Detection of plaque-forming cells in the peripheral blood of actively immunized humans.

Ten adult human volunteers were immunized with Salmonella typhi and their peripheral blood leukocytes were collected for 14 days after immunization. These peripheral blood leukocyted, rich in lymphocytes, were plaqued in a modified Jerne assay against sheep erythrocytes coated with either Salmonella or Escherichia lipopolysaccharide. A specific direct and indirect PFC response developed in immunized individuals by day 7 and peaked at day 10. This vigorous PFC response rapidly declined to normal levels by day 14. This marked and specific PFC response of human peripheral blood leukocytes may be developed as a useful tool for monitoring the humoral immune response of patients with Gram-negative bacterial infections.     

Experimental studies on bacterial product cantastim derived from Pseudomonas aeruginosa. VII. Activation of immune cells of healthy controls and cancer patients

CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa with beneficial effects related to enhancing the immune responses in conditions such as chronic viral and bacterial infections, immunodeficiencies and cancer immunosuppression. The aim of this study was to determine the capacity of this biological product to stimulate in vitro human leukocytes in whole blood. Blood samples from healthy donors and cancer patients were incubated with CS for 24 h and leukocytes were assessed for induction of inflammatory cytokines (TNF-alpha and IL-1beta) by ELISA and expression of early activation marker CD69 by flow-cytometry. For both groups of investigated subjects, the levels of TNF-alpha and IL-1beta in the supernatants of whole blood culture stimulated with CS were significantly higher than in unstimulated cultures, although lower than in LPS-stimulated samples. Stimulation of whole blood cultures with CS increased both the frequency and the expression of CD69 on the surface of T lymphocytes and NK cells. Importantly, this was noticed not only for healthy controls, but also for cancer patients. These data demonstrate the capacity of bacterial immunomodulator CS to activate human leukocytes of healthy subjects and cancer patients.     

Impaired blood clearance of bacteria and phagocytic activity in vitamin A-deficient rats

The effect of vitamin A deficiency on the functional integrity of the reticuloendothelial system and the phagocytic capacity of circulating polymorphonuclear leukocytes was evaluated in retinoate-cycled vitamin A-deficient rats under conditions such that secondary dietary imbalances were eliminated. Kinetics of blood clearance of 2 X 10(7) Escherichia coli injected intravenously was depressed within 8 days of the withdrawal of retinoic acid; all animals were profoundly affected by Day 12 of deficiency. In vitro, the phagocytic activity of polymorphonuclear leukocytes was similarly affected; by Day 12 of deficiency, phagocytic capacity in all deficient animals was less than 40% of the appropriate control values (P less than 0.01). Animals rendered vitamin A deficient by this procedure also displayed marked susceptibility to endogenous bacterial infection, as judged from the proportion of deficient rats that spontaneously developed bacteremia during the later stages of deficiency. These data together demonstrate unequivocally that reticuloendothelial and polymorphonuclear leukocytic functions are impaired in vitamin A deficiency in the absence of other dietary imbalances.     

HIV and other lentiviral infections cause defects in neutrophil chemotaxis, recruitment, and cell structure: immunorestorative effects of granulocyte-macrophage colony-stimulating factor

Patients with HIV infection exhibit deficits in bacterial and fungal clearance, and possibly depressed innate immunity. In this study, we observed that neutrophils from HIV-infected patients have a profound defect in chemotaxis in response to endogenous (IL-8) and bacterial (fMLP) chemoattractants, which was directly correlated with peripheral CD4(+) lymphocyte levels but not plasma viral load. A similar chemotactic defect was observed in the feline immunodeficiency virus (FIV) model of HIV infection. Intravital microscopy of FIV-infected animals revealed marked impairment in the in vivo recruitment of leukocytes; specifically integrin-dependent neutrophil adhesion and emigration induced by bacterial products. Treatment of FIV-infected animals with GM-CSF re-established both neutrophil recruitment (rolling, adhesion, and emigration) and in vitro chemotaxis to the levels seen in uninfected animals. This restoration of neutrophil responses was not due to GM-CSF-mediated priming. Rather, HIV and FIV infections resulted in defective neutrophil development, with an ensuing reduction in neutrophil granularity and chemotactic receptor expression. GM-CSF therapy restored neutrophil granularity, implying restoration of normal neutrophil development. Together, our findings underscore the fundamental defects in innate immunity caused by lentivirus infections, while also indicating that GM-CSF may be a potential immunorestorative therapy for HIV-infected patients.     

Increased recruitment but impaired function of leukocytes during inflammation in mouse models of type 1 and type 2 diabetes

Background

Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation.

