Willias Masocha

Cerebral vessel laminins and IFN-γ define Trypanosoma brucei brucei penetration of the blood-brain barrier

Subspecies of Trypanosoma brucei cause severe brain diseases after penetration of the blood-brain barrier. We investigated whether cytokines that modulate inflammatory cell infiltration into the brain also influence T. brucei neuroinvasion. Migration of a rodent pathogenic T. brucei strain from the cerebral blood vessels into the brain parenchyma was impeded in IFN-gamma(-/-), IFN-gamma receptor(-/-) (IFN-gammaR(-/-)), IL-12p40(-/-), and recombinant activating gene-1(-/-) (RAG-1(-/-)) mice as compared with their WT littermates despite higher levels of parasitemia in the mutant strains. Parasites accumulated in the perivascular compartment, confined between the endothelial and the parenchymal basement membranes, in certain areas of the brains of IFN-gamma(-/-), IFN-gammaR(-/-), and RAG-1(-/-) mice. This accumulation occurred around endothelial basement membranes containing the laminin alpha4 chain, while blood vessels showing robust laminin alpha5 chain immunostaining were not associated with parasite infiltration. The number of CD4+ and CD8+ T cells infiltrating the brain parenchyma was also reduced in the IFN-gamma(-/-) and IFN-gammaR(-/-) mice. Our findings suggest that lymphocyte-derived IFN-gamma facilitates trypanosome penetration across cerebral blood vessels and that the site of penetration is determined by the composition of the basement membranes of these vessels.

Migration of African trypanosomes across the blood-brain barrier

Subspecies of the extracellular parasite, Trypanosoma brucei, which are spread by the tsetse fly in sub-Saharan Africa, cause in humans Sleeping Sickness. In experimental rodent models the parasite can at a certain stage of disease pass through the blood-brain barrier across or between the endothelial cells and the vessel basement membranes. The laminin composition of the basement membranes determines whether they are permissive to parasite penetration. One cytokine, interferon-gamma, plays an important role in regulating the trypanosome trafficking into the brain. Treatment strategies aim at developing drugs that can impede penetration of trypanosomes into the brain and/or that can eliminate trypanosomes once they are inside the brain parenchyma, but have lower toxicity than the ones presently in use.

Expression and Role of CXCL10 during the Encephalitic Stage of Experimental and Clinical African Trypanosomiasis

BACKGROUND Human African trypanosomiasis, caused by Trypanosoma brucei, involves an early hemolymphatic stage followed by a late encephalitic stage. METHODS We studied the expression of chemokines with use of microarray and enzyme-linked immunosorbent assay in T. brucei brucei-infected mice and in patients with human African trypanosomiasis and examined their role in controlling brain accumulation of T cells and parasites. RESULTS The messenger RNAs (mRNAs) encoding CXCR3 ligands CXCL9 and CXCL10 demonstrated the greatest increases among chemokines in brain specimens of infected mice, as determined by microarray. CXCL9 and CXCL10 mRNA accumulation was interferon (IFN)-gamma-dependent. Expression of CXCL10 was predominantly observed in astrocytes. Weight loss was registered in wild-type but not in CXCL10(-/-) and CXCR3(-/-) infected mice. Infected CXCL10(-/-) or CXCR3(-/-) mice demonstrated reduced accumulation of trypanosomes and T cells in the brain parenchyma but similar parasitemia levels, compared with wild-type mice. CXCL10 and IFN-gamma levels were increased in the cerebrospinal fluid of patients with late stage but not early stage human African trypanosomiasis. Levels of CXCL10 in patients with late stage human African trypanosomiasis were associated with somnolence, low body weight, and trypanosomes in the cerebrospinal fluid. CONCLUSION IFN-gamma-dependent CXCL10 is critical for accumulation of T cells and trypanosomes in the brain during experimental African trypanosomiasis. Data suggest CXCL10 as a candidate marker for late stage human African trypanosomiasis.

