Liv Ingunn Bjoner Sikkeland

Resuscitation with 100% O2 Increases Cerebral Injury in Hypoxemic Piglets

Perinatal asphyxia is a major cause of immediate and postponed brain injury in the newborn. We hypothesized that resuscitation with 100% O2 compared with ambient air is detrimental to the cerebral tissue. We assessed cerebral injury in newborn piglets that underwent global hypoxia and subsequent resuscitation with 21 or 100% O2 by extracellular glycerol, matrix metalloproteinase (MMP) expression levels, and oxidative stress. Extracellular glycerol was sampled by cerebral microdialysis. MMP levels were analyzed in cerebral tissue by gelatin zymography, broad matrix degrading capacity, and real-time PCR. Total endogenous antioxidant capacity was measured by the oxygen radical absorbance capacity assay. Extracellular glycerol increased 50% after resuscitation with 100% O2 compared with 21% O2. Total MMP activity was doubled in resuscitated animals at endpoint compared with baseline (p = 0.018), and the MMP-2 activity was significantly increased in piglets that were resuscitated with 21% O2 (p = 0.003) and 100% O2 (p = 0.001) compared with baseline. MMP-2 mRNA level was 100% increased in piglets that were resuscitated with 100% O2 as compared with 21% O2 (p < 0.05). Oxygen radical absorbance capacity values in piglets that were resuscitated with 100% O2 were considerably reduced compared with both baseline (p = 0.001) and piglets that were resuscitated with 21% O2 (p = 0.001). In conclusion, our data show increased MMP-2 activity at both gene and protein levels, accompanied with cerebral leakage of glycerol, presumably triggered by augmented oxidative stress. Our findings suggest that resuscitation of asphyxiated piglets with 100% O2 is detrimental to the piglet brain compared with resuscitation with 21% O2.

Resuscitation of hypoxic piglets with 100% O2 increases pulmonary metalloproteinases and IL-8.

We hypothesized that resuscitation with 100% O2 compared with 21% O2 is detrimental to pulmonary tissue. The pulmonary injury was assessed by matrix metalloproteinase (MMP) activity, oxidative stress, IL-8, and histology 2.5 h after resuscitation from a hypoxic state. In pulmonary tissue extracts, MMP activity was analyzed by broad matrix–degrading capacity (total MMP) and zymography. MMP-2 mRNA expression was evaluated by quantitative real-time PCR. Total endogenous antioxidant capacity was measured by the oxygen radical absorbance capacity (ORAC) assay, and IL-8 was analyzed by ELISA technique. In bronchoalveolar lavage (BAL) fluid, MMPs were analyzed by zymography. In pulmonary tissue, pro- and active MMP-2 levels were increased in piglets that were resuscitated with 100% O2 compared with 21% O2. Pro–MMP-9, total MMP activity, and MMP-2 mRNA levels were significantly increased in resuscitated piglets compared with baseline. Net gelatinolytic activity increased in submucosa and blood vessels after 100% O2 and only in the blood vessels after 21% O2. Compared with baseline, ORAC values were considerably lowered in the resuscitated piglets and significantly reduced in the 100% O2 versus 21% O2 group. In BAL fluid, both pro–MMP-9 and pro–MMP-2 increased 2-fold in the 100% O2 group compared with 21% O2. Moreover, IL-8 concentration increased significantly in piglets that were resuscitated with 100% O2 compared with 21% O2, suggesting a marked proinflammatory response in the pulmonary tissue. Altogether, these data strongly suggest that caution must be taken when applying pure O2 to the newborn infant.

DNA transfection of mononuclear cells in muscle tissue

Genes encoding non‐self proteins may be injected into skeletal muscles in vivo to obtain induction of cellular and humoral immune responses against the encoded antigens (DNA vaccination). Bone marrow derived professional antigen‐presenting cells (APCs) play a key role in the induction of immunity by DNA vaccination. In the present work we have investigated whether the APCs are transfected by DNA injection into muscle.