Methodology/Principal Findings

Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62±18% and 85±21%, respectively) and high fat diet-induced (77±25% and 86±17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3±1.0) compared to control (8.0±1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice.

Conclusions/Significance

These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.     

Semi-stochastic cell-level computational modeling of the immune system response to bacterial infections and the effects of antibiotics

A mathematical model for the immune system response to bacterial infections is proposed. The formalism is based on modeling the chemokine-determined transmigration of leukocytes from a venule through the venule walls and the subsequent in-tissue migration and engulfment of the pathogens that are responsible for the infection. The model is based on basic principles, such as Poiseuille blood flow through the venule, fundamental solutions of the diffusion–reaction equation for the concentration field of pathogen-released chemokines, linear chemotaxis of the leukocytes, random walk of pathogens, and stochastic processes for the death and division of pathogens. Thereby, a computationally tractable and, as far as we know, original framework has been obtained, which is used to incorporate the interaction of a substantial number of leukocytes and thereby to unravel the significance of biological processes and parameters regarding the immune system response. The developed model provides a neat way for visualization of the biophysical mechanism of the immune system response. The simulations indicate a weak correlation between the immune system response in terms of bacterial clearing time and the leukocyte stiffness, and a significant decrease in the clearing time with increasing in-blood leukocyte density, decreasing pathogen motility, and increasing venule wall transmissivity. Finally, the increase in the pathogen death rate and decrease in pathogen motility induce a decrease in the clearing time of the infection. The adjustment of the latter two quantities mimic the administration of antibiotics.     

Immune depression--a possible cause of the unfavorable course of pneumonia in chronic alcoholics

In 27 patients, suffering with chronic alcoholism and hospitalized for pulmonary diseases in the Clinic of Pulmonology and Phthisiology, the following immunological characteristics were checked up: the functional activity of polymorphonuclear leukocytes and in 12 patients also that of alveolar macrophages were evaluated on the basis of the study of the phagocytic index and the phagocytic number, myeloperoxidase and the nitro blue tetrazolium test; the levels of serum IgG, IgA and IgM, the titer of the complement, E-rosette-forming cells (active and total) were also evaluated; the deficiency of cell-mediated immune response was determined by means of intradermal tests with the use of P.P.D., phytohemagglutinin, candidin, trichophytin. In all these investigations the depression of the functional activity of polymorphonuclear leukocytes and alveolar macrophages, dysimmunoglobulinemia, the increased level of circulating immune complexes and the suppression of cell-mediated immunity characteristics were revealed in the patients. Frequent infections and the severe course of bacterial and viral infections observed in such patients can be probably attributed to deficient cell-mediated immune response and to disturbances in phagocytosis.     

Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.     

Recruited macrophages control dissemination of group A Streptococcus from infected soft tissues

Group A Streptococcus (GAS) causes diverse infections in humans, ranging from mild to life-threatening invasive diseases, such as necrotizing fasciitis (NF), a rapidly progressing deep tissue infection. Despite prompt treatments, NF remains a significant cause of morbidity and mortality, even in previously healthy individuals. The early recruitment of leukocytes is crucial to the outcome of NF; however, although the role of polymorphonuclear neutrophils (PMNs) in host defense against NF is well established, the role of recruited macrophages remains poorly defined. Using a cutaneous murine model mimicking human NF, we found that mice deficient in TNF-α were highly susceptible to s.c. infections with GAS, and a paucity of macrophages, but not PMNs, was demonstrated. To test whether the effects of TNF-α on the outcome of infection are mediated by macrophages/monocytes, we systemically depleted C57BL/6 mice of monocytes by pharmacological and genetic approaches. Systemic monocyte depletion substantially increased bacterial dissemination from soft tissues without affecting the number of recruited PMNs or altering the bacterial loads in soft tissues. Enhanced GAS dissemination could be reverted by either i.v. injection of monocytes or s.c. administration of peritoneal macrophages. These experiments demonstrated that recruited macrophages play a key role in defense against the extracellular pathogen GAS by limiting its spread from soft tissues.     

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans ^1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages ³ and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.Unlike other infections, i.e neutrotopic viruses^4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.     

