USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

Genetic analysis of cerebral malaria in the mouse model infected with Plasmodium berghei.

Malaria is a common and sometimes fatal disease caused by infection with Plasmodium parasites. Cerebral malaria (CM) is a most severe complication of infection with Plasmodium falciparum parasites which features a complex immunopathology that includes a prominent neuroinflammation. The experimental mouse model of cerebral malaria (ECM) induced by infection with Plasmodium berghei ANKA has been used abundantly to study the role of single genes, proteins and pathways in the pathogenesis of CM, including a possible contribution to neuroinflammation. In this review, we discuss the Plasmodium berghei ANKA infection model to study human CM, and we provide a summary of all host genetic effects (mapped loci, single genes) whose role in CM pathogenesis has been assessed in this model. Taken together, the reviewed studies document the many aspects of the immune system that are required for pathological inflammation in ECM, but also identify novel avenues for potential therapeutic intervention in CM and in diseases which feature neuroinflammation.

Cysteamine broadly improves the anti-plasmodial activity of artemisinins against murine blood stage and cerebral malaria.

BACKGROUND: The potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis. METHODS: The ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals. RESULTS: Cys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite. CONCLUSION: These findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.

THEMIS is required for pathogenesis of cerebral malaria and protection against pulmonary tuberculosis.

We identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Themis(I23N) homozygous mice show reduced CD4(+) and CD8(+) T lymphocyte numbers. ECM resistance in P. berghei ANKA-infected Themis(I23N) mice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMIS(I23N) protein expression is absent from mutant mice, concurrent with the decreased THEMIS(I23N) stability observed in vitro. Biochemical studies in vitro and functional complementation in vivo in Themis(I23N/+):Lck(-/+) doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses in Themis(I23N) mice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells. Themis(I23N) mice can be used to study the newly discovered association of THEMIS (6p22.33) with inflammatory bowel disease and multiple sclerosis.

Increased resistance to malaria in mice with methylenetetrahydrofolate reductase (Mthfr) deficiency suggests a mechanism for selection of the MTHFR 677C>T (c.665C>T) variant.

The polymorphism 677C>T (NM_005957.4:c.665C>T/p.Ala222Val, rs1801133:C>T) in methylenetetrahydrofolate reductase (MTHFR) results in mild enzymatic deficiency and increased risk for several complex traits including adverse reproductive outcomes, birth defects, and heart disease. Despite these deleterious effects, homozygosity is high (5%-15%) in many populations, and among the highest in Mediterranean regions, where malaria was historically endemic and may have conferred a selective advantage for other mutations. We infected Mthfr-deficient (Mthfr(+) (/-) ) and MTHFR overexpressing (MTHFR(Tg) ) mice with Plasmodium berghei ANKA to induce cerebral malaria. Mthfr(+/-) mice survived longer (P < 0.02, log-rank test), and MTHFR(Tg) mice died earlier (P < 0.05, log-rank test) after infection compared with wild-type littermates. Flow cytometry revealed increased lymphocyte populations and increased CCR4(+) NK cells in spleen of Mthfr(+) (/-) mice; MTHFR(Tg) animals had decreased numbers of these NK cells. Interferon-γ and interleukin-10 immunoreactive proteins were increased and decreased, respectively, in brain of Mthfr(+/-) mice compared with wild-type. We suggest that mild MTHFR deficiency protects against malarial infection and that this phenomenon may have led to the high frequency of the 677C>T/c.665C>T variant in human populations.

Outreach Training and Supportive Supervision for Quality Malaria Service Delivery: A Qualitative Evaluation in 11 Sub-Saharan African Countries.

Quality improvement of malaria services aims to ensure that more patients receive accurate diagnosis, appropriate treatment, and referral. The Outreach Training and Supportive Supervision Plus (OTSS+) approach seeks to improve health facility readiness and provider competency through onsite supportive supervision, troubleshooting, and on-the-job training. As part of a multicomponent evaluation, qualitative research was conducted to understand the value of the OTSS+ approach for malaria quality improvement. Semistructured key informant interviews, focus group discussions, and structured health facility-based interviews were used to gather stakeholder perspectives at subnational, national, and global levels. Data were collected globally and in 11 countries implementing OTSS+; in-depth data collection was done in four: Cameroon, Ghana, Niger, and Zambia. Study sites and participants were selected purposively. Verbatim transcripts were analyzed thematically, following the Framework approach. A total of 262 participants were included in the analysis; 98 (37.4%) were supervisees, 99 (37.8%) were supervisors, and 65 (24.8%) were other stakeholders. The OTSS+ approach was perceived to improve provider knowledge and skills in malaria service delivery and to improve data and supply management indirectly. Improvements were attributed to a combination of factors. Participants valued the relevance, adaptation, and digitization of supervision checklists; the quality and amount of contact with problem-solving supervisors; and the joint identification of problems and solutions, and development of action plans. Opportunities for improvement were digitized checklist refinement, assurance of a sufficient pool of supervisors, prioritization of health facilities, action plan dissemination and follow-up, and data review and use. The OTSS+ approach was perceived to be a useful quality improvement approach for malaria services.

