Mouse Peripheral Blood Leukocytes Research Articles

Screening of potential key ferroptosis-related genes in sepsis.

BackgroundSepsis leads to multiple organ dysfunction caused by a dysregulated host response to infection with a high incidence and mortality. The effect of ferroptosis on the development of sepsis remains unclear. In this study, we aimed to identify the key ferroptosis-related genes involved in sepsis and further explore the potential biological functions of these ferroptosis-related genes in sepsis using bioinformatics analysis.MethodsThe GSE13904 (from children) and GSE28750 (from adults) datasets were downloaded from the Gene Expression Omnibus (GEO). The ferroptosis-related genes were obtained from the FerrDb database. The ferroptosis-related differentially expressed genes (DEGs) were screened by the limma R package. The DAVID online database or clusterProfiler R package was used for the functional enrichment analysis. Then, the STRING database was used to predict the interactions of proteins, and the CytoHubba plugin of Cytoscape was used to confirm key clustering modules. Then, the miRNAs and lncRNAs associated with the key clustering modules were predicted by miRWalk 2.0 and LncBase v.2 respectively. Finally, we generated a cecal ligation and puncture (CLP) polymicrobial sepsis model in C57 male mice and examined the expression of the mRNAs and noncoding RNAs of interest in peripheral blood leukocytes by PCR during the acute inflammation phase.ResultsIn total, 34 ferroptosis-related DEGs were identified in both adult and pediatric septic patients. These ferroptosis-related DEGs were mainly enriched in inflammatory pathways. Then, a significant clustering module containing eight genes was identified. Among them, the following five genes were closely associated with the MAPK signaling pathway: MAPK14, MAPK8, DUSP1, MAP3K5 and MAPK1. Then, crucial miRNAs and lncRNAs associated with biomarker MAPK-related genes were also identified. In particular, let-7b-5p and NEAT1 were selected as noncoding RNAs of interest because of their correlation with ferroptosis in previous studies. Finally, we examined the mRNAs, miRNAs and lncRNAs of interest using CLP-induced sepsis in peripheral blood leukocytes of mice. The results showed that MAPK14, MAPK8, MAP3K5, MAPK1 and NEAT1 were upregulated, while DUSP1 and let-7b-5p were downregulated in the CLP group compared with the sham group.ConclusionsThe MAPK signaling pathway may play a key role in regulating ferroptosis during sepsis. This study provides a valuable resource for future studies investigating the mechanism of MAPK-related ferroptosis in sepsis.

The study of the influence of exogenous factors on Saccharomyces cerevisiae DNA spheroplast

The possibility of fixing genotoxic changes in Saccharomyces cerevisiae yeast under the influence of physicochemical factors was evaluated using the DNA-comet assay. Peripheral blood leukocytes of mice subjected to similar effects were used as an object of comparison. The data obtained showed that under the action of both the alkylating agent and X-ray irradiation, the changes were more pronounced in experiments with leukocytes of the blood of mice. At a methyl-methane sulphonate concentration of 10 mM, the comet tail length in mouse peripheral blood leukocytes was about 40 μm, while the yeast spheroplasts tail length of a comet was 0.16 μm. The DNA content in the tail of the comet did not exceed 5 % for yeast after treatment with an alkylating agent at a concentration of 40 mM, which is 10 times less than in peripheral blood leukocytes of mice under similar conditions. Under the action of X-ray radiation, the length of the comet’s tail and the DNA content in it for spheroplasts of yeast also differed significantly from the leukocytes of the peripheral blood of the mouse. Thus, the comet test allows to register a significant increase in the level of DNA damage in yeast spheroplasts under the action of physicochemical factors in relatively high doses.

