Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis.

The membrane glycoprotein CD36 binds nanomolar concentrations of long chain fatty acids (LCFA) and is highly expressed on the luminal surface of enterocytes. CD36 deficiency reduces chylomicron production through unknown mechanisms. In this report, we provide novel insights into some of the underlying mechanisms. Our in vivo data demonstrate that CD36 gene deletion in mice does not affect LCFA uptake and subsequent esterification into triglycerides by the intestinal mucosa exposed to the micellar LCFA concentrations prevailing in the intestine. In rodents, the CD36 protein disappears early from the luminal side of intestinal villi during the postprandial period, but only when the diet contains lipids. This drop is significant 1 h after a lipid supply and associates with ubiquitination of CD36. Using CHO cells expressing CD36, it is shown that the digestion products LCFA and diglycerides trigger CD36 ubiquitination. In vivo treatment with the proteasome inhibitor MG132 prevents the lipid-mediated degradation of CD36. In vivo and ex vivo, CD36 is shown to be required for lipid activation of ERK1/2, which associates with an increase of the key chylomicron synthesis proteins, apolipoprotein B48 and microsomal triglyceride transfer protein. Therefore, intestinal CD36, possibly through ERK1/2-mediated signaling, is involved in the adaptation of enterocyte metabolism to the postprandial lipid challenge by promoting the production of large triglyceride-rich lipoproteins that are rapidly cleared in the blood. This suggests that CD36 may be a therapeutic target for reducing the postprandial hypertriglyceridemia and associated cardiovascular risks.

Fatty acid- and cholesterol transporter protein expression along the human intestinal tract.

BACKGROUND: Protein distribution profiles along the human intestinal tract of transporters involved in the absorption of cholesterol and long-chain fatty acids (LCFA) have been scarcely evaluated. METHODOLOGY/PRINCIPAL FINDINGS: In post-mortem samples from 11 subjects, intestinal transporter distribution profiles were determined via Western Blot. Differences in transporter protein levels were statistically tested using ANOVA and Tukey's Post Hoc comparisons. Levels in all segments were expressed relative to those in duodenum. Except for ABCG5 and FATP4, levels (mean+/-SEM) were the highest in the ileum. For ABCA1, ileal levels (1.80+/-0.26) differed significantly from those in duodenum (P = 0.049) and proximal colon (0.92+/-0.14; P = 0.029). ABCG8 levels in ileum (1.91+/-0.30) differed from those in duodenum (P = 0.041) and distal colon (0.84+/-0.22; P = 0.010) and jejunum (1.64+/-0.26) tended to be higher than distal colon (0.84+/-0.22; P = 0.087). Ileal NPC1L1 levels (2.56+/-0.51) differed from duodenum levels (P = 0.019) and from distal colon (1.09+/-0.22; P = 0.030). There was also a trend (P = 0.098) for higher jejunal (2.23+/-0.37) than duodenal NPC1L1 levels. The levels of ABCG5 did not correlate with those of ABCG8. FAT/CD36 levels in ileum (2.03+/-0.42) differed from those in duodenum (P = 0.017), and proximal and distal colon (0.89+/-0.13 and 0.97+/-0.15 respectively; P = 0.011 and P = 0.014). FABPpm levels in ileum (1.04+/-0.13) differed from proximal (0.64+/-0.07; P = 0.026) and distal colon (0.66+/-0.09; P = 0.037). CONCLUSIONS/SIGNIFICANCE: The distribution profiles showed a bell-shape pattern along the GI-tract with the highest levels in ileum for ABCA1, ABCG8, NPC1L1, FATCD36 and FABPm, suggesting a prominent role for ileum in transporter-mediated uptake of cholesterol and LCFAs.

Cardiac dysfunction and cell damage across clinical stages of severity in growth-restricted fetuses.

