Inflammation and lung injury in an ovine model of extracorporeal membrane oxygenation support.

Extracorporeal membrane oxygenation (ECMO) is a life-saving treatment for patients with severe refractory cardiorespiratory failure. Exposure to the ECMO circuit is thought to trigger/exacerbate inflammation. Determining whether inflammation is the result of the patients' underlying pathologies or the ECMO circuit is difficult. To discern how different insults contribute to the inflammatory response, we developed an ovine model of lung injury and ECMO to investigate the impact of smoke-induced lung injury and ECMO in isolation and cumulatively on pulmonary and circulating inflammatory cells, cytokines, and tissue remodeling. Sheep receiving either smoke-induced acute lung injury (S-ALI) or sham injury were placed on veno-venous (VV) ECMO lasting either 2 or 24 h, with controls receiving conventional ventilation only. Lung tissue, bronchoalveolar fluid, and plasma were analyzed by RT-PCR, immunohistochemical staining, and zymography to assess inflammatory cells, cytokines, and matrix metalloproteinases. Pulmonary compliance decreased in sheep with S-ALI placed on ECMO with increased numbers of infiltrating neutrophils, monocytes, and alveolar macrophages compared with controls. Infiltration of neutrophils was also observed with S-ALI alone. RT-PCR studies showed higher expression of matrix metalloproteinases 2 and 9 in S-ALI plus ECMO, whereas IL-6 was elevated at 2 h. Zymography revealed higher levels of matrix metalloproteinase 2. Circulating plasma levels of IL-6 were elevated 1-2 h after commencement of ECMO alone. These data show that the inflammatory response is enhanced when a host with preexisting pulmonary injury is placed on ECMO, with increased infiltration of neutrophils and macrophages, the release of inflammatory cytokines, and upregulation of matrix metalloproteinases.

Mannose-binding lectin deficiency influences innate and antigen-presenting functions of blood myeloid dendritic cells.

Mannose-binding lectin (MBL) is a serum lectin that plays a significant role in innate host defence. Individuals with mutations in exon 1 of the MBL2 gene have reduced MBL ligand binding and complement activation function and increased incidence of infection. We proposed that, during infection, MBL deficiency may impact on dendritic cell (DC) function. We analysed the blood myeloid DC (MDC) surface phenotype, inflammatory cytokine production and antigen-presenting capacity in MBL-deficient (MBL-D) individuals and MBL-sufficient (MBL-S) individuals using whole blood culture supplemented with zymosan (Zy) or MBL-opsonized zymosan (MBL-Zy) as a model of infection. Zy-stimulated MDCs from MBL-D individuals had significantly increased production of interleukin (IL)-6 and tumour necrosis factor (TNF)-α. Stimulation with MBL-Zy significantly decreased IL-6 production by MDCs from MBL-D, but had no effect on TNF-α production. MDCs from both MBL-S and MBL-D individuals up-regulated expression of the activation molecule CD83, and down-regulated expression of homing (CXCR4), adhesion (CD62L, CD49d) and costimulatory (CD40, CD86) molecules in response to Zy and MBL-Zy. MDC from both MBL-D and MBL-S individuals induced proliferation of allogeneic (allo) T cells following Zy or MBL-Zy stimulation; however, MBL-D individuals demonstrated a reduced capacity to induce effector allo-T cells. These data indicate that MBL deficiency is associated with unique functional characteristics of pathogen-stimulated blood MDCs manifested by increased production of IL-6, combined with a poor capacity to induce effector allo-T-cell responses. In MBL-D individuals, these functional features of blood MDCs may influence their ability to mount an immune response.

Mannose-binding lectin status is associated with risk of major infection following myeloablative sibling allogeneic hematopoietic stem cell transplantation.

Mannose-binding lectin (MBL) is a mediator of innate immunity that influences the risk of infection in a range of clinical settings. We previously reported associations between MBL2 genotype and infection in a retrospective study of myeloablative allogeneic hematopoietic stem cell transplantation (allo-HCT). However, other studies have been inconclusive, and the role of MBL in reduced-intensity conditioning (RIC) transplantation is unknown. Here we report a prospective study examining MBL2 genotype, MBL levels, and risk of major infection following HLA-matched sibling myeloablative (n = 83) and RIC (n = 59) HCT. Baseline MBL levels were higher in recipients than donors (P < .001), and recipient MBL levels increased during the peritransplantation period (P = .001), most notably in MBL2 wild-type individuals receiving myeloablative total body irradiation (mTBI). MBL2 coding mutations were associated with major infection in recipients receiving mTBI. The cumulative incidence of major infection in recipient harboring an MBL2 mutation receiving mTBI was 70.6%, compared with 31.1% of those without mutations not receiving mTBI (P = .01). MBL status was not associated with infection in RIC transplants. These results confirm the association of MBL status with risk of infection in myeloablative, TBI-conditioned transplantation. Studies examining the role of MBL replacement therapy to prevent infection in this setting should be considered.

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells.

Dose-intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum-free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800-fold expansion in cell number was achieved for UCB, and up to 4,000-fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10-fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave-type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients.

Low mannose-binding lectin function is associated with sepsis in adult patients.

