Issue published October 1, 2005

P resident George W. Bush and Senate majority leader Bill Frist have recently publicly advocated teaching intelligent design in science classes. Their endorsement of a discredited, nonscientific view could signal a huge step backward for scientific education. It is time for educated, motivated scientists to get involved and to educate others.

S ince the first successful transplantation of umbilical cord blood in 1988, cord blood has become an important source of hematopoietic stem and progenitor cells for the treatment of blood and genetic disorders. Significant progress has been accompanied by challenges for scientists, ethicists, and health policy makers. With the recent recognition of the need for a national system for the collection, banking, distribution, and use of cord blood and the increasing focus on cord blood as an alternative to embryos as a source of tissue for regenerative medicine, cord blood has garnered significant attention. We review the development of cord blood banking and transplantation and then discuss the scientific and ethical issues influencing both established and investigational practices surrounding cord blood collection, banking, and use.

A bout 5% of American women and 12% of men will develop a kidney stone at some time in their life, and prevalence has been rising in both sexes. Approximately 80% of stones are composed of calcium oxalate (CaOx) and calcium phosphate (CaP); 10% of struvite (magnesium ammonium phosphate produced during infection with bacteria that possess the enzyme urease), 9% of uric acid (UA); and the remaining 1% are composed of cystine or ammonium acid urate or are diagnosed as drug-related stones. Stones ultimately arise because of an unwanted phase change of these substances from liquid to solid state. Here we focus on the mechanisms of pathogenesis involved in CaOx, CaP, UA, and cystine stone formation, including recent developments in our understanding of related changes in human kidney tissue and of underlying genetic causes, in addition to current therapeutics.

T his series of Reviews on cell death explores the creation of new therapies for correcting excessive or deficient cell death in human disease. Signal transduction pathways controlling cell death and the molecular core machinery responsible for cellular self-destruction have been elucidated with unprecedented celerity during the last decade, leading to the design of novel strategies for blocking pathological cell loss or for killing unwanted cells. Thus, an increasing number of compounds targeting a diverse range of apoptosis-related molecules are being explored at the preclinical and clinical levels. Beyond the agents that are already FDA approved, a range of molecules targeting apoptosis-regulatory transcription factors, regulators of mitochondrial membrane permeabilization, and inhibitors or activators of cell death–related proteases are under close scrutiny for drug development.

T he Akt and Pim kinases are cytoplasmic serine/threonine kinases that control programmed cell death by phosphorylating substrates that regulate both apoptosis and cellular metabolism. The PI3K-dependent activation of the Akt kinases and the JAK/STAT–dependent induction of the Pim kinases are examples of partially overlapping survival kinase pathways. Pharmacological manipulation of such kinases could have a major impact on the treatment of a wide variety of human diseases including cancer, inflammatory disorders, and ischemic diseases.

I κB kinase/NF-κB (IKK/NF-κB) signaling pathways play critical roles in a variety of physiological and pathological processes. One function of NF-κB is promotion of cell survival through induction of target genes, whose products inhibit components of the apoptotic machinery in normal and cancerous cells. NF-κB can also prevent programmed necrosis by inducing genes encoding antioxidant proteins. Regardless of mechanism, many cancer cells, of either epithelial or hematopoietic origin, use NF-κB to achieve resistance to anticancer drugs, radiation, and death cytokines. Hence, inhibition of IKK-driven NF-κB activation offers a strategy for treatment of different malignancies and can convert inflammation-induced tumor growth to inflammation-induced tumor regression.

I nduction of heat shock proteins (Hsps) following cellular damage can prevent apoptosis induced by both the intrinsic and the extrinsic pathways. The intrinsic pathway is characterized by mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, apoptosome assembly, and caspase activation. Hsps promote cell survival by preventing MOMP or apoptosome formation as well as via regulation of Akt and JNK activities. Engagement of the TNF death receptors induces the extrinsic pathway that is characterized by Fas-associated death domain–dependent (FADD-dependent) caspase-8 activation or induction of NF-κB to promote cellular survival. Hsps can directly suppress proapoptotic signaling events or stabilizing elements of the NF-κB pathway to promote cellular survival.

C ell death by apoptosis or necrosis is often important in the etiology and treatment of disease. Since mitochondria play important roles in cell death pathways, these organelles are potentially prime targets for therapeutic intervention. Here we discuss the mechanisms through which mitochondria participate in the cell death process and also survey some of the pharmacological approaches that target mitochondria in various ways.

