Bovine abortion and neonatal death associated with Ureaplasma diversum

Ureaplasma diversum was isolated from the lungs and/or stomach fluid and placentas of five aborted bovine fetuses and four newborn calves. All isolates were serotype D48. Placentitis was observed in all instances in which the placenta was examined. Gross lesions consisted of focal or diffuse reddening of the chorioallantois and amnion and thickening of the amnion. Microscopically there were fibrosis, edema and inflammation of the amnion. Microscopic lesions in the lung consisted of diffuse pneumonitis with thickening of the alveolar walls and in some cases peribronchiolar lymphoid accumulations. Macrophages and granulocytes were present in the alveoli. Inoculation of the vulva of a virgin heifer with one of the isolates from a fetal lung produced hyperemia and profuse purulent discharge with slight granularity.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

Spontaneous activation of circulating granulocytes in patients with acute myocardial and cerebral diseases.

Recent animal studies have suggested that there exists an activated subpopulation of circulating granulocytes which plays an important part in microvascular sequestration and tissue injury during shock and ischemia. In this respect, spontaneous granulocyte activation in form of pseudopod formation, a manifestation of actin polymerization, is a high risk for microvascular entrapment. The present investigation was carried out to determine if there is a significant difference in pseudopod formation in vitro between granulocytes obtained from healthy volunteers without symptoms and patients with acute cardiovascular illnesses. Blood samples from 25 healthy volunteers, 12 patients with acute myocardial infarction (AMI) and 12 patients with acute cerebral infarction (ACI) to determine spontaneous pseudopod formation in granulocytes with a high resolution light microscope over a period of several hours. The results revealed that the mean percentage of cells with pseudopod formation in the control group was below 10% in the first 3 hours, and increased to about 50% at 12 hours. In AMI patients, the level of activation within the first hour was not significantly different from the controls, but it rose rapidly to 90% in 4 to 5 hours. Patients with cerebral infarction, however, showed no significant difference from the control group. When the granulocytes of healthy subjects were incubated in plasma of AMI, the cells were activated similar to AMI granulocytes in their own plasma. When AMI plasma was serially diluted with Ringer's solution, the activation curve fell successively. These results indicate that AMI patients' blood contains plasma factor(s) which can activate granulocytes at a more rapid rate than controls.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Trypanosoma cruzi: experimental Chagas' disease in rhesus monkeys. II. Ultrastructural and cytochemical studies of peroxidase and acid phosphatase activities

Ultrastructural and cytochemical studies of peroxidase and acid phosphatase were performed in skin, lymph node and heart muscle tissue of rhesus monkeys with experimental Chagas' disease. At the site of inoculation there was a proliferative reaction with the presence of immature macrophages revealed by peroxidase technique. At the lymph node a diffuse inflammatory exudate with mononuclear cells, fibroblasts and immature activated macrophages reproduces the human pattern of acute Chagas' disease inflammatory lesions. The heart muscle cells present different degrees of degenerative alterations and a striking increase in the number of lysosomal profiles that exhibit acid hydrolase reaction product. A strong inflammatory reaction was present due to lymphocytic infiltrate or due to eosinophil granulocytes associated to ruptured cells. The present study provides some experimental evidences that the monkey model could be used as a reliable model to characterize histopathological alterations of the human disease.     

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

Characterization of granulocytes and mast cells in cultures of mouse bone marrow stimulated with interleukin-3

Bone marrow cells in liquid culture with interleukin 3 produce a population of non-adherent granulocytes and mast cells. Flow cytometry was used to identify granulocytes and mast cells on the basis of the physical properties of perpendicular light scatter (PLS) and coulter volume (CV) as well as the expression lgE and CR3 receptors. Multicolor analysis indicated there were subpopulations of Thy 1.2 positive cells which transiently appeared in these cultures and also expressed lgE receptors, CR3 receptors or neither of these receptors. The data suggested a differentiation scheme in which Thy 1.2 positive precursor cells give rise to granulocytes and mast cells. Further evidence for this differentiation scheme was provided from CV vs. PLS distributions which showed increases in CV and PLS as Thy 1.2 positive cells differentiated into mast cells and decreases in CV and PLS as Thy 1.2 positive cells differentiated into granulocytes.     

