Application on Timm sulphide silver method for electron microscope localization of lead ions in blood cells.

The application of the Timm sulphide silver method for the demonstration of lead ions in peripheral blood cells is investigated, using male white adult Wistar rats treated with a single dose of lead acetate, range 150 mg/kg b.w. To improve the Timm reaction for electron microscopy, fixation of whole cells with glutaraldehyde fixative solution saturated with H2S, an agarose embedding and physical development of thick sections without prior cryostat sectioning are presented. At 6.0 hours after injection both erythrocytes as well as white cells reveal the positive Timm reaction. All types of blood cells contain numerous cytoplasmic precipitates illustrating the intracellular lead accumulation. It is shown that the invaginations of white cell nuclear membrane possess a storing function as areas of lead depots. As a rule, the neutrophils display a highest amount of cytoplasmic precipitates and exclusively a low amount of reaction products in basophils is observed. At 14 days after injection, precipitates are present only in erythrocytes and monocytes. A suggestion of a possible functional significance of changes in the Timm staining pattern in blood cell types is discussed in this paper.     

Light and electron microscopic demonstration of phospholipase B activity in the mouse eosinophil

Phospholipase B (EC 3.1.1.5) which hydrolyzes phospholipids in the alpha and beta positions was demonstrated in murine leukocytes using light and electron microscopic histochemical techniques. Leukocytes (neutrophils, lymphocytes, macrophages, eosinophils) were harvested from peritoneal exudates of mice. Cells were fixed in 4% calcium-formol fixative for 10 min at 4 degrees C for light microscopy and 30 min at room temperature for electron microscopy, after which they were incubated at 37 degrees C in medium at pH 6.6 containing 2 microM lysolecithin and CaCl2. The fatty acids released during the hydrolytic reaction were trapped as a calcium precipitate and were converted to a cobalt precipitate for light microscopy by treatment with cobalt acetate or to a lead precipitate for electron microscopy by treatment with lead nitrate. The reaction products were observed to be present in eosinophils and absent in neutrophils, lymphocytes and macrophages. It is concluded that the eosinophilic leukocyte is the carrier cell for phospholipase B in inflammatory reactions.     

Abundance of Zinc Ions in Synaptic Terminals of mocha Mutant Mice: Zinc Transporter 3 Immunohistochemistry and Zinc Sulphide Autometallography

The mocha mouse is an autosomal recessive pigment mutant on mouse chromosome 10 caused by a deletion in the gene for the delta subunit of the adaptor-like complex AP-3. Based on zinc transporter 3 (ZnT3) immunohistochemistry, zinc TSQ fluorescence and a modified Timm method, previous studies found a lack of histochemically-detectable zinc and a substantial reduction in the ZnT3 immunoreactivity. It has, therefore, been suggested that the mocha mouse could serve as a model for studies of the significance of zinc ions in zinc-enriched (ZEN) neurons. We have chosen the mocha-zinc-model in a study of the significance of ZEN neurons in hypoxia-caused damage in mouse brain. In order to establish that the model was either void of zinc ions or had a significantly decreased level of zinc ions in their ZEN terminals, we repeated the studies that had lead to the above assumption, the only methodology difference being that we used the zinc specific Neo-Timm method instead of the Timm method applied in the original study. We found that, although the ZnS autometallography (AMG) technique revealed a reduction in staining intensity as compared to the littermate controls, there were still plenty of zinc ions in the ZEN terminals, in particular visible in telencephalic structures like neocortex and hippocampus. At ultrastructural levels the zinc ions were found in a pool of vesicles of the ZEN terminals as in the control animals, but additionally zinc ions could be traced in ZEN neuronal somata in the neocortex and hippocampus. The mossy fibres in the hippocampus of mocha mice also bind with TSQ, though less than in the controls. We found ZnS AMG grains in ZEN neuronal somata, which were also immunoreactive for ZnT3. Our study confirmed the decreased ZnT3 immunoreactivity in ZEN terminals of the mocha mouse found in the original study. Based on these findings, we suggest that the mocha mouse may not be an ideal model for studies of the histochemically-detectable zinc ion pool of the central nervous system.     