OBJECTIVE: The purpose of this study was to assess cardiac function and cell damage in intrauterine growth-restricted (IUGR) fetuses across clinical Doppler stages of deterioration. STUDY DESIGN: One hundred twenty appropriate-for-gestational-age and 81 IUGR fetuses were classified in stages 1/2/3 according umbilical artery present/absent/reversed end-diastolic blood flow, respectively. Cardiac function was assessed by modified-myocardial performance index, early-to-late diastolic filling ratios, cardiac output, and cord blood B-type natriuretic peptide; myocardial cell damage was assessed by heart fatty acid-binding protein, troponin-I, and high-sensitivity C-reactive protein. RESULTS: Modified-myocardial performance index, blood B-type natriuretic peptide, and early-to-late diastolic filling ratios were increased in a stage-dependent manner in IUGR fetuses, compared with appropriate-for-gestational-age fetuses. Heart fatty acid-binding protein levels were higher in IUGR fetuses at stage 3, compared with control fetuses. Cardiac output, troponin-I, and high-sensitivity C-reactive protein did not increase in IUGR fetuses at any stage. CONCLUSION: IUGR fetuses showed signs of cardiac dysfunction from early stages. Cardiac dysfunction deteriorates further with the progression of fetal compromise, together with the appearance of biochemical signs of cell damage.

Fatty acid-binding protein as marker for renal injury.

The assessment of biomarkers to detect renal injury has been studied before, but the sensitivity and specificity of urinary and serum profiles of these markers still need to be improved. One of the promising new markers for detection of renal injury is the family of 15 kDa cytoplasmic fatty acid-binding proteins (FABPs). Remarkably, however, the application of FABP as marker for renal injury due to ischaemia, toxic heavy metals or in end-stage renal failure has only recently been investigated. The present status of two types of FABP in the kidney (heart-type (H-FABP), located in the distal tubular cells, and liver-type (L-FABP), which is located in the proximal tubular cells), is reviewed for sensitive detection of renal injury. This review is based on an overview of the literature on clinical diagnostics applying plasma and urinary H-FABP as well as L-FABP levels in renal toxicity, kidney transplantation or as clinical parameter in end-stage renal failure. H-FABP has been shown to be a proper marker for detection of ischaemic injury in tissue perfusates of non-heart-beating donor kidneys. Urinary L-FABP has been evaluated recently more explicitly and shows significant elevations in progressive end-stage renal failure as well as after ischaemic injury due to renal transplantation or cardiac bypass surgery. H- and L-FABP have been shown to be useful markers for rapid detection and monitoring of renal injury. Further study of the diagnostic and prognostic use of these FABP types will require further commercialization of automated and rapid assays for proper clinical application.

The role of membrane fatty-acid transporters in regulating skeletal muscle substrate use during exercise.

While endogenous carbohydrates form the main substrate source during high-intensity exercise, long-chain fatty acids (LCFA) represent the main substrate source during more prolonged low- to moderate-intensity exercise. Adipose tissue lipolysis is responsible for the supply of LCFA to the contracting muscle. Once taken up by skeletal muscle tissue, LCFA can either serve as a substrate for oxidative phosphorylation or can be directed towards esterification into triacylglycerol. Myocellular uptake of LCFA comprises a complex and incompletely understood process. Although LCFA can enter the cell via passive diffusion, more recent reports indicate that LCFA uptake is tightly regulated by plasma membrane-located transport proteins (fatty acid translocase [FAT/CD36], plasmalemmal-located fatty acid binding protein [FABPpm] and fatty acid transport protein [FATP]). Depending on cardiac and skeletal muscle energy demands, some of these LCFA transporters can translocate rapidly from intracellular pools to the plasma membrane to allow greater LCFA uptake. This translocation process can be induced by insulin and/or muscle contraction. However, the precise signalling pathways responsible for activating the translocation machinery remain to be elucidated. This article will provide an overview on the effects of diet, acute exercise and exercise training on the expression and/or translocation of the various LCFA transporters in skeletal muscle tissue (FAT/CD36, FABPpm, FATP).

Plasma heart-type fatty acid binding protein is superior to troponin and myoglobin for rapid risk stratification in acute pulmonary embolism.