T he commitment to programmed cell death involves complex interactions among pro- and antiapoptotic members of the Bcl-2 family of proteins. The physiological result of a decision by these proteins to undergo cell death is permeabilization of the mitochondrial outer membrane. Pharmacologic manipulation of proteins in this family appears both feasible and efficacious, whether the goal is decreased cell death, as in ischemia of the myocardium or brain, or increased cell death, as in cancer.

D isturbances in the normal functions of the ER lead to an evolutionarily conserved cell stress response, the unfolded protein response, which is aimed initially at compensating for damage but can eventually trigger cell death if ER dysfunction is severe or prolonged. The mechanisms by which ER stress leads to cell death remain enigmatic, with multiple potential participants described but little clarity about which specific death effectors dominate in particular cellular contexts. Important roles for ER-initiated cell death pathways have been recognized for several diseases, including hypoxia, ischemia/reperfusion injury, neurodegeneration, heart disease, and diabetes.

C aspases, a family of cysteine proteases, play a central role in apoptosis. During the last decade, major progress has been made to further understand caspase structure and function, providing a unique basis for drug design. This Review gives an overview of caspases and their classification, structure, and substrate specificity. We also describe the current knowledge of how interference with caspase signaling can be used to pharmacologically manipulate cell death.

R ecent studies have shown that members of the inhibitor of apoptosis (IAP) protein family are highly expressed in several classes of cancer. The primary implication of these findings is that the elevated expression of IAPs is not coincidental but actually participates in oncogenesis by helping to allow the malignant cell to avoid apoptotic cell death. This concept, together with the discovery of several IAP-regulatory proteins that use a conserved mode of action, has stimulated a major effort by many research groups to devise IAP-targeting strategies as a means of developing novel antineoplastic drugs. In this Review, we consider the evidence both for and against the IAPs being valid therapeutic targets, and we describe the types of strategies being used to neutralize their functions.

T he visualization of autophagosomes in dying cells has led to the belief that autophagy is a nonapoptotic form of programmed cell death. This concept has now been evaluated using cells and organisms deficient in autophagy genes. Most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro-survival rather than a pro-death mechanism. This review summarizes the evidence linking autophagy to cell survival and cell death, the complex interplay between autophagy and apoptosis pathways, and the role of autophagy-dependent survival and death pathways in clinical diseases.

N ADH:ubiquinone oxidoreductase (complex I) of the electron transport chain is a multimeric mitochondrial enzyme of approximately 1000 kDa consisting of 46 different proteins encoded by both the mitochondrial and nuclear genomes. Little is known about the cellular mechanisms and protein chaperones that guide its assembly. In this issue of the JCI, Ogilvie et al. use genomic sequence data to compare the proteins produced by yeasts with and without complex I in order to generate a list of proteins whose human orthologs might serve as complex I assembly proteins. The gene encoding one of these candidate proteins, B17.2L, was found to harbor a nonsense mutation in one of 28 patients with a deficiency of complex I. B17.2L associated with subcomplexes that are seen when complex I assembly is incomplete. The research described here combines clever model organism genomics and bioinformatics with sophisticated human molecular and biochemical genetics to identify the first mammalian protein required for the normal assembly of complex I.

T he pathobiology of pulmonary arterial hypertension (PAH) includes endothelial cell dysfunction and proliferation and migration of VSMCs. As PDGF has been implicated in these processes, Schermuly et al. hypothesized that altered PDGF signaling may be involved in the vascular remodeling observed in PAH. To explore this notion further, the authors evaluated the effects of the PDGF receptor inhibitor STI571 in 2 different animal models of pulmonary hypertension. In both models, after development of pulmonary vascular disease, administration of STI571 reversed pulmonary vascular changes. These studies provide preclinical proof of concept for the clinical development of a PDGF inhibitor as a targeted therapy for PAH patients.

A POA5 is a newly identified apolipoprotein that plays a crucial role in the regulation of plasma triglyceride levels. In several human studies, common APOA5 single nucleotide polymorphisms have been strongly associated with elevated plasma triglyceride levels. In this issue of the JCI, Marçais et al. report that the rare Q139X mutation in APOA5 leads to severe hypertriglyceridemia by exerting a dominant-negative effect on the plasma lipolytic system for triglyceride-rich lipoproteins. The presented data support the idea that the molecular mechanism of APOA5 function may include the enhancement of binding between lipoproteins and proteoglycans at the vascular wall and activation of proteoglycan-bound lipoprotein lipase.

