Intravitreal melphalan hydrochloride vs propylene glycol-free melphalan for retinoblastoma vitreous seeds: Efficacy, toxicity and stability in rabbits models and patients.

The use of intravitreal chemotherapy has revolutionized the treatment of advanced intraocular retinoblastoma, as intravitreal melphalan has enabled difficult-to-treat vitreous tumor seeds to be controlled, leading to many more eyes being saved. However, melphalan hydrochloride (MH) degrades rapidly in solution, increasing logistical complexity with respect to time between medication preparation and administration for intravitreal administration under anesthesia for retinoblastoma. A new propylene glycol-free melphalan (PGFM) formulation has greater stability and could therefore improve access and adoption of intravitreal chemotherapy, allowing more children to retain their eye(s). We compared the efficacy and toxicity of both formulations, using our rabbit xenograft model and clinical patient experience. Three weekly 12.5 µg intravitreal injections of MH or PGFM (right eye), and saline (left eye), were administered to immunosuppressed rabbits harboring human WERI-Rb1 vitreous seed xenografts. Residual live cells were quantified directly, and viability determined by TUNEL staining. Vitreous seeds were reduced 91% by PGFM (p = 0.009), and 88% by MH (p = 0.004; PGFM vs. MH: p = 0.68). All residual cells were TUNEL-positive (non-viable). In separate experiments to assess toxicity, three weekly 12.5 µg injections of MH, PGFM, or saline were administered to non-tumor-bearing rabbits. Serial electroretinography, optical coherence tomography (OCT) and OCT-angiography were performed. PGFM and MH both caused equivalent reductions in electroretinography amplitudes, and loss of retinal microvasculature on OCT-angiography. The pattern of retinal degeneration observed on histopathology suggested that segmental retinal toxicity associated with all melphalan formulations was due to a vitreous concentration gradient-effect. Efficacy and toxicity were assessed for PGFM given immediately (within 1 h of reconstitution) vs. 4 h after reconstitution. Immediate- and delayed-administration of PGFM showed equivalent efficacy and toxicity. In addition, we evaluated efficacy and toxicity in patients (205 eyes) with retinoblastoma vitreous seeds, who were treated with a total of 833 intravitreal injections of either MH or PGFM as standard of care. Of these, we analyzed 118 MH and 131 PGFM monotherapy injections in whom serial ERG measurements were available to model retinal toxicity. Both MH and PGFM caused reductions in electroretinography amplitudes, but with no statistical difference between formulations. Comparing those patient eyes treated exclusively with PGFM versus those treated exclusively with MH, efficacy for tumor control and globe salvage was equivalent (PGFM vs. MH: 96.2% vs. 93.8%, p = 0.56), but PGFM-treated eyes received fewer injections than MH-treated eyes (average 3.2 ± 1.9 vs. 6.4 ± 2.1 injections, p < 0.0001). Taken together, these rabbit experiments and our clinical experience in retinoblastoma patients demonstrate that MH and PGFM have equivalent efficacy and toxicity. PGFM was more stable, with no decreased efficacy or increased toxicity even 4 h after reconstitution. We therefore now use PGFM over traditional MH for our patients for intravitreal treatment of retinoblastoma.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy.

Purpose: Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Methods: Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. Results: VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Conclusions: Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.

Evaluation of intravitreal topotecan dose levels, toxicity and efficacy for retinoblastoma vitreous seeds: a preclinical and clinical study.

BACKGROUND: Current melphalan-based intravitreal regimens for retinoblastoma (RB) vitreous seeds cause retinal toxicity. We assessed the efficacy and toxicity of topotecan monotherapy compared with melphalan in our rabbit model and patient cohort. METHODS: Rabbit experiments: empiric pharmacokinetics were determined following topotecan injection. For topotecan (15 µg or 30 µg), melphalan (12.5 µg) or saline, toxicity was evaluated by serial electroretinography (ERG) and histopathology, and efficacy against vitreous seed xenografts was measured by tumour cell reduction and apoptosis induction. PATIENTS: retrospective cohort study of 235 patients receiving 990 intravitreal injections of topotecan or melphalan. RESULTS: Intravitreal topotecan 30 µg (equals 60 µg in humans) achieved the IC90 across the rabbit vitreous. Three weekly topotecan injections (either 15 µg or 30 µg) caused no retinal toxicity in rabbits, whereas melphalan 12.5 µg (equals 25 µg in humans) reduced ERG amplitudes 42%-79%. Intravitreal topotecan 15 µg was equally effective to melphalan to treat WERI-Rb1 cell xenografts in rabbits (96% reduction for topotecan vs saline (p=0.004), 88% reduction for melphalan vs saline (p=0.004), topotecan vs melphalan, p=0.15). In our clinical study, patients received 881 monotherapy injections (48 topotecan, 833 melphalan). Patients receiving 20 µg or 30 µg topotecan demonstrated no significant ERG reductions; melphalan caused ERG reductions of 7.6 µV for every injection of 25 µg (p=0.03) or 30 µg (p<0.001). Most patients treated with intravitreal topotecan also received intravitreal melphalan at some point during their treatment course. Among those eyes treated exclusively with topotecan monotherapy, all eyes were salvaged. CONCLUSIONS: Taken together, these experiments suggest that intravitreal topotecan monotherapy for the treatment of RB vitreous seeds is non-toxic and effective.

