Number Of Polymorphonuclear Granulocytes Research Articles

Comparison of systemic trimethoprim-sulfadimethoxine treatment and intrauterine ozone application as possible therapies for bacterial endometritis in equine practice.

Bacterial endometritis is one of the major problems in equine reproduction and usually treated with antimicrobial drugs. The study aimed to compare the effects of intrauterine ozone application and systemic antibiotic treatment (trimethoprim-sulfadimethoxine) on intrauterine bacterial growth and possible side effects on the endometrium in a clinical setting. Mares (n = 30) with signs of endometritis (positive uterine bacterial culture and cytological findings) were assigned randomly to different treatments: intrauterine insufflation of an ozone-air-mix (240 ml, 80 μg ozone/ml) twice at a 48 h-interval (Ozone; n = 10), systemic antibiotic therapy with trimethoprim-sulfadimethoxine (30 mg/kg, p.o., twice daily) for 5 days (TMS; n = 10), or intrauterine insufflation of air (240 ml, sterile-filtered) twice at a 48 h-interval (air; n = 10). Endometrial biopsy for histological examination was obtained before the treatment. Histological examination revealed no differences among groups. A control examination, including transrectal ultrasound, bacterial culture, cytological evaluation, and biopsy, was performed 7 days after the last treatment. Overall bacterial growth was reduced in every group after the treatment (p < 0.05), irrespective of the therapy [Ozone: 4/9 (positive culture after treatment/number of mares), TMS: 3/10 and Air: 6/10; p > 0.05]. However, Ozone and TMS (p < 0.05) were more effective in reducing growth of gram-negative bacteria as compared to Air (p > 0.05). No effects on the number of polymorphonuclear granulocytes (cytology) were observed (p > 0.05). In conclusion, trimethoprim-sulfadimethoxine and intrauterine ozone insufflation are safe treatment options for bacterial endometritis in mares but the efficacy of both treatments in reducing bacterial growth did not result in a complete absence of intrauterine bacterial growth.

Changes in blood pressure, heart rate, and blood profile in mares during the last 3 months of gestation and the peripartum period

In this study, peripartum changes in complete blood count, plasma electrolyte concentrations, blood pressure, heart rate, and heart rate variability (HRV) were determined in pregnant Warmblood mares (n = 10). Blood was collected from Days 245 to 330 of gestation (phase A), 2Days 3 to 1 before foaling (phase B), repeatedly within 12 hours after foaling (phase C), and on Days 1 to 3 postpartum (phase D). On the same days as blood collection, blood pressure and cardiac beat-to-beat intervals were measured and time domain HRV variables were calculated. Blood pressure decreased during the past 3 months of gestation, reached a nadir at foaling and increased rapidly thereafter (P < 0.001). Heart rate increased in phase A, reached a maximum in phase B, and decreased directly after foaling (P < 0.001) while HRV increased transiently after foaling (P < 0.001). The number of polymorphonuclear granulocytes was higher during phases B, C, and D than that during phase A (P < 0.001) and total leukocyte numbers peaked in phase C (P < 0.001). Erythrocyte counts and hematocrit increased immediately after foaling with a rapid decrease thereafter (P < 0.001), while plasma total protein was highest 1 hour after foaling (P < 0.001). Sodium and chloride concentrations increased from phases A to C and decreased in phase D (P < 0.001). Calcium concentrations were lowest immediately after foaling. In conclusion, changes in cardiovascular function and a transient hemoconcentration occur in peripartum mares. These changes may favor adequate uterine perfusion.

Peripheral Blood Neutrophil Granulocytes from Patients with Head and Neck Squamous Cell Carcinoma Functionally Differ from Their Counterparts in Healthy Donors

Solid tumors such as head and neck squamous cell carcinoma (HNSCC) display an intense interaction between tumoral factors and the immune system. Functional modulation of tumor-infiltrating and peripheral blood immune cells plays an important role during tumor progression. In this pilot study we compared biological functions of polymorphonuclear granulocytes (PMN) from the peripheral blood of HNSCC patients and healthy subjects. PMN were simultaneously isolated from the peripheral blood of HNSCC patients and healthy donors for functional analysis (apoptosis, production of reactive oxygen species (ROS), cytokine release and immunophenotyping). PMN from HNSCC patients showed a significantly lower inducible production of ROS (P = 0.02) and reduced spontaneous apoptosis (P= 0.008) compared with PMN from healthy donors. Under standard culture conditions, there was no significant difference regarding the release of inflammatory cytokines between PMN from HNSCC patients and PMN from healthy donors. Confirming previous observations, serum concentrations of PMN-related cytokines were significantly higher in the peripheral blood of HNSCC patients than in that of controls. Importantly, immunophenotyping revealed an increased number of immature PMN in PMN fractions isolated from HNSCC patients. Peripheral blood PMN from HNSCC patients and healthy donors show distinct functional differences. The presence of increased numbers of immature stages of PMN in HNSCC patients may partly contribute to the changes observed. After recruitment to and infiltration of the tumor, PMN may be further modulated in the local tumor microenvironment. This pilot study justifies functional analyses of myeloid cells in larger cohorts of patients with HNSCC.

