Cytokine release in healthy donors and patients with chronic granulomatous disease upon stimulation with Aspergillus fumigatus

The release of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-10 upon stimulation with non-viable conidia and hyphal fragments from Aspergillus fumigatus was investigated in an ex vivo whole-blood model. In healthy volunteers, high numbers of conidia (between 10(6) and 3 x 10(8)/ml) induced a moderate release of TNF-alpha and IL-6. Hyphal fragments (2.5 x 10(5)/ml) were more potent in stimulating the release of these pro-inflammatory cytokines. Although some IL-10 release was observed upon stimulation with either conidia or hyphal fragments, it was not significantly different from that in unstimulated controls. In comparison, in whole blood obtained from 4 patients with chronic granulomatous disease (CGD), a high release of pro-inflammatory cytokines together with a significantly higher IL-10 release than in the healthy controls was seen after stimulation with A. fumigatus. In conclusion, A. fumigatus can trigger the release of pro-inflammatory cytokines in a human whole-blood system, which is likely to be central to the activation of antifungal defence mechanisms. In contrast, A. fumigatus stimulates a higher release of anti-inflammatory cytokines in CGD patients, which may suggest that a dysregulation between pro- and anti-inflammatory cytokines contributes to the increased susceptibility to invasive aspergillosis in this patient group.     

Tumor-targeted immune complex formation: effects on myeloid cell activation and tumor-directed immune cell migration

The effectiveness of cellular immunotherapy of solid tumors is often hampered by the lack of specific infiltration of immune effector cells into the tumor mass. Therefore, we studied the potential of tumor antigen-specific antibodies to elicit tumor-specific myeloid cell activation, to induce or enhance tumor infiltration by immune cells. To this end, we developed an in vitro model system using the human myeloid cell line MonoMac-6. Incubation of IFN-gamma-primed MonoMac-6 cells with serum-opsonized zymosan or EGP-2-directed, mouse IgG2a-opsonized, EGP-2-positive tumor cells resulted in the production of ROS and TNF-alpha and induced E-selectin and ICAM-1 expression on HUVECs. FcR-mediated MonoMac-6 cell activation was strictly dependent on the activation of MonoMac-6 cells with IFN-gamma. In addition, no myeloid cell activation was observed in the presence of human serum or using tumor antigen-specific mouse antibody subclasses other than IgG2a, suggesting the crucial involvement of CD64 (FcgammaR1) in the effects observed. However, serum-inhibited myeloid cell activation was completely restored employing a 2-step targeting approach in which tumor cell opsonization with mouse anti-EGP-2 antibodies was followed by incubation with human antimouse Ig antibodies. Moreover, using this 2-step approach, not only anti-EGP-2-directed mouse IgG2a but also mouse IgG1 antibodies effectively induced tumor-specific myeloid cell activation. In conclusion, we describe a method to induce efficient and tumor-specific activation of myeloid cells based on the sequential use of mouse tumor antigen-specific and human antimouse Ig antibodies. Targeted myeloid cell activation may provide a means to aid in the induction of a tumor-directed immune response and as such, the method described here could be of clinical significance.     

Proinflammatory Cytokines in the Treatment of Bacterial and Fungal Infections

Mortality due to severe bacterial infections has not been markedly effected by the introduction of new antimicrobial drugs over the last 30-40 years. This has emphasized the need for development of new therapeutic strategies to combat sepsis. The outcome of an infection depends on two factors: the growth of the microorganisms (including the effect of antibacterial drugs), and the host's defensive response to the invading organism. It is known that injection of bacterial products into experimental animals leads to enhanced nonspecific resistance to a variety of microorganisms. The discovery of the specific mediators responsible for modulation of host defense has created new possibilities for the development of alternative treatment strategies. Molecules such as interleukins, interferons, tumor necrosis factors and hematopoietic growth factors have become available in recombinant form, and their therapeutic potential in various infectious diseases has been tested in various experimental models of infections. Initial data in various patient groups indicate that adjunctive therapy with recombinant proinflammatory cytokines may have beneficial effects in the treatment of bacterial and fungal infections.     

Potential and limitations of wave intensity analysis in coronary arteries

Wave intensity analysis (WIA) is beginning to be applied to the coronary circulation both to better understand coronary physiology and as a diagnostic tool. Separation of wave intensity (WI) into forward and backward traveling components requires knowledge of pulse wave velocity at the point of measurement, which at present cannot accurately be determined in human coronary vessels. This prompted us to study the sensitivity of wave separation to variations in wave speed. An estimate of wave speed (SPc) was calculated based on measured distal intracoronary pressure and Doppler velocity in normal and diseased coronary vessels of patients during hyperemia. Changes of the area under separated WI waveforms were determined for a range of wave speeds from 25 to 200% of the calculated value. Variations in wave speed between half to twice the calculated value did not substantially alter separated WI. In conclusion, although SPc lacks accuracy in determining local coronary wave speed it is within limits still applicable for wave separation in coronary WIA.     

