Bio

Clinical Focus


  • Molecular Genetics
  • Clinical Pathology
  • Residency

Publications

All Publications


  • A Survey of Somatic Mutations in 41 Genes in a Cohort of T-Cell Lymphomas Identifies Frequent Mutations in Genes Involved in Epigenetic Modification APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Fernandez-Pol, S., Ma, L., Joshi, R. P., Arber, D. A. 2019; 27 (6): 416–22
  • Targeted deep sequencing of cell-free DNA from body cavity fluids with malignant, suspicious, and benign cytology Yang, S., Libiran, P., Jones, C., Joshi, R., Lau, H., Stehr, H., Longacre, T., Allison, K., Zehnder, J., Long, S., Berry, G., Kunder, C. NATURE PUBLISHING GROUP. 2019
  • Transfusion Management of Conjoined Twins Undergoing Surgical Separation: A Single Center Experience with Three Sets of Thoraco-Omphalopagus Twins over Ten Years Tsang, H., Kuan, C., Boltz, M., Hartman, G., Joshi, R. P., Panigrahi, A., Andrews, J. WILEY. 2018: 122A–123A
  • The Brief Case: Confirmed Positive HIV-1 Serologic Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D., Aziz, N., Pinsky, B. A. 2017; 55 (12): 3316–20

    View details for PubMedID 29180504

  • Falsely high sirolimus concentrations due to everolimus cross-reactivity in the Siemens sirolimus immunoassay: Corrective actions implemented. Clinica chimica acta; international journal of clinical chemistry Bowen, R., Rieta, R., Joshi, R., Lee, R. C. 2017

    View details for PubMedID 29080689

  • Closing the Brief Case: Confirmed Positive HIV-1 Serological Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D., Aziz, N., Pinsky, B. A. 2017; 55 (12): 3566–67

    View details for PubMedID 29180505

  • Multiplexed detection of viral infections using rapid in situ RNA analysis on a chip LAB ON A CHIP Shaffer, S. M., Joshi, R. P., Chambers, B. S., Sterken, D., Biaesch, A. G., Gabrieli, D. J., Li, Y., Feemster, K. A., Hensley, S. E., Issadore, D., Raj, A. 2015; 15 (15): 3170–82

    Abstract

    Viral infections are a major cause of human disease, but many require molecular assays for conclusive diagnosis. Current assays typically rely on RT-PCR or ELISA; however, these tests often have limited speed, sensitivity or specificity. Here, we demonstrate that rapid RNA FISH is a viable alternative method that could improve upon these limitations. We describe a platform beginning with software to generate RNA FISH probes both for distinguishing related strains of virus (even those different by a single base) and for capturing large numbers of strains simultaneously. Next, we present a simple fluidic device for reliably performing RNA FISH assays in an automated fashion. Finally, we describe an automated image processing pipeline to robustly identify uninfected and infected samples. Together, our results establish RNA FISH as a methodology with potential for viral point-of-care diagnostics.

    View details for DOI 10.1039/c5lc00459d

    View details for Web of Science ID 000358022900010

    View details for PubMedID 26113495

    View details for PubMedCentralID PMC4670042

  • Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice NATURE COMMUNICATIONS Min, S. H., Suzuki, A., Stalker, T. J., Zhao, L., Wang, Y., McKennan, C., Riese, M. J., Guzman, J. F., Zhang, S., Lian, L., Joshi, R., Meng, R., Seeholzer, S. H., Choi, J. K., Koretzky, G., Marks, M. S., Abrams, C. S. 2014; 5: 4691

    Abstract

    PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

    View details for DOI 10.1038/ncomms5691

    View details for Web of Science ID 000342838800001

    View details for PubMedID 25178411

    View details for PubMedCentralID PMC4369914

  • Diacylglycerol Kinase zeta Limits the Generation of Natural Regulatory T Cells SCIENCE SIGNALING Schmidt, A. M., Zou, T., Joshi, R. P., Leichner, T. M., Pimentel, M. A., Sommers, C. L., Kambayashi, T. 2013; 6 (303): ra101

