OBJECTIVE

To investigate the expression of the apoptotic mediators in the retinas from human subjects with diabetes mellitus.

METHODS

Ten donor eyes from five subjects with diabetes mellitus, and eight eyes from four nondiabetic subjects without known ocular disease serving as control subjects were examined. Immunohistochemical techniques were used with antibodies directed against glial fibrillary acidic protein (GFAP), caspase-3, Fas, Fas ligand (FasL), Bax, Bcl-2, survivin, p53, extracellular signal-regulated kinases (ERK1/2), and p38.

RESULTS

In retinas from all subjects without diabetes, weak Bcl-2 immunoreactivity was confined to GFAP-positive glial cells in the nerve fiber layer. Weak immunoreactivity for ERK1/2 was noted in a few nuclei in the inner nuclear layer and in a few Müller cell processes. Cytoplasmic immunostaining for survivin was noted in the retinal pigment epithelial cells. There was no immunoreactivity for the other antibodies tested. All diabetic retinas showed cytoplasmic immunoreactivity for caspase-3, Fas, and Bax in ganglion cells. FasL immunoreactivity was detected in GFAP-positive cells. Upregulation of Bcl-2 immunoreactivity was noted in GFAP-positive cells in nerve fiber and ganglion cell layers, and Bcl-2 induction was noted in Müller cell processes. Strong immunoreactivity for ERK1/2 was observed in many nuclei in the inner nuclear layer in GFAP-positive cells in the nerve fiber and ganglion cell layers and numerous Müller cell processes. Survivin immunoreactivity was not altered in the diabetic retinas. There was no immunoreactivity for p53 and p38.

CONCLUSIONS

Ganglion cells in diabetic retinas express several proapoptosis molecules, suggesting that these cells are the most vulnerable population. Glial cells in diabetic retinas are activated and express several antiapoptosis molecules in addition to the cytotoxic effector molecule FasL, suggesting a possible role of glial cells in induction of apoptosis in ganglion cells.

White matter (WM) fractional anisotropy (FA) is thought to be related to WM integrity and decline in FA is often used as an index of decreasing WM health. However, the relationship of FA to other structural indices of cerebral health has not been well studied. We hypothesized that the decline in WM health will be associated with changes in several other indices of cerebral health. In this manuscript we studied the correlation between whole-brain/hemispheric/corpus callosum FA and gray matter (GM) thickness, sulcal span, and the volume of T2-hyperintense WM in a group of 31 healthy aging individuals (12 males/19 females) aged 57-82 years old. Individual subjects' FA measures were calculated from diffusion tracing imaging (DTI) data using tract-based spatial statistics--an approach specifically designed and validated for voxel-wise multi-subject FA analysis. Age-controlled correlation analysis showed that whole-brain average FA values were significantly and positively correlated with the subject's average GM thickness and negatively correlated with hyperintense WM volume. Intra-hemispheric correlations between FA and other measures of cerebral health had generally greater effect sizes than inter-hemispheric correction, with correlation between left FA and left GM thickness being the most significant (r=0.6, p<0.01). Regional analysis of FA values showed that late-myelinating fiber tracts of the genu of corpus callosum had higher association with other cerebral health indices. These data are consistent with the hypothesis that late-myelinating regions of the brain bear the brunt of age-related degenerative changes.

Several sources of information suggest that human beings evolved on a diet that had a ratio of omega-6 to omega-3 fatty acids (FA) of about 1/1; whereas today, Western diets have a ratio of 10/1 to 20-25/1, indicating that Western diets are deficient in omega-3 FA compared with the diet on which humans evolved and their genetic patterns were established. Omega-6 and omega-3 FA are not interconvertible in the human body and are important components of practically all cell membranes. Studies with nonhuman primates and human newborns indicate that docosahexaenoic acid (DHA) is essential for the normal functional development of the brain and retina, particularly in premature infants. DHA accounts for 40% of the membrane phospholipid FA in the brain. Both eicosapentaenoic acid (EPA) and DHA have an effect on membrane receptor function and even neurotransmitter generation and metabolism. There is growing evidence that EPA and DHA could play a role in hostility and violence in addition to the beneficial effects in substance abuse disorders and alcoholism. The balance of omega-6 and omega-3 FA is important for homeostasis and normal development throughout the life cycle.