Heavy metals in the spinal cord of normal rats and of animals treated with chelating agents: a quantitative (zinc, copper, and lead) and histochemical study

The amounts of zinc, copper, and lead in the rat spinal cord were determined by means of flameless atomic absorption spectrophotometry. Zinc was present in a concentration about 100 p.p.m. (dry weight), copper in a concentration about 5 p.p.m., and lead in slightly more than 1 p.p.m. Analysis of various levels along the cranio-caudal axis of the rat spinal cord revealed differences in the heavy metal content. The Timm sulfide silver staining method has demonstrated that metals in the spinal cord have a distinct regional distribution. To obtain a differentiation between the stainable metals, the effects of six chelating agents (DEDTC, dithizone, oxine, EDTA, dipyridyl, and phenantroline) on the Timm pattern were tested. EDTA left the pattern unchanged, while the other compounds showed individual differences in their influence on the Timm pattern, suggesting that the heavy metal pattern of the spinal cord consists of multiple compartments. The effect of intravital multiple low dose treatment with three of the chelating agents on the histochemical pattern and the metal content of the spinal cord was also investigated. It was found that a decrease in the metal content was not followed by reduction of stainability and vice versa.     

Heavy metals in the spinal cord of normal rats and of animals treated with chelating agents: A quantitative (Zinc,copper, and lead) and histochemical study

Summary The amounts of zinc, copper, and lead in the rat spinal cord were determined by means of flameless atomic absorption spectrophotometry. Zinc was present in a concentration about 100 p.p.m. (dry weight), copper in a concentration about 5 p.p.m., and lead in slightly more than 1 p.p.m. Analysis of various levels along the cranio-caudal axis of the rat spinal cord revealed differences in the heavy metal content.The Timm sulfide silver staining method has demonstrated that metals in the spinal cord have a distinct regional distribution. To obtain a differentiation between the stainable metals, the effects of six chelating agents (DEDTC, dithizone, oxine, EDTA, dipyridyl, and phenantroline) on the Timm pattern were tested. EDTA left the pattern unchanged, while the other compounds showed individual differences in their influence on the Timm pattern, suggesting that the heavy metal pattern of the spinal cord consists of multiple compartments.The effect of intravital multiple low dose treatment with three of the chelating agents on the histochemical pattern and the metal content of the spinal cord was also investigated. It was found that a decrease in the metal content was not followed by reduction of stainability and vice versa.     

Improvements in the method for the electron microscopic localization of arylsulphatase activity

Summary The optimal conditions for the demonstration of arylsulphatase activity in the proximal convoluted tubule cells of the rat kidney were studied at light and electron microscopic level. 8-hydroxyquinoline sulphate, p-nitrophenyl sulphate and 2-hydroxy-5-nitrophenylsulphate were used as substrates and barium and lead as capturing ions. The effect of fixation, capturing ions, substrate concentration and pH was studied biochemically. The results of these biochemical studies were then verified histochemically. Finally a recommended method for the light and electron microscopic demonstration of arylsulphatase activity was presented.     

Stimulating Effects of Mercuric-and Silver Ions on the Superoxide Anion Production in Human Polymorphonuclear Leukocytes

In a survey of a number of heavy metal ions for effects on the oxidative metabolism (respiratory burst) of human polymorphonuclear leukocytes (neutrophils) we have found that mercury(II) and silver ions in micromolar concentration significantly increase the production of superoxide anions in cells, initiated by formyl-methionyl-leucylphenylalanine (fMLP). The stimulation of radical formation induced by a certain ion concentration varied considerably in cells isolated from different blood donors, from a moderate increase to a very large (up to 400% of control values). When the soluble stimulator phorbol myristate acetate (PMA) or the particulate stimulator Zymosan were used to initiate the cell respiratory burst, no additional stimulating effects by the metal ions on superoxide anion formation were observed. This fact might indicate that the effect of the metal ions on the fMLP-dependent initiation of cell activity is a mechanism coupled to the interaction between the chemotactic peptide and its corresponding receptor molecules on the cell surface.

By increasing the concentration of silver ions during pre-incubation of resting neutrophils, a spontaneous activation of the cells could be recorded at a concentration exceeding 5 μM. However, the silver ion concentration at which such spontaneous initiation of the respiratory burst occurred varied significantly between blood samples from different donors with a concentration range of 5 to 15 μM. This effect could not be shown for mercuric ions due to the toxicity of the metal above 5 μM. Blood samples from some donors contained neutrophils that could be activated by either mercuric- or silver ions at concentration as low at 1 μM.

The spontaneous activation of neutrophils with elevated concentrations of silver ions is kinetically similar to the PMA-induced. The onset of superoxide anion formation is preceeded by a lag period whose length varies in time with the concentration of agent applied to the cells. It is a known fact that once the neutrophils have been activated with fMLP it is not possible to reactivate the cells by a second supplementation of fMLP. However, after cessation of the fMLP-induced activation, addition of PMA or silver ions gives rise to renewed production of superoxide anions.

