Chandini M. Thirukkumaran

Microbial respiration, biomass, metabolic quotient and litter decomposition in a lodgepole pine forest floor amended with nitrogen and phosphorous fertilizers

In order to elucidate the mechanisms by which N and P fertilizers affect below ground microbial processes, ammonium nitrate (AN) and urea (U) (0, 188, 300 kg N ha−1) singly and in combination with triple super phosphate (TSP), (0, 94, 150 kg P ha−1) were added to pine F/H material under laboratory conditions and certain microbial variables were monitored over 120 d. Ammonium nitrate significantly suppressed basal and substrate induced respiration (SIR) when added singly or in combination with TSP at all sampling times. A temporary (d 10) increase in qCO2 values were produced by AN, but significantly lower values were detected after 40 d of incubation. Litter decomposition was suppressed by AN after 40 d. Although transient enhancements in basal respiration and SIR were caused by U, significant suppressions of these measurements were detected in the later phases of the experiment. Urea caused significant elevations in qCO2 values and enhanced rates of litter decomposition only at d 20 and 40. Inhibitory effects of TSP on microbial respiration and SIR were observed only when added singly and no effects on litter decomposition were found. The observed changes in microbial indices as a result of fertilizer addition could not be directly related to changes in soil pH. These laboratory results suggest that the present recommended rates of fertilizers suppress microbial activity.

Oncolytic Viral Therapy for Prostate Cancer: Efficacy of Reovirus as a Biological Therapeutic

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Oncolytic Viral Therapy Using Reovirus

Current mainstays in cancer treatment such as chemotherapy, radiation therapy, hormonal manipulation, and even targeted therapies such as Trastuzumab (herceptin) for breast cancer or Iressa (gefitinib) for non-small cell lung cancer among others are limited by lack of efficacy, cellular resistance, and toxicity. Dose escalation and combination therapies designed to overcome resistance and increase efficacy are limited by a narrow therapeutic index. Oncolytic viruses are one such group of new biological therapeutics that appears to have a wide spectrum of anticancer activity with minimal human toxicity. Since the malignant phenotype of tumors is the culmination of multiple mutations that occur in genes eventually leading to aberrant signaling pathways, oncolytic viruses either natural or engineered specifically target tumor cells taking advantage of this abnormal cellular signaling for their replication. Reovirus is one such naturally occurring double-stranded RNA virus that exploits altered signaling pathways (including Ras) in a myriad of cancers. The ability of reovirus to infect and lyse tumors under in vitro, in vivo, and ex vivo conditions has been well documented previously by us and others. The major mechanism of reovirus oncolysis of cancer cells has been shown to occur through apoptosis with autophagy taking place during this process in certain cancers. In addition, the synergistic antitumor effects of reovirus in combination with radiation or chemotherapy have also been demonstrated for reovirus resistant and moderately sensitive tumors. Recent progress in our understanding of viral immunology in the tumor microenvironment has diverted interest in exploring immunologic mechanisms to overcome resistance exhibited by chemotherapeutic drugs in cancer. Thus, currently several investigations are focusing on immune potentiating of reovirus for maximal tumor targeting. This chapter therefore has concentrated on immunologic cell death induction with reovirus as a novel approach to cancer therapy used under in vitro and in vivo conditions, as well as in a clinical setting. Reovirus phase I clinical trials have shown indications of efficacy, and several phase II/III trials are ongoing at present. Reoviruss extensive preclinical efficacy, replication competency, and low toxicity profile in humans have placed it as an attractive anticancer therapeutic for ongoing clinical testing that are highlighted in this chapter.

Reovirus as a Viable Therapeutic Option for the Treatment of Multiple Myeloma

Purpose: Despite the recent advances made in the treatment of multiple myeloma, the disease still remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human multiple myeloma in vitro, ex vivo, and in vivo without affecting human hematopoietic stem cell (HHSC) re-population/differentiation in a murine model that partially recapitulates human multiple myeloma. Experimental Design: Human myeloma cell lines and ex vivo tumor specimens were exposed to reovirus and oncolysis and mechanisms of cell death were assessed. RPMI 8226GFP+ cells were injected intravenously to non-obese diabetic/severe combined immune deficient (NOD/SCID) mice and treated with live reovirus (LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and re-population/differentiation was monitored. Results: A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to reovirus showed significant in vitro sensitivity. Tumor response of multiple myeloma by LV, but not DV, was confirmed by comparison of total tumor weights (P = 0.05), and bone marrow infiltration (1/6, LV; 5/6, DV). Mice injected with LV- or DV-exposed HHSCs maintained in vivo re-population/lineage differentiation showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by activation of the apoptotic pathways. Conclusions: The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs places it as a promising systemic multiple myeloma therapeutic for clinical testing. Clin Cancer Res; 18(18); 4962–72. ©2012 AACR.

Microbial activity, nutrient dynamics and litter decomposition in a Canadian Rocky Mountain pine forest as affected by N and P fertilizers

To study the specific effects of N and P fertilizers on soil microbial processes under field conditions, a pine forest in southern Alberta was fertilized with ammonium nitrate and urea (0 and 188 kg N ha � 1 , respectively) singly and in combination with triple super phosphate (0 and 94 kg P ha � 1 , respectively). Microbial respiration (BR), substrate induced respiration (SIR), metabolic quotient (qCO2) and rates of microbially mediated key processes were monitored in the forest floor FH material during the growing periods of spring 1990 to fall 1992. A transient increase in soil NH4 þ availability was detected following N addition but significant increases in PO4 3� availability were detected throughout the study as a result of P enhancement. Microbial variables were unaffected by N addition, whereas soil organic matter and SIR was increased with P fertilization. Microbial BR and qCO2 were reduced with P fertilization suggesting more efficient utilization of C. Increased net mineralization of P in the P-fertilized plots was found during the second and third summers after fertilization, following immobilization of P during the first year. In contrast, NH4 þ -N mineralization in the N-fertilized plots was significantly increased only during the first year. Rates of nitrification were unaffected by either N or P addition. Decomposition of pine litter was enhanced with N addition only during the first year and P had no effect on decomposition. Addition of N and P at these rates does not appear to impose a major ecological stress to the soil ecosystem. # 2002 Elsevier Science B.V. All rights reserved.

