In vitro determination of phagocytic indices of Candida berkhout species by rat peritoneal macrophages

The aims of this investigation were to study and describe the behaviour of 13 different species of Candida, as compared with C. albicans, by means of phagocytosis assays in vitro.Tests were carried out with rat peritoneal macrophages in contact with quantified suspensions of live yeasts. Phagocytic indices, candidacidal activity and filamentation rat were tested microscopically after 3 h incubation at 37 ° C.The phagocytic indices obtained allowed us to separate the fungi into four groups. Candida albicans and tropicalis belong to Group I; diddensii and shehatae, among others, belong to Group II; sake, krusei, viswanathii, etc., Group III; and C. glaebosa and haploid strains of Pichia ohmeri (C. guilliermondii var. membranaefaciens), Group IV. These data would suggest a possible correlation between pathogenesis and phagocytic indices.There were no evidences of any phagocytes ability to kill yeasts. Candidacidal activity was absent in the species assayed. Yeast lysis may have been observed if our assays would have taken longer than 3 h.     

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.     

In vitro phagocytosis by Limulus blood cells.

Limulus blood cells maintained in culture are able to phagocytose particles under conditions where bacterial endotoxin is absent. In the presence of endotoxin, phagocytosis is inhibited because the cells are immobile under these conditions and because the extracellular gel found in the presence of endotoxin prevents cell-particle contact. It is suggested that Limulus blood cells respond to Gram-negative organisms by the formation of an extracellular gel matrix that entraps the bacteria and handles other types of foreign particles by phagocytosis.     

Rhythmic patterns in phagocytosis and the production of reactive oxygen species by zebrafish leukocytes

Multiple components of vertebrate immune systems have been shown to exhibit circadian fluctuations. While the zebrafish is currently generating a wealth of information on the molecular pacemakers that may control circadian rhythms, there have been no reports of rhythmic activity in zebrafish leukocytes. In this study, we found that phagocytosis and the production of reactive oxygen species by zebrafish leukocytes varied significantly throughout twenty-four hour periods. A distinct peak in cellular ROS levels occurred before dawn, while the kinetics of respiratory burst responses were least rapid at this time of day. Phagocytosis of E. coli peaked late in the day, whereas there was no daily variation in phagocytosis of S. aureus. As seen in other species, the number of bacteria ingested per cell peaked during the night. These data provide direct evidence of rhythmic immune system activity, and demonstrate that zebrafish can be a valuable model in which to study the relationships between circadian gene expression, systemic pacemakers, and the activity of vertebrate immune system cells.     

Leishmania adleri: in vitro phagocytosis by lizard leukocytes and peritoneal exudate cells

The interaction of iguanid lizard (Dipsosaurus dorsalis) blood cells and promastigote forms of Leishmania adleri Heisch 1954 was observed in vitro by means of wet mount preparations. Some parasites were phagocytized within a short time after their addition to the preparations. Parasites were engaged and engulfed either individually or in masses. Rapid decrease in size of the parasites occurred. The process of phagocytosis of the parasites was found to be very similar to that of mammalian leishmanias in similar preparations with mammalian cells. Cell cultures prepared from peritoneal exudates of lizards (Basiliscus vittatus) were inoculated with L. adleri promastigotes. Resultant intracellular amastigotes were observed up to 36 hr at 25 and 35 C.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.     

In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows

A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.     

Opsonin-dependent phagocytosis of bacteria by murine macrophage-like cells

We have investigated the phagocytic properties of the macrophage-like cell line DCH-7, derived from fusion of mouse macrophages with a mouse T-lymphoma cell line. These cells phagocytosed opsonized bacteria. IgG appeared to be the major opsonin forStaphylococcus aureus Wood 46 as well as for threeEscherichia coli strains; complement components were not required as opsonins. Intracellular bacteria survived to a large extent. This model system should be a useful tool for studying the process of phagocytosis and phagocytic killing of bacteria.     

Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice. AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMphi or PMphi were recovered 30-240 min later. Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.