Doxorubicin Hydrochloride Loaded Zymosan-Polyethylenimine Biopolymeric Nanoparticles for Dual 'Chemoimmunotherapeutic' Intervention in Breast Cancer.

OBJECTIVE: To utilize nanoparticles produced by condensation of zymosan (an immunotherapeutic polysaccharide) with pegylated polyethylenimine (PEG-PEI) for dual intervention in breast cancer by modulating tumor microenvironment and direct chemotherapy. METHOD: Positively charged PEG-PEI and negatively charged sulphated zymosan were utilized for electrostatic complexation of chemoimmunotherapeutic nanoparticles (ChiNPs). ChiNPs were loaded with doxorubicin hydrochloride (DOX) for improved delivery at tumor site and were tested for in-vivo tolerability. Biodistribution studies were conducted to showcase their effective accumulation in tumor hypoxic regions where tumor associated macrophages (TAMs) are preferentially recruited. RESULTS: ChiNPs modulated TAMs differentiation resulting in decrement of CD206 positive population. This immunotherapeutic action was furnished by enhanced expression of Th1 specific cytokines. ChiNPs also facilitated an anti-angiogenetic effect which further reduces the possibility of tumor progression and metastasis.

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

In vitro culture of murine peritoneal and alveolar macrophages modulates phagocytosis of Pseudomonas aeruginosa and glucose transport.

Phagocytosis by murine peritoneal macrophages (PM phi) of unopsonized Pseudomonas aeruginosa is a novel, glucose-dependent process occurring in concert with glucose or mannose transport via the GLUT1 facilitative transporter. The mechanism by which this transport triggers phagocytosis is not understood. The purpose of these investigations was to improve our understanding of this mechanism by delivery of an alternative sugar (fructose) to PM phi and to murine alveolar macrophages (AM phi). Fructose-cultured PM phi developed fructose-dependent phagocytosis of P. aeruginosa with increased glucose-dependent phagocytosis, GLUT1 expression, and [14C]glucose transport. Freshly explanted AM phi, which were unable to transport [14C]glucose or to ingest P. aeruginosa acquired the ability to transport glucose and to phagocytose P. aeruginosa with culture in either glucose or fructose. Both fructose- and glucose-cultured AM phi remained viable but incapable of measurable fructose transport or fructose-dependent phagocytosis. These studies suggest that an intracellular metabolite of fructose, glucose, and mannose is involved in triggering macrophage phagocytosis of P. aeruginosa. We demonstrate that delivery of appropriate substrates can substantially improve AM phi phagocytic function and may therefore possibly improve pulmonary host defense against P. aeruginosa.

Protein kinase C and intracellular pH regulate zymosan-induced lysosomal enzyme secretion in macrophages.

Binding of zymosan particles to macrophage beta-glucan receptors has previously been shown to trigger exocytosis of preformed lysosomal contents. In the present study, the involvement of Ca(2+)-, PKC-, and pH-dependent processes in the signaling to macrophage lysosomal secretion by zymosan was investigated. Also, the PKC dependence of lysosomal secretion in response to some soluble agents that directly alters intracellular pH was considered. Signaling to macrophage lysosomal secretion differs from that of many other secretory systems, because an elevation of cytosolic Ca2+ did not trigger a large secretory response, nor did attempts to reduce cytosolic Ca2+ affect the lysosomal secretory response to other stimuli. PKC activation by phorbol diester was also a poor stimulus of lysosomal secretion. However, when triggered by zymosan or by soluble stimuli raising lysosomal pH, the secretory response could be down-regulated by a prior prolonged incubation with phorbol diester. Such treatment also had marked effects on the binding and uptake of zymosan particles, the study of which was made possible by a novel approach. Furthermore, a synergistic effect on lysosomal secretion was obtained when stimuli that elevated lysosomal pH and stimuli that activated PKC were combined. This is of likely relevance for the secretory response to zymosan particles, a stimulus that both activates PKC and elevates lysosomal pH. The secretory response to zymosan was furthermore shown to be inhibited by a reduction of extracellular pH or [Na+], conditions that impair macrophage extrusion of acid equivalents. Earlier studies using soluble stimuli have shown a sensitivity of the secretory response to changes in cytosolic pH. We suggest a model in which the lysosomal secretory response to an elevation of lysosomal pH (1) is dependent on basal PKC activity and (2) can be enhanced further by activation of PKC. We consider PKC activity and elevation of lysosomal pH as independent and necessary signals, while cytosolic pH has a modulatory effect on some component(s) in the signal transduction pathway or in the secretory apparatus itself.

