Autoantibodies against bactericidal/permeability-increasing protein (BPI) in children with acute pneumonia

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein (BPI), an inhibitor of a lipopolysaccharide of gram-negative bacteria, are a common feature of chronic neutrophilic inflammatory processes such as cystic fibrosis. We investigated whether serum and salivary anti-BPI autoantibodies also appear in the course of acute pneumonia in 24 otherwise healthy children. Nine (38%) and four (17%) patients had detectable serum anti-BPI immunoglobulin G (IgG) (≥4 IU mL⁻¹) and IgA (ratio≥1.2), respectively, on the day of hospital admission (day 0). There was no increase in the rate of occurrence or the concentration of these antibodies in the convalescent sera obtained on day 30. The presence of anti-BPI IgG on admission did not correlate with inflammatory markers (peripheral white blood cell count, C-reactive protein) or temperature on admission. Also, salivary anti-BPI IgA, determined on days 0, 3–5 and 30, did not appear during the course of acute pneumonia. In summary, a substantial proportion of previously healthy children have pre-existing anti-BPI IgG autoantibodies. Acute neutrophilic infection, i.e. pneumonia, however, neither triggered the appearance of new antibodies nor boosted the concentrations of pre-existing ones. Thus, in typical acute pneumonia in children, autoantibodies directed against BPI may not have clinical significance.     

Determination of pneumococcus antibodies in secretions in acute pneumonia in children

IgA-antibodies to pneumococcal antigens were measured by enzyme immunoassay in saliva and nasal washings, obtained from 32 children aged 6 months to 14 years with acute pneumonia and from 25 healthy children aged 2.5-11 years. In the secretions of children with acute pneumonia an essential increase in the levels of IgA-antibodies in comparison with those observed in healthy children was detected. The levels of antibodies to pneumococci in saliva and in nasal washings were in direct correlation. These data are indicative of a protective role played, probably, by secretory antibodies at the portals of infection in pneumococcal pneumonia.     

Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.     

ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.     

GM-CSF transgene-based adjuvant allows the establishment of protective mucosal immunity following vaccination with inactivated Chlamydia trachomatis

Cellular and humoral immune responses induced following murine Chlamydia trachomatis infection confer almost sterile protection against homologous reinfection. On the other hand, immunization with inactivated organism induces little protective immunity in this model system. The underlying mechanism(s) that determines such divergent outcome remains unclear, but elucidating the mechanism will probably be important for chlamydial vaccine development. One of the distinct differences between the two forms of immunization is that chlamydia replication in epithelial cells causes the secretion of a variety of proinflammatory cytokines and chemokines, such as GM-CSF, that may mobilize and mature dendritic cells and thereby enhance the induction of protective immunity. Using a murine model of C. trachomatis mouse pneumonitis lung infection and intrapulmonary adenoviral GM-CSF transfection, we demonstrate that the expression of GM-CSF in the airway compartment significantly enhanced systemic Th1 cellular and local IgA immune responses following immunization with inactivated organisms. Importantly, immunized mice had significantly reduced growth of chlamydia and exhibited less severe pulmonary inflammation following challenge infection. The site of GM-CSF transfection proved important, since mice immunized with inactivated organisms after GM-CSF gene transfer by the i.p. route exhibited little protection against pulmonary challenge, although i.p. immunization generated significant levels of systemic Th1 immune responses. The obvious difference between i.p. and intrapulmonary immunization was the absence of lung IgA responses following i.p. vaccination. In aggregate, the findings demonstrate that the local cytokine environment is critical to the induction of protective immunity following chlamydial vaccination and that GM-CSF may be a useful adjuvant for a chlamydial vaccine.     

Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: effect on epithelial barrier function and leukocyte survival

This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0 x 10(6) and 1.0 x 10(6)cells/ml, respectively, appeared to affect both the survival of leukocytes and epithelial barrier function. Cell viability counts of immune cells showed 95% and 72.5% cell survival after isolation and after 4 days in co-culture with EEC, respectively, but only 11% cell survival when cultured alone for 4 days without EEC. Short-circuit current (I(sc)) results also showed that EEC and PBL co-culture exhibited a four-fold increase in the transepithelial resistance (TER) as compared to EEC culture alone, indicating enhanced protective barrier function. Taken together, the currently established in vitro co-culture model of endometrial epithelial cells and immune cells may provide a means to investigate local cellular immune responses upon uterine infections.     

Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia.

We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.     

Expression and modulation of C3 receptors during early T-cell ontogeny.

