Con A-induced hepatitis (Con A-hepatitis) is a hepatitis model in which hepatic injury is supposed to be caused by cytokines from activated T cells. To elucidate the pathogenesis of this disease, we analyzed the roles of IFN-gamma and TNF-alpha using deficient mice of these cytokines. Development of hepatitis was reduced significantly in IFN-gamma(-/-) mice, while susceptibility of TNF-alpha(-/-) mice was not changed. Interestingly, apoptotic cell death was observed in the affected livers of control or TNF-alpha(-/-) mice, but not in those of IFN-gamma(-/-) mice. Fas mRNA expression was increased in the livers of hepatitis mice, but less abundantly in those of IFN-gamma(-/-) mice. Since apoptosis of liver cells was rarely observed in Con A-treated lpr/lpr mice, involvement of the Fas-Fas ligand system in this apoptotic process was suggested. These observations suggest that IFN-gamma plays a central role in Con A-hepatitis by activating Fas-induced apoptosis of liver cells.

Exoenzyme S (ExoS), which has been implicated as a virulence factor of Pseudomonas aeruginosa, catalyzes transfer of the ADP-ribose moiety of NAD+ to many eukaryotic cellular proteins. Its preferred substrates include Ras and several other 21- to 25-kDa GTP-binding proteins. ExoS absolutely requires a ubiquitous eukaryotic protein factor, termed FAS (factor activating ExoS), for enzymatic activity. Here we describe the cloning and expression of a gene encoding FAS from a bovine brain cDNA library and demonstrate that purified recombinant FAS produced in Escherichia coli activates ExoS in a defined cell-free system. The deduced amino acid sequence of FAS shows that the protein (245 residues, calculated molecular mass 27,743 Da) belongs to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. Various functions have been reported for members of the 14-3-3 family, including phospholipase A2 activity and regulation of tyrosine hydroxylase, tryptophan hydroxylase, and, possibly, protein kinase C activities. Identification of FAS as a 14-3-3 protein establishes an additional function for this family of proteins--the activation of an exogenous ADP-ribosyltransferase. Elucidation of the precise role of FAS in activating ExoS will contribute to understanding the molecular mechanisms by which P. aeruginosa causes disease.

RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.

Recent studies have indicated that lipid rafts (LRs) in the cell membrane are clustered in response to different stimuli to form signaling platforms for transmembrane transduction. It remains unknown whether this LR clustering participates in redox signaling in endothelial cells. The present study tested a hypothesis that clustering of LRs on the membrane of coronary endothelial cells produces aggregation and activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, thereby forming a redox signaling platform. By confocal microscopic analysis of agonist-stimulated rafts patch formation, we found that several death receptor ligands or apoptotic factors, including tumor necrosis factor alpha, Fas ligand, or endostatin, stimulated the clustering and trafficking of individual LRs on the plasma membrane of coronary endothelial cells. Interestingly, double labeling of a membrane-bound NADPH oxidase subunit, gp91phox, and LRs showed that gp91phox colocalized within the LR patches when endothelial cells were stimulated by Fas ligand. In isolated LR fractions from Fas-stimulated endothelial cells, gp91phox, p47phox (a crucial cytosolic regulatory subunit of NADPH oxidase), and Rac GTPase were markedly increased and blocked by nystatin, a compound that disrupts LRs. These clustered LRs contained high NADPH oxidase activity, which increased in response to Fas stimulation. Functionally, Fas ligand-induced inhibition of endothelium-dependent vasorelaxation was reduced if LRs were disrupted or NADPH oxidase was inhibited. These results suggest that LR clustering occurs in coronary endothelial cells. The formation of redox signaling platforms on the cell membrane mediates transmembrane signaling of death receptors, resulting in endothelial dysfunction.

OBJECTIVE

To assess the efficacy and safety of intravitreal inserts releasing 0.2 μg/day (low dose) or 0.5 μg/day (high dose) fluocinolone acetonide (FA) in patients with diabetic macular edema (DME).

METHODS

Two parallel, prospective, randomized, sham injection-controlled, double-masked, multicenter clinical trials.

