Assessment of neutrophil function in canine cancer patients undergoing chemotherapy and correlation with neutrophil numbers.

Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.

Une diminution de la fonction des neutrophiles après l'administration d'une chimiothérapie a été rapportée chez des chiens atteints de lymphome. Le premier objectif de notre étude était de déterminer si la stimulation oxydative des neutrophiles et l'activité phagocytaire sont affectées par la chimiothérapie 7 à 10 jours après le début du traitement chez les chiens atteints de lymphomes et de tumeurs malignes non lymphomateuses. Le deuxième objectif était de déterminer s'il existe une corrélation entre les nombres de neutrophiles et la fonction des neutrophiles avant ou après le début de la chimiothérapie. L'évaluation par cytométrie en flux de la stimulation oxydative des neutrophiles et de la phagocytose après stimulation par Escherichia coli a été réalisée chez neuf chiens diagnostiqués avec un lymphome et 17 chiens avec des tumeurs non lymphomateuses avant et après la chimiothérapie, ainsi que 14 chiens témoins sans tumeur. Une corrélation des rangs de Spearman a été effectuée pour déterminer si les nombres de neutrophiles sanguins et la fonction des neutrophiles étaient significativement corrélés. Les patients atteints de lymphome ont montré un pourcentage significativement réduit de stimulation oxydative des neutrophiles après la chimiothérapie par rapport aux témoins sains ainsi que par rapport aux valeurs pré-chimiothérapie (P = 0,0022 et P = 0,0020, respectivement). Les patients atteints de lymphome ont également présenté une activité de phagocytose par les neutrophiles significativement réduite après la chimiothérapie par rapport aux témoins et aux valeurs pré-chimiothérapie (P = 0,0016 et P = 0,014, respectivement). Les chiens atteints de tumeurs malignes non lymphomateuses ont également montré une diminution significative du pourcentage de stimulation oxydative et de la phagocytose post-chimiothérapie par rapport aux valeurs pré-chimiothérapie (P = 0,00040 et P = 0,029, respectivement). Les nombres et la fonction des neutrophiles n'étaient pas significativement corrélés. Les résultats de l'étude suggèrent que le traitement chimiothérapeutique diminue la poussée oxydative des neutrophiles et l'activité phagocytaire 7 à 10 jours après le traitement chez les chiens atteints de diverses tumeurs malignes. De plus, les nombres de neutrophiles ne peuvent pas être utilisés pour prédire la fonction des neutrophiles.(Traduit par Docteur Serge Messier).

Revealing the Role of Epithelial Mechanics and Macrophage Clearance during Pulmonary Epithelial Injury Recovery in the Presence of Carbon Nanotubes.

Wound healing assays are extensively used to study tissue repair mechanisms; they are typically performed by means of physical (i.e., mechanical, electrical, or optical) detachment of the cells in order to create an open space in which live cells can lodge. Herein, an advanced system based on extensive photobleaching-induced apoptosis; providing a powerful tool to understand the repair response of lung epithelial tissue, consisting of a small injury area where apoptotic cells are still intact, is developed. Notably, the importance of epithelial mechanics and the presence of macrophages during the repair can be understood. The findings reveal that individual epithelial cells are able to clear the apoptotic cells by applying a pushing force, whilst macrophages actively phagocytose the dead cells to create an empty space. It is further shown that this repair mechanism is hampered when carbon nanotubes (CNTs) are introduced: formation of aberrant (i.e., thickening) F-actins, maturation of focal adhesion, and increase in traction force leading to retardation in cell migration are observed. The results provide a mechanistic view of how CNTs can interfere with lung repair.

Modulation of macrophage phagocytosis in vitro-A role for cholinergic stimulation?