Using a corosette assay, optimal conditions were established for the detection of C3 receptors on T lymphocytes. E⁺-C3⁺ corosetting cells were demonstrated in four T-cell lines and six patients with E-rosetting acute lymphoblastic leukemia. Small numbers were detected in normal lymphoid tissues whereas thoracic duct lymph contained a large number of these cells. Following incubation of these tissues with thymic humoral factors, there was a decrease in corosetting cells with an increase in cells rosetting SRBC exclusively. Similar results were observed in vivo in a patient with severe combined immunodeficiency following a thymic epithelial cell transplant. Our data suggest that C3 receptor-bearing T lymphocytes occur early in T-cell ontogeny and can be modulated by thymic humoral factors.     

Qualitative and quantitative behavior of B-lymphocytes and immunoglobulins in the serum of children with acute leukemia after a single immunoglobulin concentrate substitution

In 19 children affected with acute leukaemia the serum immunoglobulin level was examined. An Ig deficiency treated with a single administration of IgA or IgM concentrate could be detected in 16 of these children. The impact of this single Ig substitution on the serum immunoglobulin level and on B-lymphocytes in the peripheral blood was thrice examined after this substitution. A significant increase of the corresponding Ig class in the serum and a significant decrease of B-lymphocytes in the peripheral blood could be observed.     

Ethanol-induced dysfunction of hepatocytes and leukocytes in patients without liver failure

The repeated intake of a great amount of ethanol is followed by functional and organic changes in the body. The intestinal absorption of alcohol is accompanied by an increased absorption of Gram negative bacteria endotoxins in the portal blood. In the liver, endotoxins stimulate CD14 receptors on the membrane of Kupffer cells, with a secondary inflammatory liver response, consisting in the secretion of proinflammatory cytokines and acute phase proteins. Simultaneously, alcohol metabolism in the hepatocytes by alcohol dehydrogenase, microsomal enzymes and catalase pathways determines a large production of ROS (reactive oxygen species), with secondary oxidative aggression on all liver cells: hepatocytes, Kupffer cells, endothelial sinusoidal cells, hepatic stellate cells and liver s lymphocytes. The oxidative aggression, as well as the intermediary products of the alcohol metabolism, cause a structural change of the antigenic structures of the liver and of the released proteins, that induces an immune response on the both pathways (humoral and cellular). The pathophysiological mechanisms and the paraclinical characteristics of the ethanol-induced liver failure are well known, so we were interested to study the patients with chronic alcoholism, but no clinical or paraclinical sign of liver failure, in order to describe the liver's protective mechanisms. For this reason, 153 patients with chronic alcoholism were divided into four test lots, in order to determine: the activity and the serum level of ceruloplasmin, plasma level of MDA (malondialdehyde), lactic and pyruvic acids, serum level of transferrin, alpha1-antitrypsin, CRP (C reactive protein), C3 fraction of the complement, IgA, IgG, IgM, IL-1beta, IL-6 and IL-8, cytosolic level of the cytochrome c in the circulating leukocytes. An immunophenotype study (as normal markers) on the peripheral blood lymphocytes was performed, too. The results demonstrate an important oxidative aggression induced by three sources: the alcohol metabolism in the hepatocytes, activated Kupffer cells and activated neutrophils that have infiltrated the liver, due to the chemoattractant effect of IL-8. This aggression induces apoptosis and necrosis of the liver cells. The major liver protective factor is, in our opinion, IL-6, due to its important antioxidant, antiapoptotic and proregenerative demonstrated actions. This protective effect of IL-6 is accompanied by antioxidant and antiprotease actions of ceruloplasmin, alpha1-antitrypsin and transferrin. We consider that an increased serum level of IL-6 accompanied by a decreased level of IL-1beta signify that antiapoptotic, antioxidant and proregenerative liver mechanisms prevail against proapoptotic and necrotic mechanisms. On the other hand, the ethanol-induced apoptosis of leukocytes (especially of the B cells) is very important, probably due to the absence of IL-6 protective action on these cells. The apoptosis of the circulating leukocytes is proved by their significant increase of the cytochrome c cytosolic level. The ethanol-induced liver immune response is predominantly cellular, as proved by the decreased ratio T helper (CD4+)/T cytotoxic (CD8+) in the peripheral blood. It is very important to observe that these significant immunologic changes appear before clinical or paraclinical signs of hepatic failure start. All these parameters were investigated in three groups of patients: chronic alcoholics, chronic alcoholics in the first 24 hours of the withdrawal and chronic alcoholics with acute alcohol intoxication, so the aggression types and the protective mechanisms were measured and differentiated in each "ethanolic status".     