A new model of sciatic inflammatory neuritis (SIN): induction of unilateral and bilateral mechanical allodynia following acute unilateral peri-sciatic immune activation in rats.

Immune activation near healthy peripheral nerves may have a greater role in creating pathological pain than previously recognized. We have developed a new model of sciatic inflammatory neuritis to assess how such immune activation may influence somatosensory processing. The present series of experiments reveal that zymosan (yeast cell walls) acutely injected around the sciatic nerve of awake unrestrained rats rapidly (within 3h) produces low threshold mechanical allodynia in the absence of thermal hyperalgesia. Low (4 microg) doses of zymosan produce both territorial and extra-territorial allodynia restricted to the ipsilateral hindpaw. Higher (40-400 microg) doses of zymosan again produce both territorial and extra-territorial allodynia. However, allodynia is now expressed both in the ipsilateral as well as contralateral hindpaws. Several lines of evidence are provided that the appearance of this contralateral ('mirror') allodynia reflects local actions of zymosan on the sciatic nerve rather than spread of this immune activator to the general circulation. Since many clinical neuropathies result from inflammation/infection of peripheral nerves rather than frank physical trauma, understanding how immune activation alters pain processing may suggest novel approaches to pain control.

Tumor necrosis factor-enhanced leukotriene B4 generation and chemotaxis in human neutrophils.

In an in vivo study of five normal volunteers infused with endotoxin (20 U/kg of US reference endotoxin lot EC-5), increased neutrophil (PMN) generation of leukotriene B4 and chemotaxis to leukotriene B4 were found concomitantly with elevated plasma tumor necrosis factor (TNF) levels. To clarify the role of TNF in PMN activation, neutrophil responsiveness after in vitro treatment with TNF was examined. Neutrophils from seven normal subjects were incubated with TNF for 30 minutes and tested for chemotaxis to leukotriene B4, formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, or the calcium ionophore A23187 to assess leukotriene B4 generation. A range of 10(-13) to 10(-9) mol/L of TNF was used for these assays. When 10(-9) mol/L of TNF was used, the amount of leukotriene B4 that was produced was significantly greater than in control cells. The effect of TNF on PMN chemotaxis was uniformly inhibitory for the three stimuli at 10(-10) mol/L compared with untreated cells. At a picomolar range, PMN migration to leukotriene B4, but not to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine, was significantly increased over that of PMNs not exposed to TNF. This suggests that TNF has a specific facilitatory effect on PMN responsiveness for both leukotriene B4 production and chemotaxis to leukotriene B4 and may be the same signal for this phenomenon in endotoxemic patients.

Superoxide production by neutrophils in a model of adult respiratory distress syndrome.

Neutrophils (polymorphonuclear leukocytes [PMNs]) are thought to contribute to the pathophysiology of adult respiratory distress syndrome (ARDS) by the release of toxic oxygen metabolites. This study investigated superoxide production by circulating and bronchoalveolar lavage (BAL) PMNs in a rat model of ARDS induced by chronic Escherichia coli (lipopolysaccharide) endotoxemia. Superoxide production was stimulated by fmet-leu-phe, opsonized zymosan, and phorbol myristate acetate. Circulating and BAL PMNs from lipopolysaccharide-infused rats compared with PMNs from control rats are primed for nonselective increased superoxide production. The BAL PMNs are not only partially primed to release superoxide on adherence, they concomitantly have a depressed superoxide response to a phagocytic (opsonized zymosan) stimulus. These PMN responses may partially explain both the pulmonary injury and the increased susceptibility to pulmonary infection seen in patients with ARDS.

Propentofylline inhibits polymorphonuclear leukocyte recruitment in vivo by a mechanism involving adenosine A2A receptors.

