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Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine–glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2(ne/ne) and 1% of Fras1(Q1263*/Q1263*) mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

Head and neck cancer patients taking NSAIDs with PIK3CA tumor alterations demonstrate improved survival. Studies in relevant preclinical models implicate signaling via COX2-mediated production of PGE2 as an underlying mechanism for this survival benefit., PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA-characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09–0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14–0.69) compared with nonregular NSAID users. For PIK3CA-altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

Deletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4. This article has an associated First Person interview with the first author of the paper., Summary: In the developing atrioventricular canal, RERE promotes endothelial-to-mesenchymal transition and mesenchymal cell proliferation by positively regulating Gata4. Tissue-specific ablation of Rere in the endocardium causes ventricular septal defects.

Efficient clearance of apoptotic cells (AC) is pivotal in preventing autoimmunity and is a potent immunosuppressive stimulus. However, activation of cells prior to apoptosis abolishes their immunoregulatory properties. Here we show using the antigen-induced model of arthritis that the degree of DNA methylation within AC confers their immunomodulatory plasticity. DNA isolated from resting and activated AC mimicked their respective immune effects. Demethylation of DNA abrogated the protective effect of AC whereas remethylation of AC DNA reversed the effects of activation and restored the ability to inhibit inflammation. Disease suppression or lack thereof was associated with TGFβ and IL-6 production respectively. Apoptotic CD4+ T cells from patients with rheumatoid arthritis and systemic lupus erythematosus were demethylated compared to healthy controls and favoured production of IL-6 when cultured with healthy macrophages, in contrast to the TGFβ produced in response to healthy AC. Our data implicate AC DNA methylation as the molecular switch that imprints their regulatory properties.

The high immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were efficiently suppressed by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of 6-, 8- and 10-amino acid insertions. Not only do MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, but they also encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.

The mechanisms underlying insufficient insulin secretion and loss of beta-cell mass in feline and human type 2 diabetes mellitus are incompletely understood. However, islet amyloid polypeptide (IAPP)-derived islet amyloidosis (IA) has been linked to increased rates of beta-cell apoptosis and, therefore, our goal was to develop an in vitro model of IAPP fibrillogenesis using isolated pancreatic islets from mice transgenic for human IAPP (hIAPP Tg mice). Islets from hIAPP Tg mice, from mice transgenic for non-amyloidogenic murine IAPP (mIAPP Tg mice), and from the FVB background strain were exposed to normal (5.5 mM) or high (28 mM) glucose conditions in cell culture for 8 days. On days 0 and 8, islets were collected for electron microscopy (EM). EM showed no abnormalities in the mIAPP Tg or FVB islets at either time point. On day 8, hIAPP Tg islets cultured at high glucose concentration formed extracellular IAPP-derived flocculent deposits. No significant differences in rates of apoptosis were found between groups. Our findings, therefore, show that in vitro culture of hIAPP Tg mouse islets under high glucose conditions produces a readily available and rapidly inducible model of IAPP-derived fibrillogenesis and enables the study of early phases of the molecular pathogenesis of IA.

Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.

Targeted delivery of intravenously administered genetically altered cells or stem cells is still in an early stage of investigation. We developed a method of delivering iron oxide (ferumoxide)-labeled mesenchymal stem cells (MSCs) to a targeted area in an animal model by applying an external magnet. Rats with or without an external magnet placed over the liver were injected intravenously with ferumoxide-labeled MSCs and magnetic resonance imaging (MRI) signal intensity (SI) changes, iron concentration, and concentration of MSCs in the liver were monitored at different time points. SI decreased in the liver after injection of MSCs and returned gradually to that of control rat livers at approximately day 29. SI decreases were greater in rats with external magnets. Higher iron concentration and increased labeled cell numbers were detected in rat livers with external magnets. The external magnets influenced the movement of labeled MSCs such that the cells were retained in the region of interest. These results potentially open a new area of investigation for delivering stem cells or genetically altered cells.

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease with early lesions characterized by mononuclear cellular infiltrate, edema, demyelination, and axonal loss that contribute to the clinical course of the disease. Experimental autoimmune encephalomyelitis (EAE) in the mouse is a valuable model with a similar disease course to relapsing-remitting MS. The ability to detect the migration of encephalitogenic T cells into the central nervous system in EAE and MS would provide key information on these cells role in the development of lesions observed on magnetic resonance imaging (MRI). T cells were labeled for detection by magnetic resonance imaging using Food and Drug Administration-approved, superparamagnetic iron oxide nanoparticles (Ferumoxides) complexed to poly-L-Lysine (FE-PLL). EAE was induced by adoptive transfer of either labeled or unlabeled T cells. After disease onset, FE-PLL-labeled T cells were detected in the mouse spinal cord using in vivo and ex vivo cellular MRI. Excellent correlation was seen between MRI-visible lesions in the spinal cord and histopathology. The results demonstrate that T cells labeled with FE-PLL can induce EAE disease and can be detected in vivo in the mouse model. The magnetic labeling of cells opens the possibility of monitoring specific cellular phenotypes or pharmacologically or genetically engineered cells by MRI.

Phagocytosis of apoptotic cells (ACs) is usually a potent immunoregulatory signal but can also promote inflammation. In this article, we show that administration of apoptotic dendritic cells (DCs) inhibited inflammation in vivo through increasing production of TGF-β from intrinsic DCs and B cells. However, ACs derived from LPS-activated DCs failed to restrain inflammation because of a short-lived but marked IL-6 response, which abolished the increase in TGF-β. Inhibition of IL-6 restored the protective anti-inflammatory properties of aACs and the TGF-β response. DCs isolated from mice that had received resting but not activated ACs could transfer the suppression of inflammation to recipient mice. These transferred DCs stimulated B cell TGF-β production and relied on an intact B cell compartment to limit inflammation. These results highlight how the activation state of AC governs their ability to control inflammation through reciprocal regulation of IL-6 and TGF-β.