Effects of interferon-α on gene expression of chemokines and members of the tumour necrosis factor superfamily in HIV-infected patients

We examined the effect of interferon (IFN)‐α on the expression of 375 genes relevant to inflammatory and immunological reactions in peripheral blood mononuclear cells (PBMC) from HIV‐infected patients by cDNA expression array and real‐time quantitative RT‐PCR. Our main findings were: (i) IFN‐α induced up‐regulation of several genes in the tumour necrosis factor (TNF) superfamily including the ligands APRIL, FasL, TNF‐α and TRAIL, with particularly enhancing effects on the latter in HIV‐infected patients. (ii) While IFN‐α markedly up‐regulated the expression of anti‐angionetic ELR– CXC‐chemokines (e.g. MIG and IP‐10), it suppressed the expression of angiogenic ELR+ CXC‐chemokines (e.g. GRO‐α, IL‐8 and ENA‐78), with similar patterns in both patients and controls. (iii) IFN‐α induced a marked increase in gene expression of the HIV co‐receptor CCR5 in both patients and controls. We suggest that these effects may contribute to both the therapeutic and toxic effects of IFN‐α. Moreover, our findings underscore that the biological effects of IFN‐α in HIV infection are complex and that the clinical net effects of IFN‐α treatment may be difficult to predict. However, the potent enhancing effect of IFN‐α on several pro‐apoptotic genes in the TNF superfamily and the enhancing effect on CCR5 expression suggest a possible pathogenic role of IFN‐α in the progression of HIV‐related immunodeficiency and suggests caution in the therapeutic use of IFN‐α in HIV‐infected individuals.

Airway inflammation in cement production workers

Objective Cement aerosol exposure is associated with increased morbidity of airway disease among exposed workers. Our aim was to compare levels of inflammatory cells and soluble inflammatory markers in induced sputum samples from cement production workers between exposed and unexposed periods, and to compare these variables between cement workers and references. Methods 35 healthy, non-smoking aerosol-exposed cement production workers from Norway provided a blood sample and performed induced sputum and spirometry after 5 days without exposure and during a period of exposure. These values were compared with those from an internal low-exposed reference group of 15 office workers and an external reference group of 39 non-exposed workers. Differential cell counts and inflammatory markers were assessed. Results Median thoracic aerosol concentration over one work shift (8 h) was 0.6 mg/m3 (range 0.2–8.1) in maintenance workers and 1.75 mg/m3 (0.2–15.5) in furnace department workers. The median percentage of airway neutrophils in both groups combined was 51% (32–66) in the exposed period, which was significantly higher than in both the unexposed period (38%; 23–55) (p=0.04) and the external reference group (30%; 19–44) (p=0.001). Median interleukin-1β concentration was elevated compared with both office workers (p=0.05) and the external reference group (p=0.006). Conclusions A significantly higher percentage of neutrophils was observed in cement production workers during the exposed period compared with both the non-exposed period and the external reference group, and corresponded with elevated IL-1β concentration. These data indicate that cement aerosol exposure in concentrations below the Norwegian occupational limits (respirable dust 5 mg/m3; total dust 10 mg/m3) may cause airway inflammation.

Long-term persistence of human donor alveolar macrophages in lung transplant recipients

Background Alveolar macrophages (AMFs) are critical regulators of lung function, and may participate in graft rejection following lung transplantation. Recent studies in experimental animals suggest that most AMFs are self-maintaining cells of embryonic origin, but knowledge about the ontogeny and life span of human AMFs is scarce. Methods To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation for X/Y chromosomes and immunofluorescence staining for macrophage markers. Moreover, development of AMFs in humanised mice reconstituted with CD34+ umbilical cord-derived cells was assessed. Results The number of donor-derived AMFs was unchanged during the 2 year post-transplantation period. A fraction of the AMFs proliferated locally, demonstrating that at least a subset of human AMFs have the capacity to self-renew. Lungs of humanised mice were found to abundantly contain populations of human AMFs expressing markers compatible with a monocyte origin. Moreover, in patients with lung transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period. Conclusions The finding that human AMFs are maintained in the lung parenchyma for several years indicates that pulmonary macrophage transplantation can be a feasible therapeutic option for patients with diseases caused by dysfunctional AMFs. Moreover, in a lung transplantation setting, long-term persistence of donor AMFs may be important for the development of chronic graft rejection.

