuclear leukocytes of a two-year-old boy who had multiple episodes of bacterial infections demonstrated defective oxidative metabolism with phagocytic, but not with soluble (non-phagocytic), metabolic stimuli. We used a chemiluminescence assay to examine the patient's polymorphonuclear leukocyte responses to numerous particulate and soluble stimuli. The patient's polymorphonuclear leukocytes had substantially depressed chemiluminescent responses during phagocytosis of opsonized particles (latex, pneumococci, pseudomonas, streptococci and zymosan); however, we observed normal chemiluminescent responses when these leukocytes were stimulated with soluble agents (sodium fluoride, concanavalin A, cytochalasin E, calcium ionophore A23187 or phorbol myristate acetate). Polymorphonuclear leukocyte oxygen consumption and superoxide production were impaired during phagocytosis, even though phagocytosis was normal. In addition to the metabolic defect, this patient's polymorphonuclear leukocytes had depressed chemotactic and bactericidal activities. This study provides evidence that polymorphonuclear leukocytes have more than one mechanism for initiating oxidative metabolism.

Blood Bactericidal Activity↗
PubMed1979

Light generation with Fenton's reagent. Its relationship to granulocyte chemiluminescence.

A simple chemical system consisting of FeSO4 and H2O2 (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H2O2 was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH.) and singlet oxygen (1 deltagO2) quenchers suppressed chemiluminescence of the FeSO4 + tryptophan + H2O2 system; and, deuterium oxide (2H2O) enhanced chemiluminescence of both FeSO4 reactions. These observations suggest that a radical chain reaction involving both OH. and 1 deltag O2 is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H2O2. Since iron and H2O2 are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.

Chemical Phenomena↗
PubMed1979

The collection, preservation and function of peripheral blood hematopoietic cells in dogs.

Semicontinuous flow centrifugation (SFC) was employed in canines to obtain adequate numbers of cells for autologous marrow repopulation following supralethal cyclophosphamide administration (100 mg per kg). Four cycles using a 225 ml bowl and 30 ml per minute flow rate were carried out for procurement. Collections averaged 8.4 +/- 0.4 x 10(9) (n = 30) leukocytes. Mononuclear cells (MNC) comprised 75 +/- 2% of the population and granulocyte colony-forming units (CFU-C) totaled 1.9 +/- 0.09 x 10(5) colonies per collection. Eighty-six percent of mononuclear cells were "T" cells in the 30 to 90 second fraction compared to 49% at 150 to 120 seconds. CFU-C fractionation revealed a peak at 30-210 seconds and a second peak at 120-210 seconds. Following programed freezing and rapid thawing 77.7 +/- 11.4% of CFU-C were recovered. Using a dose of 1 x10(9) MNC per kg for reinfusion, marrow repopulation and clinical recovery occurred in four out of four dogs. It was concluded that 1) SFC was effective for obtaining adequate numbers of peripheral blood stem cells for autologous marrow repopulation. 2) A rapid thawing and direct transfusion technique appears satisfactory for administration. 3) Some separation of "T", "B" and CFU-C peripheral blood components is possible by centrifugation.

Animals↗
PubMed1978

Chemiluminescence of human and canine polymorphonuclear leukocytes in the absence of phagocytosis.

Polymorphonuclear leukocytes (PMNs) have increased oxidative metabolism during phagocytosis and emit light (chemiluminescence, CL) as a result of metabolic activation. The present study examined PMN CL in the absence of phagocytosis using sodium fluoride (NaF), a nonparticulate agent and known stimulator of cellular oxidative metabolism. Normal human and canine PMNs were assayed in a CL spectrometer which permitted continuous sample mixing and constant temperature regulation during CL measurement. PMNs treated with 20 mM NaF demonstrated maximum CL responses of 10,000-20,000 cpm above background, 13-17 min after addition of NaF at 37 degrees C. Temperature regulation of reaction mixtures was found to be a critical factor in assaying PMN CL responses to NaF, because a small decrease in temperature (i.e. 1.5 degrees C) substantially depressed and delayed the CL response. Superoxide anion production correlated closely with CL responses in NaF-treated human PMNs. CL responses were completely suppressed in the presence of the oxidative metabolic inhibitors, iodoacetamide, and N-ethylmalemide; and were partially suppressed in the presence of either superoxide dismutase or sodium azide.CL responses of NaF-treated PMNs were significantly lower than responses generated by PMNs phagocytizing opsonized yeast. When NaF was evaluated for its effect on light generation from a singlet oxygen dependent CL reaction, it was found that NaF did not quench singlet oxygen light. This study demonstrates that PMN CL can occur in the absence of phagocytosis, and it proposes that a nonphagocytic PMN CL assay may be useful in evaluating leukocyte metabolic defects.