Interleukin (IL)-18 is a newly discovered cytokine, structurally similar to IL-1, with profound effects on T-cell activation. This short review summarizes the present knowledge on IL-18, to give an insight into the future perspectives for its possible use as vaccine adjuvant. Formerly called interferon (IFN) gamma inducing factor (IGIF), IL-18 is the new name of a novel cytokine that plays an important role in the T-cell-helper type 1 (Th1) response, primarily by its ability to induce IFNgamma production in T cells and natural killer (NK) cells. Mice deficient in IL-18 have suppressed IFNgamma production despite the presence of IL-12 IL-18 is related to the IL-1 family in terms of structure, receptor family, and function. In terms of structure, IL-18 and IL-1beta share primary amino acid sequences of the so-called "signature sequence" motif and are similarly folded as all-beta pleated sheet molecules. Also similar to IL-1beta, IL-18 is synthesized as a biologically inactive precursor molecule lacking a signal peptide which requires cleavage into an active, mature molecule by the intracellular cysteine protease called IL-1beta-converting enzyme (ICE, also called caspase-1). The activity of mature IL-18 is closely related to that of IL-1. IL-18 induces gene expression and synthesis of tumor necrosis factor (TNF), IL-1, Fas ligand, and several chemokines. The activity of IL-18 is via an IL-18 receptor (IL-18R) complex. This IL-18R complex is made up of a binding chain termed IL-18Ralpha, a member of the IL-1 receptor family previously identified as the IL-1 receptor-related protein (IL-1Rrp), and a signaling chain, also a member of the IL-1R family. The IL-18R complex recruits the IL-1R-activating kinase (IRAK) and TNFR-associated factor-6 (TRAF-6) which phosphorylates nuclear factor kappaB (NFkappaB)-inducing kinase (NIK) with subsequent activation of NFkappaB. Thus on the basis of primary structure, three-dimensional structure, receptor family, signal transduction pathways and biological effects, IL-18 appears to be a new member of the IL-1 family. Similar to IL-1, IL-18 participates in both innate and acquired immunity.

The androgen-regulated enzyme fatty acid synthase (FAS), required for de novo lipogenesis, is overexpressed in several cancers including prostate carcinoma and has been associated with aggressive disease. FAS expression was assessed in 81 prostate carcinomas, both by immunohistochemistry in tissue microarrays and by Affymetrix Hu95Av2 oligonucleotide arrays. Both FAS mRNA and protein were significantly overexpressed in prostate carcinomas compared with the corresponding normal tissue. FAS mRNA and protein expression increased substantially from normal to prostatic intraepithelial neoplasia, to low grade, to high grade, and to androgen-independent bone metastases. A significant correlation between FAS mRNA and protein expression was found in two thirds of the cases. In 17% of the cases, FAS protein levels were high despite low mRNA levels, and these tumors exhibited a distinct molecular signature when compared with tumors that did not express FAS protein. Whereas the latter group of tumors expressed some proapoptotic genes, tumors with high FAS levels overexpressed, among other genes, its transcriptional regulator, steroid regulator binding protein, and apolipoprotein E. These data demonstrate (1) the consistent overexpression of FAS in prostate carcinoma compared with the adjacent normal tissue, (2) a strong association between FAS and prostate tumor initiation and progression, (3) the highest FAS expression occurring in androgen-independent bone metastases, (4) the transcriptional and posttranscriptional regulation of FAS in the majority and in a subset of prostate cancers, respectively, and (5) most importantly, the identification by FAS expression of prostate tumors with unique molecular signatures and potentially diverse biologic behavior.

We report the immunopathological analysis of the brain and tumor of two patients who died of anti-NMDAR-associated encephalitis, and of the tumor of nine patients who recovered. Findings included prominent microgliosis and deposits of IgG with rare inflammatory infiltrates in the hippocampus, forebrain, basal ganglia, and spinal cord. Detection of cells expressing markers of cytotoxicity (TIA, granzyme B, perforin and Fas/Fas ligand) was extremely uncommon. All tumors showed NMDAR-expressing neurons and inflammatory infiltrates. All patients’ NMDAR antibodies were IgG1, IgG2, or IgG3. No complement deposits were observed in any of the central nervous system regions examined. Overall, these findings coupled with recently reported in vitro data showing that antibodies downregulate the levels of NMDA receptors suggest that the antibody immune-response is more relevant than cytotoxic T-cell mechanisms in the pathogenesis of anti-NMDAR-associated encephalitis.

