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Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human β-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC50 of 12 μg/ml and 2.8 μg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.

Bone marrow transplantation (BMT) is mainly performed to restore an anti-tumor immune response, called the graft-versus-tumor (GVT) effect, against leukemia, myeloma and lymphoma. This GVT reactivity is driven by donor T cells, and it can also cause lethal graft-versus-host disease (GVHD). We previously demonstrated that the colonization of mice with helminths preserves the GVT response while suppressing GVHD. As the T helper-2 (Th2) pathway is critical to helminthic immune regulation, we asked whether the genetic induction of Th2 signaling in donor T cells can restore helminthic immune regulation after BMT. Our studies utilized transgenic donor T lymphocytes that overexpress a constitutively active form of the Th2-associated transcription factor STAT6. Constitutively active STAT6 sustained the GVT response without causing severe acute GVHD, where transgenic T cells generated robust quantities of cytotoxic proteins important in GVT response, such as granzymes A and B, interferon-γ and Fas ligand, in addition to generating high quantities of Th2/regulatory cytokines. Bioinformatic analysis based on chromosome immune precipitation experiments indicated that STAT6 stimulates the expression of granzymes directly. Thus, in preserving the GVT response without causing GVHD mortality, our results indicate the therapeutic potential of restoring helminthic immune modulation by targeting STAT6 and STAT6-dependent T cell maturation. [ABSTRACT FROM AUTHOR]

Author(s): Samarchith P Kurup [1]; Nyamekye Obeng-Adjei [2]; Scott M Anthony [1]; Boubacar Traore [3]; Ogobara K Doumbo [3]; Noah S Butler [1, 4]; Peter D Crompton [2]; John T [...]

Using a murine model of sepsis, we found that the balance of tissue pro- to anti-inflammatory cytokines directly correlated with severity of infection and mortality. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Liver tissue was analyzed for levels of IL-1 [beta], IL-1 receptor antagonist (IL-1ra), tumor necrosis factor (TNF)-[alpha], and soluble TNF receptor l by ELISA. Bacterial DNA was measured using quantitative real-time PCR. After CLP, early predominance of proinflammatory cytokines (6 h) transitioned to anti-inflammatory predominance at 24 h. The elevated anti-inflammatory cytokines were mirrored by increased tissue bacterial levels. The degree of anti-inflammatory response compared with proinflammatory response correlated with the bacterial concentration. To modulate the timing of the anti-inflammatory response, mice were treated with IL-1ra before CLP. This resulted in decreased proinflammatory cytokines, earlier bacterial load, and increased mortality. These studies show that the initial tissue proinflammatory response to sepsis is followed by an anti-inflammatory response. The anti-inflammatory phase is associated with increased bacterial load and mortality. These data suggest that it is the timing and magnitude of the anti-inflammatory response that predicts severity of infection in a routine model of sepsis. bacterial load; anti-inflammation