Ultrastructural study of the host-parasite interface of Moniliformis moniliformis (Archiacanthocephala) in laboratory-infected rats

The fine structure of the host-parasite interface of Moniliformis moniliformis in rats is described, comprising investigations on the ontogenic development of the presomal tegument, on the lesions caused by the worms, and on the host cellular reactions at the points of attachment of the worm. The presomal tegument of the worm contained more fibrous elements than the metasomal tegument. The sclerotization increased with the ages of the worms and toward the tip of the proboscis. The presomal surface coat was more coarsely structured and osmiophilic than that of the metasoma and was neither continuous with the contents of the tegumental crypts nor supported by lipids from necrotic host tissue. The surface coat occasionally detached from the proboscis, probably due to the activity of the surrounding granulocytes of the host. The proboscis hooks lost their tegumental covering during their larval development. Hooks of all worms from rats were invested with a semiliquid lipid coat, apparently derived from tegumental excretions at the base of the hooks. The invaginated area of excretion around the base of the hooks was rich in endoplasmic reticulum. The hooks seemed to renew their lipid coats at certain intervals by dipping into the excreted lipid. In rats all worms, irrespective of their age, attached superficially, penetrating only the intestinal mucosa and the tunica propria. No fibrous connective tissue was found in the lesions caused by the worms, indicating that they frequently changed their site of attachment. At 3-10 days postinfection the host's defense cells observed in the lesions consisted mainly of granulocytes, whereas plasma cells were the predominant leukocytes in lesions of older infections.     

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.     

Blood cells of Fundulus heteroclitus (L.)

Much of the parenchyma of the normal pronephros of the adult Fundulus heterodilus , a euryhaline teleost, is haematopoietic tissue which was examined in cytocentrifuge preparations and plastic embedded thick sections. As we have not characterized many of the blood cells functionally, the terminology used is based on their morphological resemblance to similarly named cells in higher vertebrates. Approximately 80% of the non-erythroid elements observed in the pronephros are mature eosinophilic granulocytes (48%), immature eosinophilic granulocytes (25%), or cells likely to be their precursors [e.g. small blast cells (6%) and large blast cells (2%)]. Although there are macrophages in the pronephros that are capable of endocytotic activity, the mature and immature granulocytes are not. The granulocytes are non-specific esterase positive, PAS positive, acid phosphatase negative, and are capable of being mobilized by a RES activating agent, Ecteinascidia turbinata .     

Altered oxidative metabolism in selenium-deficient rat granulocytes

Rats fed a selenium-deficient diet for 12 to 15 wk became selenium-depleted, measured by the selenium content of liver and granulocytes. The activity of granulocyte glutathione peroxidase, a selenoenzyme, in deficient rats was 11% of the activity in replete rat granulocytes. When stimulated with an H2O2 generating system, the HMPS activity of the deficient granulocytes was 50% of replete; however, when stimulated with methylene blue, the HMPS activity in deficient and replete granulocytes was the same. When granulocytes were incubated with PMA or OPZ, deficient granulocytes initially had the same O-2-generating activity as replete granulocytes; however, with increasing duration of stimulation, granulocytes from deficient rats generated less O-2 than replete rats. After 20 min in an H2O2-generating system, deficient granulocytes stimulated with PMA or OPZ generated less O-2 than replete granulocytes. These results indicate that deficient granulocytes did not metabolize H2O2 as well as replete granulocytes and that H2O2 caused damage to the O2-generating system. Measurement of O-2 generation in membrane-enriched particles showed the above effects were due to inactivation of the NADPH-dependent O2-generating system. Deficient granulocytes stimulated with OPZ for 20 min had 70% less membrane O-2-generating activity than controls. In addition, when membrane-enriched particles were made from cells that had been stressed with an H2O2-generating system, NADPH-dependent O-2-generating activity in deficient granulocytes was 50% of replete. In selenium-deficient granulocytes with low GSH-Px activity, prolonged incubation with stimulants and prior incubations with an H2O2-generating system caused loss of activity of the membrane-bound, NADPH-dependent, O-2-generating system.     