MSC can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of MSC to many growth factors and cytokines is unknown. Many envisioned applications of MSC, such as treating large defects in bone, involve in vivo implantation of MSC attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to MSC survival. Here, we investigated cellular responses of MSC on a biomaterial surface covalently modified with epidermal growth factor (EGF). We found that surface-tethered EGF promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining mitogen-activated protein kinase kinase-extracellular-regulated kinase signaling, tethered EGF increases the contact of MSC with an otherwise moderately adhesive synthetic polymer and confers resistance to cell death induced by the proinflammatory cytokine, Fas ligand. We concluded that tethered EGF may offer a protective advantage to MSC in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tethered EGF-modified polymers described here could be used together with structural materials to construct MSC scaffolds for the treatment of hard-tissue lesions, such as large bony defects. Disclosure of potential conflicts of interest is found at the end of this article.

Focal adhesions (FA) are large, multiprotein complexes that provide a mechanical link between the cytoskeletal contractile machinery and the extracellular matrix. FA exhibit mechanosensitive properties; they self-assemble and elongate upon application of pulling forces and dissociate when these forces are decreased. We propose a thermodynamic model for the mechanosensitivity of FA, according to which a molecular aggregate, subjected to pulling forces, tends to grow in the direction of force application by incorporating additional molecules. We demonstrate that this principle is consistent with the phenomenology of FA dynamics by considering a one-dimensional protein aggregate subjected to pulling forces and anchored to the substrate. Depending on the force level, force distribution along the aggregate, and the character of its anchoring to the substrate, the aggregate is predicted to exhibit distinct modes of assembly that are largely consistent with the experimentally observed FA behavior. We define here specific conditions that can lead to the different regimes of FA assembly, including growth, steady state, and disassembly.

Intraepithelial lymphocytes (IELs) are abundant, evolutionarily conserved T cells, commonly enriched in T cell receptor (TCR) gammadelta expression. However, their primary functional potential and constitutive activation state are incompletely understood. To address this, serial analysis of gene expression (SAGE) was applied to murine TCRgammadelta+ and TCRalphabeta+ intestinal IELs directly ex vivo, identifying 15,574 unique transcripts that collectively portray an "activated yet resting," Th1-skewed, cytolytic, and immunoregulatory phenotype applicable to multiple subsets of gut IELs. Expression of granzymes, Fas ligand, RANTES, prothymosin beta4, junB, RGS1, Btg1, and related molecules is high, whereas expression of conventional cytokines and high-affinity cytokine receptors is low. Differentially expressed genes readily identify heterogeneity among TCRalphabeta+ IELs, whereas differences between resident TCRgammadelta+ IELs and TCRalphabeta+ IELs are less obvious.

Neuroimaging studies using diffusion tensor imaging (DTI) have revealed regions of cerebral white matter with decreased microstructural organization (lowerfractional anisotropy or FA) among poor readers. We examined whether 100 hr of intensive remedial instruction affected the white matter of 8- to 10-year-old poor readers. Prior to instruction, poor readers had significantly lower FA than good readers in a region of the left anterior centrum semiovale. The instruction resulted in a change in white matter (significantly increased FA), and in the very same region. The FA increase was correlated with a decrease in radial diffusivity (but not with a change in axial diffusivity), suggesting that myelination had increased. Furthermore, the FA increase was correlated with improvement in phonological decoding ability, clarifying the cognitive locus of the effect. The results demonstrate the capability of a behavioral intervention to bring about a positive change in cortico-cortical white matter tracts.

Poly(amidoamine) (PAMAM) dendrimer-based multifunctional cancer therapeutic conjugates have been designed and synthesized. The primary amino groups on the surface of the generation 5 (G5) PAMAM dendrimer were neutralized through partial acetylation, providing enhanced solubility of the dendrimer (in conjugation of FITC (fluorescein isothiocyanate)) and preventing nonspecific targeting interactions (in vitro and in vivo) during delivery. The functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and paclitaxel (taxol, a chemotherapeutic drug) were conjugated to the remaining nonacetylated primary amino groups. The appropriate control dendrimer conjugates have been synthesized as well. Characterization of the G5 PAMAM dendrimer and its nanosize conjugates, including the molecular weight and number of primary amine groups, has been determined by multiple analytical methods such as gel permeation chromatography (GPC), nuclear magnetic resonance spectroscopy (NMR), potentiometric titration, high-performance liquid chromatography (HPLC), and UV spectroscopy. These multifunctional dendrimer conjugates have been tested in vitro for targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. We present here the synthesis, characterization, and functionality of these dendrimer conjugates.