Diet Quality Affects Allergic Peritonitis in Mice

Reasons for the increased prevalence of allergies observed in recent decades remain elusive. Here, we used a murine model to investigate the effect of nutrition on ovalbumin-induced allergic peritonitis. Compared to the organs of mice kept on a standard diet, mice exposed to a low quality diet during the growth period showed a decrease in the mass of metabolic (liver and heart) and, to a much larger extent, in lymphatic (thymus and spleen) organs, but not in testes or intestine mass. Moreover, diet manipulation affected the number ofpolymorphonuclear granulocytes as well as mast cell number and/or their responsiveness. During allergic peritonitis, mast cells from animals kept on the standard diet reacted to an allergen with degranulation, while the reaction in mice kept on the low quality diet was significantly weaker. However, the immunomodulators of this process remain unidentified as diet quality affected neither anti-ovalbumin IgE level nor synthesis/release of anti-inflammatory IL-10. Further work is needed to identify underlying immunomodulators.

Polymicrobial sepsis induces organ changes due to granulocyte adhesion in a murine two hit model of trauma

Polytrauma patients, who develop organ dysfunction, have often undergone multiple subsequent insults ("hits"). The sequence of organs that show a dysfunction mostly is lung, liver, kidney and heart. The aim of the present study was to investigate whether a second hit after trauma induces organ changes. Furthermore, it was of interest to identify possible pathogenic mediators such as polymorphonuclear granulocytes (PMN) and cytokines. For this purpose, a two hit model of systemic damage in mice was developed. Sepsis was induced by caecal ligation and puncture (CLP), which was preceded 48 hours by a femur fracture, the most common fracture of long bones in trauma patients. This fracture was combined with a haemorrhagic shock. In both mouse groups studied, a standardized femur fracture was produced using a blunt guillotine device with a weight of 500 g. This was followed by a haemorrhagic shock with substitution of ringer's lactate after 1 hour. In the study group, CLP was induced by puncturing the caecum using a 21G needle. As a control, sham animals underwent a laparotomy without CLP. Both groups were sacrificed after 48 or 96 hours. Clinical parameters were investigated on a daily basis to evaluate the animals' status. Lung, liver and kidney morphology was studied by light microscopy. PMN adhesion was determined by counting the number of adherent PMN per 100 microm of endothelium. Serum levels of TNF-alpha were measured after 48 and 96 hours. In the group submitted to laparotomy, all animals survived. The induction of polymicrobial sepsis by CLP resulted in an 85% (34/40) mortality within 96 hours after surgery (p < 0.05). The induction of a polymicrobial sepsis resulted in a significantly steady worsening of the clinical situation compared to the sham animals (p < 0.05). Lung morphology demonstrated significant changes at the end of the experimental period after 96 h in the two hit group. The alveolar septa were thickened and in all lungs haemorrhagic foci were observed. The number of PMN adhering to the pulmonary endothelium significantly increased at 96 hours. Some of the liver specimens in the two hit group showed focal hydropic degeneration and PMN infiltration. No kidney pathology was observed. This result coincided with an increase in TNF-alpha serum levels. A new rodent model mimicking the situation in the polytraumatized patient was developed. Although the animals showed minimal organ manifestation, a high percentage died probably due to cytokinemia. Furthermore, the increased TNF-alpha levels may lead to increased adhesion of PMN in the lung venules. This adhesion developed four days after the second hit. This might be the initial step for the development of extensive lung lesions in later phases. This model represents the SIRS more than MODS. This is a model for devolopment of posttraumatic disease due to cytokinemia and less for chronic multiple organ dysfunction and failure.

Uptake of pp65 in in vitro Generated pp65-Positive Polymorphonuclear Cells Mediated by Phagocytosis and Cell Fusion?

Objective: The cytomegalovirus (CMV) antigenemia consists of the detection of CMV pp65 in the nucleus of polymorphonuclear granulocytes (PMN), but it is unclear where and how PMN pick up virus particles or proteins. In an in vitro model for CMV antigenemia we investigated the mechanism of pp65 uptake by PMN that results in its expression in the nucleus. Methods: A series of inhibitors of different mechanisms was used to study the uptake of pp65 by PMN during coculture with CMV-infected endothelial cells and we performed a morphological analysis by light and transmission electron microscopy. Results: Nocodazole and cytochalasin B inhibited uptake of pp65 by PMN with 59.4 ± 14.1 and 73.3 ± 12.7%, respectively. The presence of anti-CMV hyperimmune globulin or lactoferrin during coculture reduced the number of pp65-positive PMN with 45.8 ± 7.0 or 40.6 ± 3.2%. Furthermore, a small number of the pp65-positive PMN obtained after coculture had fused to large cells with multilobed nuclei. PMN were observed that enclosed viral particles as well as free viral particles containing PMN in the cytoplasm. Conclusion: Fusion of viral particles with PMN and phagocytosis are both involved in the uptake of pp65.