BACKGROUND: Irreversible right ventricular (RV) failure with myocardial damage may precipitate fatal outcome in acute pulmonary embolism (APE). Cytoplasmic heart-type fatty acid binding protein (H-FABP) is a sensitive and specific biomarker of myocardial damage. We assessed which biomarker of myocardial damage or RV stretching is the most useful for short-term risk stratification in APE. METHODS: We analyzed 77 patients (51 F, 26 M) aged 65.3+/-16.0 years with confirmed APE. On admission, systemic blood pressure and transthoracic echocardiography (for RV overload) were recorded and plasma concentrations of myoglobin (Mb), cardiac troponin T (cTnT), N-terminal fragment of proBNP (NT-proBNP) and H-FABP were evaluated. RESULTS: Fifteen (19.5%) patients died and 24 (31.2%) experienced complicated clinical course (CCC)-death/thrombolysis/cardiopulmonary resuscitation/intravenous vasopressors. Hazard ratio analysis demonstrated that plasma H-FABP, Mb, cTnT and NT-proBNP concentrations predicted fatal outcome. When only APE-related deaths were considered, plasma H-FABP concentrations indicated fatal outcome. Multivariate hazard ratio analysis revealed H-FABP as the only 30-day mortality predictor (HR 1.02 CI 95% 1.01-1.05). CONCLUSIONS: H-FABP measured on admission is useful for short-term risk stratification in APE. It appears to be superior to cTnT, NT-proBNP and Mb in the prediction of 30-day APE-related mortality.

Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36).

Evidence is accumulating that the heavily glycosylated integral membrane protein fatty acid translocase (FAT/CD36) is involved in the transport of long-chain fatty acids across the sarcolemma of heart muscle cells. The aim of this study was to analyse the distribution between FAT/CD36 present in cardiac myocytes and endothelial cells. We therefore developed a method to purify FAT/CD36 from total rat heart and isolated cardiomyocytes, and used the proteins as standards in an immunochemical assay. Two steps, chromatography on wheat germ agglutinin-agarose and anion-exchange chromatography on Q-Sepharose fast flow, were sufficient for obtaining the protein in a > 95% pure form. When used to isolate FAT/CD36 from total heart tissue, the FAT/CD36 yield of the method was 9% and the purification factor was 64. Purifying FAT/CD36 from isolated cardiomyocytes yielded the same 88 kDa protein band on SDS-PAGE gels and reactivity of this band on western blots was comparable to that of the FAT/CD36 isolated from total hearts. Quantifying FAT/CD36 contents by western blotting showed that the amounts of FAT/CD36 that are present in isolated cardiomyocytes (10 +/- 3 microg/mg protein) and total hearts (14 +/- 4 microg/mg protein) are of comparable magnitude. Immunofluorescence labelling showed that at least a part of the FAT/CD36 present in the cardiomyocyte is associated with the sarcolemma. This study established that FAT/CD36 is a relatively abundant protein in the cardiomyocyte. In addition, the further developed purification procedure is the first method for isolating FAT/CD36 from rat heart and cardiomyocyte FAT/CD36.

Unbound free fatty acids and heart-type fatty acid-binding protein: diagnostic assays and clinical applications.

BACKGROUND: A biomarker that reliably detects myocardial ischemia in the absence of necrosis would be useful for initial identification of unstable angina patients and for differentiating patients with chest pain of an etiology other than coronary ischemia, and could provide clinical utility complementary to that of cardiac troponins, the established markers of necrosis. Unbound free fatty acids (FFA(u)) and their intracellular binding protein, heart-type fatty acid-binding protein (H-FABP), have been suggested to have clinical utility as indicators of cardiac ischemia and necrosis, respectively. METHODS: We examined results of clinical assessments of FFA(u) and H-FABP as biomarkers of cardiac ischemia and necrosis. Data published on FFA(u) and H-FABP over the past 30 years were used as the basis for this review. RESULTS: Although little clinical work has been done on FFA(u) since the initial reports, recent studies documented an association between increased serum FFAs and ventricular dysrhythmias and death in patients with acute myocardial infarction (AMI). Recent data suggest that serum FFA(u) concentrations increase well before markers of cardiac necrosis and are sensitive indicators of ischemia in AMI. H-FABP is abundant in cardiac muscle and is presumed to be involved in myocardial lipid homeostasis. Similar to myoglobin, plasma H-FABP increases within 3 h after AMI and returns to reference values within 12-24 h. CONCLUSIONS: FFA(u) may have a potential role in identifying patients with cardiac ischemia. H-FABP is useful for detecting cardiac injury in acute coronary syndromes and predicting recurrent cardiac events in acute coronary syndromes and in congestive heart failure patients. Assays are available for both markers that could facilitate further clinical investigations to assess their possible roles as markers of cardiac ischemia and/or necrosis.

