Activated Pulmonary Macrophages Are Insufficient for Resistance against Pneumocystis carinii

CD4⁺ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ^−/−), T-cell receptor αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R^−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI^−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ^−/− and RAG-1^−/− mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ^−/− mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4⁺ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4⁺ TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4⁺ T lymphocytes, must contribute to successful resistance against P. carinii.Immunocompromised patients, especially those suffering from AIDS, are at elevated risk of acquiring Pneumocystis carinii pneumonia (PCP), a major cause of premature mortality among AIDS patients (8, 35, 53). Various studies have emphasized that CD4⁺ T lymphocytes play a pivotal role in the orchestration of resistance to P. carinii (22, 43, 45), an opportunistic fungus, but the mechanisms underlying protection remain a conundrum. Pulmonary macrophages are considered the main effector cells in clearing the immunocompetent host from invading P. carinii organisms (25). It seems conceivable, therefore, that macrophage-activating functions mediated by CD4⁺ T cells are central to resistance. Impaired gamma interferon (IFN-γ) production by T cells from AIDS patients is thought to enhance susceptibility to P. carinii (34, 41). This notion is supported by reports that application of exogenous IFN-γ ameliorates disease in experimental animal models (2, 45). In contrast, in vivo neutralization of IFN-γ in spleen cell-reconstituted severe combined immunodeficiency (SCID) mice by a specific monoclonal antibody (MAb) does not affect parasite clearance (5). Further studies point to a critical role of tumor necrosis factor alpha (TNF-α) (5) and interleukin-1 (IL-1) (6) in maintaining an immunocompetent state. Both cytokines are mainly produced by macrophages and induce inflammatory responses (4, 10, 26). Overall, these findings support involvement of macrophage-derived cytokines in successful host resistance against P. carinii.To analyze in more depth the role of inflammatory and Th1/Th2-related pulmonary defense mechanisms in control of aerogenically acquired PCP, we took advantage of naturally susceptible gene disruption mutant mice lacking major histocompatibility complex (MHC) class II molecules (and therefore conventional CD4⁺ T cells) (Aβ^−/−), T-cell receptor (TCR) αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) (19). We further exploited mice deficient in the IFN-γ receptor (IFN-γ-R^−/−) or the TNF-α type I receptor (p55) (TNF-α-RI^−/−) to analyze their capacity to cope with aerogenic P. carinii organisms.Bronchoalveolar lavage (BAL) cells of healthy and diseased mice were investigated for expression of the proinflammatory cytokines IL-1, TNF-α, IFN-γ, and IL-12, as well as IL-4, IL-5, and IL-10. The latter three cytokines counteract IFN-γ- and IL-12-mediated responses but participate in protection against certain extracellular pathogens (9). Moreover, production of superoxide (SO) and nitric oxide (NO), putative effector molecules of antimicrobial defense, was taken as a further indicator of macrophage activation. Contact with foreign material induces a rapid respiratory burst in professional phagocytes which results in SO production as a first line of defense. SO has been implicated in destruction of P. carinii (31), whereas NO produced by IFN-γ-stimulated macrophages encountering pathogens (4, 18, 30) does not appear to participate in control of P. carinii infection (47). Of further interest was the role of macrophage-expressed mannose receptors (MR) and scavenger receptors (SR). MR were previously found crucial for mediating P. carinii internalization (11, 37). The relevance of SR with respect to PCP has not been evaluated, but they are mainly expressed by tissue macrophages (36) and nonspecifically bind a large array of molecules, including surface molecules of microorganisms (39). Receptors with such broad pattern reactivity may be involved in direct differentiation of self from non-self, and recent data suggest that not only MR but also SR aid pattern recognition by macrophages and subsequent internalization of invading pathogens (27).We found that BAL cells from P. carinii-diseased RAG-1^−/− and TCRβ^−/− mutants secreted elevated IFN-γ, TNF-α, IL-12, NO, and SO levels and expressed IL-1 mRNA. In contrast, cells from morbid Aβ^−/− mice produced IL-1 mRNA and high levels of NO only, whereas all other parameters were low to absent in these mutants. SR were expressed on pulmonary macrophages of all diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants, whereas MR were also expressed by macrophages of healthy animals. Yet, the apparently activated phagocyte system in the lung, most pronounced in morbid TCRβ^−/− and RAG-1^−/− mutant mice, was insufficient for protection against natural P. carinii infection. Elevated levels of IFN-γ and TNF-α in morbid mutants (not in Aβ^−/− mice) and the naturally resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mice further argue not only for independence from IFN-γ and TNF-α. Our findings indicate that CD4⁺ αβ T lymphocytes prevent and clear infection with P. carinii by mechanisms distinct from, or in addition to, pulmonary macrophage activation.(This study is part of the Ph.D. thesis of R. Hanano.)     