Complete preclinical platform for intravitreal chemotherapy drug discovery for retinoblastoma: Assessment of pharmacokinetics, toxicity and efficacy using a rabbit model.

Current melphalan-based intravitreal chemotherapy regimens for retinoblastoma vitreous seeds are effective, but cause significant ocular toxicity. We describe protocols for each step of a drug discovery pipeline for preclinical development of novel drugs to maximize efficacy and minimize toxicity. These protocols include: 1) determination of vitreous pharmacokinetics in vivo, 2) in vitro assessment of drug cytotoxicity against retinoblastoma based on empiric pharmacokinetics, 3) back-calculation of minimum injection dose to achieve therapeutic concentrations, 4) in vivo determination of maximum-tolerable intravitreal dose, using a multimodal, structural and functional toxicity-assessment platform, and 5) in vivo determination of drug efficacy using a rabbit orthotopic xenograft model of retinoblastoma vitreous seeds. We likewise describe our methodology for direct quantitation of vitreous seeds, and the statistical methodology for assessment of toxicity and efficacy in evaluating novel drugs, as well as for comparisons between drugs.•Multi-step pipeline for intravitreal chemotherapy drug discovery for retinoblastoma, using novel rabbit models.•Detailed protocols for determination of vitreous pharmacokinetics, calculation of optimal dose to inject to achieve therapeutic vitreous levels, determination of maximum tolerable dose using a novel complete toxicity-assessment platform, and in vivo efficacy against retinoblastoma using methodology to directly quantify vitreous tumor burden.•Associated statistical methodology is also presented.

Intravitreal HDAC Inhibitor Belinostat Effectively Eradicates Vitreous Seeds Without Retinal Toxicity In Vivo in a Rabbit Retinoblastoma Model.

Purpose: Current melphalan-based regimens for intravitreal chemotherapy for retinoblastoma vitreous seeds are effective but toxic to the retina. Thus, alternative agents are needed. Based on the known biology of histone deacetylases (HDACs) in the retinoblastoma pathway, we systematically studied whether the HDAC inhibitor belinostat is a viable, molecularly targeted alternative agent for intravitreal delivery that might provide comparable efficacy, without toxicity. Methods: In vivo pharmacokinetic experiments in rabbits and in vitro cytotoxicity experiments were performed to determine the 90% inhibitory concentration (IC90). Functional toxicity by electroretinography and structural toxicity by optical coherence tomography (OCT), OCT angiography, and histopathology were evaluated in rabbits following three injections of belinostat 350 µg (2× IC90) or 700 µg (4× IC90), compared with melphalan 12.5 µg (rabbit equivalent of the human dose). The relative efficacy of intravitreal belinostat versus melphalan to treat WERI-Rb1 human cell xenografts in rabbit eyes was directly quantified. RNA sequencing was used to assess belinostat-induced changes in RB cell gene expression. Results: The maximum nontoxic dose of belinostat was 350 µg, which caused no reductions in electroretinography parameters, retinal microvascular loss on OCT angiography, or retinal degeneration. Melphalan caused severe retinal structural and functional toxicity. Belinostat 350 µg (equivalent to 700 µg in the larger human eye) was equally effective at eradicating vitreous seeds in the rabbit xenograft model compared with melphalan (95.5% reduction for belinostat, P < 0.001; 89.4% reduction for melphalan, P < 0.001; belinostat vs. melphalan, P = 0.10). Even 700 µg belinostat (equivalent to 1400 µg in humans) caused only minimal toxicity. Widespread changes in gene expression resulted. Conclusions: Molecularly targeted inhibition of HDACs with intravitreal belinostat was equally effective as standard-of-care melphalan but without retinal toxicity. Belinostat may therefore be an attractive agent to pursue clinically for intravitreal treatment of retinoblastoma.

