Are viruses associated with disc herniation? A clinical case series.

BACKGROUND: There is some limited evidence for the presence of viruses in herniated disc material including a previous case series that claimed to provide "unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease". This study has not been replicated. The objective of our study was to determine if viruses were present in herniated disc fragments in participants with a prior history of back pain. METHODS: We recruited fifteen participants with a history of prior low-back pain prior to undergoing disc herniation surgery in the lumbar spine. Harvested disc samples were subject to next generation sequencing for detection of both RNA and DNA viral pathogens. Additionally, samples were analysed by a broadly reactive PCR targeting herpesviral DNA. Ethics approval was granted by the Human Research Ethics Committees of both Murdoch University, and St John of God Hospital, Western Australia. RESULTS: Of the fifteen research participants, 8 were female. Mean age was 49.4 years (SD 14.5 yrs) with a range of 24-70 years. All participants had prior back pain with mean time since first ever attack being 8.8 years (SD 8.8 yrs). No samples contained significant DNA sequences relating to known human viral agents. Inconsequential retroviral sequences were commonly found and were a mixture of putative animal and human retroviral protein coding segments. All samples were negative for herpesvirus DNA when analysed by pan-herpesvirus PCR. CONCLUSIONS: This study found no viral pathogens in any intervertebral disc fragments of patients who had previous back pain and underwent discectomy for disc herniation and thus it is unlikely that viruses are associated with disc herniation, however given the contradiction between key studies enhanced replication of this experiment is recommended.

The cost of treatment failure: resource use and costs incurred by hepatitis C virus genotype 1-infected patients who do or do not achieve sustained virological response to therapy.

Chronic hepatitis C virus (HCV) infection places a considerable economic burden on health services. Cost-effectiveness analyses of antiviral treatment for patients with chronic HCV infection are dependent on assumptions about cost reductions following sustained virological response (SVR) to therapy. This study quantified the medium-term difference in health resource usage and costs depending on treatment outcome. Retrospective chart review of patients with HCV genotype 1 infection who had received at least 2 months pegylated interferon and ribavirin therapy, with known treatment outcome was conducted. Disease status was categorized as chronic hepatitis, cirrhosis or decompensated liver disease. Health resource use was documented for each patient in each disease state. Unit costs were from the NHS 'Payment by Results' database and the British National Formulary. One hundred and ninety three patients (108 SVR, 85 non-SVR) with mean follow-up of 3.5 (SVR) and 4.9 (non-SVR) years were enrolled. No SVR patient progressed to a more severe liver disease state. Annual transition rates for non-SVR patients were 7.4% (chronic hepatitis to cirrhosis) and 4.9% (cirrhosis to decompensated liver disease). By extrapolation of modelled data over a 5-year post-treatment period, failure of patients with chronic hepatitis to achieve SVR was associated with a 13-fold increase (roughly £2300) in costs, whilst for patients who were retreated, the increase was 56-fold, equating to more than £10 000. Achievement of an SVR has significant effects on health service usage and costs. This work provides real-life data for future cost-effectiveness analyses related to the treatment for chronic HCV infection.

Co-enrichment of cancer-associated bacterial taxa is correlated with immune cell infiltrates in esophageal tumor tissue.

Esophageal carcinoma (ESCA) is a leading cause of cancer-related death worldwide, and certain oral and intestinal pathogens have been associated with cancer development and progression. We asked if esophageal microbiomes had shared alterations that could provide novel biomarkers for ESCA risk. We extracted DNA from tumor and non-tumor tissue of 212 patients in the NCI-MD case control study and sequenced the 16S rRNA gene (V3-4), with TCGA ESCA RNA-seq (n = 172) and WGS (n = 123) non-human reads used as validation. We identified four taxa, Campylobacter, Prevotella, Streptococcus, and Fusobacterium as highly enriched in esophageal cancer across all cohorts. Using SparCC, we discovered that Fusobacterium and Prevotella were also co-enriched across all cohorts. We then analyzed immune cell infiltration to determine if these dysbiotic taxa were associated with immune signatures. Using xCell to obtain predicted immune infiltrates, we identified a depletion of megakaryocyte-erythroid progenitor (MEP) cells in tumors with presence of any of the four taxa, along with enrichment of platelets in tumors with Campylobactor or Fusobacterium. Taken together, our results suggest that intratumoral presence of these co-occurring bacterial genera may confer tumor promoting immune alterations that allow disease progression in esophageal cancer.