Gait analysis of C57BL/6 mice with complete Freund's adjuvant-induced arthritis using the CatWalk system

BackgroundThe CatWalk system, a video based automated gait analysis system developed to evaluate footfall and gait changes in rodents, has been used for studying rodent models of arthritis, mainly the rat model. However, it has not been used to study static and dynamic gait parameters in mice with Complete Freund’s adjuvant (CFA). CFA is used extensively to induce arthritis in rodents including mice.MethodsThe CatWalk system was used to study gait of freely moving mice with CFA-induced monoarthritis and evaluate pharmacological pain relief in this model of arthritis. CFA (20 μl) was injected intra-articularly into the right hind (RH) limb ankle joint through the Achilles tendon of C57BL/6 mice.ResultsMice had less regularity in their walking patterns after CFA inoculation compared to baseline walking patterns, which was significant at 2 days post inoculation (dpi). The mice also showed changes in static parameters (paw pressure (light intensity) and print area) as well as dynamic parameters (stance phase duration, swing phase duration and speed, and duty cycle). The ratio of the RH limb (ipslateral) to the left hind (LH) limb (contralateral) for paw pressure, print area, stance phase duration, duty cycle (stance phase duration/sum of stance and swing phase duration), and swing speed were significantly reduced compared to baseline ratios at 1–6 and/or 7 dpi. On the other hand, RH/LH limb ratio of the swing phase duration increased at 3 dpi compared to baseline values. Treatment with indomethacin (10 mg/kg) improved or restored the gait parameters of CFA inoculated mice i.e. similar to baseline values or LH limb.ConclusionsThese data show that the CatWalk system can be used to assess static and dynamic gait changes and pharmacological pain relief in freely moving mice with CFA-induced monoarthritis.

Minocycline Impedes African Trypanosome Invasion of the Brain in a Murine Model

ABSTRACT Passage of Trypanosoma brucei across the blood-brain barrier (BBB) is a hallmark of late-stage human African trypanosomiasis. In the present study we found that daily administration of minocycline, a tetracycline antibiotic, impedes the penetration of leukocytes and trypanosomes into the brain parenchyma of T. brucei brucei-infected C57BL/6 mice. The trypanosome-induced astrocytic and microglial reactions were reduced in the minocycline-treated mice, as were the levels in the brain of transcripts encoding adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and endothelial-leukocyte adhesion molecule 1 (E-selectin); the inflammatory cytokines tumor necrosis factor alpha, interleukin-1α (IL-1α), IL-1β, IL-6, and gamma interferon; and matrix metalloprotease 3 (MMP-3), MMP-8, and MMP-12. Loss of weight occurring during infection with T. b. brucei was not observed after treatment of the mice with minocycline; these mice also survived longer than nontreated mice. Invasion of trypanosomes and leukocytes into the brain parenchyma most likely triggered the loss of weight and death of infected animals, since minocycline did not affect the growth of T. b. brucei either in vitro or in vivo or the levels of the transcripts encoding the cytokines and MMPs in the spleen. In conclusion, our data show that T. b. brucei invasion of the brain is related to that of leukocytes and that minocycline can ameliorate the disease in trypanosome-infected mice.

Systemic lipopolysaccharide (LPS)-induced microglial activation results in different temporal reduction of CD200 and CD200 receptor gene expression in the brain

LPS activates microglia, which are normally maintained in a quiescent state by CD200-CD200 receptor (CD200R) interaction. MAC-1 (a microglia marker) mRNA expression was increased in mice brains up to 1 year post LPS administration (i.p.). Minocycline treatment did not prevent LPS (5 mg/kg)-induced increase in MAC-1 mRNA but reduced that induced by 0.1 mg/kg LPS. CD200R mRNA decreased starting at 4 h, whereas CD200 mRNA increased at 4 h and decreased at 1 year post LPS inoculation. Thus, LPS-induced changes in CD200-CD200R equilibrium might keep microglia chronically activated. Minocycline does not effectively inhibit microglia activation induced by high-dose LPS.

Passage of parasites across the blood-brain barrier

The blood-brain barrier (BBB) is a structural and functional barrier that protects the central nervous system (CNS) from invasion by blood-borne pathogens including parasites. However, some intracellular and extracellular parasites can traverse the BBB during the course of infection and cause neurological disturbances and/or damage which are at times fatal. The means by which parasites cross the BBB and how the immune system controls the parasites within the brain are still unclear. In this review we present the current understanding of the processes utilized by two human neuropathogenic parasites, Trypanosoma brucei spp and Toxoplasma gondii, to go across the BBB and consequences of CNS invasion. We also describe briefly other parasites that can invade the brain and how they interact with or circumvent the BBB. The roles played by parasite-derived and host-derived molecules during parasitic and white blood cell invasion of the brain are discussed.