    Abstract

    Natural regulatory T (nT(reg)) cells are important for maintaining tolerance to self- and foreign antigens, and they are thought to develop from thymocytes that receive strong T cell receptor (TCR)-mediated signals in the thymus. TCR engagement leads to the activation of phospholipase C-γ1, which generates the lipid second messenger diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. We used mice that lack the ζ isoform of DAG kinase (DGKζ), which metabolizes DAG to terminate its signaling, to enhance TCR-mediated signaling and identify critical signaling events in nT(reg) cell development. Loss of DGKζ resulted in increased numbers of thymic CD25(+)Foxp3(-)CD4(+) nT(reg) cell precursors and Foxp3(+)CD4(+) nT(reg) cells in a cell-autonomous manner. DGKζ-deficient T cells exhibited increased nuclear translocation of the nuclear factor κB subunit c-Rel, as well as enhanced extracellular signal-regulated kinase (ERK) phosphorylation in response to TCR stimulation, suggesting that these downstream pathways may contribute to nT(reg) cell development. Indeed, reducing c-Rel abundance or blocking ERK phosphorylation abrogated the increased generation of nTreg cells by DGKζ-deficient thymocytes. The extent of ERK phosphorylation correlated with TCR-mediated acquisition of Foxp3 in immature thymocytes in vitro. Furthermore, the development of nT(reg) cells was augmented in mice in which ERK activation was selectively enhanced in T cells. Together, these data suggest that DGKζ regulates the development of nT(reg) cells by limiting the extent of activation of the ERK and c-Rel signaling pathways.

    View details for DOI 10.1126/scisignal.2004411

    View details for Web of Science ID 000327730000001

    View details for PubMedID 24280042

    View details for PubMedCentralID PMC4103616

  • The zeta Isoform of Diacylglycerol Kinase Plays a Predominant Role in Regulatory T Cell Development and TCR-Mediated Ras Signaling SCIENCE SIGNALING Joshi, R. P., Schmidt, A. M., Das, J., Pytel, D., Riese, M. J., Lester, M., Diehl, J., Behrens, E. M., Kambayashi, T., Koretzky, G. A. 2013; 6 (303): ra102

    Abstract

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)-stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (T(reg)) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating T(reg) cell development.

    View details for DOI 10.1126/scisignal.2004373

    View details for Web of Science ID 000327730000002

    View details for PubMedID 24280043

    View details for PubMedCentralID PMC4096120

  • Enhanced Effector Responses in Activated CD8(+) T Cells Deficient in Diacylglycerol Kinases CANCER RESEARCH Riese, M. J., Wang, L. S., Moon, E. K., Joshi, R. P., Ranganathan, A., June, C. H., Koretzky, G. A., Albelda, S. M. 2013; 73 (12): 3566–77

    Abstract

    Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs.

    View details for DOI 10.1158/0008-5472.CAN-12-3874

    View details for Web of Science ID 000320380300010

    View details for PubMedID 23576561

    View details for PubMedCentralID PMC3686869

  • Diacylglycerol Kinases: Regulated Controllers of T Cell Activation, Function, and Development INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Joshi, R. P., Koretzky, G. A. 2013; 14 (4): 6649–73

    Abstract

    Diacylglycerol kinases (DGKs) are a diverse family of enzymes that catalyze the conversion of diacylglycerol (DAG), a crucial second messenger of receptor-mediated signaling, to phosphatidic acid (PA). Both DAG and PA are bioactive molecules that regulate a wide set of intracellular signaling proteins involved in innate and adaptive immunity. Clear evidence points to a critical role for DGKs in modulating T cell activation, function, and development. More recently, studies have elucidated factors that control DGK function, suggesting an added complexity to how DGKs act during signaling. This review summarizes the available knowledge of the function and regulation of DGK isoforms in signal transduction with a particular focus on T lymphocytes.