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β3-integrins are enriched and stationary within FAs, whereas immobilized β1-integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs.

We have previously identified a 94- to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins. However, using sensitive techniques such as flow cytometry (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA/11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was greatly enhanced with thioglycollate elicitation. Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10-15% of the total MS content of elicited macrophages on the plasma membrane. Similar results were obtained with untreated RAW 264.7 cells. FACS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4 degrees C and uptake at 37 degrees C of 125I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface expression of MS/CD68 at steady-state represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.

The aims of this study were to (1) determine the activity profiles of a large sample of English FA Premier League soccer players and (2) examine high-intensity running during elite-standard soccer matches for players in various playing positions. Twenty-eight English FA Premier League games were analysed during the 2005-2006 competitive season (n=370), using a multi-camera computerised tracking system. During a typical match, wide midfielders (3138 m, s=565) covered a greater distance in high-intensity running than central midfielders (2825 m, s= 73, P=0.04), full-backs (2605 m, s=387, P < 0.01), attackers (2341 m, s=575, P < 0.01), and central defenders (1834 m, s=256, P < 0.01). In the last 15 min of a game, high-intensity running distance was approximately 20% less than in the first 15-min period for wide midfielders (467 m, s=104 vs. 589 m, s=134, P < 0.01), central midfielders (429 m, s=106 vs. 534 m, s=99, P < 0.01), full-backs (389 m, s=95 vs. 481 m, s=114, P < 0.01), attackers (348 m, s=105 vs. 438 m, s=129, P < 0.01), and central defenders (276 m, s=93 vs. 344 m, s=80, P < 0.01). There was a similar distance deficit for high-intensity running with (148 m, s=78 vs. 193 m, s=96, P < 0.01) and without ball possession (229 m, s=85 vs. 278 m, s=97, P < 0.01) between the last 15-min and first 15-min period of the game. Mean recovery time between very high-intensity running bouts was 72 s (s=28), with a 28% longer recovery time during the last 15 min than the first 15 min of the game (83 s, s=26 vs. 65 s, s=20, P < 0.01). The decline in high-intensity running immediately after the most intense 5-min period was more evident in attackers (216 m, s=50 vs. 113 m, s=47, P < 0.01) and central defenders (182 m, s=26 vs. 96 m, s=39, P < 0.01). The results suggest that high-intensity running with and without ball possession is reduced during various phases of elite-standard soccer matches and the activity profiles and fatigue patterns vary among playing positions. The current findings provide valuable information about the high-intensity running patterns of a large sample of elite-standard soccer players, which could be useful in the development and prescription of specific training regimes.

White matter microstructure is under strong genetic control, yet it is largely unknown how genetic influences change from childhood into adulthood. In one of the largest brain mapping studies ever performed, we determined whether the genetic control over white matter architecture depends on age, sex, socioeconomic status (SES), and intelligence quotient (IQ). We assessed white matter integrity voxelwise using diffusion tensor imaging at high magnetic field (4-Tesla), in 705 twins and their siblings (age range 12-29; 290 M/415 F). White matter integrity was quantified using a widely accepted measure, fractional anisotropy (FA). We fitted gene-environment interaction models pointwise, to visualize brain regions where age, sex, SES and IQ modulate heritability of fiber integrity. We hypothesized that environmental factors would start to outweigh genetic factors during late childhood and adolescence. Genetic influences were greater in adolescence versus adulthood, and greater in males than in females. Socioeconomic status significantly interacted with genes that affect fiber integrity: heritability was higher in those with higher SES. In people with above-average IQ, genetic factors explained over 80% of the observed FA variability in the thalamus, genu, posterior internal capsule, and superior corona radiata. In those with below-average IQ, however, only around 40% FA variability in the same regions was attributable to genetic factors. Genes affect fiber integrity, but their effects vary with age, sex, SES and IQ. Gene-environment interactions are vital to consider in the search for specific genetic polymorphisms that affect brain integrity and connectivity.