HEY2 is a basic helix-loop-helix (bHLH) transcription factor that plays an important role in the developing mammalian heart and brain. In humans, nonsynonymous mutations in HEY2 have been described in patients with atrial ventricular septal defects, and a subset of individuals with chromosomal deletions involving HEY2 have cardiac defects and cognitive impairment. Less is known about the potential effects of HEY2 overexpression. Here, we describe a female child with tetralogy of Fallot who developed severe right ventricular outflow tract obstruction due to a combination of infundibular and valvular pulmonary stenosis. She was also noted to have hypotonia, lower extremity weakness, fine motor delay and speech delay. A copy number variation (CNV) detection analysis followed by real-time quantitative PCR analysis revealed a single gene duplication of HEY2. This is the only duplication involving HEY2 identified in our database of over 70,000 individuals referred for CNV analysis. In the developing heart, overexpression of HEY2 is predicted to cause decreased expression of the cardiac transcription factor GATA4 which, in turn, has been shown to cause tetralogy of Fallot. In mice, misexpression of Hey2 in the developing brain leads to inhibition of neurogenesis and promotion of gliogenesis. Hence, duplication of HEY2 may be a contributing factor to both the congenital heart defects and the neurodevelopmental problems evident in our patient. These results suggest that individuals with HEY2 duplications should be screened for congenital heart defects and monitored closely for evidence of developmental delay and/or cognitive impairment.

Background— The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca 2+ leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca 2+ release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. Methods and Results— CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca 2+ release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca 2+ /calmodulin-dependent protein kinase II–mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca 2+ release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca 2+ -dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. Conclusions— RyR2-mediated SR Ca 2+ leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca 2+ release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) using an electrospray ionization (ESI) interface provides a sensitive method for the quantitative analysis of peptide drugs in complex biological matrixes. ESI HPLC-MS was applied to the analysis of a pentapeptide drug (IRI-514) in rabbit and human plasma. Prior to analysis, the plasma samples were prepared using protein precipitation followed by solid-phase extraction. The lower limit of quantitation using selected ion monitoring was determined to be 2 ng/mL, when 8 mL of human plasma spiked with 1-40 ng/mL was extracted. Rabbit plasma (1 mL) samples spiked with 10-40,000 ng of authentic drug/mL gave a linear response when a deuterated peptide analog was employed as an internal standard. A commercial ESI interface was modified to permit higher flow rates (10-20 microL/min) to enter the mass spectrometer source. The revised interface provided a 10-fold increase in sensitivities and permitted the use of standard HPLC columns (2.0-mm i.d.) and HPLC instrumentation. ESI HPLC-MS analysis was automated to provide unattended, precise, and sensitive detection of small peptides in both human and rabbit plasma. Using this methodology, a toxicokinetic study of intravenously administered IRI-514 at three dose levels indicated that the area under the curve values were dose proportional.

Verocytotoxigenic Escherichia coli (VTEC) are highly significant zoonotic threats to public health, and have been the causative agent implicated in numerous high-profile outbreaks affecting large numbers of people. Serovar O157 is most frequently linked with human illness; however, other serovars, such as O26, O103, O111 and O145, have also been implicated. This study aimed to characterize the prevalence and virulence determinants of these five serovars in Irish dairy farm herds, and their milk. Using real-time PCR (RTi-PCR), bovine rectal faecal swabs and raw milk samples, along with milk filters, were screened for the presence of vt genes. Positive samples were then screened for the five serovars using sero-specific PCR. Serovar-positive samples were subjected to immunomagnetic separation, to isolate viable VTEC strains. These isolates were subsequently screened for four virulence factors: vt1, vt2, eaeA and hlyA. Three hundred and eighty six of the 600 rectal faecal swabs, 85 of the 117 milk-filters and 43 of the 120 bulk-tank milk samples, were positive for vt genes. From these 514 total vt-positive samples, 58 O26, 162 O103, 1 O111, 324 O145 and 26 O157 positives were detected by sero-specific RTi-PCR. Immunomagnetic separation yielded 12 O26, 26 O103, 0 O111, 19 O145 and 10 O157 isolates. Ten of these isolates contained at least one of the four virulence determinants screened for (i.e. vt1, vt2, eaeA and hlyA). Of these 10 isolates, pulsed-field gel electrophoresis showed that two of the O26 isolates from different farms were indistinguishable. Two O157 isolates were also indistinguishable. This study found serovars O103 and O145 to be the most prevalent in samples tested. Apart from the occurrence of VTEC in dairy herds, this study shows a high occurrence of vt genes in the environment, creating the possibility of horizontal gene transfer and emergence of new VTEC strains.

To evaluate and compare circulating concentrations of islet amyloid polypeptide (IAPP), insulin, and glucose in nondiabetic cats classified by body condition score (BCS) and in cats with naturally occurring diabetes mellitus.109 (82 nondiabetic, 21 nonketoacidotic diabetic, and 6 ketoacidotic diabetic) cats.Cats were examined and BCSs were assessed on a scale of 1 to 9. After food was withheld for 12 hours, blood was collected and plasma concentrations of IAPP and serum concentrations of insulin and glucose were measured. Differences in these values were evaluated among nondiabetic cats grouped according to BCS and in diabetic cats grouped as ketoacidotic or nonketoacidotic on the basis of clinicopathologic findings. Correlations were determined among variables.In nondiabetic cats, BCS was significantly and positively correlated with circulating IAPP and insulin concentrations. Mean plasma IAPP concentrations were significantly different between cats with BCSs of 5 and 7, and mean serum insulin concentrations were significantly different between cats with BCSs of 5 and 8. Serum glucose concentrations were not significantly different among nondiabetic cats. Mean IAPP concentrations were similar between nonketoacidotic diabetic cats and nondiabetic cats with BCSs of 8 or 9. Mean IAPP concentrations were significantly reduced in ketoacidotic diabetic cats, compared with those of nondiabetic cats with BCSs of 6 through 8 and of nonketoacidotic diabetic cats.Results indicated that increased BCS (a measure of obesity) is associated with increased circulating concentrations of IAPP and insulin in nondiabetic cats.