We propose two different mechanisms of action of silver ions on oxidative metabolism of neutrophils. At a low concentration the metal ions are thought to interact with an activating agent and a corresponding cell surface receptor molecule, while at elevated ion concentrations, we postulate an action like that of phorbol-esters on neutrophils, (i.e., an interaction between activating agent and the enzyme protein kinase C of the cells).     

The effect of lead ions on the energy metabolism of human erythrocytes in vitro

The aim of this work was to evaluate the influence of chronic exposure to lead ions on the parameters of energetic status of human erythrocytes in vitro. Umbilical cord erythrocytes were incubated with lead acetate at final lead ion concentrations ranging from 10 to 200 microg/dl. ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, NAD and NADP concentrations in erythrocytes were determined using HPLC. Scanning electron micrographs of erythrocytes were taken. The mean concentrations of ATP, GTP, NAD and NADP, and mean values of adenylate energy charge (AEC) and GEC in cells incubated at the presence of lead ions were significantly lower after 20 h of incubation. Concentrations of purine degradation products (Ado, Guo, Ino) and Hyp were significantly higher. It is suggested that lead ions affect the energy metabolism of erythrocytes. Morphological changes in erythrocytes correspond to the increase of lead ions in the incubation mixture and to the decrease of ATP concentration in erythrocytes. A decrease in NAD and ATP concentration in erythrocytes could be a sensitive indicator of energy process disturbance, useful in monitoring in case of chronic lead exposure.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

Mass spectrometry of the phosphatidylcholines: fragmentation processes for dioleoyl and stearoyl-oleoyl glycerylphosphorylcholine

Mass spectra for the various phosphatidylcholines, together with accurate mass measurements on the more abundant fragment ions, have been described in a previous paper (Ref. 5). No detailed fragmentation sequence was proposed on the evidence available. In the case of dioleoyl glycerylphosphorylcholine, some question arose as to whether certain ions were produced by electron impact or by pyrolysis. In this paper, results are reported which enable a more detailed fragmentation sequence to be proposed. By observing metastable transitions in the first field free region of a double-focusing mass spectrometer, it can be shown that the major ions in the spectrum are produced by electron impact processes, and not by pyrolysis; moreover, many of these ions are directly related to one another by metastable processes. In particular, it has been demonstrated that the ions at m/e 603 for dioleoyl glycerylphosphorylcholine and at m/e 604 for stearoyl-oleoyl glycerylphosphorylcholine are derived from the appropriate molecular ions by an electron impact-induced process. From measurements of the metastable ion intensities, as well as from the appearance potentials and ionization efficiency curves, conclusions may be drawn about many of the fragmentation mechanisms, allowing a distinction to be made between rearrangement and cleavage reactions.     

The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.     

Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo. Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells.

We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection. The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay. We present direct evidence that neutrophils are involved in this phenomenon. We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c. injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice. The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells. We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells. To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios. Histologic and EM analysis of the tumors growing at the site of injection were carried out. Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells. In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils. In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells. Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition. The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present.     

Absence of right-left asymmetries in the rat hippocampus as demonstrated by Timm staining.

Although biochemical and behavioural studies have shown right-left differences in several areas of the rat limbic system, some anatomical studies reported no significant right-left differences in several morphological parameters of the hippocampus. The purpose of the present study was to determine whether there are asymmetries in the micro-anatomy of the rat hippocampus by examining the intensity of Timm staining in various hippocampal fields and the area occupied by mossy fibres by the use of combined microdensitometric and quantitative image analysis techniques. Timm staining demonstrates the distribution of intrahippocampal association pathways because it is a histochemical marker of zinc and other heavy transition metals. There were no right-left differences in the density of Timm staining at the level of the dentate gyrus, in the dendritic layer of CA1 and CA2 fields, in the mossy fibre area or in the subiculum. These findings provide further evidence of a lack of morphological asymmetry in the rat hippocampus.     

Pyridine-Functionalized Fe3O4 Nanoparticles as a Novel Sorbent for the Preconcentration of Lead and Cadmium Ions in Tree Leaf as a Bioindicator of Urban Traffic Pollution

We have developed a facile and highly sensitive sorbent for cadmium and lead ions. It is based on Fe₃O₄ nanoparticles functionalized with a derivative of picoline and was characterized by scanning electron microscopy, differential thermographic analysis, and elemental analysis. The material can be applied to the preconcentration of lead and cadmium ions. Factors such as the type, concentration and volume of eluent, the pH of the sample solution, the time for extraction, and the volume of the sample were studied. The effects of a variety of ions on preconcentration and recovery of these ions were also investigated. The ions were determined by FAAS, and the limits of detection are <0.8 and <0.061???g?L^?1 for lead and cadmium, respectively. Recoveries and precisions are >98.0?% and <1.3?%, respectively. The method was validated by analyzing several certified leaf reference materials.     