Immunopathological responses in women with chronic inflammatory diseases of the uterus and appendages and their therapeutic correction

A total of 145 women with chronic inflammatory diseases of uterus and appendages (IDUA) were examined. Bacterioscopy of smears and culture of vaginal, cervical canal and uterine cavity contents were performed in all patients. Direct immunofluorescence and PCR were used for detection of chlamydiae in scrapes and smears and in biopsy specimens taken from endometrium. The number of T lymphocytes and their subpopulations, B lymphocytes as well as the level of plasmatic cells synthesizing IgA, IgM and IgG and secretory IgA (sIgA), immune complexes and C3 component of the complement were determined in biopsy specimens of the endometrium. Pathomorphological examination revealed structural changes characteristic of the immunocomplex disease (secondary deficiency of sIgA, pronounced suppression of IgA production by plasmocytes along with an essential increase in the number of IgG synthesizing cells and immune complexes fixed to the venule endothelium) were detected. Lectin-histochemical study revealed considerable changes in secretory activity of endometrial epitheliocytes manifested by severe suppression of bactericidal activity of the uterine mucus. The detected disturbances of local protective reactions in the endometrium of women IDUA gave grounds for including immunomodulators into the traditional treatment scheme. The latter made it possible to increase essentially the therapeutic effect.     

Functions and regulation of NF-kappaB RelA during pneumococcal pneumonia

Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation. To determine the roles of the NF-kappaB subunit RelA in facilitating these events, we infected RelA-deficient mice (generated on a TNFR1-deficient background) with Streptococcus pneumoniae. RelA deficiency decreased cytokine expression, alveolar neutrophil emigration, and lung bacterial killing. S. pneumoniae killing was also diminished in the lungs of mice expressing a dominant-negative form of IkappaBalpha in airway epithelial cells, implicating this cell type as an important locus of NF-kappaB activation during pneumonia. To study mechanisms of epithelial RelA activation, we stimulated a murine alveolar epithelial cell line (MLE-15) with bronchoalveolar lavage fluid (BALF) harvested from mice infected with S. pneumoniae. Pneumonic BALF, but not S. pneumoniae, induced degradation of IkappaBalpha and IkappaBbeta and rapid nuclear accumulation of RelA. Moreover, BALF-induced RelA activity was completely abolished following combined but not individual neutralization of TNF and IL-1 signaling, suggesting either cytokine is sufficient and necessary for alveolar epithelial RelA activation during pneumonia. Our results demonstrate that RelA is essential for the host defense response to pneumococcus in the lungs and that RelA in airway epithelial cells is primarily activated by TNF and IL-1.     

Mycoplasma pneumoniae induces interleukin-8 production via the epidermal growth factor receptor pathway

In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.     

A mucosal IgA-mediated excretory immune system in vivo

The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge, Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions.     

Cellular and humoral immunodeficiency in children with vaccine-associated paralytic poliomyelitis

Markers of humoral and cellular immunity in 16 patients with vaccine-associated paralytic poliomyelitis (VAPP) were evaluated. Signs of immunodeficiency (decrease of T- and B-lymphocytes counts, impaired synthesis of immunoglobulins, defects of phagocytosis, decrease of NK number) were revealed in all of the patients. Majority of them (81.3%) had defects in humoral immunity. Decrease of CD31, CD4+ and CD8+ was detected in 86.7, 35.7 and 91.7% of the patients respectively. Study of serum immunoglobulins performed in 15 patients showed decrease of IgG, IgM and IgA levels in 6 (40%), 1 (6.7%) and 6 (40%) of the patients respectively. Agammaglobulinemia was diagnosed in one patient in which only trace quantities of IgA and IgG were detected and IgM level was well below the normal. Congenital deficiency of IgA was diagnosed in 3 children. Majority of the children (11 from 12) had comorbidities (frequent respiratory infections, dermatitis, changes of intestinal microflora). Thus, immunocompromised condition of a child is a risk factor for VAPP after administration of alive oral poliovaccine.     

The dynamics of the bacterial associations in the vaginal mucus of female Syrian hamsters during the sexual cycle]

Autofluctuations in the numbers of bacteria, leukocytes and epithelial cells were observed in the vaginal secretion of Syrian female hamsters. The amplitude of fluctuations depended both on individual features of females and on environmental factors like light conditions. It was suggested that vaginal bacteria perform functions of a stimulator for local immunity to protect against infections and play a sanitary role to eliminate epithelial cells and remains of spermatozoids. A case is possible that products of the bacterium vital activity contribute also to the formation of female sex pheromone.     