High throughput screening (HTS) of our compound file provided an attractive lead compound with modest P2X(7) receptor antagonist potency and high selectivity against a panel of receptors and channels, but also with high human plasma protein binding and a predicted short half-life in humans. Multi-parameter optimization was used to address the potency, physicochemical and pharmacokinetic properties which led to potent P2X(7)R antagonists with good disposition properties. Compound 33 (CE-224,535) was advanced to clinical studies for the treatment of rheumatoid arthritis.

Behavioral and kinematic properties and capacities of wild migratory salmonid fishes swimming upstream and jumping up waterfalls generally have played only minor roles in the design and construction of passageways intended to help these fishes get past dams and other human-made obstacles blocking their movements. This paper reports the results of an experimental study of relevant behavioral and kinematic properties of adult kokanee salmon (Oncorhynchus nerka) jumping up waterfalls as they migrate upstream. We used a portable, adjustable apparatus to study in the field fish responding to artificial waterfalls under a range of flow conditions. We observed fish under conditions of varying water flow rates, pool depths, fall heights and fall angles. We analyzed digital video recordings of their behaviors. Kokanee salmon spontaneously jump up waterfalls within a relatively narrow range of conditions, including low flow speeds, near vertical angles and pool depth to fall height ratios near 1.0. Preferred values for each parameter are, to some extent, dependent on other parameters. In contrast to previous misconceptions, jumping behavior is initiated by running S-start accelerations from beneath the boils formed in the plunge pools below waterfalls, as opposed to C-start standing jumps from the surface. S-starts are immediately followed by burst swimming to the point of takeoff at the surface. These results can contribute to an improved basis for developing designs of fish passageways that may ultimately make them more effective and efficient.

To determine growth initiation differences of Listeria monocytogenes between a cheesemaking context, milk and tryptic soy broth (TSB).A laboratory-scale cheese was made with a mix of two strains of L. monocytogenes at four initial pH values, five water activity (a(w)) values and two contamination levels at 30°C. Counts of L. monocytogenes were determined at time 0 and after 8h of cheese manufacture. Milk and TSB at the same pH and a(w) conditions were inoculated with the L. monocytogenes mix in multi-well plates. Growth was determined by plating each well onto AgostiOttaviani Listeria Agar after 8h of incubation at 30°C. Each condition was repeated six times, and growth initiation probability was modelled with logistic regression models. Growth initiation boundaries were obtained for each matrix type. The results showed that the growth limits were matrix dependent. In the three matrix types, a(w) was the most important factor affecting the probability of growth initiation. Contamination level affected growth TSB and cheesemaking conditions.The interface wideness and position in cheese, milk and TSB were dissimilar, indicating that the use of models evaluated in TSB or milk could not be used to predict the behaviour of L. monocytogenes under cheesemaking conditions.Predictive models generated in liquid media are not necessarily adaptable to solid food, and the generation of real food models is necessary.

Background: Degenerative features, such as neuronal, glial, synaptic and axonal loss, have been identified in neocortical and other grey matter structures in patients with multiple sclerosis, but mechanisms for neurodegeneration are unclear. Cortical demyelinating lesions are a potential cause of this degeneration, but the pathological and clinical significance of these lesions is uncertain as they remain difficult to identify and study in vivo. In this study we aimed to describe and quantify cellular and subcellular pathology in the cortex of myelin oligodendrocyte glycoprotein-induced marmoset experimental autoimmune encephalomyelitis using quantitative immunohistochemical methods. Results: We found evidence of diffuse axonal damage occurring throughout cortical grey matter with evidence for synaptic loss and gliosis and a 13.6% decrease in neuronal size and occurring in deep cortical layers. Evidence of additional axonal damage and a 29.6—36.5% loss of oligodendrocytes was found in demyelinated cortical lesions. Leucocortical lesions also showed neuronal loss of 22.2% and a 15.8% increase in oligodendrocyte size. Conclusions: The marmoset experimental autoimmune encephalomyelitis model, therefore, shows both focal and generalized neurodegeneration. The generalized changes cannot be directly related to focal lesions, suggesting that they are either a consequence of diffusible inflammatory factors or secondary to remote lesions acting through trans-synaptic or retrograde degeneration.

This study uses quantitative T(2)* imaging to track ferumoxides--protamine sulfate (FEPro)-labeled MDA-MB-231BR-Luc (231BRL) human breast cancer cells that metastasize to the nude rat brain. Four cohorts of nude rats were injected intracardially with FEPro-labeled, unlabeled or tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-treated (to induce apoptosis) 231BRL cells, or saline, in order to develop metastatic breast cancer in the brain. The heads of the rats were imaged serially over 3-4 weeks using gradient multi-echo and turbo spin-echo pulse sequences at 3 T with a solenoid receive-only 4-cm-diameter coil. Quantitative T(2)* maps of the whole brain were obtained by the application of single-exponential fitting to the signal intensity of T(2)* images, and the distribution of T(2)* values in brain voxels was calculated. MRI findings were correlated with Prussian blue staining and immunohistochemical staining for iron in breast cancer and macrophages. Quantitative analysis of T(2)* from brain voxels demonstrated a significant shift to lower values following the intracardiac injection of FEPro-labeled 231BRL cells, relative to animals receiving unlabeled cells, apoptotic cells or saline. Quartile analysis based on the T(2)* distribution obtained from brain voxels demonstrated significant differences (p 0.0083) in the number of voxels with T(2)* values in the ranges 10-35 ms (Q1), 36-60 ms (Q2) and 61-86 ms (Q3) from 1 day to 3 weeks post-infusion of labeled 231BRL cells, compared with baseline scans. There were no significant differences in the distribution of T(2)* obtained from serial MRI in rats receiving unlabeled or TRAIL-treated cells or saline. Histologic analysis demonstrated isolated Prussian blue-positive breast cancer cells scattered in the brains of rats receiving labeled cells, relative to animals receiving unlabeled or apoptotic cells. Quantitative T(2)* analysis of FEPro-labeled metastasized cancer cells was possible even after the hypointense voxels were no longer visible on T(2)*-weighted images.

