Immunity against fungal beta 1,3 glucan carbohydrate in the gastrointestinal tract

Inflammatory Bowel Disease (IBD) is a debilitating, life- long disease that affects about 1.4 millions Americans. Little is known about the pathogenesis of IBD and an effective cure still remains to be discovered. While there are numerous T cell targeting therapies for IBD, more research is still needed. Bispecific T Cell Engagers, BiTES, is a modified protein capable of engaging two antigens simultaneously; it is capable of activating T cells by circumventing the MHC protein molecule. This provides an alternative to the current molecular therapies for IBD. In addition to monoclonal therapy research, there has been a plethora of research on immunomodulatory molecules, such as β- glucan. The benefit of β-glucan has been shown with supplements and food sources alike in animal models. In this study, we used BiTES, CMPD-1, with an anti-CD3/ Dectin-1 epitopes capable of engaging T Cells and β-glucan in beads and fungi cell wall. CMPD-1 is capable of engaging Splenic and Lamina Propia T Cells from a C57BL/6 mice. Likewise, CMPD-1 engaged T cells to hyphae of C. albicans and A. fumigatus, which have a higher concentration of β-glucan than in the candida form. The data show a delayed in hyphae growth in yeast with CMPD-1 and a decrease in yeast growth for the first four hours when compared to non- BiTES molecules. Additionally, qualitative analysis of CMPD-1 shows a decrease A. fumigatus growth after a 72-hour incubation period. Splenic T cells from mice lacking Dectin-1 and Wild-type (WT) mouse strains where incubated with BiTES compound and yeast for 23 hours followed by a PrestoBlue killing assay to assess yeast cell viability. The PrestoBlue assay showed that CMPD-1 killed more A. fumigatus in both T cell subsets; although, the difference lacked statistical significance. The applications of this molecule as a therapeutic agent for IBD are promising, although, still in its infancy. An alternative use for this molecule is to train the immune system with the BiTES molecule in conjunction with β-glucan supplements to build immunity against opportunistic pathogens such as A. fumigatus and C. albicans that often cause havoc in IBD patients as a result of the changes in microbiota, and compromised integrity of the GI tract.2017-06-16T00:00:00

Human Myo19 Is a Novel Myosin that Associates with Mitochondria

Mitochondria are pleomorphic organelles [1, 2] that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease [3-5]. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport [6]. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues and antibodies to human Myo19 detect a ∼109kD band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain-of-function where the majority of the mitochondria move continuously. Moving mitochondria travel for many microns with an obvious leading end and distorted shape. The motility and shape-change are sensitive to latrunculin B, indicating that both are actin-dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells

Biguanides enhance antifungal activity against Candida glabrata

Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections

Biguanides enhance antifungal activity against <i>Candida glabrata</i>

<p><i>Candida spp</i>. are the fourth leading cause of nosocomial blood stream infections in North America. <i>Candida glabrata</i> is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against <i>C. glabrata</i>. Both wild type and antifungal-resistant isolates of <i>C. glabrata</i> were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against <i>C. glabrata</i>, with MIC₅₀ of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC₅₀ in voriconazole and fluconazole-resistant clinical isolates of <i>C. glabrata</i>. We also observed growth reduction of <i>C. glabrata</i> with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in <i>C. glabrata</i> may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant <i>C. glabrata</i> isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.</p

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.This work was supported by U.S. National Institutes of Health, National Institute of Allergy and Infectious Diseases Grants R01 AI092084 and R01 AI097519 (to J.M.V.) and R01 AI025780 and R01 AI139615 (to S.M.L.). This work was also supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001076), the UK Medical Research Council (FC001076), and the Wellcome Trust (FC001076)