Efficacy, Toxicity, and Pharmacokinetics of Intra-Arterial Chemotherapy Versus Intravenous Chemotherapy for Retinoblastoma in Animal Models and Patients.

Purpose: Through controlled comparative rabbit experiments and parallel patient studies, our purpose was to understand mechanisms underlying differences in efficacy and toxicity between intra-arterial chemotherapy (IAC) and intravenous chemotherapy (IVC). Methods: In rabbits, ocular tissue drug levels were measured following IAC and IVC. Retinal toxicity was assessed using electroretinography, fluorescein angiography, optical coherence tomography (OCT) and OCT angiography. Efficacy to eradicate retinoblastoma orthotopic xenografts was compared. In IAC and IVC patients, we measured blood carboplatin pharmacokinetics and compared efficacy and toxicity. Results: In rabbits receiving IAC, maximum carboplatin levels were 134 times greater in retina (P = 0.01) and 411 times greater in vitreous (P < 0.001), and total carboplatin (area under the curve) was 123 times greater in retina (P = 0.005) and 131 times greater in vitreous (P = 0.02) compared with IVC. Melphalan levels were 12 times greater (P = 0.003) in retina and 26 times greater in vitreous (P < 0.001) for IAC. Blood levels were not different. IAC melphalan (but not IV melphalan or IV carboplatin, etoposide, and vincristine) caused widespread apoptosis in retinoblastoma xenografts but no functional retinal toxicity or cytopenias. In patients, blood levels following IVC were greater (P < 0.001) but, when adjusted for treatment dose, were not statistically different. Per treatment cycle in patients, IVC caused higher rates of anemia (0.32 ± 0.29 vs. 0.01 ± 0.04; P = 0.0086), thrombocytopenia (0.5 ± 0.42 vs. 0.0 ± 0.0; P = 0.0042), and neutropenia (0.58 ± 0.3 vs. 0.31 ± 0.25; P = 0.032) but lower treatment success rates (P = 0.0017). Conclusions: The greater efficacy and lower systemic toxicity with IAC appear to be attributable to the greater ocular-to-systemic drug concentration ratio compared with IVC. Translational Relevance: Provides an overarching hypothesis for a mechanism of efficacy/toxicity to guide future drug development.

NFAT4 deficiency results in incomplete liver regeneration following partial hepatectomy.

BACKGROUND: Liver regeneration following partial hepatectomy requires the orchestration of highly regulated molecular pathways; a change in the abundance or activity of a specific gene product has the potential to adversely affect this process. The nuclear factor of activated T-cells (NFAT) transcription factors represent a family of gene transcription signaling intermediates that translate receptor-dependent signaling events into specific transcriptional responses using the Ras/Raf pathway. MATERIALS AND METHODS: Eight-week old NFAT4 knockout (KO) mice and their wild type counterparts (Balb-c) underwent two-thirds partial hepatectomy. The animals were sacrificed and their livers were harvested at specific time points during regeneration. Recovery of liver mass was measured for each time point. PCR analysis was used to analyze expression levels of the immediate early genes c-fos, c-jun and c-myc as well as downstream effectors of NFAT4 including FGF-18 and BMP-4. RESULTS: Hepatocyte proliferation and thus liver regeneration following hepatectomy was suppressed in NFAT4 knockout (KO) mice. Statistical significance was reached at 1 h, 7 d, and 10 d (P < 0.05) with a 22% median reduction in regeneration of liver mass in the NFAT4 KO mice by 10 d, at which time liver regeneration should be complete in mice. The immediate early gene c-fos was elevated in NFAT4 KO mice during early regeneration with a median value at 1 h and 1 d of 1.60E-08 and 1.09E-08 versus 6.10E-09 and 1.55E-09 in the Balb-c mice. C-jun, in contrast, was elevated during late regeneration in the NFAT4 KO mice (3.40E-09 and 5.67E-09 at 7 and 10 d, respectively) in comparison with the Balb-c mice (7.76E-10 and 1.24E-09, respectively.). NFAT2 was also up-regulated in the NFAT4 KO mice; however, no changes were detected in its downstream effectors, CCR1 and CCL3. CONCLUSIONS: We demonstrated that NFAT4 deficiency impairs hepatic regeneration in a murine model proving that NFAT4 plays an important yet unclear role in liver regeneration; its absence may be compensated by c-fos, c-jun, and NFAT2 expression changes.

