BACKGROUND

Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data.

RESULTS

A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results.

CONCLUSIONS

Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.

Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L-isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory-grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature-responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets.

Transplantable cell sheets containing osteoblasts were fabricated from periostea on temperature-responsive culture dishes. This study demonstrated the time-course of bone regeneration in living small animals. This continuous observation of bone regeneration was achieved by micro-computed tomography (µCT), which assessed the osteogenic capability of periosteal cells without biodegradable scaffolds. Real-time bone regeneration was non-invasively monitored in a rat calvarial bone defect model, using µCT. Three-dimensional (3D) images obtained over time by µCT clearly showed that two different bone regeneration modes, specific to the control and experimental groups, were observed. In the control group, bone was regenerated only from the periphery of the defect edges. In the experimental group, bone regeneration was observed in several small regions within the central portions of the defects that were covered by the transplanted cell sheets. However, bone regeneration observed after periosteal cell sheet transplantation was limited. The results of ALP staining and the time-course observations concluded that periosteal cell sheets contained a small fraction of cells that could differentiate osteoblasts. Fibroblasts in transplanted cell sheets or from around subcutaneous tissues suppressed bone regeneration. The periosteal cell sheets had a capability to produce ectopic regenerated bones. Therefore, to increase the content of osteogenic cells in harvested cell sheets, the enrichment of cells that could produce osteoblasts was expected by the modification of the initial cell preparation and the culture conditions. With further possible improvements, scaffold-free periosteal cell sheet fabricated on temperature-responsive culture dishes will be a valuable method for inducing and accelerating bone regeneration.

BACKGROUND

Compared with controls, human immunodeficiency virus (HIV)-infected persons have a greater prevalence of kidney disease, assessed according to high cystatin C level and albuminuria, but not according to creatinine level. However, the clinical importance of increased cystatin C level and albuminuria in the HIV-infected population has not been studied.

STUDY DESIGN

We conducted an observational cohort study to determine the association of kidney disease (measured according to albuminuria, cystatin C, and serum creatinine) with mortality.

SETTING & PARTICIPANTS

922 HIV-infected persons enrolled in the FRAM (Fat Redistribution and Metabolic Change in HIV Infection) Study.

PREDICTOR

Serum cystatin C and serum creatinine levels were used to estimate glomerular filtration rates (eGFR(SCysC) and eGFR(SCr), respectively). Albuminuria was defined as a positive urine dipstick result (≥ 1+) or urine albumin-creatinine ratio >30 mg/g.

OUTCOME

5-Year mortality.

RESULTS

At baseline, decreased kidney function (eGFR(SCysC) <60 mL/min/1.73 m(2)) or albuminuria was present in 28% of participants. After 5 years of follow-up, mortality was 48% in those with both eGFR(SCysC) < 60 mL/min/1.73 m(2) and albuminuria, 23% in those with eGFR(SCysC) < 60 mL/min/1.73 m(2) alone, 20% in those with albuminuria alone, and 9% in those with neither condition. After multivariable adjustment for demographics, cardiovascular risk factors, HIV-related factors, and inflammatory marker levels, eGFR(SCysC) < 60 mL/min/1.73 m(2) and albuminuria were associated with a nearly 2-fold increase in mortality, whereas eGFR(SCr) < 60 mL/min/1.73 m(2) did not appear to have a substantial association with mortality. Together, eGFR(SCysC) <60 mL/min/1.73 m(2) and albuminuria accounted for 17% of the population-level attributable risk of mortality.

LIMITATIONS

Vital status was unknown in 261 participants from the original cohort.

CONCLUSIONS

Kidney disease marked by albuminuria or increased cystatin C level appears to be an important risk factor for mortality in HIV-infected individuals. A substantial proportion of this risk may be unrecognized because of the current reliance on serum creatinine to estimate kidney function in clinical practice.

Based on in vitro rat and human hepatocyte uptake studies showing inhibition of warfarin uptake in the presence of the nonspecific organic anion-transporting polypeptide (OATP) inhibitor rifampin, a clinical study was conducted in 10 healthy volunteers to examine the in vivo relevance of OATP hepatic uptake on the pharmacokinetics of warfarin. In a randomized, single-dose, two-period, crossover design, subjects received a 7.5-mg dose of warfarin, either alone or immediately following a 600-mg intravenous dose of rifampin. Rifampin did not significantly alter the R- or S-warfarin area under the concentration-time curves (AUCs) from 0 to 12 h (period of hepatic OATP inhibition by rifampin) or the maximum plasma concentration (C(max)) value. AUC(0-∞) was decreased on days rifampin was administered, for both R-warfarin (25% reduction; P < 0.001) and S-warfarin (15% reduction; P < 0.05). No differences were seen in the area under the international normalized ratio (INR)-time curve. Our study suggests that hepatic uptake via OATPs may not be clinically important in the pharmacokinetics of warfarin.