T he amygdala is believed to play a key role in assigning emotional significance to specific sensory input, and conditions such as anxiety, autism, stress, and phobias are thought to be linked to its abnormal function. Growing evidence has also implicated the amygdala in mediation of the stress-dampening properties of alcohol. In this issue of the JCI, Pandey and colleagues identify a central amygdaloid signaling pathway involved in anxiety-like and alcohol-drinking behaviors in rats. They report that decreased phosphorylation of cAMP responsive element–binding protein (CREB) resulted in decreased neuropeptide Y (NPY) expression in the central amygdala of alcohol-preferring rats, causing high anxiety-like behavior. Alcohol intake by these animals was shown to increase PKA-dependent CREB phosphorylation and thereby NPY expression, subsequently ameliorating anxiety-like behavior. These provocative data suggest that a CREB-dependent neuromechanism underlies high anxiety-like and excessive alcohol-drinking behavior.

C holesterol efflux from macrophages, the first step in reverse cholesterol transport (RCT), is assumed to play a critical role in the pathogenesis of atherosclerosis. However, in vivo proof supporting this hypothesis is lacking, due to difficulties in determining the activity of this first step in RCT. In this issue of the JCI, Zhang et al. apply their recently developed method for measuring RCT in vivo to estimate RCT in mouse models with varying levels of HDL turnover. A surprisingly efficient clearance of cholesterol to feces is observed in mice overexpressing hepatic scavenger receptor class B type I (SR-BI), whereas in SR-BI–knockout mice, cholesterol clearance is diminished. The study demonstrates that hepatic SR-BI is a positive regulator of macrophage RCT in vivo.

D isruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell–mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

T his study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt⁴⁷³ induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival.

T ransverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.

T NF receptor–associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain–only protein that functions as a transcriptional coactivator or corepressor in a cell-type–specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-κB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2^–/– osteoclasts. FHL2 overexpression delays RANK ligand–induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2^–/– mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2^–/– osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis–stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling.

T hrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.

W e investigated the role of cAMP-responsive element–binding protein (CREB) in genetic predisposition to anxiety and alcohol-drinking behaviors using alcohol-preferring (P) and -nonpreferring (NP) rats. The levels of CREB, phosphorylated CREB, and neuropeptide Y (NPY) were innately lower in the central amygdala (CeA) and medial amygdala (MeA), but not in the basolateral amygdala (BLA), of P rats compared with NP rats. P rats displayed higher baseline anxiety-like behaviors and consumed higher amounts of alcohol compared with NP rats. Ethanol injection or voluntary intake reduced the higher anxiety levels in P rats. Ethanol also increased CREB function in the CeA and MeA, but not in the BLA, of P rats. Infusion of the PKA activator Sp-cAMP or NPY into the CeA decreased the alcohol intake and anxiety-like behaviors of P rats. PKA activator infusion also increased CREB function in the CeA of P rats. On the other hand, ethanol injection or voluntary intake did not produce any changes either in anxiety levels or on CREB function in the amygdaloid structures of NP rats. Interestingly, infusion of the PKA inhibitor Rp-cAMP into the CeA provoked anxiety-like behaviors and increased alcohol intake in NP rats. PKA inhibitor decreased CREB function in the CeA of NP rats. These novel results provide the first evidence to our knowledge that decreased CREB function in the CeA may be operative in maintaining the high anxiety and excessive alcohol-drinking behaviors of P rats.

D eficiency in docosahexaenoic acid (DHA), a brain-essential omega-3 fatty acid, is associated with cognitive decline. Here we report that, in cytokine-stressed human neural cells, DHA attenuates amyloid-β (Aβ) secretion, an effect accompanied by the formation of NPD1, a novel, DHA-derived 10,17S-docosatriene. DHA and NPD1 were reduced in Alzheimer disease (AD) hippocampal cornu ammonis region 1, but not in the thalamus or occipital lobes from the same brains. The expression of key enzymes in NPD1 biosynthesis, cytosolic phospholipase A₂ and 15-lipoxygenase, was altered in AD hippocampus. NPD1 repressed Aβ42-triggered activation of proinflammatory genes while upregulating the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1). Soluble amyloid precursor protein-α stimulated NPD1 biosynthesis from DHA. These results indicate that NPD1 promotes brain cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity.

N ADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.

D rusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (Aβ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Aβ on cultured human RPE cells as well as ocular findings in neprilysin gene–disrupted mice, which leads to an increased deposition Aβ. The results showed that Aβ treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Aβ-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Aβ accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Aβ reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD.

