Defective neutrophil motility and recurrent infection. In vitro and in vivo effects of levamisole.

Eighteen patients with primary abnormalities of neutrophil chemotaxis are described. The most common clinical presentation was one of recurrent upper respiratory tract infection (nine patients) or recurrent pyoderma (seven patients), and two children had a history of oropharyngeal candidiasis and recurrent skin sepsis. Of these eighteen patients, sixteen had intrinsic polymorphonuclear leucocyte (PMN) defects as shown by diminished random migration and movement towards endotoxin-activated serum. PMN chemotaxis towards casein was, however, normal. In nine out of the latter patients, there was an associated inability of the serum to generate chemotactic factors. PMN from two adult patients, both suffering from recurrent boils, moved normally both in random and directed systems, but sera from these patients contained heat-stable inhibitors of neutrophil chemotaxis. In vitro levamisole treatment (10(-3) M) markedly improved the PMN function. When patients were treated with levamisole, however, no clinical response was noted, although PMN movement improved in a number of cases.     

Interaction of mouse peritoneal macrophages with fixed rabies virus in vivo and in vitro.

The resistance of mice to intraperitoneal and intramuscular infection with fixed rabies virus increases with age. Treatment of mature animals with either silica, indian ink or antimacrophage serum, which are cytotoxic for macrophages, reduced their resistance to intraperitoneal, but not to intramuscular or intracerebral infection. Transfer of peritoneal macrophages from adults to syngeneic suckling mice delayed but did not prevent mortality from intraperitoneal infection: transfer of peritoneal macrophages to intramuscular sites of infection did not protect adult mice. Rabies virus was phagocytosed by peritoneal macrophages in culture but neither replicated nor induced interferon. Evidence of active intracellular destruction of virus was obscured by thermal inactivation at 37 degrees C. Less inactivation occurred at 33 degrees C. Infected macrophages from suckling mice, but not those from adult mice, spread infection to susceptible cells.     

Effect of levamisole on E-rosette-forming cells in vivo and in vitro in Hodgkin's disease.

Incubation in vitro of lymphocytes from patients with Hodgkin's disease with 40 mug per milliliter of levamisole resulted in a rise in the number of E-rosette-forming cells from 33.6 +/- 12.5 per cent (mean +/- S.D.) to 56.7 +/- 14.6 per cent. The drug had no effect on normal lymphocytes. Ten patients with Hodgkin's disease treated six months previously with levamisole were restudied. The positive skin tests to PPD, candida and mumps persisted. However, the E-rosette-forming cells decreased to the pretreatment levels (34.7 +/- 6.4 per cent). Readministration of 150 mg of levamisole for three days raised the number of E-rosette-forming cells to 54.1 +/- 5.6 per cent. This effect was observed for at least two months. However, the drug had no effect in vitro as long as the in vivo effect persisted. These results demonstrate a clear immunologic effect of levamisole in Hodgkin's disease and indicate that the low number of E-rosette-forming cells is not due to a real T-cell depletion.     

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

Candidacidal activity of mouse macrophages in vitro.

Mouse peritoneal macrophages were infected in vitro with Candida albicans, and the phagocytic and candidacidal activities were estimated by microscopic examination of Giemsa-stained cells. Activated macrophages obtained from either BCG-vaccinated animals or by in vitro exposure of normal macrophages to phytohemagglutinin-induced lymphokines exhibited higher phagocytic and candidacidal activities than did normal macrophages. However, activated macrophages obtained by in vitro exposure of macrophages to candida-induced lymphokines exhibited the highest phagocytic and candidacidal activities. The incorporation of immune mouse serum into the culture medium also enhanced the phagocytic and candidacidal activities of the normal macrophages but failed to improve the function of the activated macrophages. These results suggest that both activated macrophages and antibodies may be required for controlling candida infections in mice.     

The long-term impaired macrophages functions are already observed early after high-dose ethanol administration

Herein, we described an experimental model of high-dose ethanol (EtOH) administration, able to induce in vitro impairment in macrophage phagocytic capacity, already observed at 24 h after the last EtOH administration. This phenomenon was characterized by enlarged time required for adhesion and internalization events. Parallel studies documented an overall impaired production of interleukin (IL)-6 and nitric oxide (NO) production by peritoneal macrophages in EtOH-treated mice following interferon (IFN)-γ and lipopolysaccharide (LPS) stimuli. Although the impaired IL-6 response could not be restored by any of the experimental conditions tested, the lower NO response to INF-γ and LPS was overturned by simultaneous IFN-γ/LPS stimuli. It was interesting to notice that high-dose EtOH administration drives peritoneal macrophages towards long-term impairment in phagocytosis capacity with slower adhesion time, but with no impact on the time required for internalization. Moreover, 30 days after the last EtOH administration, lower IL-6 response to INF-γ and impaired NO production were still observed in response to IFN-γ/LPS stimuli, with the IL-6 response to IFN-γ being restored by IFN-γ/LPS stimuli. Histological studies showed that high-dose EtOH administration led to long-term in vivo impairment of antigen-clearance following OVA-driven delayed-type-hypersensitivity induction, characterized by the presence of a large amount of unprocessed OVA surrounded by dermal inflammatory infiltrate, suggesting defective activity of antigen-presenting cells. Together, these findings supported our hypothesis that the poor antigen clearance in vivo may be related to the impaired macrophage function in vitro . These observations in the murine experimental model may reflect some of the consequences of EtOH consumption by humans.     