Acetylcholine is synthetized and released from neural cells, but also by non-neuronal cells such as epithelial cells or keratinocytes. Cholinergic agonists enhance the phagocytosis of zymosan particles in primary peritoneal macrophages. The aim of this study was to investigate the effect of carbachol stimulation on phagocytosis in a macrophage cell line using microspheres. The murine cell line MH-S was used in a phagocytosis assay with fluorescent latex beads. The amount of the ingested beads was determined using flow cytometry. Gene expression was investigated using polymerase chain reaction. Gene expression of the muscarinic receptors M1, M3, M4 and M5 but not M2 was found. Carbachol slightly increased the phagocytosis of microspheres in the macrophages. A co-stimulation using lipopolysaccharide and carbachol did not increase the effect of lipopolysaccharide alone. In conclusion, cholinergic stimulation in vitro only moderately modulates the phagocytosis of microspheres. M2 might have a role in stimulation of macrophage phagocytosis.

The assessment of serum-mediated phagocytosis of necrotic material by polymorphonuclear leukocytes to diagnose and predict the clinical features of systemic lupus erythematosus: an observational longitudinal study.

BACKGROUND: Serum-mediated phagocytosis of antibody- and complement-opsonized necrotic cell material (NCM) by polymorphonuclear leukocytes can be quantified by using a flow cytometry-based assay. The phagocytosis of necrotic cell material (PNC) assay parallels the well-known lupus erythematosus cell test. In this study, we aimed to investigate the diagnostic accuracy of the assay and the relationship with clinical manifestations and disease activity in systemic lupus erythematosus (SLE). METHODS: The diagnostic accuracy for SLE diagnosis of the PNC assay was studied by cross-sectional assessment of blood samples from 148 healthy control subjects and a multicenter rheumatic group (MRG) of 529 patients with different rheumatic symptoms. A cohort of 69 patients with an established SLE diagnosis (SLE cohort) underwent longitudinal clinical and laboratory follow-up for analysis of the temporal relationships between PNC positivity and specific clinical manifestations. RESULTS: In 35 of 529 MRG patients, 13 of whom had SLE, the PNC assay result was positive. Combined positivity of the PNC assay and anti-double-stranded DNA antibodies increased specificity and positive predictive value for SLE diagnosis to 0.99 and 0.67, respectively. In the longitudinal study, 42 of 69 SLE cohort patients had positive results in the PNC assay at least once. PNC assay positivity was associated with current hematological manifestations and could predict mucocutaneous manifestations. When combined with hypocomplementemia, PNC positivity preceded increased Systemic Lupus Erythematosus Disease Activity Index 2000 score, glomerulonephritis, and alopecia. CONCLUSIONS: Serum-mediated PNC by polymorphonuclear leukocytes is commonly but not exclusively seen in patients with SLE. The PNC assay may be used in follow-up of patients with SLE and, especially in combination with other routinely assessed laboratory tests, may help to predict flares and different clinical manifestations, including glomerulonephritis. Our results encourage further development of the PNC assay as a complementary laboratory tool in management of patients with SLE.

Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

Assessment of neutrophil apoptosis.

Timely neutrophil apoptosis and cell clearance by surrounding phagocytes are essential components of the resolution phase of acute inflammation. Programmed cell death by apoptosis occurs with maintenance of an intact cell membrane in order to prevent the release of histotoxic intracellular products such as proteases and reactive oxidant species into the extracellular surroundings as occurs during necrosis. Macrophage phagocytosis results in attenuation of toll-like receptor-driven proinflammatory mediator production further promoting inflammation resolution. Failures in this cascade of events can result in tissue damage, chronic inflammation and disease. By studying human neutrophil apoptosis and phagocytic clearance in vitro, it is possible to delineate key control mechanisms in the regulation of these processes and therefore also identify potential therapeutic targets. Apoptotic signalling pathways are well described in the literature using a variety of laboratory techniques. In this paper, we outline the key in vitro assays used to assess neutrophil apoptosis, activation of key components of the apoptotic machinery, and phagocytic clearance of these cells.

