Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.     

Influence of external concentration fluctuations on leukocyte chemotactic orientation

The quantitative dependence of leukocyte chemotactic orientation on imprecision in the measurement of chemoattractant concentrations from thermal fluctuations is analyzed. First, a mathematical model relating orientation to differences in receptor occupancy across cell dimensions is developed. This is then coupled with an extension of Berg and Purcell's analysis (1) of the precision of attractant concentration measurements by means of receptor occupancy. Our results show that thermal fluctuations in external concentrations can limit the accuracy of orientation, unless the measurement noise is reduced by averaging the measurements over a period of time. Comparison of our model predictions to experimental orientation data suggests that leukocytes do overcome this limitation, and allows estimation of the time-averaging period necessary to do so. For the orientation observed in a visual bridge assay by Zigmond (2) using the attractant peptide FNLLP, we estimate that receptor occupancy measurements for spatial comparison across cell dimensions must be averaged for a few minutes. Otherwise, the fluctuations in the attractant concentration near the cell will be too great to allow the observed degree of orientation. Our analysis also suggests that the ratio of signal-to-signal noise does not adequately characterize orientation accuracy. Accurate orientation can, in some situations, occur when this ratio is substantially less than unity; in other situations, a ratio much greater than unity is required for accurate orientation.     

Abnormalities in lymphocyte proliferation in classical and atypical measles infection

Impaired in vitro lymphocyte responses to PHA stimulation have been observed in seven patients with acute classical measles infection, and three patients with atypical measles. The impairment is evident only at suboptimal concentrations of PHA and disappears in convalescence. Spontaneous DNA synthesis in peripheral leukocytes was elevated in both types of patients, but the classical measles patients demonstrated a delayed return to normal values as compared to the atypical measles group. We failed to demonstrate a serum inhibitory factor as an explanation for the impaired PHA responses of lymphocytes in these patients.     

Cystic fibrosis serum promotes [45Ca] uptake by normal human leukocytes

A two-fold increase in [⁴⁵Ca] labeling was observed when normal human leukocytes were incubated in the presence of serum obtained from patients with cystic fibrosis. The degree of [⁴⁵Ca] labeling of leukocytes isolated from patients with cystic fibrosis was also significantly greater than that observed for leukocytes that had been isolated from normal individuals.     

Aphid transmission of Beet Yellows Virus affected by homologous antiserum

A thin layer of homologous antiserum (against the beet yellows virus - BYV) between the leaf surface and a Parafilm membrane totally inhibited the acquisition of BYV by aphidsMyzus persicae (Sulz.), but it did not affect the inoculation of BYV by infective aphids. BYV transmission decreased with aphids picking up the virus from leaves coated with a normal rabbit serum. Aphids sucking on purified BYV suspension through the Parafilm membrane as well as aphids allowed to probe into leaves of healthy plants spread with an infectious purified BYV suspension failed to transmit BYV. No BYV particles could be detected in eluates from stylets and labia cut off from aphids which had probed on BYV infected plants by electron microscopic examination. The acquisition seems to be the most important phase for the aphid transmission of BYV which is apparently carried on the stylet surface.     

TLR2/TLR4-independent neutrophil activation and recruitment upon endocytosis of nucleosomes reveals a new pathway of innate immunity in systemic lupus erythematosus

The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.     

“Pseudochemotaxis” by micro-organisms in an attractant gradient

The random behavior of microorganisms in defined attractant gradients may exhibit apparent chemotaxis, or pseudochemotaxis, if the random motility of the population is a function of the attractant concentration. Several examples are studied using computer techniques to solve the differential equations of motion for populations of micro-organisms responding chemokinetically to variable attractant concentrations. The chemokinetic coefficient is a convenient parameter to characterize the time course of experiments in which micro-organisms move in attractant gradients. Measurements of the chemokinetic coefficient as a function of time may be used to directly determine the random mobility as a function of attractant concentration.     