Mannose-binding lectin (MBL) is an innate immune system pattern recognition molecule that kills a wide range of pathogens via the lectin complement pathway. MBL deficiency is associated with severe infection but the best measure of this deficiency is undecided. We investigated the influence of MBL functional deficiency on the development of sepsis in 195 adult patients, 166 of whom had bloodstream infection and 35 had pneumonia. Results were compared with 236 blood donor controls. MBL function (C4b deposition) and levels were measured by enzyme-linked immunosorbent assay. Using receiver-operator characteristics of MBL function in healthy controls, we identified a level of <0.2 U microL(-1) as a highly discriminative marker of low MBL2 genotypes. Median MBL function was lower in sepsis patients (0.18 U microL(-1)) than in controls (0.48 U microL(-1), P<0.001). MBL functional deficiency was more common in sepsis patients than controls (P<0.001). MBL functional deficient patients had significantly higher sequential organ failure assessment (SOFA) scores and higher MBL function and levels were found in patients with SOFA scores predictive of good outcome. Deficiency of MBL function appears to be associated with bloodstream infection and the development of septic shock. High MBL levels may be protective against severe sepsis.

Impact of mannose-binding lectin on susceptibility to infectious diseases.

When the adaptive immune response is either immature or compromised, the innate immune system constitutes the principle defense against infection. Mannose-binding lectin (MBL) is a C-type serum lectin that plays a central role in the innate immune response. MBL binds microbial surface carbohydrates and mediates opsonophagocytosis directly and by activation of the lectin complement pathway. A wide variety of clinical isolates of bacteria, fungi, viruses, and parasites are bound by MBL. Three polymorphisms in the structural gene MBL2) and 2 promoter gene polymorphisms are commonly found that result in production of low serum levels of MBL. Clinical studies have shown that MBL insufficiency is associated with bacterial infection in patients with neutropenia and meningococcal sepsis. Low MBL levels appear to predispose persons to HIV infection. Numerous other potential infectious disease associations have been described. Therapy to supplement low MBL levels is being explored using either plasma-derived or recombinant material.

Cardiopulmonary bypass induces enduring alterations to host neutrophil physiology: a single-center longitudinal observational study.

Studies during and immediately post-cardiopulmonary bypass (CPB) surgery have revealed that neutrophils (PMNs) are pivotal to post-CPB inflammation and innate immunity. The aim of this study was to investigate the effects of CPB on the PMN phenotype and respiratory burst function over a longer post-CPB period (up to day 5). Blood samples were collected pre-CPB and on days 1, 3, and 5 post-CPB from 20 patients. Changes to PMN surface expression of CD16, CD62L, CD11b, CD18, and CD43, and PMN respiratory burst activity were measured, together with the white blood cell count and absolute PMN count. Cardiopulmonary bypass induced neutrophilia on days 1 and 3. One day post-CPB, CD16 expression reached a nadir (P = 0.001), and platelet-activating factor-induced CD18 increase was depressed (P < 0.05). Three days post-CPB, CD43 expression peaked (P < 0.05), with a concomitant resistance to N-formyl-Met-Leu-Phe-induced CD11b upregulation (P < 0.05). The PMN respiratory burst activity declined continuously post-CPB until day 5. Neutrophilia on days 1 and 3 was associated with changes to surface molecules expression that may reduce PMN activation response. This study demonstrated that CPB depresses the respiratory burst activity of host PMNs for an extraordinarily longer period of at least 5 days even after neutrophilia had resolved. Collectively, the changes portray an autoprotective yet responsive homeostatic balance.

Mannose-binding lectin deficiency does not appear to predispose to cryptococcosis in non-immunocompromised patients.

While most patients with cryptococcosis have obvious cellular immune deficiency, a minority have no apparent predisposing factors. However, in the latter there may be subtle innate immune system deficiencies which go unrecognized. Mannose-binding lectin (MBL) deficiency is associated with increased susceptibility to infectious diseases and may predispose to cryptococcosis, particularly when it disseminates to the central nervous system (CNS) in apparently immunocompetent patients. MBL function and levels, as well as MBL2 genotype were determined in 36 HIV-negative cryptococcosis patients (25 with CNS involvement) using C4 deposition and mannan-binding ELISA. MBL deficiency was defined using C4 deposition level < 0.2 U/microl or mannan-binding level < 0.5 microg/ml. MBL results were compared between patients with cryptococcosis and healthy controls and among the cryptococcosis patients according to the site of their disease. There was no difference in MBL function, mannan-binding level or increase in the frequency of MBL deficiency or low producing MBL2 genotypes in any of these comparisons. Patients with CNS cryptococcosis were no more likely to be MBL deficient than those with non-CNS disease. It appears that MBL deficiency is not associated with cryptococcosis in non-immunocompromised hosts. Beta errors consequent on the small number of patients studied may account for the lack of association.

The systemic inflammatory response syndrome induces functional changes and relative hyporesponsiveness in neutrophils.

PURPOSE: To study the effects of systemic inflammatory response syndrome (SIRS) on polymorhonuclear neutrophil (PMN) function and phenotype by comparing neutrophils from critically ill patients with SIRS against those from healthy blood donors. MATERIAL AND METHODS: Intensive care unit patients (n = 110) who met at least one SIRS criterion were recruited to the study. One hundred healthy blood donors were recruited as normal controls. RESULTS: Polymorphonuclear cells from critically ill patients with SIRS were more resistant to activation than PMNs from healthy donors, but when stimulated had an exaggerated microbicidal response. Buffer-treated PMNs from patients with SIRS had significantly higher CD43 surface expression that may inhibit heterotypic cellular contact or ligand stimulation of membrane receptors, had significantly lower expression of IgG receptor CD16, demonstrated resistance to shedding of L-selectin when primed by platelet-activating factor which could be pro-inflammatory, and had reduced respiratory burst when primed by platelet-activating factor than activated by formyl-Met-Leu-Phe. CONCLUSION: The phenotypic and functional changes observed in neutrophils in the critically ill indicate that they require a higher level of stimulus to become activated. This may represent an auto-protective mechanism where the neutrophils in the already inflamed host may, by this mechanism, avoid excessive inflammation reducing the risk of further host cell injury and death.