BACKGROUND

Plasmodium falciparum can cause a diffuse encephalopathy known as cerebral malaria (CM), a major contributor to malaria associated mortality. Despite treatment, mortality due to CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM and other forms of severe malaria is multi-factorial and appear to involve cytokine and chemokine homeostasis, inflammation and vascular injury/repair. Identification of prognostic markers that can predict CM severity will enable development of better intervention.

METHODS

Postmortem serum and cerebrospinal fluid (CSF) samples were obtained within 2-4 hours of death in Ghanaian children dying of CM, severe malarial anemia (SMA), and non-malarial (NM) causes. Serum and CSF levels of 36 different biomarkers (IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, CRP, G-CSF, GM-CSF, IFN-gamma, TNF-alpha, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, SDF-1alpha, CXCL11 (I-TAC), Fas-ligand [Fas-L], soluble Fas [sFas], sTNF-R1 (p55), sTNF-R2 (p75), MMP-9, TGF-beta1, PDGF bb and VEGF) were measured and the results compared between the 3 groups.

RESULTS

After Bonferroni adjustment for other biomarkers, IP-10 was the only serum biomarker independently associated with CM mortality when compared to SMA and NM deaths. Eight CSF biomarkers (IL-1ra, IL-8, IP-10, PDGFbb, MIP-1beta, Fas-L, sTNF-R1, and sTNF-R2) were significantly elevated in CM mortality group when compared to SMA and NM deaths. Additionally, CSF IP-10/PDGFbb median ratio was statistically significantly higher in the CM group compared to SMA and NM groups.

CONCLUSIONS

The parasite-induced local cerebral dysregulation in the production of IP-10, 1L-8, MIP-1beta, PDGFbb, IL-1ra, Fas-L, sTNF-R1, and sTNF-R2 may be involved in CM neuropathology, and their immunoassay may have potential utility in predicting mortality in CM.

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Folic acid (FA) supplements and food fortification are used to prevent neural tube defects and to lower plasma homocysteine. Through exposure to food fortification and vitamin supplement use, large populations in the United States and elsewhere have an unprecedented high FA intake. We evaluated dietary and supplemental intakes of folate and FA in relation to an index of immune function, natural killer cell (NK) cytotoxicity, among 105 healthy, postmenopausal women. Among women with a diet low in folate (<233 microg/d), those who used FA-containing supplements had significantly greater NK cytotoxicity (P = 0.01). However, those who consumed a folate-rich diet and in addition used FA supplements>> 400 microg/d had reduced NK cytotoxicity compared with those consuming a low-folate diet and no supplements (P = 0.02). Prompted by this observation, we assessed the presence of unmetabolized FA in plasma as a biochemical marker of excess FA. Unmetabolized folic acid was detected in 78% of plasma samples from fasting participants. We found an inverse relation between the presence of unmetabolized FA in plasma and NK cytotoxicity. NK cytotoxicity was approximately 23% lower among women with detectable folic acid (P = 0.04). This inverse relation was stronger among women>>or= 60 y old and more pronounced with increasing unmetabolized FA concentrations (P-trend = 0.002). Because of the increased intake of FA in many countries, our findings highlight the need for further studies on the effect of long-term high FA intake on immune function and health.

Lymphomas were studied in kindreds with autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome), a disorder of lymphocyte homeostasis usually associated with germline Fas mutations. Fas (CD95/APO-1) is a cell surface receptor that initiates programmed cell death, or apoptosis, of activated lymphocytes. Lymphoma phenotype was determined by immunohistochemistry, frequency of CD3(+)CD4(-)CD8(-) T-cell-receptor alpha/beta cells by flow cytometry, nucleotide sequences of the gene encoding Fas (APT1, TNFRSF6), and the percentage of lymphocytes undergoing apoptosis in vitro. Of 223 members of 39 families, 130 individuals possessed heterozygous germline Fas mutations. Eleven B-cell and T-cell lymphomas of diverse types developed in 10 individuals with mutations in 8 families, up to 48 years after lymphoproliferation was first documented. Their risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected (each P <.001). Investigation of these 10 patients and their relatives with Fas mutations revealed that all had defective lymphocyte apoptosis and most had other features of ALPS. The tumor cells retained the heterozygous Fas mutations found in the peripheral blood and manifested defective Fas-mediated killing. These data implicate a role for Fas-mediated apoptosis in preventing B-cell and T-cell lymphomas. Inherited defects in receptor-mediated lymphocyte apoptosis represent a newly appreciated risk factor for lymphomas.