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Studying the spectrum of immunotropic action of Pletnev drops No 60 (Phospholiten)

The purpose of the present work was studying of immunomodulating activity of Pletnev drops No 60. On the basis of the received results it is established that Pletnev drops No 60 possess immunomodulatory properties, stimulates humoral and cellular immunity, the bactericidal activity of peritoneal exudate cells of mice, bactericidal activity of phagocytes of peripheral blood, promote production of TNF-α by mononuclear cells, the number of peripheral blood leukocytes of mice. Pletnev drops No 60 increase the survival of animals. Pletnev drops No 60 possess antioxidant properties and, under his influence, the observed suppression of spontaneous and simhasan-induced of chemiluminescence of peripheral blood leukocytes of mice and suppression of luminol-dependent and lucigenin-dependent spontaneous and simhasan-induced of chemiluminescence of leukocytes of man.

Pulse-modulated extremely high-frequency electromagnetic radiation protects cellular DNA from the damaging effects of physical and chemical factors in vitro

Using an alkaline version of the comet assay, we investigated the protective effects of extremely high-frequency electromagnetic radiation for DNA in mouse peripheral blood leukocytes on the damaging effects of X-ray radiation, hydrogen peroxide, and the alkylating agent methyl methanesulfonate. It was shown that the pre-exposure of cells to pulse-modulated low-intensity electromagnetic radiation (42.2 GHz, 100 µW/cm2, exposure for 20 min, and the modulation frequency of 1 and 16 Hz) exerted a protective effect by reducing the level of DNA damage by 20–45% depending on the type of genotoxic agent. The effectiveness of pulse-modulated radiation increases in the following series: methyl methanesulfonate–X-rays–hydrogen peroxide. Continuous electromagnetic radiation does not have such a protective effect. The mechanisms of the detected protective effects may be associated with the induction of an adaptive response by reactive oxygen species that are formed by the action of pulse-modulated radiation in nanomolar concentrations.

Effect of oral administration of commercial gold nanocolloid on peripheral blood leukocytes in mice.

During the last few decades, owing to their unique properties, gold nanoparticles (AuNPs) have found numerous biomedical applications. Studies on rodents prove that AuNPs entering an organism easily reach the bloodstream and undergo wide tissue distribution. The presence of nanoparticles inside blood and bone marrow cells of exposed animals may implicate its influence on hematopoesis and the functions of peripheral blood leukocytes. The aim of this study was to determine the effect of oral administration of commercial gold nanocolloid, recommended by the producer as a dietary supplement, on the percentage of lymphocyte populations and proliferative response, as well as the activity of phagocytes in the peripheral blood of mice. The colloid was given to the animals in three different doses (0.25, 2.5, 25 ppm), for three different time periods (7, 14, 28 days). Mice given nanoparticles showed increased activity of phagocytes and some changes in the lymphocyte phenotypes. The elevated activity of granulocytes and monocytes, in terms of both phagocytic and respiratory burst activity, was transient and noticed only after a short time of administration, which may indicate some adaptability of blood phagocytes to prolonged presence of gold nanoparticles in the body. However, phenotypic modifications among lymphocytes in the group of animals given the middle dose of colloid (i.e. increased percentage of B and CD4+CD8+ DP T cells) did not occur until after the 28-day administration, which in turn seems indicative of some immune dysregulation due to the prolonged contact with nanogold.

Age dynamics of DNA damage and CpG methylation in the peripheral blood leukocytes of mice

Certain types of DNA damage are known to accumulate with age. Here we present the quantitative data describing the extent of the spontaneous DNA damage in 144 SHK mice of various ages. In each animal, we assessed double strand breaks, single strand breaks and alkali-labile sites, as well as amounts of oxidized purines, oxidized pyrimidines and misincorporated uracils. In addition, overall levels of genome DNA methylation were evaluated. The amounts of oxidized pyrimidines were correlated with age in males only, while the amounts of double strand breaks (DSB) in DNA samples were correlated with age in females only (R=0.26; p<0.035). No age-related accumulation of single-strand breaks (SSB) was observed. The hypomethylation of DNA was significant in aging females, but not in aging males. Various types of DNA damage were correlated to each other. In attempt to develop more stable indicator of age-dependent alterations in DNA, the DNA Damage Level Differential (DDLD) indices was developed for comet assaying of peripheral blood leukocytes. As expected, DDLD index was shown to be better correlated with age than any single quantitative measure reflecting certain type of DNA damage. A variability of effectiveness of various kinds of DNA repair in individual animals was larger than expected. This conclusion may have a substantial impact on subsequent studies of the mutagens and other kinds of environmental stressors in animal populations. Nor DDLD neither individual quantitative measures of DNA damage were capable of prediction post-sampling survival time.

