Haem oxygenase protects against thrombocytopaenia and malaria-associated lung injury.

BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.

Integrin αDß2 influences cerebral edema, leukocyte accumulation and neurologic outcomes in experimental severe malaria.

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.

Integrin αDß2 (CD11d/CD18) mediates experimental malaria-associated acute respiratory distress syndrome (MA-ARDS).

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a potentially lethal complication of clinical malaria. Acute lung injury in MA-ARDS shares features with ARDS triggered by other causes, including alveolar inflammation and increased alveolar-capillary permeability, leading to leak of protein-rich pulmonary oedema fluid. Mechanisms and physiologic alterations in MA-ARDS can be examined in murine models of this syndrome. Integrin αDß2 is a member of the leukocyte, or ß2 (CD18), sub-family of integrins, and emerging observations indicate that it has important activities in leukocyte adhesion, accumulation and signalling. The goal was to perform analysis of the lungs of mice wild type C57Bl/6 (a D (+/+) ) and Knockout C57Bl/6 (a D (-/-) ) with malaria-associated acute lung injury to better determine the relevancy of the murine models and investigate the mechanism of disease. METHODS: C57BL/6 wild type (a D (+/+) ) and deficient for CD11d sub-unit (a D (-/-) ) mice were monitored after infection with 10(5) Plasmodium berghei ANKA. CD11d subunit expression RNA was measured by real-time polymerase chain reaction, vascular barrier integrity by Evans blue dye (EBD) exclusion and cytokines by ELISA. Protein and leukocytes were measured in bronchoalveolar lavage fluid (BALF) samples. Tissue cellularity was measured by the point-counting technique, F4/80 and VCAM-1 expression by immunohistochemistry. Respiratory function was analysed by non-invasive BUXCO and mechanical ventilation. RESULTS: Alveolar inflammation, vascular and interstitial accumulation of monocytes and macrophages, and disrupted alveolar-capillary barrier function with exudation of protein-rich pulmonary oedema fluid were present in P. berghei-infected wild type mice and were improved in αDß2-deficient animals. Key pro-inflammatory cytokines were also decreased in lung tissue from α D (-/-) mice, providing a mechanistic explanation for reduced alveolar-capillary inflammation and leak. CONCLUSIONS: The results indicate that αDß2 is an important inflammatory effector molecule in P. berghei-induced MA-ARDS, and that leukocyte integrins regulate critical inflammatory and pathophysiologic events in this model of complicated malaria. Genetic deletion of integrin subunit αD in mice, leading to deficiency of integrin αDß2, alters lung inflammation and acute lung injury in a mouse model of MA-ARDS caused by P. berghei.

Dysregulated coagulation associated with hypofibrinogenaemia and plasma hypercoagulability: implications for identifying coagulopathic mechanisms in humans.

Identifying coagulation abnormalities in patients with combined bleeding and thrombosis history is clinically challenging. Our goal was to probe the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis. The patient is a 56-year-old female with a history of haematomas, poor wound healing, and thrombosis (retinal artery occlusion and transient cerebral ischaemia). She had a normal activated partial thromboplastin time, prolonged thrombin and reptilase times, and decreased functional and antigenic fibrinogen levels, and was initially diagnosed with hypodysfibrinogenaemia. This diagnosis was supported by DNA analysis revealing a novel FGB mutation (c.656A>G) predicting a Q189R mutation in the mature chain that was present in the heterozygote state. However, turbidity analysis showed that purified fibrinogen polymerisation and degradation were indistinguishable from normal, and Bß chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Interestingly, plasma thrombin generation testing revealed the patient's thrombin generation was higher than normal and could be attributed to elevated levels of factor VIII (FVIII, 163-225%). Accordingly, in an arterial injury model, hypofibrinogenaemic mice (Fgn(+/-)) infused with factor VIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn(+/-) mice. Together, these data associate the complex bleeding and thrombotic presentation with combined hypofibrinogenaemia plus plasma hypercoagulability. These findings suggest previous cases in which fibrinogen abnormalities have been associated with thrombosis may also be complicated by co-existing plasma hypercoagulability and illustrate the importance of "global" coagulation testing in patients with compound presentations.