Propentofylline is an atypical xanthine derivative that blocks adenosine uptake and has been shown to protect against ischemia-induced cerebral damage. We have studied the effect of propentofylline on recruitment of polymorphonuclear leukocytes during acute peritonitis induced by zymosan in mice. Following i.p. injection of zymosan, recruitment of polymorphonuclear leukocytes, reflected by myeloperoxidase activity in the peritoneal cavity, increased from 2 h onwards, peaked at 4 h and then decreased gradually. Propentofylline antagonized the zymosan-induced peritoneal myeloperoxidase accumulation in a concentration-dependent manner. This effect of propentofylline was counteracted by the non-selective adenosine receptor antagonist theophylline (50 mg/kg), and by the selective adenosine A2A receptor antagonists, 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo[4,3-a]quinoxaline (CP 66713) and 1,3-dipropyl-8-[3,4-dimethoxystyryl]-7-methylxanthine (KF 17387) (both at 2 mg/kg). The results indicate that propentofylline can reduce polymorphonuclear leukocyte recruitment in vivo and that this effect is related to an action on adenosine A2A receptors.

Blockade of PAF receptors controls interleukin-8 production by regulating the activation of neutrophil CD11/CD18.

The production of interleukin-8 by neutrophils in response to particulate stimuli may play a role in the recruitment and activation of further neutrophils in an inflammatory reaction. Here, we have evaluated the sequence of early events leading to interleukin-8 production by phagocytosing neutrophils. Kinetic experiments showed that the phagocytosis of zymosan particles by human neutrophils was rapid in onset. In contrast, interleukin-8 production was more protracted and only detectable 6 h later. Nevertheless, inhibition of phagocytosis with cytochalasins B or D suppressed the late interleukin-8 production. Activation of neutrophils with zymosan failed to enhance CD11/CD18 expression on the neutrophil surface but led to an increase in the expression of an activation-dependent epitope on CD11/CD18. Pretreatment with the platelet-activating factor (PAF) receptor antagonist, UK-74505 (4-(2-chlorophenyl)-1,4-dihydro-3-ethoxycarbonyl-6-methyl-2-[4-(2-methylimidazol[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2-pyridyl)carbamoyl]pyridine), significantly blocked the increase in the expression of the activation epitope, resulting in inhibition of the phagocytosis of zymosan and interleukin-8 production. In conclusion, the activation of neutrophils with zymosan leads to the activation of PAF receptors and this is followed by activation of CD11/CD18, phagocytosis of zymosan particles and subsequent interleukin-8 release.

HUVEC take up opsonized zymosan particles and secrete cytokines IL-6 and IL-8 in vitro.

Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined. HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles. The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated. It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out. Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged. Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals. The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process. Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.

A new assay to monitor the degranulation process in phagocytizing human neutrophils.

oxidation, we set up a method for measuring MPO intraphagosomal release in human neutrophils. The method is based on the passive engulfment of DAB together with the phagocytosable particle. Inside the vacuole, this substrate is oxidized by MPO released from the azurophilic granules. The colorimetrical evaluation of the amount of DAB oxidized allows for cheap, rapid quantification of MPO intraphagosomal secretion in whole cells. Using this method, we show that the degranulation process, involving azurophilic granules, can be monitored carefully during phagocytosis. It takes place after the ingestion of zymosan particles opsonized with normal human serum, as well as during IgG-mediated phagocytosis and under conditions where beta2 integrins are blocked. However our findings also show that the extent of intraphagosomal secretion depends on either the extent of opsonization or the type of receptor engaged during the phagocytic event.

Intravenous zymosan-A challenge induces an alveolar inflammatory response.