Attempt to restore lymphocytes with defective T-cell function by mixing with normal cells in vitro

Blood leukocytes from a patient with T-cell immunodeficiency failed to respond to stimulation with phytohemagglutinin (PHA) in vitro although ample lymphocytes were present. Leukocytes from this patient were mixed in various proportions with normal leukocytes, and the mixtures were stimulated with PHA and cultured for three days. There was no restoration of PHA responsiveness of the patient''s cells in the presence of normal cells. This would indicate that the defect in the patient''s cell population was not due to the absence of any facilitating factor or cooperative function that could be provided by normal T cells or other blood leukocytes. Since in thymic aplasia the patient''s own cells are rapidly restored to normal PHA responsiveness by an unknown facilitating factor after thymic transplantation, we suggest that this mixed-cell culture method might differentiate between patients who need a bone marrow transplant and those who will respond to a thymus transplant.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Schistosoma mansoni: reduction in clinical manifestations and in worm burdens conferred by serum and transfer factor from immune or normal rhesus monkeys.

Although delayed hypersensitivity to Schistosoma mansoni was conferred on rhesus monkeys (Macaca mulatta) by means of dialyzable transfer factor prepared from peripheral leukocytes or lymph node cells of infected immune donors, when such animals were challenged with 1000 cercariae of S. mansoni they developed worm burdens similar to those of nontreated controls. However, recipients of transfer factor that, in addition, received hyperimmune serum showed minimal clinical symptoms and significantly reduced worm burdens after subsequent infection with S. mansoni irrespective of whether the donors used for the transfer factor were immune or uninfected. A significant but lower degree of protection was conferred by combinations of either S. mansoni transfer factor or normal transfer factor and normal serum. Neither transfer factor nor hyperimmune serum alone conferred protection to recipients. Susceptibility to infection was assessed by observing the signs of the disease, determining the worm burdens by perfusion 10 weeks after exposure, and by observing the appearance of the intestine at autopsy. The animals which received transfer factor and immune serum were protected against clinical disease. Good correlation between worm burdens and severity of disease was observed.     

Studies on the Longevity,Sequestration and Release of the Leukocytes in Chronic Myelogenous Leukemia

The intravascular life-span of leukocytes labelled in vitro with radioactive di-isopropylfluorophosphate was studied in 12 patients with chronic myelogenous leukemia (CML). In relapse, leukocyte specific activity (LSA) disappeared slowly; in remission, LSA curves approached normal and only a small proportion of LSA disappeared slowly. The level of maturation of the leukocytes that persisted in the blood was investigated by a leukocyte fractionation technique which excluded immature myeloid cells from leukocyte samples. The influence of extracorpuscular factors upon the pattern of disappearance of LSA was investigated by means of cross-transfusion experiments, and LSA curves obtained with in vitro and in vivo labelling were compared. The results suggest that: (1) the intravascular life-span of the mature leukemic neutrophil is prolonged in relapse and in remission; (2) intrinsically abnormal leukocytes are sequestered in an extravascular pool(s) but recycling occurs; (3) extracorpuscular factors modify the LSA curves; (4) exchange of leukocytes between intravascular and extravascular pools may not occur in relapse; and (5) the intravascular and extravascular pools constitute a self-sustaining pool(s) not replenished from a non-miscible precursor pool.     

Effects of serum on phagocytic activity and proteomic analysis of tilapia (Oreochromis mossambicus) serum after acute osmotic stress

The objective of the study was to analyze the effect of serum from freshwater (FW) exposed tilapia or from 25 ppt seawater (SW) exposed tilapia on the ability to mediate the phagocytic activity of tilapia phagocytes. To analyze the phagocytic activity, head kidney (HK) and spleen leukocytes were tested in 300 or 500 mOsm medium using three different treatment groups (a) control, (b) addition of 25% serum from freshwater (FW) exposed tilapia, and (c) addition of 25% of serum from 25 ppt seawater (SW) exposed tilapia. HK leukocytes cultured in 300 and 500 mOsm media for 4 h showed an increase of phagocytic ability in the control group as compared to the addition of serum from either FW or SW exposed tilapia. HK leukocytes exposed to 500 mOsm medium showed a higher phagocytic ability than those leukocytes exposed to 300 mOsm medium in each corresponding group. Concurrently, spleen leukocytes in the control group showed a higher phagocytic ability than those leukocytes with the addition of serum from FW or SW exposed tilapia. As compared to spleen leukocytes cultured in 300 mOsm medium, leukocytes cultured in 500 mOsm medium showed an increase of phagocytic ability within their respective group. To further investigate the observed phenomenon, 2D-gel electrophoresis was performed for analyzing the differentially expressed proteins in serum that was thought to influence the phagocytic ability. Up-regulated serum proteins in SW exposed tilapia contained complement C3 protein, NADH dehydrogenase (Ubiquinone) Fe–S protein 3, Mg²⁺-dependent neutral sphingomyelinase, Semaphorins, and Caspase 3. Taken together these results suggest that addition of serum decreased the phagocytic activity in HK and spleen leukocytes in vitro, furthermore, induced proteins semaphorin, complement C3, Mg²⁺-dependent neutral sphingomyelinase, and Caspase 3 are up-regulated in the serum, which might have decreased the phagocytic activity upon exposure to hyperosmotic solutions.     