Effect of 28-day oral administration of silver nanocolloid on the peripheral blood leukocytes in mice.

Silver nanoparticles, which have found a wide range of applications owing to their antimicrobial properties, are also recommended as dietary supplements in alternative medicine. Studies on rodents confirm that nanosilver is absorbed from the digestive tract into the bloodstream, which implies its possible interactions with leukocytes. The objective of the experiment discussed herein has been to determine the effect of 28-day oral administration of different doses (0.25, 2.5, 25 ppm) of commercial silver nanocolloid on hematological parameters, percentages of particular lymphocyte populations and activity of the peripheral blood leukocytes in mice. All the tested colloid doses decreased the counts of monocytes in the animals' blood and induced phenotypic modifications among lymphocytes: an increase in CD4+/CD8+ T cell distribution, a decrease in NK and NKT cell distribution (doses of 0.25 and 2.5 ppm) and an increased CD4+:CD8+ ratio (25 ppm). Silver nanocolloid also affected the activity of cells, depressing the proliferation of lymphocytes (0.25 ppm) and stimulating phagocytosis as well as the respiratory burst of granulocytes and monocytes (all doses). The results verify the influence of orally administered silver colloid on the peripheral blood leukocytes, at the same time implying the potential risk of developing an inappropriate immune response of an organism exposed to prolonged administration of this substance.

Effect of oral administration of kynurenic acid on the activity of the peripheral blood leukocytes in mice.

Kynurenic acid (KYNA), an endogenous tryptophan metabolite, is a selective ligand of the GPR35 receptor, expressed mainly on the immune cells. In inflammatory conditions, by affecting this receptor, KYNA inhibits the synthesis of pro-inflammatory cytokines and probably protects tissues from oxidative damage. However, we lack data regarding the effect of exogenous KYNA on the activity of immune cells in healthy individuals. The objective of this study has been to determine the influence of kynurenic acid administered to mice in different doses (2.5, 25 or 250 mg/l) and for different time periods (3, 7, 14, 28 days) in drinking water, on the activity of their peripheral blood leukocytes. The determinations comprised the proliferative activity of lymphocytes (MTT assay) and the phagocytic activity as well as the respiratory burst activity of granulocytes and monocytes (Phagotest, Phagoburst). It was only the lowest KYNA dose that influenced the mitogenic response of lymphocytes, namely by increasing the proliferation of T cells. The impact on the phagocytic activity was varied with KYNA dose and administration time. However, all the KYNA doses significantly lowered the activity of oxidative burst in phagocytes, which was probably associated with its antioxidant properties. In the light of the research results, kynurenic acid may find applications as an immuno-modulating agent able to correct an excessive or insufficient response of phagocytizing cells, protecting an organism from oxidative stress.

Radioprotective Effects of Gallic Acid in Mice

Radioprotecting ability of the natural polyphenol, gallic acid (3,4,5-trihydroxybenzoic acid, GA), was investigated in Swiss albino mice. Oral administration of GA (100 mg/kg body weight), one hour prior to whole body gamma radiation exposure (2–8 Gy; 6 animals/group), reduced the radiation-induced cellular DNA damage in mouse peripheral blood leukocytes, bone marrow cells, and spleenocytes as revealed by comet assay. The GA administration also prevented the radiation-induced decrease in the levels of the antioxidant enzyme, glutathione peroxidise (GPx), and nonprotein thiol glutathione (GSH) and inhibited the peroxidation of membrane lipids in these animals. Exposure of mice to whole body gamma radiation also caused the formation of micronuclei in blood reticulocytes and chromosomal aberrations in bone marrow cells, and the administration of GA resulted in the inhibition of micronucleus formation and chromosomal aberrations. In irradiated animals, administration of GA elicited an enhancement in the rate of DNA repair process and a significant increase in endogenous spleen colony formation. The administration of GA also prevented the radiation-induced weight loss and mortality in animals (10 animals/group) exposed to lethal dose (10 Gy) of gamma radiation. (For every experiment unirradiated animals without GA administration were taken as normal control; specific dose (Gy) irradiated animals without GA administration serve as radiation control; and unirradiated GA treated animals were taken as drug alone control).