    View details for DOI 10.3390/ijms14046649

    View details for Web of Science ID 000318017100008

    View details for PubMedID 23531532

    View details for PubMedCentralID PMC3645659

  • AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species BLOOD Juntilla, M. M., Patil, V. D., Calamito, M., Joshi, R. P., Birnbaum, M. J., Koretzky, G. A. 2010; 115 (20): 4030–38

    Abstract

    Although AKT is essential for multiple cellular functions, the role of this kinase family in hematopoietic stem cells (HSCs) is unknown. Thus, we analyzed HSC function in mice deficient in the 2 isoforms most highly expressed in the hematopoietic compartment, AKT1 and AKT2. Although loss of either isoform had only a minimal effect on HSC function, AKT1/2 double-deficient HSCs competed poorly against wild-type cells in the development of myeloid and lymphoid cells in in vivo reconstitution assays. Serial transplantations revealed an essential role for AKT1 and AKT2 in the maintenance of long-term HSCs (LT-HSCs). AKT1/2 double-deficient LT-HSCs were found to persist in the G(0) phase of the cell cycle, suggesting that the long-term functional defects are caused by increased quiescence. Furthermore, we found that the intracellular content of reactive oxygen species (ROS) is dependent on AKT because double-deficient HSCs demonstrate decreased ROS. The importance of maintaining ROS for HSC differentiation was shown by a rescue of the differentiation defect after pharmacologically increasing ROS levels in double-deficient HSCs. These data implicate AKT1 and AKT2 as critical regulators of LT-HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

    View details for DOI 10.1182/blood-2009-09-241000

    View details for Web of Science ID 000277923600007

    View details for PubMedID 20354168

    View details for PubMedCentralID PMC2875090

  • Lytic Granule Loading of CD8(+) T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control IMMUNITY Migueles, S. A., Osborne, C. M., Royce, C., Compton, A. A., Joshi, R. P., Weeks, K. A., Rood, J. E., Berkley, A. M., Sacha, J. B., Cogliano-Shutta, N. A., Lloyd, M., Roby, G., Kwan, R., McLaughlin, M., Stallings, S., Rehm, C., O'Shea, M. A., Mican, J., Packard, B. Z., Komoriya, A., Palmer, S., Wiegand, A. P., Maldarelli, F., Coffin, J. M., Mellors, J. W., Hallahan, C. W., Follman, D. A., Connors, M. 2008; 29 (6): 1009–21

    Abstract

    Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

    View details for DOI 10.1016/j.immuni.2008.10.010

    View details for Web of Science ID 000262012400019

    View details for PubMedID 19062316

    View details for PubMedCentralID PMC2622434

  • Pretreatment photosensitizer dosimetry reduces variation in tumor response INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Zhou, X. D., Pogue, B. W., Chen, B., Demidenko, E., Joshi, R., Hoopes, J., Hasan, T. 2006; 64 (4): 1211–20

    Abstract

    To compensate for photosensitizer uptake variation in photodynamic therapy (PDT), via control of delivered light dose through photodynamic dose calculation based on online dosimetry of photosensitizer in tissue before treatment.Photosensitizer verteporfin was quantified via multiple fluorescence microprobe measurements immediately before treatment. To compensate individual PDT treatments, photodynamic doses were calculated on an individual animal basis, by matching the light delivered to provide an equal photosensitizer dose multiplied by light dose. This was completed for the lower quartile, median, and upper quartile of the photosensitizer distribution. PDT-induced tumor responses were evaluated by the tumor regrowth assay.Verteporfin uptake varied considerably among tumors and within a tumor. The coefficient of variation in the surviving fraction was found significantly decreased in groups compensated to the lower quartile (CL-PDT), the median (CM-PDT), and the upper quartile (CU-PDT) of photosensitizer distribution. The CL-PDT group was significantly less effective compared with NC-PDT (Noncompensated PDT), CM-PDT, and CU-PDT treatments. No significant difference in effectiveness was observed between NC-PDT, CM-PDT, and CU-PDT treatment groups.This research suggests that accurate quantification of tissue photosensitizer levels and subsequent adjustment of light dose will allow for reduced subject variation and improved treatment consistency.

    View details for DOI 10.1016/j.ijrobp.2005.11.019

    View details for Web of Science ID 000235897100032

    View details for PubMedID 16504761

  • Photosensitizer dosimetry controlled PDT treatment planning reduces inter-individual variability in response to PDT Zhou, X., Pogue, B. W., Chen, B., Demidenko, E., Joshi, R., Hoopes, J., Hasan, T., Kessel, D. SPIE-INT SOC OPTICAL ENGINEERING. 2006: 157-+

    View details for DOI 10.1117/12.647439

    View details for Web of Science ID 000237680100018