Warm hepatic ischemia-reperfusion promotes growth of colorectal carcinoma micrometastases in mouse liver via matrix metalloproteinase-9 induction.

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromal-derived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery.

FXR1 expression domain in Wilms tumor.

BACKGROUND/PURPOSE: Wilms tumor (WT) is the most common childhood kidney cancer globally. Our prior unbiased proteomic screen of WT disparities revealed increased expression of Fragile X-Related 1 (FXR1) in Kenyan specimens where survival is dismal. FXR1 is an RNA-binding protein that associates with poor outcomes in multiple adult cancers. The aim of this study therefore was to validate and characterize the FXR1 expression domain in WT. METHODS: Quantitative FXR1 gene expression was compared between WT, adjacent, adult, and fetal kidney specimens. The cellular and subcellular expression domain of FXR1 was characterized across these tissues using immunoperoxidase staining. RNA-sequencing of FXR1 was performed from WT and other pediatric malignancies to examine its broader target potential. RESULTS: FXR1 was detected in all clinical WT specimens evaluated (n = 82), and as a result appeared independent of demographic, histology, or adverse event. Specific cytosolic staining was strongest in blastema, intermediate and variable in epithelia, and weakest in stroma. When present, areas of skeletal muscle differentiation stained strongly for FXR1. qPCR revealed increased FXR1 expression in WT compared to adult and adjacent kidney (p < 0.0002) but was similar to fetal kidney (p = 0.648). RNA-sequencing revealed expression of FXR1 in multiple pediatric tumors, greatest in rhabdomyosarcoma and WT. CONCLUSIONS: FXR1 was expressed consistently across this broad sampling of WT and most robustly in the primitive blastema. Notably, FXR1 labeled a specific self-renewing progenitor population of the fetal kidney.

Rabbit Model of Intra-Arterial Chemotherapy Toxicity Demonstrates Retinopathy and Vasculopathy Related to Drug and Dose, Not Procedure or Approach.

Purpose: To use our intra-arterial chemotherapy (IAC) rabbit model to assess the impact of IAC procedure, drug, dose, and choice of technique on ocular structure and function, to study the nature and etiology of IAC toxicity, and to compare to observations in patients. Methods: Rabbits received IAC melphalan (0.4-0.8 mg/kg), carboplatin (25-50 mg), or saline, either by direct ophthalmic artery cannulation, or with a technique emulating nonocclusion. Ocular structure/function were assessed with examination, electroretinography (ERG), fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT angiography, prior to and 5 to 6 weeks after IAC. Blood counts were obtained weekly. We reviewed our last 50 IAC treatments in patients for evidence of ocular or systemic complications. Results: No toxicity was seen in the saline control group. With standard (0.4 mg/kg) melphalan, no vascular/microvascular abnormalities were seen with either technique. However, severe microvascular pruning and arteriolar occlusions were seen occasionally at 0.8 mg/kg doses. ERG reductions were dose-dependent. Histology showed melphalan dose-dependent degeneration in all retinal layers, restricted geographically to areas of greatest vascular density. Carboplatin caused massive edema of ocular/periocular structures. IAC patients experienced occasional periocular swelling/rash, and only rarely experienced retinopathy or vascular events/hemorrhage in eyes treated multiple times with triple (melphalan/carboplatin/topotecan) therapy. Transient neutropenia occurred after 46% of IAC procedures, generally after triple therapy. Conclusions: IAC toxicity appears to be related to the specific drug being used and is dose-dependent, rather than related to the IAC procedure itself or the specific technique selected. These rabbit findings are corroborated by our clinical findings in patients.

Organ-specific roles for transcription factor NF-kappaB in reovirus-induced apoptosis and disease.

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Pharmacokinetics, Tissue Localization, Toxicity, and Treatment Efficacy in the First Small Animal (Rabbit) Model of Intra-Arterial Chemotherapy for Retinoblastoma.

Purpose: Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy. Methods: Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity. Results: OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 µM and area under the curve (AUC0→∞) was 5.26 µM·h. Treated eye vitreous Cmax was 2.24 µM and AUC0→∞ was 4.19 µM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 µM and AUC0→∞ was 2.07 µM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan. Conclusions: This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

Molecular and epidemiologic characterization of Wilms tumor from Baghdad, Iraq.