Magnitude of radiation-induced DNA damage in peripheral blood leukocytes and its correlation with aggressiveness of thymic lymphoma in Swiss mice

Purpose: The present study is aimed to investigate the magnitude and kinetics of DNA damage in peripheral blood leukocytes of mice exposed to whole body gamma irradiation (WBI; 3 Gy) and its correlation with aggressiveness of thymic lymphoma (TL).Materials and methods: DNA damage was monitored in peripheral blood cells of individual mice by comet assay at different intervals of post-irradiation, which were correlated with weight of TL in respective mice at 120th day. To further study genomic radiosensitivity in TL development, peripheral blood samples collected at the 15th and 90th day of post-irradiation from control and WBI animals were irradiated (0.5 Gy) ex vivo followed by assessment of DNA damage by comet assay.Results: The maximum DNA damage (tail moment) was observed at 5 min after WBI, which decreased at longer period, and was minimum at the 7th day after WBI. However, residual damage was observed in comparison to control and it persisted up to 90 days of irradiation. Tail moment values observed at an early time (5 min) of post-irradiation was better correlated (correlation coefficient, r = 0.84) with weight of TL than at longer time period (60 days; r = 0.21). Our results showed that in ex vivo irradiated (0.5 Gy) peripheral blood, the magnitude of DNA damage was higher in samples obtained from WBI mice than sham-irradiated controls suggesting enhanced genomic radiosensitivity in WBI mice. Genomic susceptibility to radiation observed in peripheral blood from WBI animals showed better correlation with weight of TL at the 15th day (r = 0.9) post-irradiation period than at the 90th day (r = 0.44).Conclusion: These results suggest that the magnitude of radiation-induced initial DNA damage in peripheral blood leukocytes and genomic radiosensitivity could be an indicator of TL aggressiveness in mice.

CXCR7 Protein Is Not Expressed on Human or Mouse Leukocytes

Since the discovery that CXCR7 binds to CXCL12/SDF-1α, the role of CXCR7 in CXCL12-mediated biological processes has been under intensive scrutiny. However, there is no consensus in the literature on the expression of CXCR7 protein by peripheral blood cells. In this study we analyzed human and mouse leukocytes and erythrocytes for CXCR7 protein expression, using a competitive CXCL12 binding assay as well as by flow cytometry and immunohistochemistry using multiple CXCR7 Abs. CXCR7(-/-) mice were used as negative controls. Together, these methods indicate that CXCR7 protein is not expressed by human peripheral blood T cells, B cells, NK cells, or monocytes, or by mouse peripheral blood leukocytes. CXCR7 protein is, however, expressed on mouse primitive erythroid cells, which supply oxygen to the embryo during early stages of development. These studies therefore suggest that, whereas CXCR7 protein is expressed by primitive RBCs during murine embryonic development, in adult mammals CXCR7 protein is not expressed by normal peripheral blood cells.