Reovirus modulates autophagy during oncolysis of multiple myeloma

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10–15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.

Viral purging of haematological autografts: should we sneeze on the graft?

High-dose cytotoxic chemotherapy followed by autologous haematopoietic stem cell transplantation (ASCT) is extensively used for the treatment of many haematopoietic, as well as several epithelial cancers. Disease relapse may be the result of tumour contamination within autograft as evidenced by gene marking studies. The multiple purging strategies that have been described to date have not proven effective in most ASCT settings. This review addresses the possibility of using oncolytic viruses as a novel purging strategy. DNA viruses such as genetically engineered adenoviral vectors have widely been used to deliver either a prodrug-activating enzyme or express wild-type p53 selectively in tumour cells in ex vivo purging protocols. In addition, conditionally replicating adenoviruses that selectively replicate in tumour cells and herpes simplex virus type 1 are other DNA viruses that have been tested as ex vivo purging agents under laboratory conditions. Vesicular stomatitis virus (VSV) and reovirus are naturally occurring RNA viruses that appear to hold promise as purging agents under ex vivo and in vivo settings. Preclinical data demonstrate reoviruss purging potential against breast, monocytic and myeloma cell lines as well as patient-derived tumours of diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, Waldenstrom macroglobulinemia and small lymphocytic lymphoma. In addition, VSV has shown effective killing of leukaemic cell lines and multiple myeloma patient specimens. Given the increasing interest in the utilization of viruses as purging agents, the following review provides a timely summary of the potential and the challenges of oncolytic viruses as purging modalities during ASCT.

Reovirus as a successful ex vivo purging modality for multiple myeloma

Autologous stem cell rescue (ASCT) following high-dose myeloablative chemotherapy is considered to be a therapeutic option for many multiple myeloma (MM) patients; however relapse post ASCT presents a major challenge. The oncolytic potential of reovirus has been previously demonstrated and is currently undergoing phase I monotherapy clinical trials for MM and phase II/III clinical trials for solid tumors. Here we tested the hypothesis that reovirus can successfully purge MM in a murine model that partially recapitulates human MM. RPMI 8226, MM1S, H929 and U266 human myeloma cell lines were exposed to reovirus and oncolysis was assessed. Apheresis product admixed with MM cells was purged with live reovirus (LV) or dead virus (DV) and purging efficacy was monitored via flow cytometry, reverse transcribed–PCR (RT–PCR) and disease relapse in non obese diabetic/severe combined immune deficient (NOD/SCID) mice. Significant LV purging was seen with MM1S, H929 and U266 and the complete ex vivo purging achieved with RPMI 8226 was confirmed by flow cytometry, RT–PCR and absence of disease relapse in vivo. Mice that received LV-purged autografts exhibited 100% survival in comparison to mice that received DV-purged controls. Reovirus’s unique ability to kill MM while sparing hematopoietic stem cells places it as an attractive purging agent for MM during ASCT.

Current Immunotherapeutic Strategies to Enhance Oncolytic Virotherapy

Oncolytic viruses (OV) represent a promising strategy to augment the spectrum of cancer therapeutics. For efficacy, they rely on two general mechanisms: tumor-specific infection/cell-killing, followed by subsequent activation of the host’s adaptive immune response. Numerous OV genera have been utilized in clinical trials, ultimately culminating in the 2015 Food and Drug Administration approval of a genetically engineered herpes virus, Talminogene laherparepvec (T-VEC). It is generally accepted that OV as monotherapy have only modest clinical efficacy. However, due to their ability to elicit specific antitumor immune responses, they are prime candidates to be paired with other immune-modulating strategies in order to optimize therapeutic efficacy. Synergistic strategies to enhance the efficacy of OV include augmenting the host antitumor response through the insertion of therapeutic transgenes such as GM-CSF, utilization of the prime-boost strategy, and combining OV with immune-modulatory drugs such as cyclophosphamide, sunitinib, and immune checkpoint inhibitors. This review provides an overview of these immune-based strategies to improve the clinical efficacy of oncolytic virotherapy.

Oncolytic virotherapy as an immunotherapeutic strategy for multiple myeloma

Multiple Myeloma (MM), a clonal malignancy of antibody-producing plasma cells, is the second most common hematologic malignancy and results in significant patient morbidity and mortality. The high degree of immune dysregulation in MM, including T cell imbalances and up-regulation of immunosuppressive checkpoint proteins and myeloid derived suppressor cells, allows this malignancy to escape from host immune control. Despite advances in the therapeutic landscape of MM over the last decade, including the introduction of immunomodulatory drugs, the prognosis for this disease is poor, with less than 50% of patients surviving 5 years. Thus, novel treatment strategies are required. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumour specific oncolysis and the generation of a potent adaptive anti-tumour immune response for efficacy. To date, a number of OV have shown efficacy in pre-clinical studies of MM with three reaching early phase clinical trials. OVs represent a rational therapeutic strategy for MM based on (1) their tumour tropism, (2) their ability to potentiate anti-tumour immunity and (3) their ability to be rationally combined with other immunotherapeutic agents to achieve a more robust clinical response.