Proteasomal degradation of the papillomavirus E2 protein is inhibited by overexpression of bromodomain-containing protein 4.

The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.

Embedding Carbon Dots in Superabsorbent Polymers for Additive Manufacturing.

A type of orange carbon dots (O-CDs) synthesized via an ultrasonication route with citric acid and 1,2-phenylenediamine as precursors was embedded into sodium polyacrylate (SPA) as the ink for 3D printing. Characterizations of these spherical O-CDs revealed an ultra-small size (~2 nm) and excitation-independent, but solvent dependent, emission. The O-CDs were evenly distributed with low degree of aggregation in sodium polyacrylate (SPA), which was achieved due to the property that SPA can absorb water together with O-CDs. The 3D printed photoluminescent objective with the ink revealed a great potential for high yield application of these materials for additive manufacturing. This also represents the first time, bare CDs have been reported as a photoluminescent material in 3D printing, as well as the first time SPA has been reported as a material for 3D printing.

CCDC88B is a novel regulator of maturation and effector functions of T cells during pathological inflammation.

We used a genome-wide screen in mutagenized mice to identify genes which inactivation protects against lethal neuroinflammation during experimental cerebral malaria (ECM). We identified an ECM-protective mutation in coiled-coil domain containing protein 88b (Ccdc88b), a poorly annotated gene that is found expressed specifically in spleen, bone marrow, lymph nodes, and thymus. The CCDC88B protein is abundantly expressed in immune cells, including both CD4(+) and CD8(+) T lymphocytes, and in myeloid cells, and loss of CCDC88B protein expression has pleiotropic effects on T lymphocyte functions, including impaired maturation in vivo, significantly reduced activation, reduced cell division as well as impaired cytokine production (IFN-γ and TNF) in response to T cell receptor engagement, or to nonspecific stimuli in vitro, and during the course of P. berghei infection in vivo. This identifies CCDC88B as a novel and important regulator of T cell function. The human CCDC88B gene maps to the 11q13 locus that is associated with susceptibility to several inflammatory and auto-immune disorders. Our findings strongly suggest that CCDC88B is the morbid gene underlying the pleiotropic effect of the 11q13 locus on inflammation.

An N-ethyl-N-nitrosourea (ENU)-induced dominant negative mutation in the JAK3 kinase protects against cerebral malaria.

Cerebral malaria (CM) is a lethal neurological complication of malaria. We implemented a genome-wide screen in mutagenized mice to identify host proteins involved in CM pathogenesis and whose inhibition may be of therapeutic value. One pedigree (P48) segregated a resistance trait whose CM-protective effect was fully penetrant, mapped to chromosome 8, and identified by genome sequencing as homozygosity for a mis-sense mutation (W81R) in the FERM domain of Janus-associated kinase 3 (Jak3). The causative effect of Jak3(W81R) was verified by complementation testing in Jak3(W81R/-) double heterozygotes that were fully protected against CM. Jak3(W81R) homozygotes showed defects in thymic development with depletion of CD8(+) T cell, B cell, and NK cell compartments, and defective T cell-dependent production of IFN-γ. Adoptive transfer of normal splenocytes abrogates CM resistance in Jak3(W81R) homozygotes, an effect attributed to the CD8(+) T cells. Jak3(W81R) behaves as a dominant negative variant, with significant CM resistance of Jak3(W81R/+) heterozygotes, compared to CM-susceptible Jak3(+/+) and Jak3(+/-) controls. CM resistance in Jak3(W81R/+) heterozygotes occurs in presence of normal T, B and NK cell numbers. These findings highlight the pathological role of CD8(+) T cells and Jak3-dependent IFN-γ-mediated Th1 responses in CM pathogenesis.