H uman noncollagenous domain 1 of the α1 chain of type IV collagen [α1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human α1(IV)NC1 binds to α1β1 integrin, competes with type IV collagen binding to α1β1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, α1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, α1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in α1 integrin receptor knockout ECs. Consistent with this, α1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in α1 integrin receptor knockout mice. This suggests a highly specific, α1β1 integrin–dependent antiangiogenic activity of α1(IV)NC1. In addition, α1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1α and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human α1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that α1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.

P rogression of pulmonary hypertension is associated with increased proliferation and migration of pulmonary vascular smooth muscle cells. PDGF is a potent mitogen and involved in this process. We now report that the PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension. In rats with monocrotaline-induced pulmonary hypertension, therapy with daily administration of STI571 was started 28 days after induction of the disease. A 2-week treatment resulted in 100% survival, compared with only 50% in sham-treated rats. The changes in RV pressure, measured continuously by telemetry, and right heart hypertrophy were reversed to near-normal levels. STI571 prevented phosphorylation of the PDGF receptor and suppressed activation of downstream signaling pathways. Similar results were obtained in chronically hypoxic mice, which were treated with STI571 after full establishment of pulmonary hypertension. Moreover, expression of the PDGF receptor was found to be significantly increased in lung tissue from pulmonary arterial hypertension patients compared with healthy donor lung tissue. We conclude that STI571 reverses vascular remodeling and cor pulmonale in severe experimental pulmonary hypertension regardless of the initiating stimulus. This regimen offers a unique novel approach for antiremodeling therapy in progressed pulmonary hypertension.

X -linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber–FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome–specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.

M MPs, which degrade components of the ECM, have roles in embryonic development, tissue repair, cancer, arthritis, and cardiovascular disease. We show that a missense mutation of MMP13 causes the Missouri type of human spondyloepimetaphyseal dysplasia (SEMD_MO), an autosomal dominant disorder characterized by defective growth and modeling of vertebrae and long bones. Genome-wide linkage analysis mapped SEMD_MO to a 17-cM region on chromosome 11q14.3–23.2 that contains a cluster of 9 MMP genes. Among these, MMP13 represented the best candidate for SEMD_MO, since it preferentially degrades collagen type II, abnormalities of which cause skeletal dysplasias that include Strudwick type SEMD. DNA sequence analysis revealed a missense mutation, F56S, that substituted an evolutionarily conserved phenylalanine residue for a serine in the proregion domain of MMP13. We predicted, by modeling MMP13 structure, that this F56S mutation would result in a hydrophobic cavity with misfolding, autoactivation, and degradation of mutant protein intracellularly. Expression of wild-type and mutant MMP13s in human embryonic kidney cells confirmed abnormal intracellular autoactivation and autodegradation of F56S MMP13 such that only enzymatically inactive, small fragments were secreted. Thus, the F56S mutation results in deficiency of MMP13, which leads to the human skeletal developmental anomaly of SEMD_MO.

D ietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element–binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes.

< i>Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor–positive and –negative (PAF-R–positive and –negative) cells and PAF-R–deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca²⁺ flux only in PAF-R–positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R–deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R–expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

W hile type 1 hyperlipidemia is associated with lipoprotein lipase or apoCII deficiencies, the etiology of type 5 hyperlipidemia remains largely unknown. We explored a new candidate gene, APOA5, for possible causative mutations in a pedigree of late-onset, vertically transmitted hyperchylomicronemia. A heterozygous Q139X mutation in APOA5 was present in both the proband and his affected son but was absent in 200 controls. It was subsequently found in 2 of 140 cases of hyperchylomicronemia. Haplotype analysis suggested the new Q139X as a founder mutation. Family studies showed that 5 of 9 total Q139X carriers had hyperchylomicronemia, 1 patient being homozygote. Severe hypertriglyceridemia in 8 heterozygotes was strictly associated with the presence on the second allele of 1 of 2 previously described triglyceride-raising minor APOA5 haplotypes. Furthermore, ultracentrifugation fraction analysis indicated in carriers an altered association of Apoa5 truncated and WT proteins to lipoproteins, whereas in normal plasma, Apoa5 associated with VLDL and HDL/LDL fractions. APOB100 kinetic studies in 3 severely dyslipidemic patients with Q139X revealed a major impairment of VLDL catabolism. Lipoprotein lipase activity and mass were dramatically reduced in dyslipidemic carriers, leading to severe lipolysis defect. Our observations strongly support in humans a role for APOA5 in lipolysis regulation and in familial hyperchylomicronemia.