Animals↗
PubMed1976

Evaluation of type-specific and non-type-specific pseudomonas vaccine for treatment of pseudomonas sepsis during granulocytopenia.

The protective role of serotype-specific and non-type-specific active immunity against Pseudomonas aeruginosa infection was assessed in granulocytopenic dogs. Dogs were preimmunized with either specific serotype 6 vaccine (SI) or nonspecific serotype 3 vaccine (NSI) and challenged intravenously with 10(7) viable serotype 6 P. aeruginosa during granulocytopenia. Control dogs (C) having insignificant anti-pseudomonas antibody levels were also tested. Results showed: (i) significant increase in survival of SI dogs (P less than 0.05) compared to C and NSI dogs, with no significant difference between C and NSI animals; (ii) lower febrile responses in SI dogs; and (iii) markedly reduced bacteremia in SI dogs compared to C and NSI animals. SI dog sera from survivor animals did not kill the infecting pseudomonas strain in vitro. The study demonstrated that type-specific immunity to P. aeruginosa induced by active immunization is effective in protection against pseudomonas during granulocytopenia and that non-type-specific immunity offers no cross-reactive protection. The findings suggest that the reticuloendothelial system in conjunction with specific immunity constitute an important defense against pseudomonas infections.

Agranulocytosis↗
PubMed1976

Passive immunity against pseudomonas sepsis during granulocytopenia.

Specific passive immunity against Pseudomonas aeruginosa sepsis was assessed in granulocytopenic dogs. Dogs were infused with either normal or antipseudomonas immune plasma 24 h before pseudomonas challenge. They were challenged intravenously with 10(7) serotype 6 P. aeruginosa during granulocytopenia. Treatment was evaluated by observation of survival periods, febrile responses, type 6 pseudomonas antibody titers, and quantitative cultures of blood and tissues. The results demonstrated that passively immunized dogs did not survive infection. Both normal-plasma and immune-plasma recipients had bacteremia at death, with median values of 980 and 470 pseudomonas per ml of blood, respectively. All dogs had marked febrile responses 24 h after pseudomonas challenge and had high concentrations of pseudomonas in their lung tissue at death, with median values of 10(8) pseudomonas per g of wet tissue weight. After plasma infusion, immune-plasma recipients had high concentrations of anti-pseudomonas antibody, with total antibody titers ranging from 256 to 1,024 and a median value of 1,024. These titers were comparable to titers attained in a previous study from our laboratory using active immunization with pseudomonas lipopolysaccharide vaccine, where the median total anti-pseudomonas antibody titer was 2,048. Actively immunized animals, however, were significantly protected against pseudomonas sepsis and had prolonged survival periods and prevention of bacteremia. The present study demonstrates that circulating type-specific antibody is not solely responsible for the protection afforded to granulocytopenic dogs actively immunized against pseudomonas.

Agranulocytosis↗
PubMed1976

Combined pre-immunization and granulocyte transfusion therapy for treatment of pseudomonas septicemia in neutropenic dogs.

An experimental model was designed to evaluate a combined protocol of active immunization and granulocyte transfusions for treatment of Pseudomonas aeruginosa sepsis in the neutropenic host. One member of a pair of dogs was immunized with P. aeruginosa vaccine. Both dogs were then rendered transiently neutropenic with a single intravenous dose of cyclophosphamide (40 mg. per kilogram) and challenged with an intravenous inoculum of P. aeruginosa. Twenty-four and 48 hours after pseudomonas challenge each animal received granulocyte transfusions. Effectiveness of therapy was evaluated by observation of survival time, febrile response, and quantitative blood cultures. Results showed a significant increase in the survival period (P is less than 0.05), a lower febrile response (P is less than 0.025), negative blood cultures, and a greater recovery rate in the immune group. Immune dogs that died had negative blood cultures or less than or equal to 10 pseudomonas per milliliter of blood despite the presence of P. aeruginosa in tissues. In contrast, control dogs had septic deaths within 67 hours of pseudomonas challenge, marked febrile responses with 24 hours of infection, and positive blood cultures with 4,000 to 25,800 pseudomonas per milliliter of blood. These data show that combined therapy with immunization and granulocyte transfusions is effective in reducing the severity of P. aeruginosa infection and in preventing bacteremia during periods of leukopenia.

Agranulocytosis↗