Assessment of tumor antigen-loaded solid lipid nanoparticles as an efficient delivery system for dendritic cell engineering.

AIM: The work attempts to overcome tumor-associated immune tolerance using a surface-modified solid lipid nanoparticle (SLNP) delivery system for dendritic cell (DC) immunotherapy. MATERIALS & METHODS: Different formulations of SLNPs (SLNPs-alone, cationic SLNPs and mannosylated SLNPs) were prepared using tumor cell lysates. Prepared nanoparticles were characterized and their ability to activate DCs to induce a tumor cell-specific response was assessed. RESULTS: SLNPs induced a strong phagocytic signal to DCs without any significant toxicity. Comparatively, mannosylated SLNPs evoked an optimum and effective cell-mediated immune response with no significant toxicity. CONCLUSION: Surface-modified SLNPs may play a pivotal role in designing a clinically translatable DC-based immunotherapy for gastrointestinal malignancies. This novel approach may also facilitate the treatment of residual disease, following standard therapy.

[M1 and M2 macrophage phenotypes functional activity as essential components in innate immune response assessment].

Macrophage capacity to phagocytosis and migration activity are crucial components in innate immune response assessment. Differences in functional responses of two macrophage phenotypes were detected. Phagocytic activity of proinflammatory alveolar M1 phenotype in relation to S. aureus is more expressed than of antinflammatory M2 phenotype. Comparative analysis of migration activity showed alternative dependence of migration index on the type of used chemoattractant.

A much convenient and economical method to harvest a great number of microglia.

Microglia, implicating in such neuro-pathologies as brain inflammation, neurodegeneration, glioma, and neurogenesis, play an important role in central nervous system. Advanced research on microglia is crucial in exploring the neuro-pathology and neuro-physiology of these diseases, so how to culture large numbers of microglia in vitro becomes the base of a research. The wildly used method, at present, obtaining microglia from murine cannot fulfill the requirement of research, costing too much time and needing too many rats. We intend to introduce an optimized method that can harvest large quantities of microglia with high purity. Neonatal 2-3 days old Wistar rats were sacrificed and the cerebral cortices were trypsinized. We primarily cultured mixed cortical cells for 8-10 days. The microglia were harvested from the liquid supernatant; the left cells in the mixed cortical glial culture were passaged at a 1:2 density. After another 8-10 days of culture, microglia were collected again. And then, we passaged the left cells again for acquiring microglia from the third collection. We did not add additional mitogens in the experiment. At last, on average, 7.0 × 10(6) microglia were collected from one neonatal rat. By this modified method, much more microglia can be effectively and easily harvested comparing with the usual protocol before. We compared the characteristics of microglia harvested from these three passages, such as morphology, phenotype, purity, and abilities on proliferation, secretion, and phagocytosis. The cells presented typical microglia morphology, having phenotype markers of CD11b/c and CD45. The microglia from these three passages retained similar phagocytosis and secretion functions. Expanded population of microglia for investigation can be provided by this easy method in a short time with little cost and few rats.

Flow cytometric assessment of leukocyte function in sickle cell anemia.

Sickle cell anemia is associated with susceptibility to infection due to hyposplenism and the reduced ability of neutrophils to kill pathogenic organisms. In this study, blood samples from sickle cell anemia patients were divided into two groups: the painful crisis group and the steady state group. Flow cytometric assessment of phagocytosis and burst formation of neutrophils and monocytes as well as basophil function were performed, and these were compared to those of age- and sex-matched normal control subjects. Neutrophils and monocytes in sickle cell anemia patients were significantly different from those in the normal control subjects in the areas of weaker phagocytosis, fewer ingested bacteria and reduced burst formation. Basophil degranulation was normal. This pilot study using flow cytometry explains in part the susceptibility to infection of sickle cell anemia patients despite their high neutrophil and monocyte counts.

Assessment of liver phagocytic activity using EPR spectrometry and imaging.