Depressive symptoms and disability in acute and chronic back pain patients

Depression and level of disability are evaluated in acute and chronic low back pain (LBP) patients. To assess the possibility that some somatic symptoms are confounded with pain, the items of the Beck Depression Inventory arc divided into a cognitive-affective and somatic subscale. The sample consisted of 37 chronic LBP patients. 41 acute LBP patients, and 28 healthy participants. The level of disability was assessed by the Oswestry Low Back Pain Disability Questionnaire. Chronic LBP patients, but not acute LBP patients, have more depressive symptoms than controls. Additionally, chronic LBP patients report more somatic symptoms of depression (e.g.. emo ltional and self disturbance complaints) than cognitive-affective symptoms. Finally, correlation statistics reveal significant relations between the level of disability and depression scores. Whereas chronic patients show a significant correlation between the somatic subscale and level of disability, in acute patients the cognitive-affective subscale is significantly related to the level of disability. The findings suggest careful consideration of whether somatic symptoms of depression are related to pain when using self-report measurements of depression in pain patients. The separation of cognitive-affective and somatic symptoms of depression to evaluate pain problems seems appropriate.     

Identification of the membrane protein of porcine epidemic diarrhea virus

Sequence information on the genome of porcine epidemic diarrhea virus (PEDV) has only recently been determined. In contrast, very little is known about the viral proteins. In the present report we have identified the membrane glycoprotein (M) of PEDV by use of rabbit anti-peptide sera and transient expression of the cloned M gene in Vero cells and by expression in the baculovirus system. The native M protein of PEDV is incorporated into virions, is N-glycosylated, and migrates with a relative mobility (Mr) of 27 k in polyacrylamide gels. In contrast, the M protein synthesized by recombinant baculoviruses migrates with a Mr of 23 k, that is, with identical mobility as the deglycosylated product of PEDV. Thus, it appears that M protein specified by the recombinant baculovirus is poorly, if at all, glycosylated. Using monoclonal antibodies and rabbit antipeptide sera specific for the N and C termini of the M protein, we were able to show that a 19 k band detected in PEDV-infected cells but not in virions represented a fragment of M from which the C terminus had been cleaved off. Finally, by electron microscopy and immunogold labelling, the relative orientation of M within the virion envelope was determined as NexoCcyt. In conclusion, all of these data strongly support the hypothesis that PEDV should be classified with the group I coronaviruses.     

Alveoläre Hypoventilation und künstliche obstruktive Ventilationsstörung

Zusammenfassung Es wird über Versuche berichtet, in denen bei freier und artefiziell behinderter Ausatmung Beziehungen zwischen Atemzeitvolumen und alveolärem CO₂-Druck aufgestellt wurden. Unter Stenoseatmung werden für gleiche Änderungen des Atemzeitvolumens größere Differenzen des alveolären CO₂-Druckes benötigt. Die Verschiebungen werden als Folge der bei erschwerter Atmung erhöhten Atemarbeit gedeutet und nicht auf eine Änderung der Erregbarkeit des Atemzentrums zurückgeführt. Die alveoläre Hypoventilation bei obstruktiven Ventilationsstörungen kann daher — so wird weiter gefolgert — ihren Ursprung in der vermehrten Atemarbeit haben, ohne daß zwingend eine andere pathogenetisch wirksame Ursache diskutiert werden muß. Auf die Bedeutung von Veränderungen des Atemwiderstandes bei der experimentellen Prüfung atemwirksamer Pharmaka wird hingewiesen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Determination of Dissolved Oxygen in Heterogenous Systems Particularly in Emulsions and Oily Liquids