SUMMARY Short range hydrodynamic and electrosensory signals are important during final stages of prey capture in elasmobranchs (sharks, skates and rays), and may be particularly useful for dorso-ventrally flattened batoids with mouths hidden from their eyes. In stingrays, both the lateral line canal and electrosensory systems are highly modified and complex with significant differences on ventral surfaces that relate to feeding ecology. This study tests functional hypotheses based on quantified differences in sensory system morphology of three stingray species, Urobatis halleri, Myliobatis californica and Pteroplatytrygon violacea. Part I investigates the mechanosensory lateral line canal system whereas part II focuses on the electrosensory system. Stingray lateral line canals include both pored and non-pored sections and differ in branching complexity and distribution. A greater proportion of pored canals and high pore numbers were predicted to correspond to increased response to water flow. Behavioral experiments were performed to compare responses of stingrays to weak water jets mimicking signals produced by potential prey at velocities of 10–20 cm s–1. Bat rays, M. californica, have the most complex and broadly distributed pored canal network and demonstrated both the highest response rate and greater response intensity to water jet signals. Results suggest that U. halleri and P. violacea may rely on additional sensory input, including tactile and visual cues, respectively, to initiate stronger feeding responses. These results suggest that stingray lateral line canal morphology can indicate detection capabilities through responsiveness to weak water jets.

SUMMARY Elasmobranch fishes (sharks, skates and rays) possess highly sensitive electrosensory systems, which enable them to detect weak electric fields such as those produced by potential prey organisms. Different species have unique electrosensory pore numbers, densities and distributions. Functional differences in detection capabilities resulting from these structural differences are largely unknown. Stingrays and other batoid fishes have eyes positioned on the opposite side of the body from the mouth. Furthermore, they often feed on buried prey, which can be located non-visually using the electrosensory system. In the present study we test functional predictions based on structural differences in three stingray species (Urobatis halleri, Pteroplatytrygon violacea and Myliobatis californica)with differing electrosensory system morphology. We compare detection capabilities based upon behavioral responses to dipole electric signals(5.3–9.6 μA). Species with greater ventral pore numbers and densities were predicted to demonstrate enhanced electrosensory capabilities. Electric field intensities at orientation were similar among these species, although they differed in response type and orientation pathway. Minimum voltage gradients eliciting feeding responses were well below 1 nVcm–1 for all species regardless of pore number and density.

HLA-DR expression in neuroendothelial cells (NEC) was studied during the course of SIV encephalitis in rhesus monkeys. HLA-DR determinants were detected on NEC in monkeys with SIV encephalitis, but not in control animals. In situ hybridization with an SIV probe indicated that HLA-DR expression was not a consequence of SIV replication within NEC. Cultured rhesus NEC stimulated with gamma interferon expressed HLA-DR to a higher degree than cultured brain fibroblasts or astrocytes. These data support the contention that NEC participate in retrovirus-induced inflammation and autoimmunity within the central nervous system.

The activity of transposable elements (TEs) has had a profound impact on the evolution of eukaryotic genomes. Once thought to be purely selfish genomic entities, TEs are now recognized to occupy a continuum of relationships, ranging from parasitic to mutualistic, with their host genomes. One of the many ways that TEs contribute to the function and evolution of the genomes in which they reside is through the donation of host protein coding sequences (CDSs). In this chapter, we will describe several notable examples of eukaryotic host CDSs that are derived from TEs. Despite the existence of a number of such well-established cases, the overall extent and significance of this phenomenon remains a matter of controversy. Genome-scale computational analyses have yielded vastly different estimates for the fraction of host CDSs that are derived from TEs. We explain how these seemingly contradictory findings are the result of specific ascertainment biases introduced by the different methods used to detect TE-related sequences. In light of this problem, we propose a comprehensive and systematic framework for definitively characterizing the contribution of TEs to eukaryotic CDSs.

Elasmobranchs (sharks, skates, and rays) possess a variety of sensory systems including the mechanosensory lateral line and electrosensory systems, which are particularly complex with high levels of interspecific variation in batoids (skates and rays). Rays have dorsoventrally compressed, laterally expanded bodies that prevent them from seeing their mouths and more often than not, their prey. This study uses quantitative image analysis techniques to identify, quantify, and compare structural differences that may have functional consequences in the detection capabilities of three Eastern Pacific stingray species. The benthic round stingray, Urobatis halleri, pelagic stingray, Pteroplatytrygon (Dasyatis) violacea, and benthopelagic bat ray, Myliobatis californica, show significant differences in sensory morphology. Ventral lateral line canals correlate with feeding ecology and differ primarily in the proportion of pored and nonpored canals and the degree of branching complexity. Urobatis halleri shows a high proportion of nonpored canals, while P. violacea has an intermediate proportion of pored and nonpored canals with almost no secondary branching of pored canals. In contrast, M. californica has extensive and highly branched pored ventral lateral line canals that extended laterally toward the wing tips on the anterior edge of the pectoral fins. Electrosensory morphology correlates with feeding habitat and prey mobility; benthic feeders U. halleri and M. californica, have greater electrosensory pore numbers and densities than P. violacea. The percentage of the wing surface covered by these sensory systems appears to be inversely related to swimming style. These methods can be applied to a broader range of species to enable further discussion of the relationship of phylogeny, ecology, and morphology, while the results provide testable predictions of detection capabilities.