Kinetic of Genotoxic Expression in the Pharmacodynamics of Busulfan

Busulfan (BUS) is a highly toxic antineoplastic bifunctional-alkylating agent and has a narrow therapeutic window. Our previous study revealed a narrow dose-range of BUS, which causes a sudden dose-dependent transition from early- to late-expressing micronucleus induction and from a non-cytotoxic to a cytotoxic effect. In the present study, the kinetics of DNA-damaged cell induction by BUS and its dose-effect relationship were established. This was achieved by using the kinetics of DNA-damaged cell induction, determined by the comet assay in murine peripheral blood leukocytes of mice, after the intraperitoneal exposure to 16, 30, 45, 60 or 80 micromol/kg of BUS. Doses of 15 or 30 micromol/kg of BUS were able to increase DNA-damaged cell frequency, but doses of 45 micromol/kg body weight or higher caused a sudden drop in this frequency. This suggests that higher doses cause lesions that inhibit the expression of damage as comets, i.e., DNA-protein or interstrand crosslinks. The latter could be explained by sudden monoadduct-to-crosslink transformation due to a DNA conformational change induced by monoadduct accumulation that facilitates crosslink formation. This narrow dose-dependent transition could contribute to the narrow therapeutic window of BUS.

CpG and Non-CpG Oligodeoxynucleotides Induce Differential Proinflammatory Gene Expression Profiles in Liver and Peripheral Blood Leukocytes in Mice

Proinflammatory effects caused by oligodeoxynucleotides (ODN) include cytokine production, splenomegaly and infiltration of mononuclear cells into tissues. Presence of one or more CpG motifs in an ODN sequence confers potency for proinflammatory properties. The objective of this research was to characterize the proinflammatory effects produced by CpG containing ODN as compared to non-CpG ODN using gene array analysis. Female CD-1 mice were administered equipotent dose regimens of a CpG ODN (ISIS 12449, 4 mg/kg sc, single or repeat dose for 7 d) or a non-CpG ODN (ISIS 2302, 50 mg/kg sc, q2d for 1 or 3 weeks) and tissues (liver and peripheral blood leukocytes) were harvested for immunohistochemical analysis or gene array analysis. Splenomegaly, a marker of ODN-induced inflammation, was greatest (3-fold above control) with ISIS 12449 when given at multiple doses. Immunohistochemical staining identified mainly monocytes/macrophages as the immune cell infiltrates in the liver following ISIS 12449 or ISIS 2302 treatment. Gene analysis of liver tissue indicated enhanced expression of chemokines (MIG, MIP-2β, MCP-1, IL-1β, CCR3), cell surface markers (CD14, CD18, CD86, CD11c, P-selectin), intracellular markers (NF-κBp65, MyD88, Survivin) and markers of cell proliferation (PCNA, Ki-67, CD71) was produced with ISIS 12449 or ISIS 2302. Although CpG and non-CpG containing ODN produced similar gene expression profiles, notable differences were observed to suggest that their mechanisms of immune modulation are not completely overlapping. MIG and MIP 1β were identified as potential biomarker for immune stimulation that may be used to further study the species specificity, sequence/structure dependence and time course of proinflammatory ODN and antisense inhibitors used as therapeutics.

Transgenic mouse expressing human CCR5 as a model for in vivo assessments of human selective CCR5 antagonists

The species selectivity of receptor antagonists often hinders their preclinical assessment in vivo. In order to evaluate human selective CC chemokine receptor type 5 (CCR5) antagonists in vivo, we generated human CCR5 transgenic mice that expressed the transgene on both peripheral blood leukocytes as well as thymocytes. The selective CCR5 ligand CC chemokine ligand 4 (CCL4)/macrophage inflammatory protein (MIP)-1β induced the chemotaxis of thymocytes that had been derived from the transgenic mice, but not from littermate mice, suggesting that the human CCR5 expressed in the transgenic mice were functional. The binding of the human CCR5 specific antibody 45531 to peripheral blood granulocytes from the transgenic mice was inhibited by human selective CCR5 antagonist SCH-351125. Using this antibody, we developed an ex vivo assay system that is suitable for the evaluation of a test compound's ability to occupy the human CCR5 receptor on mouse peripheral blood leukocytes. This transgenic mouse model is useful for estimating the pharmacodynamics of human selective CCR5 antagonists in vivo.