Surface water temperature impacts on coastal wetland denitrification: Implications for river reconnection.

Louisiana, located in the southeast United States, is home to 40% of the continental US's coastal wetlands yet accounts for 80% of the nation's coastal wetland loss. This loss is generally attributed to decreased sediment supply, hydrologic alteration from levees, channelization, subsidence, sea-level rise, and wave and tidal induced marsh edge erosion. The Mid-Barataria Sediment Diversion is a US $1.3 billion coastal restoration project that will divert up to 2100 m3 s-1 of sediment-laden Mississippi River water directly into Barataria Basin. The influx of colder, nutrient-rich, springtime river water could negatively impact water quality of the receiving basin. We quantified the effects of colder, surface water temperature on the nitrate (NO3-) reduction rate in vegetated marsh and open water bay sediments. Colder water limited NO3- removal processes averaging 17.1 mg N m-2 d-1 in the range of 5-14 °C, before increasing almost 3-fold in the 20 °C treatments at 50.6 mg N m-2 d-1. Low N removal rates, especially near the project inflow where temperatures will be coldest will favor transport of NO3- further into Barataria Basin where eutrophic conditions could become expressed. These results will inform coastal managers around the world of the potential ecosystem response to coastal restoration aimed at river reconnection where colder waters enter warmer, shallow basins.

Seeking a way to promote the use of constructed wetlands for domestic wastewater treatment in developing countries.

The aim of this study was to evaluate the domestic wastewater treatment efficiency as well as the survivability of commercially valuable ornamental plants in subsurface flow wetlands (SSFW) for domestic wastewater (DWW) treatment in laboratory and pilot wetland studies. The laboratory scale study included five different species (Zantedeschia aethiopica, Strelitzia reginae, Anthurium andreanum, Canna hybrids and Hemmerocallis dumortieri) that were evaluated in horizontal flow subsurface treatment cells. All the plants survived during the 6-month experimental period demonstrating high wetland nutrient treatment efficiency. In order to validate and expand these preliminary results, a pilot-scale wetland study was carried out in SSFWs under two different flow regimes (horizontal and vertical flow). Four ornamental species were tested during a 1-year period: Zantedeschia aethiopica, Strelitzia reginae, Anthurium andreanum and Agapanthus africanus. The removal efficiencies were significantly higher in the vertical subsurface-flow constructed wetlands (VFCW) for all pollutants, except for nitrate (NO(3)-N), total nitrogen (TN) and total suspended solids (TSS). These results show that it is feasible to use select non-wetland plants with high market value in SSFWs without reducing the efficiency of the wastewater treatment system, although future work should continue in order to apply this technology in a large scale. The added value of floriculture in treatment wetlands can help to promote the use of constructed wetlands (CW) for domestic wastewater treatment in developing countries where economical resources are scarce and water pollution with DWW is common.

Treatment of wastewater ammonium under varying salinity conditions within the marshland upwelling system.

Coastal wetlands and estuaries are impacted by nutrient loads from a variety of sources including infrequently occupied hunting and fishing camps. The marshland upwelling system (MUS) was designed to treat wastewater in the coastal environment where traditional septic systems or centralized wastewater collection and treatment are not viable. A laboratory macrocosm study was designed to simulate field conditions in which domestic wastewater is treated via injection into a marsh subsurface. Treatment of wastewater nitrogen (N) utilizing the MUS was examined under high (∼20 ppt) and low (∼2 ppt) salinity conditions. Two N wastewater solutions were used, one treatment consisted of 100 mg NH4-N L-1, while a second treatment consisted of 80 mg NH4-N L-1/20 mg NO3-N L-1. The 20 ppt salinity treatment was found to have a negative impact on NH4-N sorption. The potentially mineralizable N rate was higher in the low salinity treatment, which could potentially be offset by the higher sorption capacity at lower salinities. The background salinity of the local groundwater should be considered as the salinity will play a role in the longevity of the system.

Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils.

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.

Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.

Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3-100.0) and 99.5 (95% CI 98.8-99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3-100.0) and 99.8 (95% CI 99.2-100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the test population.

The short-term effects of prescribed burning on biomass removal and the release of nitrogen and phosphorus in a treatment wetland.

Nutrient removal by constructed wetlands can decline over time due to the accumulation of organic matter. A prescribed burn is one of many management strategies used to remove detritus in macrophyte-dominated systems. We quantified the short-term effects on effluent water quality and the amount of aboveground detritus removed from a prescribed burn event. Surface water outflow concentrations were approximately three times higher for P and 1.5 times higher for total Kjeldhal nitrogen (TKN) following the burn event when compared to the control. The length of time over which the fire effect was significant (P < 0.05), 3 d for TKN and up to 23 d for P fractions. Over time, the concentration of soluble reactive phosphorus (SRP) in the effluent decreased, but was compensated with increases in dissolved organic phosphorus (DOP) and particulate phosphorus (PP), such that net total P remained the same. Total aboveground biomass decreased by 68.5% as a result of the burn, however, much of the live vegetation was converted to standing dead material. These results demonstrate that a prescribed burn can significantly decrease the amount of senescent organic matter in a constructed wetland. However, short-term nutrient releases following the burn could increase effluent nutrient concentrations. Therefore, management strategies should include hydraulically isolating the burned area immediately following the burn event to prevent nutrient export.

Review article: novel oral-targeted therapies in inflammatory bowel disease.

BACKGROUND: There is a great unmet clinical need for efficacious, tolerable, economical and orally administrated drugs for the treatment of inflammatory bowel disease (IBD). New therapeutic avenues have become possible including the development of medications that target specific genetic pathways found to be relevant in other immune mediated diseases. AIMS: To provide an overview of recent clinical trials for new generation oral targeted medications that may have a future role in IBD management. METHODS: Pubmed and Medline searches were performed up to 1 March 2018 using keywords: "IBD", "UC", "CD", "inflammatory bowel disease" "ulcerative colitis", "Crohn's disease" in combination with "phase", "study", "trial" and "oral". A manual search of the clinical trial register, article reference lists, abstracts from meetings of Digestive Disease Week, United European Gastroenterology Week and ECCO congress were also conducted. RESULTS: In randomised controlled trials primary efficacy endpoints were met for tofacitinib (JAK 1/3 inhibitor-phase III), upadacitinib (JAK 1 inhibitor-phase II) and AJM300 (α4-integrin antagonist-phase II) in ulcerative colitis. Ozanimod (S1P receptor agonist-phase II) also demonstrated clinical remission. For Crohn's disease, filgotinib (JAK1 inhibitor-phase II) met primary endpoints and laquinimod (quinolone-3-carboxide small molecule-phase II) was also efficacious. Trials using mongersen (SMAD7 inhibitor) and vidofludimus (dihydroorotate dehydrogenase inhibitor) have been halted. CONCLUSIONS: This is potentially the start of an exciting new era in which multiple therapeutic options are at the disposal of physicians to treat IBD on an individualised basis. Head-to-head studies with existing treatments and longer term safety data are needed for this to be possible.

Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells.

Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3alpha (HNF-3alpha), and HNF-3beta regulate the transcription of genes expressed in the respiratory epithelium. To test whether members of the HNF-3/forkhead family influence TTF-1 gene expression, deletion constructs containing the 5' region of the human TTF-1 gene were transfected into immortalized mouse lung epithelial (MLE) cells. DNase I protection and electrophoretic mobility shift assays identified elements in the 5' region of the TTF-1 gene that bound MLE cell nuclear proteins consistent with the binding of HNF-3 to sites at positions -135 to -124 and -14 to -3. In MLE cells, TTF-1-luciferase reporter constructs were activated by cotransfection with HNF-3beta, activated to a lesser extent by HNF-3alpha, but not activated by HFH-8. HNF-3alpha. and HFH-8 inhibited the activation of TTF-1-luciferase by HNF-3beta. Site-specific mutagenesis of each of the HNF-3 binding sites in the human TTF-1 gene inhibited the binding of MLE cell nuclear proteins and inhibited transactivation of the TTF-1-luciferase constructs after cotransfection with HNF-3beta. Immunohistochemical staining demonstrated that both HNF-3beta and TTF-1 were detected in bronchiolar and alveolar type II cells in the human lung. Modulation of TTF-1 gene expression by members of the HNF-3/forkhead family members may provide a mechanism by which distinct HNF-3/forkhead family members influence respiratory epithelial cell gene expression and cell differentiation.