Characteristics of nonspecific resistance in children with lung diseases due to opportunistic microflora

Acute destructive pneumonia in children was found to be complicated by acute pleural empyema (APE) on days 3-21 of the disease. The time of the development of this complication depended on the state of the nonspecific resistance of the body: the greater was the degree of deficiency as manifested by cell-mediated and humoral immunity indices, the earlier developed APE. Staphylococci and Pseudomonas aeruginosa infected the pleural cavity of children under the conditions of essentially decreased phagocytic activity, phagocytic index, C'H50 and one of the classes of immunoglobulin. The reaction of the body to staphylococci and P. aeruginosa took its course after the type of primary or secondary immune response, depending on the time of infection.     

Peri-epithelial origin of prostanoids in the human colon

The biology of prostanoids in the normal human colon is only beginning to be understood. We used in situ and in vitro techniques to define the lineage, number, per cell enzyme content, and epithelial functional effect of prostaglandin-generating cells, identified by the presence of cyclooxygenase 1 (COX 1). Immunohistochemical results were quantitated densitometrically, and cell surface staining in situ was verified by flow cytometry of isolated cells and by Western blotting. Three populations of COX 1(+) mucosal cells were identified, based on their morphology and local distribution in human mucosa; these were in the intra-epithelial, crypt apical, and lamina propria regions, with each containing a similar amount of COX 1 protein on a per cell basis. The most numerous were COX 1(+) mononuclear cells in the lamina propria, identified as CD3(+) T lymphocytes, both in situ and ex vivo. In toto, 21% of lamina propria mononuclear cells were COX 1(+), and over 50% of these cells were CD3(+) T cells. Findings were similar in the colon with mild-moderate inflammation due to ulcerative colitis. Using established surface markers, intra-epithelial and crypt apical COX 1(+) cells were non-lymphoid CD45(+) leukocytes; neither IgA (B-lymphocytes) nor alpha-smooth muscle actin (myelofibroblasts) was co-expressed on these COX 1(+) cells. Examining the effect of a major product of COX 1 in an in vitro system of human colonic epithelial monolayers, prostaglandin E(2) (PGE(2)) in low concentration (10(-6) M) enhanced epithelial barrier function and partially protected epithelia from the barrier-disruptive consequences of a pro-inflammatory cytokine, IFN-gamma. We conclude that the human colon contains three tiers of cell types for local synthesis of prostanoids, distinguishable by their location, morphology, and cell lineage. Further, maintenance of the barrier function of colonic epithelium may be added to other cell functions in mucosa regulated, in part, by prostanoids.     

Low-power laser irradiation induces leukocyte priming

Laser radiation (LR) of various spectral composition has been broadly used in clinical practice. However, the mechanism of the stimulating effects of LR remains obscure. The effect of He-Ne LR (633 nm) on human blood leukocytes was investigated both in the absence and presence of 8.65 nmol/l phthalocyanine (PhC). Irradiation of non-stimulated leukocytes with 0.025 to 0.5 J/cm2 did not lead to any activation of their luminol-dependent chemiluminescence (LCL). On the other hand, LR increased in most cases the subsequent CL response of the cells to opsonized zymosan (priming action of He-Ne-laser light). The effect of LR on the leukocytes was not standard. In irradiated leukocytes isolated from patients with severe acute or chronic pneumonia, or chronic bronchitis, the maximal LCL exceeded that for non-irradiated cells by 80% (0.05 J/cm2), 20-25% (0.15 J/cm2), and 0%, respectively (doses are shown in parentheses). Further increase of the exposure brought about a dose-dependent inhibition of LCL in cells from patients with severe acute and chronic pneumonia. There was an intriguing relationship between maximal CL responses of leukocytes subjected to laser irradiation in the presence and without PhC. When the priming effect of LR on isolated cells was small, it increased in the presence of exogenous photosensitizer, phthalocyanine; in cells of severely ill patients where the initial effect of LR was strong, Pc inhibited the priming action of LR. Apparently, different cells contained different amounts of endogenous photosensitizer(s); the addition of exogenous sensitizer increased the priming action of LR at low concentrations and decreased it at higher concentrations of the endogenous photosensitizer.     

Hepatitis in Selective IgA Deficiency

Hepatitis occurred in five out of 12 patients with selective IgA deficiency who had been detected by immunelectrophoretic screening of 2,600 individuals. This apparent increased susceptibility to hepatitis has not been reported before, and it may be due to the defect in their local and humoral immune mechanisms.