The aim of the present study was to evaluate the usefulness of electron paramagnetic resonance (EPR) spectroscopy and imaging in assessing the phagocytic activity of the liver after administration of India ink. We conducted experiments on livers from control rodents and from rodents in which the Kupffer cell population had been depleted by pretreatment with gadolinium chloride. The EPR signal intensity recorded in liver homogenates was about two times lower in GdCl(3) treated rats than in control rats. EPR imaging carried out on precision-cut liver slices indicated a good correlation between the depletion of Kupffer cells and the EPR signal intensity.

Simultaneous flow cytometric assessment for cellular types and phagocytic abilities of the haemocytes of the hard clam, Meretrix lusoria.

The aim of this study was to devise a simple protocol for flow cytometric analysis to separate various haemocytic populations of the hard clam Meretrix lusoria based on the mitochondrial membrane potential diversity detected by the fluorescence probe 3,3-dihexyloxacarbocyanine iodide (DiOC6). Compared with the traditional technique for separation of haemocytic populations, continuous Percoll gradient centrifugation, our novel method was more efficient and yielded a higher ratio in separating the clams' haemocytic populations. Based on fluorescence 1 (FL-1) and side scatter (SSC) analysis for haemocytes stained with various fluorescent densities of DiOC6 using flow cytometer, the data showed that there were three obvious cell regions R1, R2, and R3, identified by hyalinocytes, small granulocytes and large granulocytes, respectively. At the same time our results showed that the percentages of haemocytes in R1, R2, and R3 were 49.71+/-0.65%, 19.35+/-00.74%, 30.94+/-0.69%, respectively. After classifying the haemocytic populations, phagocytic activity of the haemocytes was simultaneously analysed with phycoerythrin (PE)-labeled Vibrio vulnificus and detected by flow cytometry. Our results demonstrated that there were higher percentages of large granulocytes compared with hyalinocytes and the percentage of small granulocytes was related to the mitochondrial membrane potential and phagocytic activities.

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.

Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.

Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells--retinal pigment epithelium (RPE)--to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (>30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts--a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.

[Assessment of selected parameters of non-specific cellular response in patients with tick borne encephalitis (TBE)]. / Ocena wybranych parametrów odpornosci komórkowej nieswoistej u chorych z kleszczowym zapaleniem mózgu (KZM).

The aim of this study was the assessment of certain PMN functions involving migration, chemotaxis, phagocytic activity and intracellular oxygen-dependent killing in patients with TBE. The examination involved 47 persons treated in the Department of Infectious and Neuroinfectious Diseases Medical Academy in Bialystok. Two examinations were done: before and just after treatment. Control group contained 29 healthy persons. Migration and chemotaxis were assessed by agarose method of Nelson and al. and Glaser and al. Phagocytic activity was examined by microscopic method and intracellular oxygen-dependent killing by reduction test of NBT by Parks method. Analysis of results showed a decrease of all examined parameters both before and after treatment. It indicates a depression of non-specific cellular response in patients with Lyme meningoencephalitis.

Differences between bacterial species shown by simultaneous assessment of neutrophil phagocytosis and generation of reactive oxygen intermediates in trauma patients.

HYPOTHESIS: Previous studies on alterations in phagocytosis and bacterial killing after trauma have yielded conflicting results. We hypothesize that these changes are variable, depending on the species of bacteria used to assay these variables. DESIGN: Blood samples from patients were assayed by means of flow cytometry for phagocytosis and reactive oxygen intermediate generation. Several common clinical pathogens were used: Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Results were compared with those from controls. SETTING: Regional level I trauma center. PATIENTS: Ten consecutive patients were studied with E. coli and K. pneumoniae. Five of these were also studied with S. aureus. Patients were 18 years of age or older, with an Injury Severity Score of 16 or more. Patients who were taking corticosteroids before hospital admission or who were administered corticosteroids before blood was drawn were not studied. Isolated head injuries or limb fractures were also excluded. Controls consisted of healthy volunteers. MAIN OUTCOME MEASURES: The ingestion of bacteria by neutrophils and the generation of reactive oxygen intermediates. RESULTS: After trauma, phagocytosis of E. coli was enhanced, whereas ingestion of K. pneumoniae was depressed. Ingestion of S aureus remained unchanged. The generation of reactive oxygen intermediates was depressed after incubation with E. coli and unchanged with K. pneumoniae, but enhanced with S. aureus. CONCLUSIONS: Neutrophil response to trauma is dependent on which bacterial species the cell is attempting to kill. This may, in part, explain why only a limited number of bacterial species cause a significant proportion of early infections after trauma.