Comparison of a New Intranasal Naloxone Formulation to Intramuscular Naloxone: Results from Hypothesis-generating Small Clinical Studies.

Easy-to-use naloxone formulations are needed to help address the opioid overdose epidemic. The pharmacokinetics of i.v., i.m., and a new i.n. naloxone formulation (2 mg) were compared in six healthy volunteers. Relative to i.m. naloxone, geometric mean (90% confidence interval [CI]) absolute bioavailability of i.n. naloxone was modestly lower (55%; 90% CI, 43-70% vs. 41%; 90% CI, 27-62%), whereas average (±SE) mean absorption time was substantially shorter (74 ± 8.8 vs. 6.7 ± 4.9 min). The opioid-attenuating effects of i.n. naloxone were compared with i.m. naloxone (2 mg) after administration of oral alfentanil (4 mg) to a separate group of six healthy volunteers pretreated with 240 mL of water or grapefruit juice. The i.m. and i.n. naloxone attenuated miosis by similar extents after water (40 ± 15 vs. 41 ± 21 h*%) and grapefruit juice (49 ± 18 vs. 50 ± 22 h*%) pretreatment. Results merit further testing of this new naloxone formulation.

Hydrogen Isotopic Composition of Arctic and Atmospheric CH4 Determined by a Portable Near-Infrared Cavity Ring-Down Spectrometer with a Cryogenic Pre-Concentrator.

In this study, near-infrared continuous wave cavity ring-down spectroscopy was applied to the measurement of the δ2H of methane (CH4). The cavity ring-down spectrometer (CRDS) system consisted of multiple DFB laser diodes to optimize selection of spectral line pairs. By rapidly switching measurements between spectral line peaks and the baseline regions, the long-term instrumental drift was minimized, substantially increasing measurement precision. The CRDS system coupled with a cryogenic pre-concentrator measured the δ2H of terrestrial atmospheric CH4 from 3 standard liters of air with a precision of ±1.7‰. The rapidity with which both C and H isotopic measurements of CH4 can be made with the CRDS will enable hourly monitoring of diurnal variations in terrestrial atmospheric CH4 signatures that can be used to increase the resolution of global climate models for the CH4 cycle. Although the current instrument is not capable of measuring the δ2H of 10 ppbv of martian CH4, current technology does exist that could make this feasible for future spaceflight missions. As biological and abiotic CH4 sources have overlapping carbon isotope signatures, dual-element (C and H) analysis is key to reliable differentiation of these sources. Such an instrument package would therefore offer improved ability to determine whether or not the CH4 recently detected in the martian atmosphere is biogenic in origin. Key Words: Arctic-Hydrogen isotopes-Atmospheric CH4-CRDS-Laser. Astrobiology 16, 787-797.

Hendra Virus Infection Dynamics in the Grey-Headed Flying Fox (Pteropus poliocephalus) at the Southern-Most Extent of Its Range: Further Evidence This Species Does Not Readily Transmit the Virus to Horses.

Hendra virus (HeV) is an important emergent virus in Australia known to infect horses and humans in certain regions of the east coast. Whilst pteropid bats ("flying foxes") are considered the natural reservoir of HeV, which of the four mainland species is the principal reservoir has been a source of ongoing debate, particularly as shared roosting is common. To help resolve this, we sampled a colony consisting of just one of these species, the grey-headed flying fox, (Pteropus poliocephalus), at the southernmost extent of its range. Using the pooled urine sampling technique at approximately weekly intervals over a two year period, we determined the prevalence of HeV and related paramyxoviruses using a novel multiplex (Luminex) platform. Whilst all the pooled urine samples were negative for HeV nucleic acid, we successfully identified four other paramyxoviruses, including Cedar virus; a henipavirus closely related to HeV. Collection of serum from individually caught bats from the colony showed that antibodies to HeV, as estimated by a serological Luminex assay, were present in between 14.6% and 44.5% of animals. The wide range of the estimate reflects uncertainties in interpreting intermediate results. Interpreting the study in the context of HeV studies from states to the north, we add support for an arising consensus that it is the black flying fox and not the grey-headed flying fox that is the principal source of HeV in spillover events to horses.

