Evidence of male-to-female sexual transmission of Zika virus (ZIKV) and viral RNA in semen and sperm months after infection supports a potential role for testicular cells in ZIKV propagation. Here, we demonstrate that germ cells (GCs) are most susceptible to ZIKV. We found that only GCs infected by ZIKV, but not those infected by dengue virus and yellow fever virus, produce high levels of infectious virus. This observation coincides with decreased expression of interferon-stimulated gene Ifi44l in ZIKV-infected GCs, and overexpression of Ifi44l results in reduced ZIKV production. Using primary human testicular tissue, we demonstrate that human GCs are also permissive for ZIKV infection and production. Finally, we identified berberine chloride as a potent inhibitor of ZIKV infection in both murine and human testes. Together, these studies identify a potential cellular source for propagation of ZIKV in testes and a candidate drug for preventing sexual transmission of ZIKV.

Alzheimer's disease (AD) is characterized by the presence of parenchymal amyloid-␤ (A␤) plaques, cerebral amyloid angiopathy (CAA) and neurofibrillary tangles. Currently there are no effective treatments for AD. Immunotherapeutic approaches under development are hampered by complications related to ineffectual clearance of CAA. Genome-wide association studies have demonstrated the importance of microglia in AD pathogenesis. Microglia are the primary innate immune cells of the brain. Depending on their activation state and environment, microglia can be beneficial or detrimental. In our prior work, we showed that stimulation of innate immunity with Toll-like receptor 9 agonist, class B CpG (cytosine-phosphate-guanine) oligodeoxynucleotides (ODNs), can reduce amyloid and tau pathologies without causing toxicity in Tg2576 and 3xTg-AD mouse models. However, these transgenic mice have relatively little CAA. In the current study, we evaluated the therapeutic profile of CpG ODN in a triple transgenic mouse model, Tg-SwDI, with abundant vascular amyloid, in association with low levels of parenchymal amyloid deposits. Peripheral administration of CpG ODN, both before and after the development of CAA, negated short-term memory deficits, as assessed by object-recognition tests, and was effective at improving spatial and working memory evaluated using a radial arm maze. These findings were associated with significant reductions of CAA pathology lacking adverse effects. Together, our extensive evidence suggests that this innovative immunomodulation may be a safe approach to ameliorate all hallmarks of AD pathology, supporting the potential clinical applicability of CpG ODN.

Accurate automated cell fate analysis of immunostained human stem cells from 2- and 3-dimensional (2D-3D) images would improve efficiency in the field of stem cell research. Development of an accurate and precise tool that reduces variability and the time needed for human stem cell fate analysis will improve productivity and interpretability of the data across research groups. In this study, we have created protocols for high performance image analysis software Volocity® to classify and quantify cytoplasmic and nuclear cell fate markers from 2D-3D images of human neural stem cells after in vitro differentiation. To enhance 3D image capture efficiency, we optimized the image acquisition settings of an Olympus FV10i® confocal laser scanning microscope to match our quantification protocols and improve cell fate classification. The methods developed in this study will allow for a more time efficient and accurate software based, operator validated, stem cell fate classification and quantification from 2D and 3D images, and yield the highest ≥94.4% correspondence with human recognized objects.

Background: BRD4 functions as an important epigenetic regulator of transcription of pathways commonly involved in oncogenesis, including cell cycle progression and apoptosis. Inhibition of BRD4 with small molecule inhibitors has demonstrated activity in various hematologic and solid malignancies. While downregulation of c-myc following BRD4 inhibition has been described, the exact mechanism of action of BRD4 inhibitors across B cell malignancies remains unclear. Here we demonstrate the efficacy of PLX51107 across a spectrum of B cell malignancies and the signaling pathways involved. Methods: A panel of 22 human multiple myeloma (MM) cell lines and 56 B cell lymphoma (BCL) cell line, were evaluated for the effects of PLX51107 on cell proliferation after a 5 day exposure to the drug. Sensitive NCI-H929, MM.1S, KMS-12BM and resistant RPMI, EJM, L363 - MM cell lines were selected for further mechanism of action (MOA) studies; along with BCL sensitive DoHH2- DLBCL GCB subtype, Ly3- DLBCL ABC subtype, MINO-mantle cell, 2A8-Burkitt. Matched resistant cell lines SUDHL-8, U2932, GRANTA, DB were also selected for the MOA studies. All MOA assays were performed by treating the MM cells at a dose of 150nM PLX51107 except the BCL cells which were exposed to 300nM. Cell cycle analysis and induction of apoptosis was performed by FACS after propidium iodide or Annexin V FITC staining respectively on cells treated for 72 hours. Cellular senescencewas determined by measurement of β-galactosidase activity in cells treated for 7 days (BCL) or 5 days (MM). Western blot analyses for c-myc, p-IRAK, p-Iκβα, p65 and p-MAPK Erk1/2 were performed at 4 and 24 hours post treatment. DMSO treatment was included as a control in the MOA experiments. Results: PLX51107 potently inhibited the in vitro proliferation and survival of a variety of B cell malignancies, including GCB and ABC subtype DLBCL, mantle cell lymphoma, Burkitt lymphoma, and multiple myeloma cell lines.Cell cycle arrest in G1 phasewas observed in 3/4 sensitive BCL, Ly3, 2A8 and MINO and 2/3 sensitive MM, NCI-H2929, MM1.S. Increased apoptosis was demonstrated in the sensitive cell lines 2A8, MINO (BCL) and NCI-H2929, KMS12BM (MM). Three sensitive BCL (DoHH2, Ly3, 2A8) and 2 sensitive MM (KMS12BM, MM1.S) displayed premature senescence. Interestingly, DoHH2 (DLBCL-GCB), Ly3 (DLBCL-ABC) and the MM cell line MM1.S, all of which were growth inhibited by PLX51107 treatment, did not demonstrate an increase in apoptosis and all were found to have the greatest degree of premature senescence. Our data indicate that these differential mechanistic findings do not correspond with a known underlying predictive biomarker. In contrast to the resistant cell lines, all sensitive lines demonstrated marked downregulation of c-myc expression after PLX51107 treatment. Downregulation of p-IRAK, a signaling intermediary of the MyD88-dependent pathway, was also observed in all sensitive BCL cell lines, while no corresponding change of this marker was observed in the sensitive MM cell lines. P-MAPK Erk 1/ 2 was downregulated in all sensitive BCL cell lines, but only 1 of 3 sensitive MM cell lines. Expression of p-Iκβα, a regulator of the NFκʙ pathway, was unchanged in all sensitive BCL cell lines but was downregulated in all 3 sensitive MM cell lines. Discussion: PLX51107 demonstrates in vitro efficacy through differential mechanisms across a maturational spectrum of B-cell malignancies. Treatment of BCL and MM cell cultures results in downregulation of c-myc in sensitive cell lines, consistent with findings from previous BRD4 inhibitors. We demonstrate the involvement of novel signaling pathways with PLX51107 treatment that vary based on the maturational stage of the malignant B cell. These data suggests treatment with PLX51107 affects the MyD88-dependent toll-like receptor pathway in the BCL cell cultures, while the NFκʙ pathway is changed in MM cell cultures. In conclusion, these results demonstrate that PLX51107 has distinct mechanisms of action beyond downregulation of the c-myc transcriptional program and is a promising targeted therapeutic agent across a broad range of clinically and histologically diverse B-cell neoplasms. Disclosures Powell: Plexxikon: Employment. Slamon:Pfizer: Equity Ownership, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Larson:BMS: Consultancy.