Detection of brain injury by fatty acid-binding proteins.

The rapid detection of brain injury (neuronal damage in general) is an important parameter in the management of cerebrovascular accidents, especially in hemorrhagic and/or ischemic events. Two types of 15-kDa cytoplasmic fatty acid-binding proteins (FABPs), brain-type FABP and heart-type FABP, have recently been postulated as novel markers for brain injury detection. Here we review the possible roles of these FABPs as rapid diagnostic markers for the detection of brain injury due to cerebrovascular accident, trauma or neurodegenerative diseases. The occurrence of brain- and heart-type FABPs in segments of the human brain is also described. Although only limited amounts of data are available, brain- and heart-type FABPs show higher sensitivities and specificities than protein S100 and neuron specific enolase in the rapid detection of brain injury in stroke, trauma and neurodegenerative diseases.

Rapid analysis of fatty acid-binding proteins with immunosensors and immunotests for early monitoring of tissue injury.

Fatty acid-binding protein (FABP) holds promise for early detection of tissue injury. This small protein (15kD) appears earlier in the blood than large proteins after cell damage. Combined its characteristics of high concentration tissue contents and low normal plasma values provide the possibility of a rapid rise above the respective reference values, and thus an early indication of the appearance of tissue injury. A general review was presented on the current status of different types of FABP for the detection of tissue injury in patients with myocardial injury, brain injury and also in athletes or horses with skeletal muscle injury. To take full advantage of the characteristics of the early marker FABP, rapid analysis is a crucial parameter. In this review, an overview of the development of immunoassay for the quantification of FABP in buffer, plasma or whole blood was outlined. The characteristics of different FABP immunosensors and immunotests were described. The feasibility of these immunoassays to be used in routine clinical practice and in emergency case was also discussed. Nowadays, the improved automated immunoassays (e.g. a microparticle-enhanced turbidimetric immunoassay), less time-consuming bedside immunosensors and immunotests (e.g. a one-step FABP lateral flow immunotest), are the main advance technology in point-of-care testing. With these point-of-care tests, the application of FABP as an early tissue injury marker has a great potential for many clinical purposes.

Release of brain-type and heart-type fatty acid-binding proteins in serum after acute ischaemic stroke.

This study aimed at an analysis of the release of Braintype and Heart-type Fatty Acid- Binding Proteins (B-FABP and HFABP) in acute ischaemic stroke and their potential value as neurobiochemical markers of brain damage. We investigated 42 consecutive patients admitted within 6 hours after ischaemic stroke. Serial venous blood samples were taken hourly between 1 to 6 hours, and at 12, 18, 24, 48, 72, 96, and 120 hours after stroke onset. In all patients lesion topography was assessed and infarct volume was calculated. The neurological deficit was quantified by the National Institutes of Health stroke scale score, and functional outcome was assessed with the modified Rankin Scale 3 months after stroke. H-FABP and B-FABP concentrations showed peak values already 2 to 3 hours after stroke onset and remained elevated up to last measurements at 120 hours.Unlike BFABP, early H-FABP concentrations were significantly associated with the severity of the neurological deficit and the functional outcome. High H-FABP release was associated with large infarction on CT. Our study shows for the first time quantitative data of serum BFABP and H-FABP being elevated early in acute ischaemic stroke indicating that especially H-FABP might have the potential to be a rapid marker of brain damage and clinical severity. As both FABPs indicate damage to neuronal and glial tissue but are not specific for cerebral infarction, further investigations are needed to better understand the prolonged release of both in ischaemic stroke which is in contrast to the transient increase after myocardial infarction and can not be explained by their renal extraction.

Fatty acid-binding proteins as plasma markers of tissue injury.