Myb is required for self-renewal in a model system of early hematopoiesis.

In hematopoiesis, self-renewal, proliferation, differentiation and apoptosis represent opposing decisions made by stem cells and progenitor cells, which when dysregulated can result in leukaemia. Here, we have investigated the function of Myb proteins in regulating these key cellular decisions, using the cell line FDCP-mix A4 as a model of early hematopoiesis. High concentrations of IL-3 in these cells favour self-renewal over differentiation and apoptosis. However when endogenous Myb activity was inhibited with an inducible dominant interfering protein, self-renewal was replaced by apoptosis and differentiation. Differentiation was to granulocytes and monocyte/macrophages and was closely associated with a G1-S phase block in the cell cycle. As for normal hematopoiesis, cytokine-induced terminal differentiation of FDCP-mix cells is associated with concomitant proliferation prior to its completion. However, when Myb activity was inhibited during this process, proliferation and survival were both reduced, resulting in a much lower yield of mature cells. These results indicate multiple cellular roles of Myb proteins during normal hematopoiesis.

Changes in streptonigrin lethality during adaptation of Escherichia coli to picolinic acid. Correlation with intracellular picolinate and iron uptake.

Uptake studies with [14C]picolinate and 55Fe3+ have provided an explanation for the change in streptonigrin killing on adaptation of Escherichia coli to picolinate, in terms of the available iron within the cell. When picolinic acid is added to a growing culture of E. coli an interval of bacteriostasis ensues; this adaptation period is followed by resumption of exponential growth. Addition of picolinate (4 mM) to a log phase culture of strain W3110 gave protection from the lethal action of streptonigrin (30 microM) when the two agents were added simultaneously. In contrast streptonigrin killed cells that had adapted to picolinate; however, a preincubation of adapted W3110 with phenethyl alcohol protected the cells from streptonigrin lethality. [14C]Picolinate uptake studies showed that initially picolinate entered the cells, but that it was excluded from adapted cells; addition of phenethyl alcohol permitted the entry of picolinate into adapted W3110. The changes in streptonigrin killing parallel the changes in concentration of intracellular picolinate, which can chelate the iron required by streptonigrin for its bactericidal action. 55Fe3+ uptake studies showed that initially picolinate prevented iron accumulation by strain W3110, whereas adapted cells did take up iron in the presence of picolinate. Addition of phenethyl alcohol prevented any observed uptake of iron by adapted W3110. This modulation of iron transport by picolinate also affects streptonigrin lethality. Experiments with iron transport mutants showed that picolinate acted on both the enterochelin and citrate routes of uptake. Therefore picolinate affects the concentration of available iron within the cell both by (a) its intracellular presence resulting in chelation of iron and (b) its action on iron uptake; these effects explain the change in streptonigrin killing on adaptation of E. coli to picolinate.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Quantitative prediction and clinical evaluation of an unexplored herb-drug interaction mechanism in healthy volunteers.

Quantitative prediction of herb-drug interaction risk remains challenging. A quantitative framework to assess a potential interaction was used to evaluate a mechanism not previously tested in humans. The semipurified milk thistle product, silibinin, was selected as an exemplar herbal product inhibitor of raloxifene intestinal glucuronidation. Physiologically based pharmacokinetic (PBPK) model simulations of the silibinin-raloxifene interaction predicted up to 30% increases in raloxifene area under the curve (AUC0-inf) and maximal concentration (Cmax). Model-informed clinical evaluation of the silibinin-raloxifene interaction indicated minimal clinical interaction liability, with observed geometric mean raloxifene AUC0-inf and Cmax ratios lying within the predefined no effect range (0.75-1.33). Further refinement of PBPK modeling and simulation approaches will enhance confidence in predictions and facilitate generalizability to additional herb-drug combinations. This quantitative framework can be used to develop guidances to evaluate potential herb-drug interactions prospectively, providing evidenced-based information about the risk or safety of these interactions.

Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 alpha and HIF-2 alpha in a primary neuronal model of hypoxia.

The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.