Myocardial gene expression of inflammatory cytokines after heart transplantation in relation to the development of transplant coronary artery disease

Long-term success of cardiac transplantation is mainly limited by the development of transplant coronary artery disease (CAD); it is generally accepted that it is immune mediated, involving cytokines and growth factors. We show that development of transplant CAD is associated with a particular cytokine profile in myocardial biopsies characterized by a late (i.e., 1 year) increase in tumor necrosis factor-alpha and interferon-gamma gene expression, which precede and potentially contribute to the development of allograft vasculopathy, further supporting a role for inflammation and the pathogenesis of transplant CAD in humans.

Circulating lipopolysaccharides in the blood from “bioprotein” production workers

Background: Workers producing bacterial single-cell protein (BSCP), “bioprotein,” are exposed to organic dust containing high levels of endoxins (lipopolysaccharides, LPS). Workers in this industry have complained of episodes of fever, fatigue, chest tightness, skin dryness and rubor. The aim of the present study was to quantify LPS and inflammatory mediators in plasma among the workers and non-exposed control subjects. Methods: We included eight non-smoking production workers, aged 32–51 (median 38), and eight non-smoking, non-exposed controls, aged 30–51 (median 39). Airborne and plasma endotoxin concentrations were measured, as well as plasma hsCRP and different cytokines, chemokines and metalloproteinases. Results: The workers who did not use personal respiratory protection were exposed to varying airborne levels of endotoxin, 430 (75–15 000) EU/m3 (median, range). The level of plasma LPS was significantly elevated (p = 0.01) among the workers compared to the non-exposed controls. The workers also had elevated levels of MCP-1 (p = 0.02), MIP-1α (p = 0.05) and MMP-3 (p = 0.04). IL-6 and hsCRP were also elevated among the exposed group, but not significantly (p = 0.10 and p = 0.07, respectively). Conclusions: In this study, we detected LPS in plasma of individuals exposed to high levels of LPS at their workplace. This finding is supported by elevated levels of several inflammatory cytokines among the workers, significantly exceeding that of the non-exposed control group. To the best of our knowledge, this is the first time that plasma LPS, together with increased inflammatory markers in plasma, has been detected in an occupational setting.

Macrophage enrichment from induced sputum

Since induced sputum has become a widely used non-invasive method of recovering cells from the surfaces of the bronchial airways, isolating specific cell populations will be necessary in order to learn more about their specific role in innate immunity and inflammation in the airways. Several studies have demonstrated the ability to conduct ex vivo analyses on sputum cells such as phagocytosis and surface marker measurements, but these have not been performed on isolated cell types.1–3 This study demonstrates the capability to isolate sputum macrophages from human volunteers in order to advance our understanding of macrophage biology in the airways. To this end, techniques that can enrich and isolate cells without significant activation would prove extremely useful. We compared two common methods for isolating and enriching macrophages in sputum: (1) magnetic bead separation; and (2) Percoll gel density gradient centrifugation. Cell purity and markers of cell activation (mRNA tumour necrosis factor α (TNFα)) …

Airway inflammation in paper mill workers.

Paper mill workers are exposed to culturable microorganisms (MOs). We hypothesized that inflammatory airway response could be detected in sputum of nonsymptomatic workers. From four paper mills, we included 29 healthy nonsmoking men. Workers exposed to high levels of MOs (HMOE, n = 17) were compared with workers exposed to low levels of MO (LMOE, n = 12). A reference group of 22 healthy, nonsmoking, nonexposed (NE) men were also included. We performed differential cell counts of induced sputum, studied gene expressions of isolated sputum macrophages and analyzed inflammatory parameters, including matrix metalloproteinases. Sputum from HMOE workers had a significantly higher percentage of neutrophils than that from LMOE workers (P < 0.05) and NE controls (P < 0.001). There was also an increased gene expression of different pro-inflammatory cytokines, interleukin-6, tumor necrosis factor-&agr;, and macrophage inflammatory protein-1&bgr; in isolated airway macrophages and increased levels of total matrix metalloprotease-9 activity in induced sputum from the HMOE group. Our findings indicate that paper industry workers exposed to MOs develop subclinical airway inflammation.