Assessment of the functional capacity for intracellular death and phagocytosis of polymorphonuclear cells in healthy neonates.

The objective of this study was to assess the functional capacity for intracellular death (ID) and/or phagocytic index (PI) of polymorphonuclear cells of 24-h-old healthy newborns with respect to the PMN cells of adults using the same standard exogenous source of opsonins. The ID and PI techniques were standardized and 2-3 ml of blood were used. No differences were found in the percentages of ID, P, PI among the PMNs of the newborns and those of the adults: 43.95 +/- 15.70 vs. 44.56 +/- 8.43 for ID; 38.96 +/- 14.34 vs. 39.00 +/- 14.54 for P; 1.71 +/- 0.54 vs. 1.73 0.45 for PI, respectively. It was concluded that the PMNs of 24-h newborns have an ID, P, PI functionality comparable to adult PMNs; differences observed in PMN function in newborns may be due to humoral deficiencies (opsonins).

Assessment of the functional capacity for intracellular dearth and phagocytosis of polymorphonuclear cells in healthy neonates

The objective of this study was to assess the functional capacity for intracellular death (ID) and/or phagocytic index (PI) of polymorphonuclear cells of 24-h-old healthy newborns with respect to the PMN cells of adults using the same standard exogenous source of opsonins. The ID and PI techniques were standardized and 2-3 ml of blood were used. No differences were found in the percentages of ID, P, PI among the PMNs of the newborns and those of the adults: 43.95 ñ 15.70 vs. 44.56 ñ 8.43 for ID; 38.96 ñ 14.34 vs. 39.00 ñ 14.54 for P; 1.71 ñ 0.54 vs. 1.73 0.45 for PI, respectively. It was concluded that the PMNs of 24-h newborns have an ID, P, PI functionality comparable to adult PMNs; differences observed in PMN function in newborns may be due to humoral deficiencies (opsonins)

Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells.

This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guérin (BCG) and Escherichia coli) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC). Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)-conjugated antibodies. Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence. The validity of the method was shown by inhibition of BCG phagocytosis at 4 degrees C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies. BCG-phagocytizing and -nonphagocytizing cell lines were discriminated by applying this technique to a series of bladder carcinoma cell lines. There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer. In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quantification of specific bacterial internalization by large numbers of (bladder) epithelial cells. This method should be generally applicable to the study of in vitro interaction between bacteria and different types of host cells.

In vitro assessment of phagocytosis of bovine collagen by human monocytes/macrophages using a spectrophotometric method.

The use of a wound dressing with covering and haemostatic properties significantly improves wound healing. In this study, a lyophilized bovine collagen sponge used for the treatment of wounds and ulcerae has been tested in a cell culture system. Phagocytosis of collagen fragments by human blood monocytes/macrophages has been investigated. For the assessment of collagen ingestion by mononuclear phagocytes, a picrosirius dye specific for collagen molecules has been used. By adapting this histochemical technique to microplate cell culture system, replicate monocyte cultures are assayed. Collagen content is determined by evaluating spectrophotometrically at 540 nm the absorbance of a sirius red/picric acid solution. Using this simple and sensitive method, the phagocytosis of bovine collagen by LPS-stimulated monocytes/macrophages has been ascertained.