Marmoset experimental autoimmune encephalomyelitis (EAE) has previously been shown to replicate the essential features of both white matter and grey matter lesions of MS. This study set out to investigate whether cortical atrophy occurs in marmoset EAE and whether cortical thinning is related to the presence of focal, demyelinated cortical lesions. Seventeen leucocortical lesions and 13 subpial lesions were identified in 6 EAE cases. Cortical thickness surrounding these lesions was recorded and compared with matched cortical areas from five control animals. We found a diffuse 13-21% loss of cortical thickness in all areas of EAE cortex compared with control animals but there was no additional loss seen in demyelinated versus myelinated EAE cortex. These findings could not be accounted for by effects of age, sex and disease duration. We conclude that localised cortical demyelination is not responsible for the major part of the atrophy observed and that cortical thinning is largely due to more diffuse or more remote factors. Marmoset EAE is an invaluable tool which can be used to further investigate the cause and the substrate of cortical loss in demyelinating diseases.

Aims: Magnetic nanoparticles have been studied widely as MRI contrast agents to increase the sensitivity of this technique. This work describes the synthesis and characterization of magnetic nanotubes (MNTs) as a novel MRI contrast agent. Methods: MNTs with high saturation magnetization were fabricated by the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) directly in the pores of silica nanotubes (SNTs). The MNTs were characterized by electron microscopy, superconducting quantum interference device and MRI. Preliminary studies on in vitro cytotoxicity and cell labeling were carried out. Results: The MNTs retained the superparamagnetic characteristics in bulk solutions with a considerably high saturation magnetization of 95 emu/gFe. The nuclear magnetic resonance (NMR) relaxivities for MNTs of 500 nm in length and of 60 nm in diameter were r1 = 1.6 ± 0.3 mM-1s-1 and r2 = 264 ± 56 mM-1s-1 and, for the MNTs of 2 µm in length and 70 nm in diameter, the r1 and r2 were 3.0 ± 1.3 and 358 ± 65 mM-1s-1, respectively. In vitro cell labeling showed promising results with excellent labeling efficiency. No cellular toxicity was observed in vitro. Conclusions: The integration of SPIONs with SNTs imparts the superparamagnetic characteristics of SPIONs onto the SNTs, creating unique magnetic nanoparticles with multifunctionality. The MNTs showed promising results as a MRI contrast agent with high NMR relaxivities, little cytotoxicity and high cell-labeling efficiency.

Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in beta cell granules. IAPP may have a significant role in the development of Type 2 diabetes mellitus due to its propensity to form islet cell-disrupting amyloid deposits, and by opposing the action of insulin in peripheral tissues. Most evidence to-date suggests that an intrinsic structural motif of IAPP is linked to the amyloidogenicity of IAPP, and that this motif occurs only in those species (e.g., humans and cats) that also develop age-associated or Type 2 diabetes. We utilized polymerase chain reaction methodology in this study to obtain the IAPP nucleotide and protein sequences of the dog, a species not known to develop islet amyloid. We show that dog IAPP contains the same putative amyloidogenic sequence (GAILS) at residues 24–28 as human and cat IAPP, and that although dogs do not develop islet amyloid they do develop IAPP-derived amyloid in association with neoplastic beta cells (i.e., insulinomas). These results provide strong evidence that the amyloidogenicity of IAPP is linked to at least two prerequisites: a species-specific amyloidogenic structural motif, and aberrations in the synthesis (or processing) of IAPP which leads to increased concentration of IAPP in the local milieau.

Islet amyloid polypeptide (IAPP) has been implicated by in vitro studies as an inhibitor of insulin-stimulated glucose utilization by skeletal muscle cells and also as an inhibitor of insulin-stimulated insulin secretion by beta cells. Increased expression and production of IAPP by beta cells, as has been suggested to occur in cats with impaired glucose tolerance, could thus contribute substantially to the development of the insulin resistance and impaired insulin release which are the hallmarks of Type 2 diabetes mellitus. The effects of IAPP with respect to glucose metabolism in living animals, however, have not been previously reported. In the present in vivo study we show that synthetic amidated IAPP induced impaired glucose tolerance in each of the 3 cats studied, with dramatic impairment (increases in glucose to T 1 2 values of 124% and 234%) in 2 of the 3 cats. Impaired insulin responses were also evident in the 2 cats with the most dramatic states of glucose intolerance. These results provide the most direct evidence to-date that IAPP may have an important role in the development of Type 2 diabetes mellitus.

The use of immunohistochemical methods has led to a new understanding of the prevalence and significance of cortical lesions in multiple sclerosis but these lesions have not yet been formally described in an animal model. In this study we have set out to use immunohistochemical techniques to identify and describe cortical lesions in marmosets with experimental autoimmune encephalomyelitis (EAE). Using antibodies to proteolipid protein (PLP), we found a total of 70 cortical lesions in 11 tissue blocks from 6 animals. These lesions were subdivided into leucocortical (40), intracortical (12) and subpial lesions (18). We quantified the density of inflammatory cells within lesions using a double labelling protocol which employed anti-PLP in addition to antibodies against markers of B-lymphocytes (CD20), T-lymphocytes (CD3), macrophages (MAC387) and MHC-II expressing cells (CR3/43). This analysis revealed that the large subpial lesions accounted for the majority of demyelinated cortex (88%) despite possessing the lowest density of inflammatory cells. This study has shown that lesions in this model share many of the major features of cortical lesions in multiple sclerosis both in terms of morphology and inflammatory cell content. We believe that this tool can be exploited in future studies to investigate the aetiology, development and clinical significance of cortical lesions in demyelinating disease.

The Swarm rat chondrosarcoma is a tumor tissue line derived from a tumor that arose spontaneously in a Sprague-Dawley rat. The original tissue has given rise to several tissue lines and cell lines that have been prepared in different laboratories. It has been observed that these lines differed in their growth rates and biochemical characteristics. We have characterized our Swarm rat chondrosarcoma tissue and cell lines currently in use in terms of their cytogenetic profiles and their tumorigenic properties in vivo. We found a wide variety of chromosomal abnormalities among cell lines, including translocations, deletions and polyploidy. There were also significant differences in their growth properties in vivo, giving rise to tumors of a few milligrams in the case of Ng cells, to 35 grams in the tissue line JWS. The cytogenetic complexity of the Swarm rat chondrosarcoma between and among different lines makes it very suitable to address questions about the changes that occur as a result of karyotypic abnormalities and to provide links between cytogenetic abnormalities and the dynamic oncogenic machinery.