METHODS

Subjects with persistent DME despite at least 1 macular laser treatment were randomized 1:2:2 to sham injection (n = 185), low-dose insert (n = 375), or high-dose insert (n = 393).

METHODS

Subjects received study drug or sham injection at baseline and after 6 weeks were eligible for rescue laser. Based on retreatment criteria, additional study drug or sham injections could be given after 1 year.

METHODS

The primary outcome was the percentage of patients with improvement from baseline best-corrected visual acuity (BCVA) in Early Treatment Diabetic Retinopathy Trial (ETDRS) letter score of 15 or more at month 24. Secondary outcomes included other parameters of visual function and foveal thickness (FTH).

RESULTS

The percentage of patients with improvement from baseline ETDRS letter score of 15 or more at month 24 was 28.7 and 28.6 in the low- and high-dose insert groups, respectively, compared with 16.2 in the sham group (P = 0.002 for each). Benefit occurred for both doses compared with sham at 3 weeks and all subsequent time points. The mean improvement in BCVA letter score between baseline and month 24 was 4.4 and 5.4 in the low- and high-dose groups, respectively, compared with 1.7 in the sham group (P = 0.02 and P = 0.016). At all time points compared with sham, there was significantly more improvement in FTH. Subjects requiring cataract surgery were more frequent in the insert groups, and their visual benefit was similar to that of subjects who were pseudophakic at baseline. Glaucoma requiring incisional surgery occurred in 3.7%, 7.6%, and 0.5% of the low-dose, high-dose, and sham groups, respectively.

CONCLUSIONS

Both low- and high-dose FA inserts significantly improved BCVA in patients with DME over 2 years, and the risk-to-benefit ratio was superior for the low-dose insert. This is the first pharmacologic treatment that can be administered by an outpatient injection to provide substantial benefit in patients with DME for at least 2 years.

Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4(-/-) cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

BACKGROUND

Although the pathogenesis of follicular thyroid carcinoma (FTC) and its relation to follicular adenoma (FA) remains unclear, detailed understanding of FTC carcinogenesis would facilitate addressing the scientific and clinical challenges, given that there are morphological and molecular similarities between FTC and the frequently occurring FA. Micro-RNAs (miRNAs) are a new class of small, noncoding RNAs implicated in development and cancer and may lend novel clues to FTC genesis. For the latter process, a deregulated miRNA can orchestrate the aberrant expression of several hundred target genes.

OBJECTIVE

The objective of the study was to identify deregulated miRNAs in FTC.

METHODS

We used two high-density expression arrays to identify miRNAs and their target genes that are differentially expressed between FTC and FA. Validation was done by quantitative RT-PCR. We further functionally characterized the effect of deregulated miRNAs in vitro using HEK293T, FTC133, and K5 cell lines.

METHODS

In total, 45 primary thyroid samples (23 FTC, 20 FA, four normal control thyroid) were analyzed.

RESULTS

Two specific miRNAs, miR-197 and miR-346, were significantly overexpressed in FTC. In vitro overexpression of either miRNA induced proliferation, whereas inhibition led to growth arrest. Overexpression of miR-197 and miR-346 repressed the expression of their predicted target genes in vitro and in vivo.

CONCLUSIONS

Our observations show that miR-197 and miR-346 contribute to FTC carcinogenesis. Both miRNAs and their target genes might potentially provide for novel molecular markers and act as novel targets for treatment by interference, which could potentially normalize the deregulated profile of many downstream target genes.