The correlation between the numbers of some bacteria in human oral cavity and blood neutrophil count

The correlation between the number of blood polymorphonuclear granulocytes (PMNs) and the counts of oral bacteria in 92 children (33 girls and 59 boys), aged 4–15 was investigated. The groups of children comprised 44 healthy individuals and 48 children suffering from acute lymphoblastic leukaemia who were given intensive antileukaemic chemotherapy. It was found that while the number of PMNs in blood decreased, the counts of Streptococcus spp., Staphylococcus spp., mutans streptococci, Lactobacillus spp., and Actinomyces spp. in the saliva tended to increase. The similar reciprocal correlation between the number of blood PMNs and the counts of these bacteria in the saliva was found in the group of healthy children. We concluded that the blood PMNs, by influencing the number of oral PMNs which control bacteria in this habitat, influence also the number of some groups of bacteria there.

The correlation between the numbers of some bacteria in human oral cavity and blood neutrophil count.

The correlation between the number of blood polymorphonuclear granulocytes (PMNs) and the counts of oral bacteria in 92 children (33 girls and 59 boys), aged 4-15 was investigated. The groups of children comprised 44 healthy individuals and 48 children suffering from acute lymphoblastic leukaemia who were given intensive antileukaemic chemotherapy. It was found that while the number of PMNs in blood decreased, the counts of Streptococcus spp., Staphylococcus spp., mutans streptococci, Lactobacillus spp., and Actinomyces spp. in the saliva tended to increase. The similar reciprocal correlation between the number of blood PMNs and the counts of these bacteria in the saliva was found in the group of healthy children. We concluded that the blood PMNs, by influencing the number of oral PMNs which control bacteria in this habitat, influence also the number of some groups of bacteria there.

Does lung contusion affect both the traumatized and the noninjured lung parenchyma? A morphological and morphometric study in the pig.

Isolated unilateral lung contusion (LC) was induced in 12 pigs to determine the pathophysiological role of LC in the high mortality after multiple injury. The Horovitz quotient, pulmonary vascular resistance, mean pulmonary artery pressure, mixed venous oxygen consumption, inspiratory pressure and compliance were significantly decreased in the LC group as compared to controls. The number of polymorphonuclear granulocytes, the microvascular permeability of albumine and the Wilhelmy balance as determined by bronchoalveolar lavage were significantly increased after lung contusion. As indicators of a systemic reaction we found elevated plasma levels of the terminal complement complex and decreased levels of the complement factor 3a after LC. The morphological assessment revealed changes such as those encountered during the early phase of adult respiratory distress syndrome, with granulocyte sticking, endothelial cell adhesion and transendothelial diapedesis. Morphometric analysis demonstrated a significant decrease in alveolar diameter in both the injured and the contralateral lung due to impaired surfactant surface activity. A distinct increase in septal diameter, related to edema and caused by increased microvascular permeability, was found in the injured lungs. These findings emphasize that LC leads to a generalized impairment of the entire lung, which may lead to progressive lung failure.

In vitro determination of the force required for detachment of human polymorphonuclear granulocytes (PMN) from cultured endothelium and immobilized albumin

Detachment of adherent human PMN from cultured endothelial cells (human umbilical vein, HUVEC) and from immobilized albumin (bovine serum albumin, BSA) was investigated as a function of shear stress. Non-activated PMN did not adhere to either of the substrates «2%). 20 min after activation with PMA (phorbol myristate acetate, 10 nM), 48 ± 5% (mean ± SEM, n=15) of the total number of PMN added had adhered to albumin, and 18 ± 4% (n=9) to HUVEC. The dishes with pre-adhered PMN were subjected to shear stress in the range occurring in venules in vivo, using a rotating plate-and-cone (cone angle 2.50 ) apparatus made of transparent plastic to allow observation of the cells during the assay. On HUVEC, the majority of adhered PMN (66 ± 3%, n=9) was removed by a shear stress of 0.12 Pa (1.2 dyn/cm2), while the same shear stress removed only 15 ± 5% (n=9) of the PMN adhered to BSA. Further increase of shear stress to 2.3 Pa (23 dyn/cm2) removed 39 ± 4% (n=14) of the PMN adhered to BSA. Using spherical and semispherical approximations for the shape of the adhered PMN, the adhesive forces between PMA-activated PMN and HUVEC were calculated to be on the order of 0.05 nN per PMN, while most PMN on BSA adhered with a force of more than 4 nN per PMN. The force needed to detach PMN from HUVEC is more than one order of magnitude lower than value reported for PMN adhesion to venular endothelium in vivo. The present results suggest that the endothelium may playa regulatory role in PMN adhesion and limit the attachment force to allow emigration.