The production of ceroid by mouse peritoneal macrophages in vitro.

Murine resident peritoneal macrophages accumulated lipid droplets and subsequently insoluble, ceroid-like material when cultured in vitro in a medium containing 33% fetal calf serum. At least some of this insoluble lipid was membrane-bound and by light microscopy it often appeared as ''rings'' with a hollow centre. It is suggested that the production of ceroid may be the consequence of the uptake of lipids from the extracellular medium and the activity of the macrophage''s membrane-bound oxidative microbicidal mechanisms. The results indicate that macrophages are capable of rendering lipids insoluble, supporting the suggestion that this might occur in the atherosclerotic plaque.     

Acetylspiramycin and the immune system. I. Effects of acetylspiramycin on phagocytosis by mouse macrophages in vitro and in vivo

A study was made of the effect of acetylspiramycin (ASPM) on the phagocytic activity of mouse macrophages in vitro and in vivo, using opsonized, 51Cr-labelled sheep red blood cells (SRBC) as test particles. Resident mouse peritoneal macrophages cultured with ASPM (25-100 micrograms/ml for 18 h) showed a 62-92% reduction in phagocytosis. This was due to decreases in both attachment and ingestion of SRBC and was additional to the detachment of some macrophages from the surface of the culture chamber. Morphologically the macrophages were vacuolated, with some nuclear condensation. When the cells were cultured for a further 48 h after removal of ASPM there was almost complete functional and morphological recovery. When mice were treated with ASPM (50-100 mg/kg orally for 7 days) their peritoneal macrophages showed increases of 57-121% in phagocytic activity in vitro. In mice treated with ASPM (200 mg/kg orally for 7-21 days) the clearance of i.v. injected opsonized SRBC was significantly accelerated. Thus, although ASPM is reversibly toxic to macrophages in vitro, it is not toxic in vivo, but actually stimulates the mononuclear phagocyte system. It is possible that metabolites of ASPM, such as neospiramycin, produced in vivo but not in vitro, are responsible for the stimulation.     

In vivo andin vitro HeNe laser effects on phagocyte functions

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's aggregation, when using PHA and PMA, presented a reduction that was statistically very significant, not as a subordinate dose. An increase of the release of ATP was demonstrated only at 4 joules and precedes granulocyte aggregation. When using Ca²⁺ ionophore A23187 as stimulus, laser irradiation at 1, 2 or 4J did not show any modification of granulocyte aggregation. The monoclonal antibody 60.1, which identifies a membrane antigen fundamental for aggregation and chemotaxis, is expressed in normal amounts on granulocyte membranes both before and after irradiation with a HeNe laser. In fact, laser irradiation preferentially attacks the area of the cellular centrosome that determines a modification of cellular morphology. The electron microscope and immunofluorescence study with a monoclonal antibody have pointed out a disorganization of the microtubles. The alteration of some of the granulocyte functions is correlated to the damage in the centrioles. The granulocyte mitocondrial system and surface membrane remain intact, and this explains the normal production and release of free radicals. Further experiments are necessary to evaluate the clinical application of lasers in various diseases with immunophagocytic pathogenesis.     

Activation process of macrophages after in vitro treatment of mouse lymphocytes with dodecylglycerol.

Alkylglycerols, inflammation products of cancerous membrane lipids, efficiently activate macrophages. A brief in vitro treatment (30 min) of peritoneal cells (mixture of non-adherent and adherent cells) with a small amount (50 ng/ml) of synthetic dodecylglycerol (DDG) resulted in greatly enhanced Fc-receptor-mediated ingestion activity of macrophages. However, treatment of adherent cells (macrophages) alone with DDG produced no significant enhancement of macrophage ingestion activity, implying that macrophage activation requires a contribution of non-adherent cells. DDG-treated non-adherent cells were found to generate a macrophage-activating signal factor. Studies with a serum free-0.1% egg albumin-supplemented RPMI 1640 medium revealed that a serum factor is essential for macrophage activation process. Time course analysis of stepwise transfers of conditioned media of DDG-treated or untreated B cells and T cells revealed that DDG-treated B cells rapidly transmit a factor to untreated T cells which yield the ultimate macrophage-activating factor. This signal transmission among these cells for the macrophage activation process is too rapid to allow time for synthesis of inducible gene products. Thus, we hypothesized that a serum factor is modified by the pre-existing function of DDG-treated B cells and further modified by the pre-existing function of untreated T cells to yield macrophage-activating factor. This hypothesis was confirmed by the demonstration that DDG-treated splenic non-adherent cell ghosts modify a serum factor to yield macrophage-activating factor.     