BACKGROUND: One of the novel and promising plasma markers for detection of tissue injury is the family of 15 kDa cytoplasmic fatty acid-binding proteins of which various tissue-specific types occur. AIMS AND OBJECTIVES: The present status of heart-type fatty acid-binding protein (H-FABP) as a diagnostic and prognostic marker for acute and chronic cardiac injury, as well as the preliminary diagnostic use of other types of FABP for detecting injury in other organs, is reviewed. METHODS: This review is based on an overview of the literature on clinical diagnostics of various forms of organ injury, and uses additional literature on physiological aspects relevant for the interpretation of plasma marker concentrations. RESULTS: H-FABP not only proves to be an excellent early marker for cardiac injury in acute coronary syndromes, but also allows detection of minor myocardial injury in heart failure and unstable angina. Preliminary results indicate that sensitivity, rule-out power and prognostic value of H-FABP in cardiac injury surpass the performance of the standard early marker myoglobin. The liver only contains liver-type FABP (L-FABP), but co-expression of H-FABP and L-FABP occurs in the kidney. Similarly, intestinal-type FABP (I-FABP) and L-FABP are found in intestines, and brain-type FABP (B-FABP) and H-FABP occur in the brain. Preliminary but promising applications of these proteins have been demonstrated for liver rejection, viability selection of kidneys from non-heart-beating donors (NHBD), inflammatory and ischemic bowel disease, traumatic brain injury and in the prevention of muscle injury in trained athletes. CONCLUSIONS: Further study of the diagnostic and prognostic use of various FABP types is warranted, but their clinical application will require further commercialization of automated and rapid assays.

Brain- and heart-type fatty acid-binding proteins in the brain: tissue distribution and clinical utility.

BACKGROUND: Detection of brain injury by serum markers is not a standard procedure in clinical practice, although several proteins, such as S100B, neuron-specific enolase (NSE), myelin basic protein, and glial fibrillary acidic protein, show promising results. We investigated the tissue distribution of brain- and heart-type fatty acid-binding proteins (B-FABP and H-FABP) in segments of the human brain and the potential of either protein to serve as plasma marker for diagnosis of brain injury. METHODS: B-FABP and H-FABP were measured immunochemically in autopsy samples of the brain (n = 6) and in serum samples from (a) patients with mild traumatic brain injury (MTBI; n = 130) and (b) depressed patients undergoing bilateral electroconvulsive therapy (ECT; n = 14). The protein markers S100B and NSE were measured for comparison. Reference values of B-FABP and H-FABP were established in healthy individuals (n = 92). RESULTS: The frontal, temporal, and occipital lobes, the striatum, the pons, and the cerebellum had different tissue concentrations of B-FABP and of H-FABP. B-FABP ranged from 0.8 microg/g wet weight in striatum tissue to 3.1 microg/g in frontal lobe. H-FABP was markedly higher, ranging from 16.2 microg/g wet weight in cerebellum tissue to 39.5 microg/g in pons. No B-FABP was detected in serum from healthy donors. H-FABP serum reference value was 6 microg/L. In the MTBI study, serum B-FABP was increased in 68% and H-FABP in 70% of patients compared with S100B (increased in 45%) and NSE (increased in 51% of patients). In ECT, serum B-FABP was increased in 6% of all samples (2 of 14 patients), whereas H-FABP was above its upper reference limit (6 microg/L) in 17% of all samples (8 of 14 patients), and S100B was above its upper reference limit (0.3 microg/L) in 0.4% of all samples. CONCLUSIONS: B-FABP and H-FABP patterns differ among brain tissues, with the highest concentrations in the frontal lobe and pons, respectively. However, in each part of the brain, the H-FABP concentration was at least 10 times higher than that of B-FABP. Patient studies indicate that B-FABP and H-FABP are more sensitive markers for minor brain injury than the currently used markers S100B and NSE.

Intestinal-type and liver-type fatty acid-binding protein in the intestine. Tissue distribution and clinical utility.