A substantial proportion of individuals infected with human immunodeficiency virus (HIV) also abuse alcohol. Given that each condition can disrupt brain structural integrity, with a predilection for white matter, we used MR diffusion tensor imaging (DTI) and quantitative fibre tracking to examine the separate and combined effects on the microstructure of the corpus callosum. Subjects were men and women with alcoholism alone (n = 87), HIV infection alone (n = 42), alcoholism and HIV infection comorbidity (n = 52) and non-affected controls (n = 88). The two alcoholism groups had similar lifetime alcohol consumption histories; the two HIV-infected groups had similar CD4+ counts and viral loads; all groups were matched in body mass index, and no participant was demented. Compared with controls, all patient groups had lower fractional anisotropy (FA) and higher mean diffusivity (MD) in callosal regions and fibre bundles coursing through the genu and splenium, but these effects were only significant in the two groups with alcoholism, which exhibited 0.65-1.2 SD abnormalities in FA and MD. The callosal regions were differentially affected by alcoholism, with the genu more affected than the splenium, a pattern even more pronounced in the fibre tracks. When the HIV-infected groups were divided by disease severity defined as an acquired immunodeficiency syndrome (AIDS)-defining event or low CD4+ counts (<200) and alcoholism comorbidity, the HIV-infected subgroup with AIDS and alcoholism exhibited approximately 2 SD FA and MD abnormalities in the callosal sectors and fibres, abnormalities that were more than twice the effect sizes observed in the other three HIV-infected subgroups. Degradation of the callosal microstructure was consistently associated with alcoholism, with evidence for compounded alcoholism-HIV effects. Functional relevance of the microstructural abnormalities was supported by associations between motor deficits and low FA or high MD within the diagnostic groups. The high prevalence of alcoholism in HIV-infected individuals and the interfering effect of alcohol on HIV pharmacological response and therapy compliance underscore the need to recognize the independent and synergistic contributions of each condition to brain structure and function.

Recently, we have identified the dramatic depletion of cardiolipin (CL) in diabetic myocardium 6 weeks after streptozotocin (STZ) injection that was accompanied by increases in triacylglycerol content and multiple changes in polar lipid molecular species. However, after 6 weeks in the diabetic state, the predominant lipid hallmarks of diabetic cardiomyopathy were each present concomitantly, and thus, it was impossible to identify the temporal course of lipid alterations in diabetic myocardium. Using the newly developed enhanced shotgun lipidomics approach, we demonstrated the dramatic loss of abundant CL molecular species in STZ-treated hearts at the very earliest stages of diabetes accompanied by a profound remodeling of the remaining CL molecular species including a 16-fold increase in the content of 18:2-22:6-22:6-22:6 CL. These alterations in CL metabolism occur within days after the induction of the diabetic state and precede the triacylglycerol accumulation manifest in diabetic myocardium. Similarly, in ob/ob mice, a dramatic and progressive redistribution from 18:2 FA-containing CL molecular species to 22:6 FA-containing CL molecular species was also identified. Collectively, these results demonstrate alterations in CL hydrolysis and remodeling at the earliest stages of diabetes and are consistent with a role for alterations in CL content in precipitating mitochondrial dysfunction in diabetic cardiomyopathy.

Activation of the Fas cell surface molecule, either by specific antibody or by its as yet unidentified ligand, has been shown to induce apoptosis. Because apoptosis is also evoked in target cells by cytolytic T cells, we investigated whether the Fas pathway is involved in CD4+ T cell-mediated cytotoxicity. Analysis of Fas expression in APC, such as the B lymphoma A20.2J and MHC class II-transfected fibroblasts RT2.3, revealed a correlation between the degree of expression and sensitivity to cytotoxic attack, high level of Fas expression in A20.2J being associated with efficient lysis. To examine whether increased Fas expression in RT2.3 would render these cells more susceptible to CD4+ CTL lysis, they were transfected with a Fas gene expression vector. Indeed, Fas- but not mock-transfected RT2.3 proved to be more sensitive to lysis by either Ag specifically or nonspecifically activated CD4+ CTL. Similarly, MHC class II-negative, Fas-transfected L1210 leukemia cells were lysed with nonspecifically activated CD4+ CTL. The importance of the Fas engagement in CD4+ CTL-mediated cytotoxicity is further substantiated by the failure of both cloned and normal CD4+ CTL to lyse B cell blasts from Ipr mice. These mice are known to have a defect in functional Fas expression. Although the bulk of CD4+ T cell-mediated lysis appears to be Fas induced, the fact that the effector phase of A20.2J lysis is only partially Ca2+ independent indicates that other pathways also contribute to target cell death.