Leukocyte lysozymes in various forms of leukemia]

The amount of lysozyme in the leukocytes of 47 patients with different forms of leukaemia and 6 healthy persons was investigated. The lysozyme determination was carried out in the lysate of isolated leukocytes obtained after freezing and thawing it seven times. The results expressed in microgram per 10(6) cells were compared with the simultaneously determined lysozyme concentration of serum and urine. A substantial reduction of the lysozyme amount as compared with the normal value (3.1 microgram/10(6) cells) was determined in the leukocytes of patients suffering from chronic lymphatic leukaemia, acute lymphatic leukaemia and the blastic crisis of chronic myeloid leukaemia. Different amounts of lysozyme ranging from extremely low ones to strongly elevated ones were found in leukocytes taken from patients with acute myeloblastic and chronic monocytic leukaemia. In many cases there was a lack of correlation between the lysozyme content of leukocytes on the one hand and that of serum and urine on the other hand. Possible causes underlying this lack of correlation are discussed.     

Chemotaxis of blood monocytes and polymorphonuclear leukocytes from patients with diabetes mellitus]

The chemotaxis of blood monocytes and polymorphonuclear leukocytes was measured in 20 adult patients with diabetes mellitus and 11 normal control subjects. In experiments dealing with monocytes, the diabetics had a chemotaxis value (125+/-10) significantly lower than that of controls (368+/-22); the polymorphonuclear leukocytes chemotaxis value of diabetics (468+/-31) was also lower than normal (1256+/-62). Adding insulin (10 unit/1000 cc) to the diabetes leukocytes suspension significantly increased the value of chemotaxis.     

Serum factors in chronic myeloid leukemia

Mononuclear cells from the peripheral blood of healthy test persons were cultivated in a methylcellulose medium with serum samples taken from 13 patients with chronic myeloid leukemia (CML) and with osteomyelosclerosis (OMS) as well as with serum samples of 6 healthy test persons. From evaluating the proliferation of granulopoietic cells quantitatively, conclusions were made concerning the concentrations of granulopoietic stimulating substances in these sera. In all cultures with the serum of patients the number of granulopoietic cell colonies was greater than that in cultures with the serum of normal persons. The stronger proliferation of granulopoietic precursor cells in cultures with serum of patients is seen to be due to an enhanced production of the granulocyte-macrophage colony stimulating factor (GM-CSF) by leukemic cells. The differential hemograms and curves indicating the course of leukocytes in patients are compared with the corresponding results of cultures. In patients with CML an increased output of GM-CSF will apparently influence the increase in size of the granulopoietic stem cell pool, which is evident in the steep increase of those curves indicating the course of leukocytes. In patients with OMS, however, there is a discrepancy between granulopoietic serum activity and proliferation in vivo. From these investigations the hypothesis is derived that an increased synthesis of GM-CSF in patients with CML may be one of the causes underlying hyperplastic granulopoiesis. A direct advantage of leukemic cells in proliferation cannot be derived from it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Models of normal and transformed cell adhesion,and capping and locomotion in vitro

