Nathalie Geurts

Zymography methods for visualizing hydrolytic enzymes

Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN‐γ‐induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T‐lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN‐γ ligand and receptor KO mice, indicating that IFN‐γ is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN‐γ and also adhesion molecules in the brain were not lower in CM‐resistant Balb/c and DBA/2 mice compared to CM‐sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM‐resistant than in CM‐sensitive mice. This difference correlated with a higher up‐regulation of the CXC chemokine receptor (CXCR)‐3 on splenic T cells and a higher chemotactic response to IFN‐γ‐inducible protein‐10 (IP‐10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite‐induced IFN‐γ in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain‐dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.

Immunopathology and Dexamethasone Therapy in a New Model for Malaria-associated Acute Respiratory Distress Syndrome

RATIONALE Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. OBJECTIVES Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. METHODS C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. MEASUREMENTS AND MAIN RESULTS In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. CONCLUSIONS We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Matrix metalloproteinases as therapeutic targets in protozoan parasitic infections

Matrix metalloproteinases (MMPs) are associated with processes of tissue remodeling and are expressed in all infections with protozoan parasites. We here report the status of MMP research in malaria, trypanosomiasis, leishmaniasis and toxoplasmosis. In all these infections, the balances between MMPs and endogenous MMP inhibitors are disturbed, mostly in favor of active proteolysis. When the infection is associated with leukocyte influx into specific organs, immunopathology and collateral tissue damage may occur. These pathologies include cerebral malaria, sleeping sickness (human African trypanosomiasis), Chagas disease (human American trypanosomiasis), leishmaniasis and toxoplasmic encephalitis in immunocompromised hosts. Destruction of the integrity of the blood-brain barrier (BBB) is a common denominator that may be executed by leukocytic MMPs under the control of host cytokines and chemokines as well as influenced by parasite products. Mechanisms by which parasite-derived products alter host expression of MMP and endogenous MMP inhibitors, have only been described for hemozoin (Hz) in malaria. Hence, understanding these interactions in other parasitic infections remains an important challenge. Furthermore, the involved parasites are also known to produce their own metalloproteinases, and this forms an extra stimulus to investigate MMP inhibitory drugs as therapeutics. MMP inhibitors (MMPIs) may dampen collateral tissue damage, as is anecdotically reported for tetracyclines as MMP regulators in parasite infections.

Beta-hematin interaction with the hemopexin domain of gelatinase B/MMP-9 provokes autocatalytic processing of the propeptide, thereby priming activation by MMP-3.

Gelatinase B or matrix metalloproteinase-9 is involved in inflammation and in autoimmune and vascular diseases. In contrast to the constitutive and homeostatic matrix metalloproteinase-2, matrix metalloproteinase-9 is an inducible enzyme. Furthermore, it needs tight regulation, and a major control mechanism of its enzymatic activity is the activation of the latent enzyme by proteolysis of the 87 residue propeptide. Activated matrix metalloproteinase-9 is detected in many vascular or hematological disease states, including in an experimental model for cerebral malaria with Plasmodium berghei ANKA. However, insight into its activation mechanism is incomplete. In view of the association with hemorrhagic and hemolytic diseases, it was studied whether and how hemoglobin and its derivatives might activate pro-matrix metalloproteinase-9. Incubation of matrix metalloproteinase-9 with hemin or beta-hematin, the core constituent of hemozoin or malaria pigment, leads to differential autocatalysis of the propeptide, mediated by allosteric interaction with the hemopexin domain. The cleavage catalyzed by beta-hematin coincides with the first cleavage by stromelysin-1/matrix metalloproteinase-3, and preincubation of matrix metalloproteinase-9 with beta-hematin enhances the activation rate by matrix metalloproteinase-3 at least 6-fold. These findings suggest that reduction of hemorrhage and hemolysis might prevent matrix metalloproteinase-9-mediated inflammatory and vascular damages.

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain.

AIM To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS Fluorogenic Dye-quenched (DQ)™-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a high-throughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of heterocyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbiturate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (k(cat)/k(M) ± 40% less than full-length MMP-9). CONCLUSION The DQ™-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.

Acute Disruption of Bone Marrow B Lymphopoiesis and Apoptosis of Transitional and Marginal Zone B Cells in the Spleen following a Blood-Stage Plasmodium chabaudi Infection in Mice

B cells and antibodies are essential for the protective immune response against a blood-stage Plasmodium infection. Although extensive research has focused on memory as well as plasma B-cell responses during infection, little is known about how malaria affects B-cell development and splenic maturation into marginal zone B (MZB) and follicular B (FoB) cells. In this study, we show that acute Plasmodium chabaudi AS infection in C57Bl/6 mice causes severe disruption of B lymphopoiesis in the bone marrow, affecting in particular pro-, pre-, and immature B cells as well as the expression of the bone marrow B-cell retention chemokine CXCL12. In addition, elevated apoptosis of transitional T2 and marginal zone (MZ) B cells was observed during and subsequent to the control of the first wave of parasitemia. In contrast, Folllicular (Fo) B cells levels were retained in the spleen throughout the infection, suggesting that these are essential for parasite clearance and proper infection control.

Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases . However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

Meprins process matrix metalloproteinase‐9 (MMP‐9)/gelatinase B and enhance the activation kinetics by MMP‐3

Meprin alpha cleaves MMP‐9 by enzymatic study (View interaction)

Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. To develop a new animal model for MA-ARDS, mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was characterized by increased lung weight, edema, leukocyte infiltration and hemorrhages (Figure ​(Figure1).1). The pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with P. chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8+ T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation. Figure 1 Histopathology of P. berghei NK65-induced MA-ARDS. Frozen sections of lungs of mice infected for 10 days with P. berghei NK65 or control mice were stained with H&E.The black bar corresponds with 100 μm. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.