Two citrus-based sodas and a known wasp attractant were compared in a field trial to assess their attractiveness to local nuisance wasp species. The wasps captured included Vespula germanica (F.), Vespula maculifrons (Buysson), Vespula vulgaris (L.), Vespula flavopilosa Jacobson, Vespula squamosa (Drury), Dolichovespula maculata (L.), Polistes fuscatus (L.), Polistes metricus Say, and Polistes dominulus (Christ). Wasps in the genus Vespula were present in significantly higher numbers in traps than Dolichovespula and Polistes. Both citrus soda products were superior to the isobutanol-acetic acid mixture as attractants for almost all of the wasp species.

Recently, there have been several reports using various superparamagnetic iron oxide (SPIO) nanoparticles to label mammalian cells for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI). The purpose of this study was to evaluate the efficiency and toxicity of labeling cells using 2 commercially available Food and Drug Administration (FDA)-approved agents, ferumoxides, a suspension of dextran-coated SPIO used as an MRI contrast agent, and protamine sulfate, conventionally used to reverse heparin anticoagulation but also used ex vivo as a cationic transfection agent. After labeling of human mesenchymal stem cells (MSCs) and hematopoietic (CD34+) stem cells and other mammalian cells with ferumoxides-protamine sulfate complexes (FE-Pro), cellular toxicity, functional capacity, and quantitative cellular iron incorporation were determined. FE-Pro-labeled cells demonstrated no short- or long-term toxicity, changes in differentiation capacity of the stem cells, or changes in phenotype when compared with unlabeled cells. Efficient labeling with FE-Pro was observed with iron content per cell varying between 2.01 ± 0.1 pg for CD34+ cells and 10.94 ± 1.86 pg for MSCs with 100% of cells labeled. Cell labeling using these agents should facilitate the translation of this method to clinical trials for evaluation of trafficking of infused or transplanted cells by MRI. (Blood. 2004;104:1217-1223)

Background Superparamagnetic iron oxides (SPIO) are being used to label cells for in vivo monitoring by magnetic resonance imaging (MRI). The purpose of this study is to present protocols using SPIO and a polycationic transfection agent for magnetic labeling of cells as a basis for cellular MRI. Methods Various concentrations of ferumoxides (FE)-poly-l-lysine (PLL) complexes were used to magnetically label cells. Iron incorporation into cells along with cell viability and short- and long-term toxicity were evaluated. Results Rapidly growing cell suspension and adherent cells were effectively labeled by means of endocytosis into endosomes at low concentrations of FE (25 microg/mL media) and PLL (0.75 microg/mL media). Hematopoietic stem cells and lymphocytes required higher concentrations of PLL (1.5 microg/mL) in serum-free media during initial FE-PLL complex formation before labeling the cells in culture. Total iron concentration in cells depended on the cell type, concentration of FE-PLL complexes in media, cellular density, and incubation time. Iron concentrations after overnight incubation with given FE at 25 microg/mL media resulted in, for example, T cells being labeled with 1 to 3 pg/cell of intracytoplasmic endosomal iron and 15 to 20 pg/cell of intracytoplasmic iron in mesenchymal stem cells compared with 0.01 to 0.1 pg/cell for unlabeled cells. Protocols developed for this study demonstrated no adverse effect on the cell viability, functional capacity, or toxicity. Conclusion This technique can be used to label cells for in vivo MRI tracking of stem cells and lymphocytes. FE at a concentration of 25 to 50 microg/mL with a ratio of SPIO to PLL of 1:0.03 to 1:0.06 would be sufficient to label cells for cellular MRI.

Mammalian stem cells or other cells are being considered for use for infusion or transplantation into tissue for purposes of repair or for revascularization or therapeutic approaches (i.e., genetically altered cells) (1–5). Dextrancoated superparamagnetic iron oxide (SPIO) nanoparticles, a distinct class of MR contrast agents, cannot be used to efficiently label stem cells or other mammalian cells in vitro in their native unmodified form (6–8). Previously, we demonstrated that by conjugating antigenspecific internalizing monoclonal antibodies to the surface, dextran coating cells could be magnetically labeled during their normal expansion in culture (6). This magnetic labeling approach is limited because it requires the availability of an internalizing monoclonal antibody that recognizes a specific cell surface antigen. It is suitable only for labeling of cells that express the targeted receptor and is commonly species specific. Other approaches have involved the synthesis and modification of ultrasmall SPIO (USPIO or MION) particles with tat-proteins facilitating the incorporation into the cells, although this involves also a (synthetic) protein derivative (7,8). An alternative approach is the complex synthesis of a superparamagnetic iron oxide (SPIO) coated with dendrimers or “magnetodendrimers”, which will non-specifically label most mammalian cells (9). The dendrimer coating of the iron oxide nanoparticles provides the needed high affinity for cellular membranes for the construction of suitable cellular contrast agents, as dendrimers are commonly used as non-viral transfection agents (10,11). Magnetodendrimers can be used to efficiently and non-specifically label mammalian stem cells and cancer cells (12,13), however, they are not widely available or approved by the Food and Drug Administration (FDA). Over the past 10 years there has been significant research in developing new transfection agents (TA) including cationic peptides, dendrimers, poly-amines and lipids for nonviral transfection of DNA into the nucleus (10,14,15). These transfection agents are being developed to overcome the problem of the endosomal capture of the TA-DNA complex and inefficient release of the targeted material into the nucleus (14,15). TA’s are macromolecules with molecular weights from 1 to 10 kilo Dalton possessing an electrostatic charge. Based upon efficient labeling of mammalian cells by magnetodendrimers (12,13), we hypothesize that commercially available macromolecular transfection agents would coat via electrostatic interaction with dextran-coated iron oxide MR contrast agents and chaperon these nanoparticles into cells. We present here the magnetic labeling results of combining dendrimers and other commercially available TA’s with (FDA-approved) dextran-coated iron oxide MR contrast agents (Feridex and MION-46L). Acad Radiol 2002; 9(suppl 2):S484–S487