OBJECTIVES: Intestinal-type fatty acid-binding protein (I-FABP) has been proposed as plasma marker for the detection of acute intestinal injury. However, intestinal mucosa also expresses liver-type FABP (L-FABP). We have investigated the tissue distribution of I-FABP and L-FABP in segments of the human intestine along the duodenal to colonal axis and the potential of both proteins to serve as plasma marker for the diagnosis of intestinal injury. DESIGN AND METHODS: I-FABP and L-FABP were measured with specific immunoassays in autopsy samples of the intestine (duodenum, jejunum, ileum and colon) of 23 subjects and in plasma samples from patients (n = 51) with intestinal and/or hepatic disease. Plasma reference values were established in normal healthy individuals (n = 92). RESULTS: The I-FABP tissue contents in duodenum, jejunum, ileum, proximal colon and distal colon amounted to 2.22, 4.79, 1.04, 0.27 and 0.25 mug/g ww, respectively. L-FABP tissue contents were markedly higher, amounting to 124 and 198 mug/g ww in duodenum and jejunum, and to 58, 26 and 44 mug/g ww in ileum, proximal colon and distal colon, respectively. Elevated plasma levels of both I-FABP and L-FABP were found in patients suffering from intestinal diseases, while only L-FABP was increased in cases of purely hepatocellular injury. CONCLUSIONS: I-FABP and L-FABP show a similar pattern of tissue distribution along the duodenal to colonal axis with highest tissue contents found in the jejunum but in each intestinal segment a >40-fold higher content of L-FABP than of I-FABP. Accordingly, besides I-FABP, also L-FABP is a useful plasma marker for the detection of intestinal injury, especially in patients undergoing intestinal surgery.

Human dendritic cells transfected with renal tumor RNA stimulate polyclonal T-cell responses against antigens expressed by primary and metastatic tumors.

Although renal cell carcinoma has been shown to respond to immunotherapy, renal cell carcinoma-specific rejection antigens and their corresponding CTL epitopes have rarely been described. The use of dendritic cells (DCs) transfected with mRNA isolated from tumor cells may allow specific immunotherapy even in cancers for which potent rejection antigens have not been identified. Here we show that DCs transfected with RNA isolated from renal cancer tissue are remarkably effective in stimulating tumor-specific T-cell response in vitro but do not cross-react with normal tissue antigens including antigens expressed by renal parenchyma. In contrast, the tumor-specific CTLs lysed allogeneic tumor but not allogeneic normal tissue targets, suggesting the presence of shared albeit unidentified antigens among renal carcinomas. CTL responses against telomerase reverse transcriptase (TERT) accounted in part for the reactivities against allogeneic tumors because renal tumor RNA-transfected DCs stimulated polyclonal CTL responses, which encompassed as a subcomponent a response against TERT. Nonetheless, the tumor-specific CTLs were consistently superior to the CTLs stimulated with TERT RNA-transfected DCs in recognizing and lysing tumor targets, suggesting that tumor-specific CTLs represent a polyclonal response providing more effective antitumor activity than T-cell responses directed against a single antigen in the form of TERT. Tumor RNA-transfected DCs were capable of stimulating T-cell reactivities not only against the primary tumor but also against metastatic tumors, although discrete differences in the antigenic repertoire expressed by these tissues were apparent. Thus, total tumor RNA-transfected DCs may represent a broadly applicable vaccine strategy to induce polyclonal and potentially therapeutic T-cell responses in renal cancer patients.

Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA.

Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.

Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro.

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.

Initial interlaboratory validation study of FETAX: phase I testing.

An interlaboratory validation study was undertaken to evaluate the repeatability and reliability of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), which is a whole embryo developmental toxicity screening assay. A three-phase experimental program with seven participants was carried out. Phase I was a training and protocol evaluation phase where the identity of the three test materials was known. Hydroxyurea, isoniazid and 6-aminonicotinamide were tested in Phase I. Because the chemicals has been tested previously in FETAX, the same concentrations needed to establish the 96-h median lethal concentration (LC50) and the concentration inducing malformations in 50% of the surviving embryos (EC50) were used by all laboratories. The results of Phase I are presented in this report, and FETAX has proved to be as repeatable and reliable as many other bioassays. Some excess variation was observed in individual laboratories. Some of this variation may have been due to training difficulties. One change in protocol design necessitated by this study was the use of 6-aminonicotinamide as a reference toxicant. While 6-aminonicotinamide provided excellent concentration-response data in most laboratories, the protocol was written too strictly based on historical FETAX data. Phases II and III are currently in progress.