The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.

The role of complement in the maintenance of self-tolerance has been examined in two models: an immunoglobulin transgenic model of peripheral tolerance and a lupus-like murine model of CD95 (Fas) deficiency. We find that self-reactive B lymphocytes deficient in complement receptors CD21/CD35 or transferred into mice deficient in the complement protein C4 are not anergized by soluble self-antigen. In the second model, deficiency in CD21/CD35 or C4 combined with CD95 deficiency results in high titers of anti-nuclear antibodies leading to severe lupus-like disease. These findings suggest a novel role for the complement system in B cell tolerance and provide insight into the genetic association of complement deficiency with susceptibility to systemic lupus erythematosus.

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.

Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highFSChigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and beta-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors are members of the tumor necrosis factor superfamily. TRAIL selectively kills cancer cells but not normal cells. We report here the cloning of the mouse homologue of the TRAIL receptor KILLER/DR5 (MK). The cDNA of MK is 1146 bp in length and encodes a protein of 381 amino acids. MK contains an extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic death-domain characteristic of Fas, tumor necrosis factor, and human TRAIL receptors. MK is highly homologous and binds TRAIL with similar affinity as human DR4 and KILLER/DR5. MK induces apoptosis in mouse and human cells and inhibits colony growth of NIH3T3 cells. Expression of MK is p53-dependent and up-regulated by tumor suppressor p53 and by DNA damaging agents in mouse cells undergoing apoptosis. This is the first report describing a mouse TRAIL receptor gene and also demonstrating that the p53-dependent regulation of KILLER/DR5-mediated apoptosis is conserved between human and mouse.

Hippocampus has been implicated in participating in the pathophysiology of schizophrenia. However, the functional and anatomical connectivities between hippocampus and other regions are rarely concurrently investigated in schizophrenia. In the present study, both functional magnetic resonance imaging (fMRI) during rest and diffusion tensor imaging (DTI) were performed on 17 patients with paranoid schizophrenia and 14 healthy subjects. Resting-state functional connectivities of the bilateral hippocampi were separately analyzed by selecting the anterior hippocampus as region of interest. The fornix body was reconstructed by diffusion tensor tractography, and the integrity of this tract was evaluated using fractional anisotropy (FA). In patients with schizophrenia, the bilateral hippocampi showed reduced functional connectivities to some regions which have been reported to be involved in episodic memory, such as posterior cingulate cortex, extrastriate cortex, medial prefrontal cortex, and parahippocampus gyrus. We speculated that these reduced connectivity may reflect the disconnectivity within a neural network related to the anterior hippocampus in schizophrenia. Meanwhile the mean FA of the fornix body was significantly reduced in patients, indicating the damage in the hippocampal anatomical connectivity in schizophrenia. The concurrence of the functional disconnectivity and damaged anatomical connectivity between the hippocampus and other regions in schizophrenia suggest that the functional-anatomical relationship need to be further investigated.

Prenatal exposure to alcohol can result in fetal alcohol syndrome (FAS), characterized by growth retardation, facial dysmorphologies, and a host of neurobehavioral impairments. Neurobehavioral effects in FAS, and in alcohol-related neurodevelopmental disorder, include poor learning and memory, attentional deficits, and motor dysfunction. Many of these behavioral deficits can be modeled in rodents. This paper reviews the literature suggesting that many fetal alcohol effects result, at least in part, from teratogenic effects of alcohol on the hippocampus. Neurobehavioral studies show that animals exposed prenatally to alcohol are impaired in many of the same spatial learning and memory tasks sensitive to hippocampal damage, including T-mazes, the Morris water maze, and the radial arm maze. Direct evidence for hippocampal involvement is provided by neuroanatomical studies of the hippocampus documenting reduced numbers of neurons, lower dendritic spine density on pyramidal neurons, and decreased morphological plasticity after environmental enrichment in rats exposed prenatally to alcohol. Electrophysiological studies also demonstrate changes in synaptic activity in in vitro hippocampal brain slices isolated from prenatal alcohol-exposed animals. Considered together, these observations demonstrate that prenatal exposure to alcohol can result in abnormal hippocampal development and function. Such studies provide a better understanding of neurological deficits associated with FAS in humans, and may also contribute to the development of strategies to ameliorate the effects of prenatal alcohol exposure on behavior.