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

MION-46L, a superparamagnetic iron oxide contrast agent, was investigated for its ability to increase the sensitivity of in vivo 3D MRI in the detection of brain lesions in a chronic experimental allergic encephalomyelitis (crEAE) mouse model. Lesion conspicuity on postcontrast 3D MRI was dramatically enhanced as compared to precontrast images corresponding to areas of inflammatory and demyelinating lesions. MION-46L could be detected on Prussian blue iron stain in the vascular endothelium, the perivascular space, and in macrophages within perivascular cuffs and areas of inflammation and demyelination. By taking advantage of the MION-46L induced macroscopic susceptibility effect, acute early lesions measuring only 100 microm in diameter could be detected. MION-46L enhanced MRI may be used to 1) provide a unique sensitivity in EAE lesion detection and correlate imaging to histopathology; 2) help to understand EAE lesion development and its underlying pathophysiology; and 3) eventually assist in preclinical screening of new experimental therapies directed at patients with multiple sclerosis (MS).

Gill epithelial cells of euryhaline crustaceans demonstrate net inward transport of sodium ions, possibly via apical Na+/H+ antiporters, Na+/K+/2Cl− cotransporters or Na+ channels working in series with the basolateral Na++K+-ATPase. We have identified and sequenced the cDNA coding for a crustacean Na+/H+ antiporter, starting with mRNA isolated from gills of the euryhaline green shore crab Carcinus maenas. The complete 2595-base-pair cDNA includes an open reading frame coding for a 673-amino-acid protein. A search of GenBank revealed more than 20 high-scoring matches, all Na+/H+ antiporter sequences from mammalian, amphibian, teleost and nematode species. Injection of Xenopus laevis oocytes with cRNA transcribed from the cloned crab sequence substantially enhanced Na+-dependent H+ efflux from the oocytes. Analysis of crab tissue antiporter mRNA levels by semi-quantitative reverse transcription–polymerase chain reaction revealed that posterior and anterior gills of Carcinus maenas expressed this antiporter the most strongly, followed in decreasing order by skeletal muscle, hepatopancreas, hypodermis and heart. Hydropathy and transmembrane α-helix analysis suggested a 10-helix membrane-spanning topology of the antiporter protein. It is clear from this study that Carcinus maenas gills vigorously transcribe a gene coding for a Na+/H+ antiporter. Whether these gills also express a gene coding for an epithelial Na+ channel or Na+/K+/2Cl− cotransporter remains to be demonstrated.

Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila copia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.

The most characteristic morphologic features of the pancreatic islets of human non-insulin-dependent diabetes mellitus (NIDDM, or type 2 diabetes mellitus) and of similar forms of diabetes in cats and macaques are the deposition of amyloid (islet amyloid) and the loss of beta cells. Islet amyloid is derived from islet amyloid polypeptide (IAPP), which is a normal secretory product of the beta cells. Therefore, knowledge of the IAPP gene is of potential importance in defining the pathogenesis of NIDDM. To identify the feline chromosome(s) on which the IAPP gene is located, we screened genomic DNA obtained from 38 feline-rodent hybrid cell lines that have a known feline chromosome content. Feline IAPP DNA was amplified and detected using the polymerase chain reaction technique. Discordancy analysis for each feline chromosome showed that chromosome B4 had the lowest discordancy ( P < 0.0001). Feline chromosome B4 shows an extensive conserved syntenic relationship with human chromosome 12, on which the human IAPP gene is located. This study therefore extends and confirms the homology between human chromosome 12 and feline chromosome B4 and provides an additional genetic marker for feline chromosome B4.

The single-stranded DNA parvoviruses occur in humans and many species of animals. In general, they are species-specific and capable of producing disease at any stage of life. Parvoviruses have a requirement to replicate in cells in a permissive S-phase of DNA mitosis. The infections may be cytolytic to select cell groups resulting in specific developmental defects or may produce more generalized effects such as anemia, pancytopenia, or hemorrhage. The fetus is at particular risk for damage because of the vast number of cells in active mitosis. The teratogenic effects may be severe, often resulting in fetal death. Infections in childhood and adulthood are more frequently mild to subclinical. Some of the teratogenic effects recognized in animal species have been identified in humans. With increased knowledge of parvovirus effects in animals, more pathogenic effects may be related to human parvoviral disease. The need for vaccination, currently used annually in many domestic animal species, continues to be evaluated for humans.

Although immature thymocytes from day 14 embryonic mice (FTC) express IL-2R, they have generally been considered to be unresponsive to IL-2. We show here that they can in fact undergo substantial and prolonged growth in vitro provided that high doses of IL-2 are present. The ability of FTC to grow in IL-2 could be enhanced slightly by PMA and also by IL-4, but dramatically by the combination of IL-4 + PMA with IL-2. Pretreatment of FTC with IL-4 + PMA for as little as 24 h primed FTC for rapid and prolonged responsiveness to IL-2, permitting the establishment of long term lines. Kinetic and clonal analysis revealed that most individual FTC could grow under these conditions. Although proliferating cells expressed functional IL-2R of only very low affinity, and IL-2R alpha chains were undetectable by immunofluorescence, blocking experiments showed unambiguously that both IL-2R alpha and IL-2R beta were involved in signal transmission. FTC lines and clones developed in this manner lacked lineage-specific markers of mature T cells, B cells, and myeloid cells, but expressed the NK cell markers NK1.1 and asialo-GM1. They displayed potent cytolytic activity against NK-sensitive targets, and, when stimulated with PMA+ionomycin, secreted IL-3 and IFN-gamma, but not IL-2 or IL-4. After intrathymic injection they showed no evidence of growth or differentiation. These results demonstrate that most, if not all, immature thymocytes have the capacity to differentiate into cells which appear to be indistinguishable from mature NK cells. They suggest that T cells and NK cells derive from a common precursor which in the thymic environment differentiates into T cells and in the extrathymic environment into NK cells.