Do bacterial infections cause reduced ejaculate quality? A meta-analysis of antibiotic treatment of male infertility

Ejaculate quality may limit male reproductive success, and consequently,sperm quality is of importance. Spermatozoa are perceived as"non-self" by the immune system and are exposed to immunologicalattacks in the male reproductive tract. To reduce immunologicalreactions against their own sperm, males are dependent on thetestis being an immunoprivileged site. Immunoprivilege is obtainedby the blood-testis barrier and by local immunosuppression byandrogens. Despite this testicular immunosuppression, an influxof leukocytes may occur in testes. The condition in which maleshave a heightened level of leukocytes in semen is called leukocytospermia,and it is associated with reduced fertility. As the abilityof immunosuppression by androgens may depend on current intensitiesof infectious organisms in the extratesticular soma, only maleswith high parasite resistance may be able to bear the cost ofimmunosuppression and consequently produce high quality ejaculates.This issue is addressed by a meta-analysis on the effects ofbroad-spectrum antibiotic treatment of male leukocytospermia-associatedinfertility. The analysis showed that antibiotic treatment ofleukocytospermic men, without diagnosed genital tract infections,resulted in a significant improvement of ejaculate quality,that is, an increase in ejaculate volume, sperm concentration,number of motile spermatozoa, and number of spermatozoa withnormal morphology. Moreover, the amount of leukocytes in semenwas also reduced. This suggests that broad-spectrum treatmenttargeted toward bacterial infections reduces the density ofleukocytes in semen and, at the same time, improves the qualityof ejaculates produced. Our results emphasize the importanceof parasitic resistance and immunity as factors that cause variationsin ejaculate quality.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigen-specific responses accelerate bacterial clearance in the bladder

Urinary tract infections (UTIs) cause patient morbidity and have a substantial economic impact. Half of all women will suffer a UTI at least once, and 25% of these women will have recurrent infections. That 75% of previously infected women do not become reinfected strongly suggests a role for an adaptive immune response. The goal of this study was to characterize the adaptive immune responses to uropathogenic Escherichia coli (UPEC), the predominant uropathogen. A novel murine model of UTI reinfection was developed using the prototypic cystitis UPEC isolate NU14 harboring a plasmid encoding OVA as a unique antigenic marker. Bacterial colonization of the bladder was quantified following one or more infections with NU14-OVA. Animals developed anti-OVA serum IgG and IgM titers after the initial infection and marked up-regulation of activation markers on splenic T cells. We observed a 95% reduction in bacterial colonization upon reinfection, and splenic leukocytes showed Ag-specific proliferation in vitro. Adoptive transfer of splenic T cells or passive transfer of serum from previously infected mice protected naive syngeneic mice from UPEC colonization. These findings support our hypothesis that adaptive immune responses to UPEC protect the bladder from reinfection and form the basis of understanding susceptibility to recurrent UTI in women.     

Management of hand and upper-extremity infections in heart transplant recipients

Hand and upper-extremity infections are routinely managed by antibiotics, immobilization, and limited incision and drainage. However, in immunocompromised patients, these infections may be more aggressive and they may require more emergent treatment. The authors performed a retrospective review of the Stanford University Medical Center experience with hand and upper-extremity infections in 911 cardiac transplant recipients over the past 30 years. Thirteen heart transplant recipients were treated for infections of the hand and upper extremity on an inpatient basis. Ten patients (77 percent) required operative debridement, and three (23 percent) required more than one operative procedure. Nine patients (69 percent) had bacterial infections, six (46 percent) had fungal infections [four of these patients (31 percent) had both bacterial and fungal infections], one (7.7 percent) had a mycobacterial infection, and one (7.7 percent) was not cultured. Hand and upper-extremity infections in transplant recipients frequently resulted in deep-space infections, tenosynovitis, and osteomyelitis. The authors believe such infections represent a surgical emergency, requiring immediate evaluation by hand surgeons and early, aggressive treatment.     

Factor associated with neutral sphingomyelinase activity mediates navigational capacity of leukocytes responding to wounds and infection: live imaging studies in zebrafish larvae

Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response.     

Introducing shear stress in the study of bacterial adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm(2). Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm(2)). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes (1). On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm(2)(2). The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium (3). To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber (4). Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells (1), to proliferate on the cell surface (5)and to detach to colonize new sites (6) (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience(1) and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.     

Effect of cytostatic drugs on fever development following administration of bacterial pyrogen]

A study was made of the development of pyretic reaction to the administration of a bacterial lipopolysaccharide (pyrogenal) after preliminary treatment of rabbits with actinomycin D and cortisone. Such treatment failed to change the reactivity of thermoregulating centres to the endogenous pyrogen. Intravenous injection of bacterial pyrogen was followed by marked shortening of pyretic reaction; the reaction was markedly inhibited in response to its intracysternal administration. An important role played by polymorphonuclear leukocytes in the formation of endogenous pyrogens in the mechanism of pyrexia induced by bacterial pyrogens was shown in this work.     

Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.