The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1alpha, FGF2, PDGF-AB, and TGFbeta(1). Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of alpha-smooth muscle actin (alpha-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1alpha increased cell proliferation (70.2 and 74.3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant (p<0.05) extracellular matrix contraction. TGFbeta(1) induced myofibroblast differentiation with prominent FA and FN assembly, expression of alpha-SMA and significantly greater (p<0.05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.

The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multigenetic. The apoptosis genes, fas and fas ligand (fasL), are candidate contributory genes in human SLE, as mutations of these genes result in autoimmunity in several murine models of this disease. In humans, fas mutations result in a familial autoimmune lymphoproliferative syndrome, but defects in FasL have not yet been identified. In this study, DNA from 75 patients with SLE was screened by single-stranded conformational polymorphism analysis for potential mutations of the extracellular domain of FasL. A heterozygous single-stranded conformational polymorphism for FasL, was identified in one SLE patient, who exhibited lymphadenopathy. Molecular cloning and sequencing indicated that the genomic DNA of this patient contained an 84-bp deletion within exon 4 of the fasL gene, resulting in a predicted 28 amino acid in-frame deletion. Analysis of PBMC from this patient revealed decreased FasL activity, decreased activation-induced cell death, and increased T cell proliferation after activation. This is the first report of defective FasL-mediated apoptosis related to a mutation of the human Fasl, gene in a patient with SLE and suggests that fasL mutations are an uncommon cause of the disease.

Diffusion tensor imaging (DTI) provides a unique source of information about the underlying tissue structure of brain white matter in vivo including both the geometry of major fiber bundles as well as quantitative information about tissue properties represented by derived tensor measures. This paper presents a method for statistical comparison of fiber bundle diffusion properties between populations of diffusion tensor images. Unbiased diffeomorphic atlas building is used to compute a normalized coordinate system for populations of diffusion images. The diffeomorphic transformations between each subject and the atlas provide spatial normalization for the comparison of tract statistics. Diffusion properties, such as fractional anisotropy (FA) and tensor norm, along fiber tracts are modeled as multivariate functions of arc length. Hypothesis testing is performed non-parametrically using permutation testing based on the Hotelling T(2) statistic. The linear discriminant embedded in the T(2) metric provides an intuitive, localized interpretation of detected differences. The proposed methodology was tested on two clinical studies of neurodevelopment. In a study of 1 and 2 year old subjects, a significant increase in FA and a correlated decrease in Frobenius norm was found in several tracts. Significant differences in neonates were found in the splenium tract between controls and subjects with isolated mild ventriculomegaly (MVM) demonstrating the potential of this method for clinical studies.

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the interleukin-1beta-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system. Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells. Importantly, the two apoptotic systems seem to be regulated by different proteases.

This study investigated the global and regional effects of aging on brain volume, mean diffusivity (MD), and fractional anisotropy (FA) in 73 normal female subjects using voxel-based analysis. On a global scale, gray matter volume and FA were negatively correlated, whereas MD was positively correlated with age. Voxel-wise analyses showed brain volume and FA were negatively correlated predominantly in anterior structures, whereas MD was positively correlated in the cortical gray matter and periventricular white matter. Volume preservation was observed in the cingulate gyrus and subjacent white matter. FA increase was observed in the putamen. Voxel-based direct comparisons of volume and diffusion properties showed FA was more strongly negatively correlated in the fronto-temporal white matter, compared with volume and MD. Stronger positive correlation of MD was observed in the thalamus, caudate nucleus, and midbrain and stronger negative correlation of brain volume was observed in the frontal lobe and basal ganglia, compared with the other. These results indicate that diffusion properties and brain volume are complementary markers to the effects of aging.