BACKGROUND: Wilms tumor (WT) is the most common childhood kidney cancer worldwide, yet its incidence and clinical behavior vary according to race and access to adequate healthcare resources. To guide and streamline therapy in the war-torn and resource-constrained city of Baghdad, Iraq, we conducted a first-ever molecular analysis of 20 WT specimens to characterize the biological features of this lethal disease within this challenged population. METHODS: Next-generation sequencing of ten target genes associated with WT development and treatment resistance (WT1, CTNNB1, WTX, IGF2, CITED1, SIX2, p53, N-MYC, CRABP2, and TOP2A) was completed. Immunohistochemistry was performed for 6 marker proteins of WT (WT1, CTNNB1, NCAM, CITED1, SIX2, and p53). Patient outcomes were compiled. RESULTS: Mutations were detected in previously described WT "hot spots" (e.g., WT1 and CTNNB1) as well as novel loci that may be unique to the Iraqi population. Immunohistochemistry showed expression domains most typical of blastemal-predominant WT. Remarkably, despite the challenges facing families and care providers, only one child, with combined WT1 and CTNNB1 mutations, was confirmed dead from disease. Median clinical follow-up was 40.5 months (range 6-78 months). CONCLUSIONS: These data suggest that WT biology within a population of Iraqi children manifests features both similar to and unique from disease variants in other regions of the world. These observations will help to risk stratify WT patients living in this difficult environment to more or less intensive therapies and to focus treatment on cell-specific targets.

Inhibition of phospholipase C attenuates liver mitochondrial calcium overload following cold ischemia.

BACKGROUND: Graft failure due to cold ischemia (CI) injury remains a significant problem during liver transplantation. During CI, the consumption of ATP and the increase in cellular Ca concentration may result in mitochondrial Ca (mCa) overload through the mCa uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa uptake in the liver, and now extend those studies to examine the role of mitochondrial PLC in liver CI injury. METHODS: Rat livers were perfused with University of Wisconsin (UW) solution. Half was homogenized immediately; the other half was cold-stored for 24 hr in UW. Mitochondria were extracted by differential centrifugation and incubated in buffer containing ATP and 0.1 or 0.2 microM Ca. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa uptake following CI. Western blots and densitometry quantified mitochondrial PLC expression. Mito Tracker Red fluorescence microscopy was used to verify mitochondrial transmembrane potential. RESULTS: Twenty-four hour CI caused a significant increase in mCa uptake (P<0.001), and increasing extramitochondrial Ca potentiated this effect. The PLC inhibitor, U-73122, decreased mCa uptake in nonischemic mitochondria (P<0.001), and had a greater effect on CI mitochondria (P<0.001). Mitochondrial PLC-dl expression increased 175+/-75% following CI (P<0.05). CONCLUSIONS: These data demonstrate that PLC-dl is essential for mCa uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-delta1 expression represents a novel mechanism whereby mCa uptake can increase independently of cytosolic conditions.

Reovirus strain-dependent inflammatory cytokine responses and replication patterns in a human monocyte cell line.

Mammalian Orthoreoviruses are important models for studies of viral pathogenesis. In the rat lung, Reovirus strain type 3 Dearing (T3D) induces substantially more inflammation than does strain type 1 Lang (T1L). To better understand mechanisms underlying differences in the host inflammatory response elicited by T1L and T3D, we characterized cytokine expression patterns induced by those strains after infection of THP-1 monocyte cells. THP-1 cells were adsorbed with either viable or ultraviolet- inactivated T1L and T3D and assayed for mRNA and protein production of growth-regulated oncogene-alpha (GRO-alpha), interleukin-8 (IL-8), or tumor necrosis factor-alpha (TNF-alpha). T3D stimulated mRNA and protein production of all three cytokines, whereas T1L stimulated mRNA and protein production of IL-8 and TNF-alpha but not GRO-alpha. In each case, T3D induced greater cytokine mRNA and protein expression than did T1L. Nonviable virus did not stimulate detectable cytokine secretion, suggesting a requirement for viral RNA synthesis in cytokine induction by THP-1 cells. A greater percentage of THP-1 cells was infected with T1L than T3D as assessed by infectious center assay, and T1L achieved higher yields of infectious progeny than did T3D in infected THP-1 cells as determined by plaque assay. These strain-dependent differences in cytokine responses and corresponding replication patterns in monocyte cells parallel findings made in studies of rat models of pneumonia and provide clues about how Reovirus interfaces with the host innate immune response to produce pulmonary disease.

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake.

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.