New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis.

This report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membrane-bound particles. Fluorescein-conjugated heat-killed Candida albicans (F-Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F-Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy-transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F-Ca particles for 20 min at 37 degrees C or, as a control, at 4 degrees C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F-Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury-lamp-powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane-bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F-Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one-step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes.

Circadian rhythms of peripheral blood leukocytes in aging mice

The effect of aging on white blood cell numbers was studied with special reference to the lability in blood cell physiology. Thus, the total numbers of leukocytes, polymorphonuclear granulocytes (PMNs) and lymphocytes were investigated in C3H mice, 3-month-old mice vs. mice aged 16, 21 and 26 months. Strong circadian rhythms were observed in the different cell classes, while there were insignificant seasonal changes. Even the oldest mice had significant variations, especially in the total leukocyte numbers and the PMNs. Subtle age-related shifts in the rhythmicity patterns were indicated. When comparing the 24-h means, there was a shift between the different white blood cell classes. A permanent reduction of the lymphocyte number was observed from the age of 16 months, while the number of PMNs steadily increased in the aging mice. The present results may explain inconsistencies among previous studies, demonstrating the significance of a chronobiological approach.

A comparative study of total body irradiation as a method of inducing granulocyte depletion in mice

Since conventional methods of inducing depletion of polymorphonuclear granulocytes (PMNs) in mice, such as treatment with cytostatic drugs and anti-PMN sera, proved to be insufficient to induce a stable PMN depletion for several days, and were accompanied by considerable toxic side effects, we induced neutrophil depletion in mice by total body irradiation (TBI) in a single dose of 6.0 Gy (600 rads.) at a dose rate of 0.20 Gy/min. This treatment reduced the number of PMNs in the peripheral circulation to values below 150/μl from day 3–10 after irradiation. The number of lymphocytes fell simultaneously. Platelet counts remained above 60% of normal values during the first 7 days after irradiation. Complement levels were not significantly affected by TBI. The results show that TBI of 6.0 Gy induces pronounced and stable PMN depletion in mice for at least 7 days. Furthermore, under an aseptic regimen the mice can be kept in good condition and losses are less than 5%.

Absence of requirement for polymorphonuclear granulocytes in the acute antibody-mediated rejection of murine skin allografts.

The role of polymorphonuclear granulocytes (PMN) in acute antibody-mediated rejection (AAR) was studied in a murine skin allograft model. B10.A skin was grafted onto B10.D2 recipients that were treated with goat antimouse lymphocyte serum, to postpone cellular rejection. This prolonged the median survival time of the grafts to 21.5 +/- 1.0 days. PMN depletion was effected by total body irradiation of 6.0 Gy (600 rads) on day 4 after grafting, which reduced the number of PMN in the circulation to levels below 150/microliters from days 3-10 after the irradiation. To induce AAR the mice received, on day 8 after grafting, an i.v. injection of a monoclonal antibody with specificity against the donor H-2Kk antigen together with 0.25 ml fresh rabbit serum. This caused acute rejection of all grafts in the control group within 72 hr, with abundant presence of PMN in the graft vessels on histologic examination. In PMN-depleted recipients the reaction in the grafts was somewhat retarded in th first 24 hr, but nevertheless acute rejection occurred within 72 hr in 9 of 11 animals, whereas PMN were virtually absent from the grafts. The results show that PMN act as accelerators and amplifiers of the acute antibody-mediated rejection process, but are not essential mediators in the damage to the vascular endothelium of the grafts.

Free secretory component and other proteins in human lung lavage.

Subsegmental bronchoalveolar lavage of the lingula and right middle lobe was performed in a group of normal subjects (32 cigarette smokers and 24 non-smokers) to analyze the recoverable fluid for its content of respiratory cells, several proteins including IgG and IgA, and free secretory component, a glycoprotein elaborated by serous bronchial epithelial cells. The lavage fluid obtained from the smokers consistently demonstrated an increased cell yield, reflecting primarily an increase in alveolar macrophages. In addition, the number of polymorphonuclear granulocytes was tenfold greater in smokers, and the total number of lymphocytes recovered was approximately twice that of nonsmokers. As in previous studies, the analysis of proteins contained in bronchoalveolar lavage (BAL) revealed an increase in the ratio of IgA to albumin in both smokers and nonsmokers, as compared with serum; but an increase in the IgG/albumin ratio in BAL was found only in smokers. The total amount of secretory component recovered ...