The purpose of this study was to evaluate the ability of intravenous zymosan-A (ZyA) challenge to induce an alveolar inflammatory response as indicated by inflammatory changes among lung lavage cells. The organ distribution of 1 mg of [51Cr]ZyA revealed that immediately following intravenous challenge of female ICR mice approximately 81% of the total cpm injected was associated with pulmonary tissue. Approximately 15% of the injected cpm was associated with the peripheral blood, liver, and spleen. ZyA translocated from the vascular compartment into pulmonary alveoli and was detected within polymorphonuclear neutrophils (PMN) and alveolar macrophages (AM) 18 h after intravenous challenge. PMN numbers among lung lavage cells increased beginning one day after challenge to a peak of approximately 5 x 10(5) PMNs by day 3 after challenge. The PMN response subsided by day 5 after challenge. There was no significant increase in the numbers of AM during the first week after intravenous ZyA; however, the number of AM increased from approximately 5 x 10(5) AM on day 1 after challenge to approximately 1.1 x 10(6) AM by day 5 after challenge. Within 24 h of intravenous ZyA, the number of AM in S phase of the cell cycle increased from approximately 2.5 x 10(4) AM one day after challenge to 1.1 x 10(5) AM in S phase five days after challenge. The data suggest that intravenous ZyA localized within pulmonary tissue immediately following intravenous challenge and translocated into the alveolar compartment where ZyA particles were found within phagocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphofunctional study of the haemocytes of the bivalve mollusc Mytilus galloprovincialis with emphasis on the endolysosomal compartment.

In the present work the haemocytes of mussels Mytilus galloprovincialis (Mollusca, Bivalvia) have been studied by light and electron microscopy in order to describe their main morphological features and to relate these to their roles in immune defence. The haemocytes belong to two definitive differentiated types, hyalinocytes and granulocytes. The former shows the presence of several fine pseudopodial protrusions, large nucleus with clumps of dense chromatin, scant cytoplasm, a well developed Golgi apparatus, lysosomes, several mitochondria (some with characteristic inclusions), coated pits and peripherally placed membrane-bound endocytic vesicles, considerable amounts of endoplasmic reticulum and ribosomes. The granulocytes generally possess an organelle-free ectoplasmic zone, numerous membrane-delimited dense granules of various types, coated pits and vesicles, endocytic and phagocytic vesicles, multivesicular bodies, several peroxisome-like organelles, mitochondria with inclusions, scant endoplasmic reticulum and small Golgi apparatus. These cells show the presence of few lipid droplets and variable amounts of glycogen particles. Some of the substructural features of the granules are documented here to indicate their probable biogenesis, growth and relationship with the endolysosomal compartment. In addition, in vitro phagocytosis experiments demonstrate that both hyalinocytes and granulocytes uptake latex and zymosan particles, granulocytes being much more active in phagocytosis than hyalinocytes.

Potential mechanisms responsible for zymosan-associated endothelial injury in rats.

OBJECTIVE: To assess alterations in endothelial barrier integrity and potential factors involved in zymosan-associated endothelial injury. DESIGN: Experimental study. SETTING: University hospital, Sweden. ANIMALS: 42 adult male Sprague-Dawley rats. INTERVENTIONS: One hour before an intraperitoneal injection of paraffin or zymosan (0.25 mg/g body weight), 1.0 ml of a solution of saline, N-acetyl-L-cysteine, dimethyl sulphoxide, indomethacin, verapamil, or allopurinol was given intravenously. MAIN OUTCOME MEASURES: Measurement of tissue water content, tissue intravascular plasma volume, interstitial fluid volume, and extravascular 125I-labelled human serum albumin distribution as well as plasma concentrations of albumin, alpha1-macroglobulin, alpha2-antiplasmin, and antithrombin III, 24 hours after the intraperitoneal injection. RESULTS: Endothelial permeability significantly increased in abdominal organs and the gastrointestinal tract, and plasma antiplasmin concentrations decreased. Pretreatment with N-acetyl-L-cysteine, dimethyl sulphoxide, or indomethacin protected against zymosan-induced endothelial barrier injury and the decline in protease inhibitors in plasma to varying degrees, while pretreatment with verapamil or allopurinol had a limited effect. CONCLUSION: Oxygen free radicals, prostaglandin, and proteases may have roles in the pathogenesis of zymosan-induced endothelial barrier injuries, implying that several mediators probably are interacting.

Fusion between large phagocytic vesicles: targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in Chinese hamster ovary cells.