Mouse Cathelin-Related Antimicrobial Peptide Chemoattracts Leukocytes Using Formyl Peptide Receptor-Like 1/Mouse Formyl Peptide Receptor-Like 2 as the Receptor and Acts as an Immune Adjuvant

Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

SERIAL PHENOTYPIC ANALYSIS OF MOUSE PERIPHERAL BLOOD LEUKOCYTES

Repeated phenotypic analysis of mouse peripheral blood leukocytes over short periods of time (2 weeks) has been difficult because of the very limited volumes of blood available under guidelines of the Institutional Animal Care and Use Committee. The loss of leukocytes and variations among laboratories during conventional flow cytometry sample preparation based on lysing and repeated washing have been limiting factors when measuring multiple parameters in small samples. We describe a method of phenotypic analysis using a no-lyse, no-wash staining technique combined with fluorescent triggering for data collection that can be performed on volumes of 20 μL or less of whole blood per set of markers in one tube. This method allows repeated phenotypic analysis of peripheral whole blood from mice. Fluorescent triggering with anti-CD45-PE/Cy5 antibody allows high-quality phenotypic data to be collected for CD4, CD8, TcR- β, CD45R (B220), CD11b, and Gr-1 epitopes on leukocytes from mouse peripheral blood without lysis. The markers selected cover the major populations in peripheral mouse blood. Reproducibility and time-course data are presented for sampling periods as long as 4 weeks. Data produced by flow cytometers manufactured by two different companies show well-correlated results. An instrument equipped with a gated amplifier or a photomultiplier tube suitable for Cy7 conjugates could measure additional parameters. Because of interference from unlysed erythrocytes, scatter parameters are not useful for identifying cell populations with this method.

Murine defense mechanism against Candida albicans infection. II. Opsonization, phagocytosis, and intracellular killing of C. albicans.

The phagocytic and intracellular killing activities of normal mouse phagocytes against Candida albicans were studied to elucidate the role of these activities in nonspecific and specific defense mechanisms. In the presence of fresh normal mouse serum, viable C. albicans cells were ingested by mouse peripheral blood leukocytes (PBLs) and peritoneal macrophages (PMPs) at the same rate. Serum-chelation experiments indicated that the factors involved in the alternative complement pathway are opsonins for C. albicans. PBLs killed intracellular C. albicans more effectively than PMPs. Lymphokine-activated PMPs manifested marked intracellular killing activity and the occurrence of increased superoxide anion- and singlet oxygen production, in the absence of increased myeloperoxidase (MPO) production, suggests that the enhanced, MPO-independent, oxidative mechanism may play an important role in the candidacidal activity. Specific rabbit antibodies played no role in the phagocytosis and intracellular killing of C. albicans. These results suggest that PMNs and factors involved in the alternative complement pathways, and lymphokine-activated macrophages play major roles in the protection of mice against C. albicans infection.

Phagocytosis in Fungus Infection

The study of phagocytosis of Candida albicans and Cryptococcus neoformans by human, guinea pig and mouse peripheral blood leukocytes (PBL) and guinea pig and mouse peritoneal macrophages (PMP) in vitro has been performed to elucidate the role of phagocytosis in host defense mechanism against systemic mycoses. Although C. albicans was ingested by guinea pig and mouse PBL and PMP in the same rates in the presense of fresh serum, the killing rates of intracellular C. albicans in these phagocytes were not the same showing a high ability of guinea pig and a low of mouse phagocytes. The human PBL showed an intermediate killing ability between them. Moreover, intracellular killing abilities of guinea pig and mouse PMP were lower than those of PBL. The killing capacity of mouse PMP increased remarkably when the PMP had been cultured previousely with lymphokines for 3 days. The PBL and PMP ingested C. albicans almost completely in the presense of fresh serum alone, and no more enhancement of ingestion was shown when specific antibody was added to this system. The results of phagocytosis in vitro using EGTA and Mg++ indicate that normal opsonin for C. albicans is factors of properdin system in fresh serum. Guinea pig PBL and PMP ingested encapsulated strain of C. neoformans more effectively than nonencapsulated strain, and specific antibody did not enhance ingestion of both strains of C. neoformans. It might been suggested that properdin system and PMP play an important role at the primary stage of Candida or Cryptococcal infections and that activated macrophages participate in the host defense mechanism after induction of sensitized lymphocytes.