Gangliosides are known as modulators of transmembrane signaling by regulating various receptor functions. We have found that insulin resistance induced by tumor necrosis factor-alpha (TNF-alpha) in 3T3-L1 adipocytes was accompanied by increased GM3 ganglioside expression caused by elevating GM3 synthase activity and its mRNA. We also demonstrated that TNF-alpha simultaneously produced insulin resistance by uncoupling insulin receptor activity toward insulin receptor substrate-1 (IRS-1) and suppressing insulin-sensitive glucose transport. Pharmacological depletion of GM3 in adipocytes by an inhibitor of glucosylceramide synthase prevented the TNF-alpha-induced defect in insulin-dependent tyrosine phosphorylation of IRS-1 and also counteracted the TNF-alpha-induced serine phosphorylation of IRS-1. Moreover, when the adipocytes were incubated with exogenous GM3, suppression of tyrosine phosphorylation of insulin receptor and IRS-1 and glucose uptake in response to insulin stimulation was observed, demonstrating that GM3 itself is able to mimic the effects of TNF on insulin signaling. We used the obese Zucker fa/fa rat and ob/ob mouse, which are known to overproduce TNF-alpha mRNA in adipose tissues, as typical models of insulin resistance. We found that the levels of GM3 synthase mRNA in adipose tissues of these animals were significantly higher than in their lean counterparts. Taken together, the increased synthesis of cellular GM3 by TNF may participate in the pathological conditions of insulin resistance in type 2 diabetes.

Cerebral white matter undergoes various changes with normal aging. This study investigated the association between age, gender, and the global and regional fractional anisotropy (FA) and mean diffusivity (MD) in 145 adults (30 to 80 years old) using diffusion tensor magnetic resonance imaging. We studied sixteen regions of interest in both hemispheres to search for regions that display age- and gender-related white matter changes and also performed a complementary voxel-based analysis without any hypothesis a priori. On a global scale, our results indicate that the full brain FA was negatively correlated with age. The regional analysis showed that the anterior corpus callosum, the bilateral anterior and posterior internal capsule, and the posterior periventricular regions had the most significant age-related FA decrease. On the other hand, the FA in the temporal and occipital regions was not correlated with age. However, in contrast to males, females overall had a significantly lower FA in the right deep temporal regions. More gender differences in precentral, cingulate, and anterior temporal white matter areas were also found, suggesting that microstructural white matter organization in these regions may have a sexual dimorphism. Such differences were mainly due to the increase in diffusion perpendicular to fiber tracts.

OBJECTIVE

The accumulation of shed plasma membrane vesicles in the peripheral circulation is unique to cancer. Because these membrane fragments (MFs) express biologically active components, such as Fas ligand (FasL), the objective of this study was to define the link between the presence of shed membrane vesicles, apoptosis, and suppression of T-cell receptor/CD3-zeta expression in T lymphocytes of patients with ovarian cancer.

METHODS

MF shedding was measured chromatographically in sera from women with ovarian cancer (n = 11) and, as controls, non-cancer-bearing females (n = 9) and women with benign ovarian disease (n = 4). FasL associated with these shed fragments was assayed by Western immunoblots, whereas HLA class I expression was defined by slot-blotting. The effect of shed MFs on CD3-zeta expression was evaluated using a T-cell bioassay, and apoptosis of circulating T cells was measured by a cell-death ELISA and electrophoretic analysis of caspase-3.

RESULTS

MFs were undetectable in control sera, and their levels were significantly elevated in sera from women with ovarian cancer. These tumor-derived MFs expressed 41-kDa FasL and HLA class I antigens. In co-incubation experiments, dose-dependent suppression of T-cell receptor/CD3-zeta expression by MFs was observed. Decreases in zeta expression correlated with the level of FasL in MFs but not with the level of HLA. The suppression of CD3-zeta by MFs appeared to be linked to the induction of apoptosis and caspase-3 within T cells.

CONCLUSIONS

Our results suggest that FasL associated with tumor-derived MFs is responsible for apoptosis of T lymphocytes and a concomitant loss of zeta-chain expression in patients with ovarian carcinoma.

The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia-related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage-inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.