The content of dissolved oxygen was determined by four independent methods in a series of non-aqueous or heterogenous systems. The Lex-O₂-Content Analyzer represents a fast and simple apparatus that employs a coulometric oxygen assay with Hersch cell detection. A comparison of the results with different methods demonstrates the reliability of the Lex-O₂ in the determination of oxygen dissolved in heterogeneous or non-aqueous systems. Therefore, this apparatus can be recommended for the measurement of oxygen in oxygenator or perfusion fluids, as well as in blood substitutes or other oxygen transporting systems.     

Radiopharmacokinetics and radiation dose from 99mTc-HM-PAO (Preliminary report)

Whole body retention measurements were performed in volunteers after i.v. injection of ^99mTc-HM-PAO (Ceretec^®). The organ accumulation was measured in mice and data were transferred to standard man according to ICRP. Absorbed dose calculations were made with these data by using the concept of absorbed fractions (MIRD method). In man, the whole body retention and the retention in the brain could be calculated by direct measurement, absorbed doses to the other organs could only be derived from animal data. The absorbed dose to the brain derived from human data (10.3 Gy/MBq) is greater by a factor of 2 than that derived from animal data. The highest absorbed dose was received by the thyroid (24.4 Gy/MBq), the absorbed dose to the ovaries, testes and whole body ranged from 2.8 to 4.2 Gy/MBq.Dedicated to Professor Dr. Guenter Liess on the occasion of his 65th anniversary     

Risk factors for Epstein–Barr virus reactivation after renal transplantation: Results of a large,multi-centre study

Epstein–Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.     

Impact of donor extracellular vesicle release on recipient cell “cross-dressing” following clinical liver and kidney transplantation

In several murine models of transplantation, the “cross-dressing” of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.     

Rapid rise in plasma glucagon induced by acute cold exposure in man and rat

The effect of acute cold exposure on the concentration of glucagon in the blood was investigated in man and in intact and adrenalectomized rats.In man fasted overnight acute cold exposure, which caused a twofold increase in O₂-consumption resulted in a rapid rise in plasma glucagon. The levels of insulin and blood glucose remained unaltered, while the concentration of serum free fatty acids and -hydroxybutyrate increased.In fasted intact rats acute cold exposure lead to similar effects. A close parallelism between the rise in plasma glucagon and the concentration of hepatic cycloAMP was observed. Adrenalectomy did not impair the cold induced rise in plasma glucagon and hepatic cycloAMP.It is concluded that acute cold exposure caused a rapid rise in the concentration of plasma glucagon leading to an increase in the concentration of hepatic cycloAMP, thus enhancing the rate of hepatic gluconeogenesis and ketogenesis. As these alterations were similar in the absence of glucocorticoids and medulla-derived catecholamines, it is suggested that glucagon may play a role in the metabolic adaptation to acute cold exposure.     

Time-resolved fluorometric assay for the detection of endostatin in chromatographically separated extracts of natural peptides

We present a heterogeneous non-competitive immunological detection assay for peptide and protein antigens from crude extracts of biological sources. This time-resolved fluoroimmunoassay (TR-FIA) has been designed in a solid-phase mode using 96-well microtiter plates. Using the rare-earth metal europium as a fluorescent marker, a highly sensitive, selective and efficient procedure was developed. This technique prevents from interferences of intrinsic protein fluorescence which is highly important for antigen measurement in complex matrices. The TR-FIA has been applied for the detection of circulating forms of the potential anti-tumor agent endostatin, a C-terminal fragment of collagen XVIII, and its close homolog collagen XV (restin) from hemofiltrate. Endostatin was detected with a limit of detection of 3 ng (150 fmol/well) and a broad dynamic range from 10-1000 ng/well.