It is proposed that patching, capping and endocytosis, and cell locomotion are manifestations of a single process whereby the cell discards foreign materials. Capping results from the binding to the cell surface of particulate (or molecular) objects which cannot function as immovable substratum. This might be described as unsuccessful or abortive cell adhesion in that the particles adhere to the cell rather than the cell adhering to the substratum. Lateral particle movements on the cell surface membrane are effected by the submembranous microfilament-microtubule system, resulting in capping without displacement of the cell. Successful adhesion of the cell to a substratum renders capping and endocytosis impossible and the cell attempts to discard the substratum by mechanisms analogous to capping. The cell achieves this by lateral movement and detachment of the trailing edge.The concept of abortive adhesion leading to capping has been amplified by the development of molecular models of normal and neoplastic cell adhesion in vitro in the presence and absence of serum. In these models, the normal cells have molecule A (adhesion sites) on their surface; they can spread on the substratum in the absence of serum. In the presence of serum, the A molecules on the normal cell surface bind with B molecules in serum, which may be substratum-bound or free in suspension. Binding of free B molecules with cell surface A molecules results in blockage of adhesion sites; these are cleared via capping. New adhesion sites (A molecules) are produced at the active edges of the cell. Binding of cell surface A molecules with the substratum bound B molecules results in cell adhesion. Transformed cells do not have A molecules on their surface; they cannot spread in the absence of serum. The transformed cells may recruit A molecules from the serum to attain deformability and spreading.These models also relate to capping of gold or resin particles, cell locomotion and regulation of cell division, and lectin-induced agglutination of transformed cells.     

Sound production in scolytidae: 'rivalry' behaviour of male Dendroctonus beetles

Intrasex response of two males at the gallery of an attractive female included ‘rivalry’ stridulation that was distinct in certain acoustic properties from chirps evoked by the female attractant. With the southern pine beetle, Dendroctonus frontalis, comparison of chirps evoked by natural and synthetic chemostimulus confirmed that myrtenol is part of the female attractant, that verbenone is multifunctional according to its concentration, and that endo-brevicomin has a rivalry function as well as its anti-aggregative effect. Considerable acoustic specificity was apparent in tested chirps of three bark beetles.     

The role of cytoplasmic microtubules in polymorphonuclear leukocyte chemotaxis : Evidence for the release hypothesis by means of time-lapse analysis of PMN movement relative to dot-like attractants

The present experiments were designed to elucidate the role of cytoplasmic microtubules in the chemotaxis of human polymorphonuclear leukocytes (PMNs) by means of the Boyden chamber technique and by means of analysis of PMN locomotion around a dot-like attractant.Casein induced positive chemotaxis in a small and variable fraction of the PMNs in the Boyden chamber.The movements of individual PMNs in coverslip preparations of clotted autoplasma were analysed as regards velocity of locomotion, locomotive index and net radial dislocation relative to the cell centre, with or without a yeast-phagocytosing leukocyte as a dot-like attractant.PMNs without obvious attractants tended to leave the visual field, i.e. they had a negative net radial dislocation relative to the centre of the visual field. Their locomotive indices suggested that their disappearance from the visual field was due to random movement. In contrast, the locomotive indices of PMNs influenced by attractants suggested the presence of both positive and negative chemotaxis in the population of moving PMNs.Yeast-phagocytosing leukocytes attracted wandering PMNs isolated by the Isopaque-Ficoll method (IF-PMNs) with a force which approximately balanced the basic tendency of the IF-PMNs to leave the visual field. Selective pretreatment of the moving IF-PMNs with podophyllic acid ethylhydrazide (SPI), 0.5 μg/ml (1.05 × 10⁻⁶ M), did not inhibit their attraction towards the central yeast phagocyte. The attraction of wandering IF-PMNs towards the central yeast phagocyte was inhibited by selective pretreatment of the phagocytes with SPI, 0.5 μg/ml. These observations indicate that cytoplasmic microtubules have an essential role in the release of chemotactic substances from phagocytosing leukocytes but not in the direction-finding of attractant-approaching PMNs.From the present observations by means of SPI, it is suggested that antitubulin inhibition of the release of chemotactic substances from phagocytosing leukocytes is the mechanism of inhibition of PMN chemotaxis by sub-antimitotic antitubulin concentrations in vitro. The latter phenomenon is thought to reflect the cellular basis of the anti-inflammatory action of the antitubulins.     

Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.     

A mathematical framework for the study of morphogenetic development in the slime mold

A mathematical continuum model of the slime mold Dictyostelium discoideum in the morphogenetic development stage is presented, which represents the amoebae as a fluid with surface tension, acted upon by a body force due to the presence of a chemical attractant. Assuming that the amoebae at a given time are in quasi-equilibrium, and that the shape of the organism is prescribed, it is possible to calculate from the model plausible concentration and source-sink distributions that are consistent with the given shape. Typical observed shapes are accounted for by the presence of very large concentrations and concentration gradients of the chemical attractant at the top of the structure.