Identification of Stage Biomarkers for Human African Trypanosomiasis

Human African trypanosomiasis (HAT), caused by infection with sub-species of Trypanosoma brucei (T. b.), manifests as a hemolymphatic stage followed by an encephalitic stage. The distinction of the two stages needs improvement as drugs used for the late stage are highly toxic. Transcripts encoding 16 secreted proteins differentially expressed in the brains of mice at late stage T. b. brucei infection when the early stage drug suramin is no longer effective and different to immunoglobulins, chemokines, and cytokines, were selected by microarray analysis. Lipocalin 2 and secretory leukocyte peptidase inhibitor (SLPI) mRNA showed the highest differential expression in mice. These transcripts were also upregulated in brains from infected rats. Lipocalin 2 was increased in cerebrospinal fluid (CSF) from rats during late stage T. b. brucei infection. Protein levels of lipocalin 2, SLPI, and the chemokine CXCL10 were found increased in CSF from Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense late stage HAT compared to early stage.

Distinct Toll-like receptor signals regulate cerebral parasite load and interferon α/β and tumor necrosis factor α-dependent T-cell infiltration in the brains of Trypanosoma brucei-infected mice.

BACKGROUND The penetration of T cells and trypanosomes into the brain parenchyma is a major pathogenetic event in African trypanosomiasis. METHODS The role of innate immune responses in the penetration of T cells and Trypanosoma brucei brucei into the brain was studied in knockout mice by using double immunofluorescent staining and real-time polymerase chain reaction. RESULTS We demonstrate that Toll-like receptor (TLR)-MyD88-mediated signaling is required for T-cell and parasite penetration into the brain and microglial activation, besides controlling parasitemia and antigen-specific T-cell activation. Among different TLR-deficient mice studied, TLR9 mediated parasitemia control and T-cell penetration into the brain. TLR-MyD88 signals increased levels of interferon (IFN) β and tumor necrosis factor (TNF) α transcripts in the brains of infected mice and both TNF-α and IFN-α/β, receptors promoted T-cell and trypanosoma infiltration into the brain parenchyma. Both resident and infiltrating inflammatory cells in the brain controlled parasite densities in a TLR2- and TLR9-MyD88-mediated manner. However, neither IFN-α/β nor TNF-α contributed to parasite control in the brain. CONCLUSIONS Our data indicate that innate immune TLR signals stimulate the expression of TNF-α and IFN-α/β that initiate brain invasion of T cells and trypanosomes, and control T. brucei brucei load in the brain by molecules distinct from these.

Assessment of weight bearing changes and pharmacological antinociception in mice with LPS-induced monoarthritis using the Catwalk gait analysis system

AIMS We evaluated the possibility of using the video-based Catwalk gait analysis method to measure weight bearing changes and for testing pharmacological antinociception in freely moving mice with lipopolysaccharide (LPS)-induced monoarthritis. MAIN METHODS LPS or its solvent (PBS) was injected intra-articularly into the right hind (RH) limb ankle joint through the Achilles tendon of C57BL/6 mice. The Catwalk system was used to assess behavioral changes in freely moving mice. The effects of indomethacin on changes in LPS-inoculated mice were examined. KEY FINDINGS Mice inoculated with LPS into the RH limb showed reduced paw pressure (measured as light intensity) and print area on the RH limb, whereas they exerted more pressure with the left hind (LH) and front limbs, showing a transfer of weight bearing from RH to LH and front limbs, which was significant at 2 days post-LPS inoculation. There were no differences between the front limbs. No changes were observed in the PBS injected controls. There were no changes in interlimb coordination (regularity index) in both PBS- and LPS-injected mice. Treatment with indomethacin (10 and 100mg/kg) restored the weight bearing (measured as the ratio of the pressure exerted by the paws) and the print area ratios of LPS-inoculated mice similar to that observed in control mice. SIGNIFICANCE This study shows that the Catwalk gait analysis system can be used to objectively quantify LPS-induced monoarthritis weight bearing changes in all four limbs and evaluate pharmacological antinociception in freely moving mice.