Immunoglobulins and complement in the skin of rhesus monkeys immunized with x-irradiated cercariae of Schistosoma japonicum

Skin sections of rhesus monkeys immunized with X-irradiated Schistosoma japonicum cercariae were stained by an unlabeled antibody enzyme method for the detection of IgG, IgM, IgA, IgE, and C3. While all of them could be found in the skin lesions, their distribution patterns varied with time and reactions. In whealing reaginic reactions, IgE appeared prominent, having an affinity to the mast cells. In Arthus-like reactions, IgG was predominant. IgG complexes were found on the schistosomula, on the walls of blood vessels, and in granulocytes near the schistosomula. In the late stage of Arthus-like reactions and in delayed hypersensitive reactions, IgA was predominant in granulocytes, mononuclear cells, and macrophages. Characteristics of each immunoglobulin pattern seemed to reflect its function in the effector mechanism. It may be speculated that these immunoglobulins and C3, together with effector cells, synergistically and sequentially destroy schistosomula in the skin.     

Purification of human granulocytes by centrifugal elutriation and measurement of transmembrane potential

Viable cell samples containing 93% pure granulocytes were obtained from human blood using the techniques of dextran sedimentation followed by centrifugal elutriation. The resting transmembrane potential (E_m) of human granulocytes was estimated using the fluorescent lipophilic cation, Di-S-C₃(5), from the null point for potassium—i.e., the external K concentration at which there is no change in E_m in response to valinomycin (a K ionophore). The E_m of human granulocytes, as calculated from the Nernst potential for K at the null point, is approximately – 100 mV. Data indicate that this large transmembrane potential is due in part to the presence of an electrogenic Na-K pump in human granulocytes which is stimulated by external potassium and inhibited by ouabain.     

烧伤后免疫抑制引起肠道细菌易位和脓毒症的病理学研究

近年来,创伤后体内细菌易位造成的内源性感染逐渐受到重视,为了探讨细菌易位的条件和途径等机理。本文利用烧伤后注射大剂量地塞米松的大鼠动物模型,模拟烧伤后应激状态下体内肾上腺皮质激素分泌增多,免疫机能下降的条件,观察肠道和其它脏器的病理改变。观察结果证实,烧伤与大剂量皮质激素确有引起肠道细菌易位和内源性感染,造成脓毒症的叠加作用。其基本条件包括肠道菌群微生态平衡的改变,宿主免疫机能下降和肠粘膜屏障的破坏。易位的肠道细菌主要是通过肠粘膜的坏死灶,上皮细胞坏变缺损处进入肠粘膜淋巴管和小静脉的。超微结构改变包括肠上皮细胞表面的微绒毛倒伏、变形、脱落、上皮细胞顶端崩解破坏,细胞间隙泡状增宽。脓肿中多数细菌微细结构完好,粒细胞和吞噬细胞胞浆中有多量糖元颗粒聚集,提示糖皮质激素可能影响吞噬细胞的糖代谢而降低其吞噬功能,使易位的细菌得以定植形成脓肿感染。     

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

The cellular composition of hemopoietic spleen colonies

The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.     

Dynamic Leukocyte Populations Are Associated With Early- and Late-stage Lesions in Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition typically targeting the axillary and anogenital regions of the body. The massive inflammatory cell infiltrate produced in this cryptogenic condition has led investigators in the attempt to link particular inflammatory cell fractions and cytokines to disease development, and ultimately to disease treatment. This study qualitatively and quantitatively analyzes the white blood cell fractions of macrophages, B-lymphocytes, T-lymphocytes, plasma cells, and granulocytes in 104 HS lesions on formalin-fixed paraffin-embedded tissues using immunohistochemistry (IHC). Four dermis-associated epithelial categories were investigated from persons with HS: 15 unaffected HS skin (US), 19 distended but unruptured follicle epithelium (UF), 62 migrating stratified squamous epithelium (MSSE) from ruptured follicles, and 35 degraded migrating epithelial sheets (DMES). In addition, 27 control skin (CS) from persons without HS were evaluated. Analysis of cell counts indicated that non-migratory dermal epithelium (CS, US, and UF) stimulated very little inflammatory response. However, contrary to previous studies which indicated macrophages to be the chief inflammatory cell in HS, this study showed that plasma cells were the primary cell type present in early-stage HS lesions (MSSE), whereas granulocytes were the major cell population seen in late-stage HS lesions (DMES):