This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetti or the Protozoan flagellate Leishmania amazonensis. Infection by these organisms is compatible with cell survival and multiplication. Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles. Donor vesicles contained heat-killed yeast, zymosan, beta-glucan or latex beads taken up by the host cells. Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp. Cell Res. 104, 325-334, 1977). Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy. Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection. Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours). Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined. The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads. In addition, only rarely did latex beads enter these vacuoles. The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals.

Determinants of the phagosomal pH in neutrophils.

Phagosomes formed by neutrophils are much less acidic than those of other phagocytic cells. The defective acidification seen in neutrophils has been attributed to consumption of protons during the dismutation of superoxide, because a large, sustained acidification is unmasked when the cells are treated with inhibitors of the NADPH oxidase. Consumption of protons transported into the phagosome by dismutation would tightly couple the activities of the NADPH oxidase and the vacuolar type H(+)-pump (or V-ATPase). We tested the existence of the predicted coupling using microfluorimetry and digital imaging and found that the rate of superoxide generation was independent of the activity of the H(+)-pump. Moreover, we failed to detect the alkalinization predicted to develop through dismutation when the pump was inhibited. Instead, two other mechanisms were found to contribute to the inability of neutrophil phagosomes to acidify. First, the insertion of V-ATPases into the phagosomal membrane was found to be reduced when the oxidase is active. Second, the passive proton (equivalent) permeability of the phagosomal membrane increased when the oxidase was activated. The increased permeability cannot be entirely attributed to the conductive H(+) channels associated with the oxidase, since it is not eliminated by Zn(2+). We conclude that the NADPH oxidase controls the phagosomal pH by multiple mechanisms that include reduced proton delivery to the lumen, increased luminal proton consumption, and enhanced backflux (leak) into the cytosol.

Differential neutrophil activation before and after endotoxin infusion in enterally versus parenterally fed volunteers.

This study was done to determine whether or not increased susceptibility to infection seen in enterally versus parenterally fed patients was caused by altered neutrophil (PMN) responsiveness. To determine the differential effects of route of feeding on human PMN activation, plasma C3a levels, circulating PMN counts, PMN migration to leukotriene B4 (LTB4), the peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan activated serum (ZAS) and generation of LTB4 were assayed before and after and infusion of endotoxin. Nine normal volunteers were enterally (n=4) or parenterally (n=5) fed a diet sufficient to maintain body weight for seven days prior to a standard challenge of endotoxin. Samples were taken prior to the infusion and hourly thereafter for six hours. Prior to the injection of endotoxin, significant differences were seen in the two feeding groups. Plasma C3a levels, absolute circulating PMN counts and chemotaxis to LTB4 were all significantly (p less than 0.02) elevated in the enterally fed group. Generation of LTB4 was higher in the intravenously fed group at base line than the orally fed group (p less than 0.05). Plasma C3a levels rose in the enterally fed group, but not in the intravenously fed group, at two hours after infusion. Neutrophil counts rose in both feeding groups after endotoxin infusion; but the change in percentage was greater in the enterally fed group than in the intravenously fed group. Chemotaxis to FMLP and ZAS was not different during the study and did not differ between the two feeding can have significant impact on neutrophil function and that parenteral nutrition may impair host responsiveness.

Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production.

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.

Phagosomal pH determination by dual fluorescence flow cytometry.

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.

Reticuloendothelial stimulation: levamisole compared.

PURPOSE: Levamisole in combination with 5-fluorouracil is an effective adjuvant for the treatment of resected Dukes stage C colon cancer. Since the mechanism of action of levamisole is not known, we have investigated its effects on hepatic and splenic reticuloendothelial system (RES) activity in the rat and compared the effect of levamisole with other known RES stimulators. METHODS: The hepatic and splenic uptake of an intravenous dose of technetium-99m-sulfur colloid has been used to measure RES activity in rats treated with levamisole, glucan, zymosan, chlormethiazole, octreotide, and saline. RESULTS: Levamisole significantly increased the hepatic uptake of technetium-99m-sulfur colloid and is comparable in its effect to the other RES stimulators. In contrast, levamisole has no effect on splenic RES activity. CONCLUSION: RES function is considered to be a potentially important factor in the development of liver metastases, and the stimulatory effect of levamisole on the hepatic RES may partly explain its efficacy as an adjuvant treatment in colon cancer.