TEMPO-mediated oxidation of native cellulose: Microscopic analysis of fibrous fractions in the oxidized products

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holocellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of cellulose I of the original native celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each cellulose microfibril or each bundle of cellulose microfibrils seemed to be present in the TEMPO-oxidized celluloses.     

The phenomenon of the formation of universal rosette-forming cells under superextreme loads

A combined immunological study of cell-mediated and humoral immunity factors in sportsmen at the period of important competitions was made. The effect of the maximum bearable load was found to lead to the formation of a great number of universal rosette-forming lymphocytes and neutrophils. In the control group their number did not exceed 1-4% of the total population of these cells. Simultaneously, the suppression of the phagocytic activity of neutrophils and some of the humoral factors of systemic and local immunity with the parallel development of compensating processes were recorded.     

Histochemical localization of zinc ions in the epididymis of the rat

Summary In the present study, the autometallograpic zinc sulphide technique, an improved version of the original Timm sulphide-silver method, was used. This technique reveals a particular pool of ionic zinc that is chelatable by diethyldithiocarbamate. At the light microscopical level, no reaction for zinc was found in tissues of young prepubertal rats. In adult mating and non-mating rats low zinc staining was found in the head and intermediate epididymis whereas the tail of the epididymis demonstrated high levels of zinc ions. Sections from the epididymal tail revealed a compartmentalization, based on pronounced differences in staining intensity along the epididymal ducts. At higher magnification zinc ions were found in the apical part of the principal cell and in the lumen. At the ultrastructural level autometallographic grains were located in vesicles and in lysosome-like structures of the apical parts of the principal cells. The luminal grains were found either associated with sperm cells, with the surface of the large microvilli (stereocilia), or free in the seminal fluid. The variation in content of zinc ions in the epididymal epithelium and lumen suggests that zinc ions are secreted into the lumen from the epididymal tail and may somehow be involved in maturation of the sperm cells.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Scanning electron microscopy study of neutrophil membrane tubulovesicular extensions (cytonemes) and their role in anchoring, aggregation and phagocytosis. The effect of nitric oxide

We have shown that human neutrophils develop dynamic thin and very long tubulovesicular extensions (cytonemes) upon adhesion to fibronectin, if cell spreading was blocked by Na(+)-free medium or by 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and cytochalasin D (S. I. Galkina, G. F. Sud'ina and V. Ullrich, (2001). Exp. Cell Res. 266, 222-228). In the present work we found that similar in size and behavior tubulovesicular extensions were formed on the neutrophil cell bodies upon adhesion to fibronectin-coated substrata in the presence of the nitric oxide donor diethylamine NONOate. In the presence of the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl ester, neutrophils were well spread and had no microextensions. Using scanning electron microscopy, we demonstrated that tubulovesicular extensions of neutrophils executed long-range adhesion and binding objects for phagocytosis, such as serum-opsonized zymosan particles and erythrocytes. Tubulovesicular extensions anchored neutrophils to substrata in a beta1 and beta2 integrin-independent, but L-selectin-dependent manner. BODIPY-sphingomyelin impaired development of tubulovesicular extension, and heparitinase 1 played a role in their destruction. Membrane tubulovesicular extensions are supposed to represent protrusions of an intracellular exocytotic traffic and serve as cellular sensory and adhesive organelles. Nitric oxide seems to play a role in regulation of tubulovesicular extensions formation, thus affecting neutrophil adhesive interactions and phagocytosis.     

Activated host neutrophils in the larval midgut lumen of the human bot fly Dermatobia hominis

Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe activated polymorphonuclear neutrophils from mammalian hosts as well as invading bacteria in the midgut lumen of larvae of the human bot fly Dermatobia hominis. Other resident or recruited cells associated with dermal myiasis were fed on by larvae and digested more rapidly than neutrophils. The latter were observed moving towards bacteria and particles of food, extending the filopodia and engulfing material to be digested within phagosomes. The larval midgut lumen, thus, appears to be a suitable environment to produce neutrophil activation at least for short periods, as seen in mammalian hosts. Although interactions between phagocytes and bacteria in the midgut lumen may be important in bot fly larval development, further studies are required to confirm this.