The influence of immunization on some functions of polymorphonuclear leucocytes in vitro.

Polymorphonuclear leucocytes (PMNL) obtained from immunized guinea pigs possess an affinity to antigen. The guinea pigs were immunized with human serum albumin (HSA) in complete Freund's adjuvant. The ability of PMNL to bind antigen was tested by means of the functional disorders induced by antigen after incubation with PMNL. The "immune" PMNL appeared to be more responsive to antigen when compared to PMNL obtained from non-immunized animals. The antigen decreases oxygen consumption, the phagocytosis coefficient and 14C-glycine incorporation in the "immune" PMNLs in vitro and also induces leakage of some lysosomal hydrolases from PMNL in the course of an 18 hrs incubation period.     

The effects of azathioprine and levamisole on lymphocyte stimulation.

Azathioprine at 10 micrograms per ml caused approximately a 50% inhibition of phytohaemagglutinin (PHA), purified protein derivative (PPD) or Veillonella induced in vitro lymphocyte stimulation. This suppression was significantly reduced in lymphocyte cultures from patients undergoing an eight week course of levamisole therapy. Azathioprine-induced suppression was also reversed when culturing lymphocytes in the presence of levamisole. On the other hand, levamisole in vitro failed to reverse the azathioprine suppression of cord blood lymphocyte responses.     

In vitro and in vivo effects of Imipenem on phagocytic activity of murine peritoneal macrophages

Imipenem, a new antibiotic beta-lactam, and Tienam (an Imipenem/Cilastatine combination) have been studied in vitro and in vivo respectively, in the phagocytic function of macrophages. In this paper we have seen the variations produced by 50 mg/l of Imipenem and 120 mg/kg of Tienam in the adherence, spontaneous mobility, chemotaxis, opsonization, phagocytosis of Candida albicans and latex beads, candidicid effect and nitroblue tetrazolium (NBT) reduction in peritoneal macrophages from BALB/C mice. This antibiotic significantly increases in vitro and in vivo the adherence, spontaneous mobility and chemotaxis, phagocytosis of latex beads and the digestion of ingested material (nitroblue tetrazolium reduction) in the above-mentioned cells. The number of Candida albicans opsonized and ingested by the macrophages is not modified in the presence of Imipenem, and neither is the candidicid effect.     

The influence of some metabolic inhibitors on in vitro phagocytizing macrophages

In the present work the uptake of foreign materials by macrophages has been studied in order to elucidate its possible energy-dependent mechanisms. We used monolayer cultures of macrophages from human peripheral venous blood, treated with the following metabolic inhibitors: iodoacetic acid, fluoroacetic acid, sodium fluoride, sodium malonate, sodium azide, 2–4-dinitrophenol, cycloheximide, and ouabain. The test assay was performed by using a zymosan particles suspension in Mc Coy 5 A medium supplemented as follows.The quantitation of phagocytosis was obtained by direct count of intracellular zymosan particles by oil 100X microscopy and the results were submitted to a statistical evaluation. The most effective inhibitor we found was iodoacetate, an inhibitor of anaerobic glycolysis, but fluoride, which acts on the same metabolic pathway at a different site, was quite ineffective. The same ineffectiveness we found for fluoracetate and malonate which act on the Krebs cycle. On the contrary, dinitrophenol (uncoupler of oxidative phosphorylation), azide (inhibitor of cytochrome linked-phosphorylation), ouabain (inhibitor of membrane ATPase activity) and cycloheximide (inhibitor of protein synthesis) give a remarkable decrease of index of phagocytosis after a 3h incubation. In conclusion, we can suppose that the energy-dependent phagocytosis is first depending on transport across the cell membrane (ATPase activity and protein synthesis) and second both on anaerobic glycolysis and oxidative phosphorylation.     

The effects of homogeneous human prealbumin on in vitro and in vivo immune responses in the mouse

A highly purified preparation of human prealbumin was shown to potentiate the sensitivity of rosette spleen forming cells of adult thymectomized mice to azathioprine in vitro and in vivo and to induce the appearance of the Thy 1, 2 antigen in vitro on spleen cells of adult thymectomized mice. Prealbumin also enhanced IgM antibody synthesis to sheep red blood cells (SRBC) in vitro in 12 week old mice and in vivo in aged (45 - 58 week old) and nude (nu/nu) mice. In vivo administration, to mice that had been pre-treated with hydrocortisone, resulted in a decrease in the specific activity of thymocyte terminal deoxynucleotidyl transferase. The data indicate that the prealbumin molecule possesses immunopotentiating properties in a number of in vitro and in vivo immunocompromised murine models and that the immuno-enhancing properties of the partially purified preparation previously described were in fact due to the prealbumin component and not to other contaminating proteins.     

The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.

The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.