OBJECTIVE In this 2.5-year study of simian immunodeficiency virus (SIVsm) infection in rhesus monkeys, quinolinic acid (QUIN) levels and virus isolation determinations were made in serial cerebrospinal fluid (CSF) and blood samples to evaluate the relationship between these parameters over the course of infection. METHODS Eight rhesus monkeys were inoculated in the saphenous vein with SIVsm. Four animals were maintained as uninoculated controls. CSF and blood samples were obtained every 1-4 weeks over the course of study. SIV isolation was determined in H9 cells for the CSF and in primary rhesus lymphocyte co-cultures for peripheral blood mononuclear cells (PBMC). QUIN was quantitated in CSF and serum by electron-capture negative chemical ionization gas chromatography mass spectrometry. RESULTS All SIV-inoculated animals became CSF and PBMC isolation-positive by 1-3 weeks post-inoculation. Control animals remained SIV-negative. One SIV-positive animal was humanely euthanized at 2 weeks post-inoculation. The three SIV-inoculated animals that were CSF isolation-negative after the fifth week post-inoculation maintained CSF QUIN values < 100 nM, remained CSF and PBMC isolation-negative, and clinically healthy in the chronic course of disease. In contrast, the four SIV-inoculated animals that were CSF isolation-positive 6-8 weeks post-inoculation had CSF QUIN levels as high as 153-565 nM during the second month post-inoculation and remained CSF virus isolation-negative, persistently PBMC isolation-positive, and experienced clinical symptoms of SIV in the chronic course of disease. Three of these four animals have succumbed to SIV infection. DISCUSSION Initial QUIN responses and viral isolation status in the first month post-inoculation were consistent among SIV-inoculated animals with CSF and serum QUIN values significantly higher than those of controls. A divergence within the SIV-inoculated group of animals became apparent within the second month of primary SIV infection and was maintained throughout the course of infection. Persistent PBMC viral isolation and marked elevations of QUIN were linked to symptomatic disease and a poor prognosis for survival. Predominantly negative PBMC viral isolation and slight, but significant, elevations of QUIN were linked to asymptomatic disease with a favorable prognosis for survival.

The transport of the neutral amino acid L-leucine as well as of choline and D-glucose across the blood-brain barrier (BBB) of male Wistar rats was studied after 14-day exposure to ethanol and treatment with the nootropic drug AWD 52-39 (1). After ethanol exposure the half-saturation constant (Km) and the maximum velocity of transport (Vmax) declined in the majority of the investigated brain regions. Also, the treatment elicited a regionally different increase of the permeability-surface area (PS) product of choline (between 10% and 33%) and glucose (between 12% and 27%). The changes in the blood-brain transfer of the three compounds were diminished or prevented by additional application of 1. The cerebral blood flow was increased by the exposure to ethanol by maximally 44%. After additional administration of 1 the changes were reversed and the blood flow reached control values. In addition, the activity of the enzyme acetylcholine esterase was determined in the striatum and the hippocampus. After ethanol exposure the enzyme activity declined by 32%. It was less diminished after treatment with 1. The latter effects let assume that the changes of the BBB permeability elicited by ethanol and 1 are related to alterations of brain metabolism.

Increased concentrations of the excitotoxin quinolinic acid (QUIN) have been implicated in the neurologic deficits and brain atrophy that may accompany infection with the human immunodeficiency virus type-1. Key neuropathologic features of the AIDS encephalitis are replicated in some macaques following infection with the simian immunodeficiency virus (SIV). In the present studies, cerebrospinal fluid (CSF) QUIN concentrations increased within 2 weeks following infection of 11 rhesus macaques (Macaca mulatta) with a neurotropic sooty mangabey isolate of the simian immunodeficiency virus (SIVsm) and were sustained to greater than 2 standard deviations above uninfected control macaques. Highest CSF QUIN concentrations (up to 400-fold above pre-inoculation levels) were observed in 6 SIVsm-infected macaques with motor and behavioral abnormalities during life, brain atrophy on MRI scan and inflammatory lesions within the brain and meninges. Four of the 6 neurologic macaques deteriorated rapidly within 12 weeks after inoculation and had substantially larger increases in CSF QUIN levels than 2 other neurologic macaques and 5 macaques without neurologic signs which survived for longer than 37 weeks. Increases in serum QUIN and CSF kynurenic acid also occurred but generally to a lesser degree than the increases in CSF QUIN. In some animals, increases in serum L-kynurenine concentrations and reductions in CSF and serum L-tryptophan occurred and were consistent with activation of indoleamine-2, 3-dioxygenase, the first enzyme of the kynurenine pathway in extrahepatic tissues. CSF QUIN exceeded serum QUIN in 8.8% of samples from macaques with neurologic signs, supporting increased QUIN synthesis within the central nervous system. Production of [13C6]QUIN was demonstrated in one SIVsm-infected macaque and one uninfected control macaque following an intracisternal injection of [13C6]L-tryptophan and suggests that L-tryptophan is a substrate for QUIN synthesis within the nervous system or meninges, although the cellular localization of QUIN synthesis remain to be determined. We conclude that increases in kynurenine pathway metabolism occur in SIV-infected macaques and are most prominent in macaques with neurologic signs. Macaques infected with SIV offer a model to investigate the relationship between the metabolism of neuroactive kynurenines and neurologic disturbances associated with retroviral infection.

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU(+), 5-10% of GFP(+) and 5-15% of dextran(+) macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.