Receptor Recognition of and Immune Intracellular Pathways for Veillonella parvula Lipopolysaccharide

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-α and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-α, IL-1β, IL-6, and IL-10 were released in wild-type and TLR2^−/−, but not TLR4^−/−, mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-α, IL-1β, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.Veillonella organisms are small, nonfermentative, strictly anaerobic, gram-negative cocci which form part of the normal flora of the oral, genitourinary, respiratory, and intestinal tracts of humans and animals (10). The genus Veillonella was first isolated by Veillon and Zuber in 1898 and currently consists of eight species (28).Veillonella species have been reported as causes of serious infections, including meningitis (6), osteomyelitis and discitis (7, 28), prosthetic joint infection (26), and acute and chronic pleuropulmonary infection (33).Risk factors for Veillonella infection include periodontal disease, immunodeficiency, intravenous drug use, and premature birth (28). V. parvula is an important pathogen implicated in periodontitis and other dental infections (3, 18), and it is one of the most common anaerobic pathogens in chronic maxillary sinusitis and deep neck infections (9, 37). V. parvula has also been reported as a pathogen for osteomyelitis (34) and abscessed orchiepididymitis with sepsis (4). Endovascular infections reportedly may range from bacteremia to severe endocarditis and fatal cases of sepsis (8, 14, 25).Lipopolysaccharides (LPS) are major pathogenic factors of gram-negative bacteria. LPS from aerobic and facultative bacteria have been extensively studied (5). On the contrary, very little is known regarding the biological activity of LPS from anaerobic microorganisms such as Veillonella (10, 24, 29, 32). In addition, little is known about cellular and molecular mechanisms responsible for innate immune response against V. parvula, as well as for inflammatory reactions leading to severe periodontitis or sinusitis. Toll-like receptors (TLRs) recognize microbial compounds (36) and trigger the inflammatory and immune responses against pathogens. Immunohistochemical localization of TLR2 and TLR4 in gingival tissue of periodontitis patients has been reported (30). In mammals, engagement of TLRs by LPS results in the recruitment of cytoplasmic signaling molecules (12, 36), which eventually activates mitogen-activated protein kinases (MAPKs), including p38, JNK, and extracellular signal-regulated kinase (ERK). MAPK activation leads to cytokine release (2).However, the interaction between oral V. parvula LPS and TLRs has not been directly studied yet. The aim of this study was to investigate the potential role of TLR2 and TLR4 for the recognition of Veillonella parvula LPS in both human peripheral blood mononuclear cells (PBMC) and in TLR2 and TLR4 knockout (KO) mouse macrophages, as well as the intracellular kinase signaling pathways induced after challenge of monocytes with Veillonella parvula LPS.     

Genetic Basis for Recurrent Vulvo-Vaginal Candidiasis

Vulvovaginal candidiasis (VVC) is a frequent disease affecting more than 75% of all women at least once in their lifetime. Up to 8% of them suffer from recurrent VVC (RVVC) characterized by at least three episodes each year. Several risk factors, such as antibiotic use, diabetes, or pregnancy, are known, but the vast majority of women with RVVC develop the infection without having any risk factor, implying that a genetic component most likely plays an important role in the susceptibility to RVVC. This review summarizes the immunogenetic alterations that lead to an increased susceptibility to vaginal infections with Candida albicans. Different mutations and polymorphisms in innate immune genes alter the mucosal immune response against fungi and are likely to have an important role in susceptibility to RVVC. A better understanding of the genetic and immunological mechanisms leading to RVVC is important for both the understanding of the pathophysiology of the disease and the design of novel therapeutic strategies.     

Quality improvement of surgical prophylaxis in Dutch hospitals: evaluation of a multi-site intervention by time series analysis

OBJECTIVES: Misuse of antibiotics in surgical prophylaxis is still quite common. The objectives of this study were to reduce the quantity and improve the quality of surgical prophylaxis and to reduce costs. METHODS: Prospective multi-site study of elective procedures in 13 Dutch hospitals. The quality of prophylaxis was audited before and after an intervention consisting of performance feedback and implementation of national clinical practice guidelines. Process outcome parameters were antibiotic choice, duration, timing, antibiotic volume and costs. Segmented regression analysis was used to estimate the effect size of the intervention. Patient outcome was documented by the incidence of surgical site infections (SSI). RESULTS: Before the intervention, 1763 procedures were recorded and 2050 thereafter. Antimicrobial use decreased from 121 to 79 DDD (defined daily doses)/100 procedures and costs reduced by 25% per procedure. After the intervention, antibiotic choice was inappropriate in only 37.5% of the cases instead of in 93.5% expected cases had the intervention not occurred. Prolonged prophylaxis was observed in 31.4% instead of 46.8% expected cases and inappropriate timing in 39.4% instead of the expected 51.8%. Time series analysis showed that all improvements were statistically significant (P < 0.01) and that they could be fully attributed to the intervention. The overall SSI rates before and after intervention were 5.4% (95% CI: 4.3-6.5) and 4.6% (95% CI: 3.6-5.4), respectively. CONCLUSIONS: The intervention led to improved quality of surgical prophylaxis and to reduced antibiotic use and costs without impairment of patient outcome.