H epatic expression of the scavenger receptor class B type I (SR-BI) promotes selective uptake of HDL cholesterol by the liver and is believed to play a role in the process of reverse cholesterol transport (RCT). We hypothesized that hepatic SR-BI expression is a regulator of the rate of integrated macrophage-to-feces RCT and used an in vivo model to test this hypothesis. Cholesterol-loaded and [³H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice, after which the appearance of the [³H]cholesterol in the plasma, liver, and feces over 48 hours was quantitated. Mice overexpressing SR-BI in the liver had significantly reduced [³H]cholesterol in the plasma but markedly increased [³H] tracer excretion in the feces over 48 hours. Conversely, mice deficient in SR-BI had significantly increased [³H]cholesterol in the plasma but markedly reduced [³H] tracer excretion in the feces over 48 hours. These studies demonstrate that hepatic SR-BI expression, despite its inverse effects on steady-state plasma HDL cholesterol concentrations, is an important positive regulator of the rate of macrophage RCT.

P athogen-induced apoptosis of lymphocytes is associated with increased susceptibility to infection. In this study, we determined whether apoptosis influenced host resistance to the fungus Histoplasma capsulatum. The level of apoptotic leukocytes progressively increased in the lungs of naive and immune mice during the course of H. capsulatum infection. T cells constituted the dominant apoptotic population. Apoptosis was diminished in H. capsulatum–infected gld/gld and TNF-α–deficient mice; concomitantly, the fungal burden exceeded that of controls. Treatment of naive and H. capsulatum–immune mice with caspase inhibitors decreased apoptosis but markedly enhanced the severity of infection. Administration of a proapoptotic dose of suramin diminished the fungal burden. The increased burden in recipients of a caspase inhibitor was associated with elevations in IL-4 and IL–10 levels. In the absence of either of these cytokines, caspase inhibition suppressed apoptosis but did not increase the fungal burden. Thus, apoptosis is a critical element of protective immunity to H. capsulatum. Production of IL-4 and IL-10 is markedly elevated when apoptosis is inhibited, and the release of these cytokines exacerbates the severity of infection.

B eryllium exposure can lead to the development of beryllium-specific CD4⁺ T cells and chronic beryllium disease (CBD), which is characterized by the presence of lung granulomas and a CD4⁺ T cell alveolitis. Studies have documented the presence of proliferating and cytokine-secreting CD4⁺ T cells in blood of CBD patients after beryllium stimulation. However, some patients were noted to have cytokine-secreting CD4⁺ T cells in blood in the absence of beryllium-induced proliferation, and overall, the correlation between the 2 types of responses was poor. We hypothesized that the relative proportion of memory T cell subsets determined antigen-specific proliferation. In most CBD patients, the majority of beryllium-specific CD4⁺ T cells in blood expressed an effector memory T cell maturation phenotype. However, the ability of blood cells to proliferate in the presence of beryllium strongly correlated with the fraction expressing a central memory T cell phenotype. In addition, we found a direct correlation between the percentage of beryllium-specific CD4⁺ T_EM cells in blood and T cell lymphocytosis in the lung. Together, these findings indicate that the functional capability of antigen-specific CD4⁺ T cells is determined by the relative proportion of memory T cell subsets, which may reflect internal organ involvement.

T LRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2^–/– and TLR2^+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2^–/– mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.

M odified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3γ1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8⁺ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8⁺ T cells that was similar in vitro and in vivo and induced regulatory CD8⁺CD25⁺ T cells. These cells inhibited the responses of CD4⁺ cells to the mAb itself and to antigen. The regulatory CD8⁺CD25⁺ cells were CTLA4⁺ and Foxp3⁺ and required contact for inhibition. Foxp3 was also induced on CD8⁺ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8⁺ T cells.

H uman monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcγ receptor (FcγR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-γ most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcγRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcγRs on human DCs to generate either antigen-specific immunity or tolerance.

W e have previously published that 2 proven treatments for acute promyelocytic leukemia, As₂O₃ and retinoic acid, can be antagonistic in vitro. We now report that As₂O₃ inhibits ligand-induced transcription of the retinoic acid receptor, as well as other nuclear receptors that heterodimerize with the retinoid X receptor α (RXRα). As₂O₃ did not inhibit transactivation of the estrogen receptor or the glucocorticoid receptor, which do not heterodimerize with RXRα. We further show that As₂O₃ inhibits expression of several target genes of RXRα partners. Phosphorylation of RXRα has been reported to inhibit nuclear receptor signaling, and we show by in vivo labeling and phosphoamino acid detection that As₂O₃ phosphorylated RXRα in the N-terminal ABC region exclusively on serine residues. Consistent with our previous data implying a role for JNK in As₂O₃-induced apoptosis, we show that pharmacologic or genetic inhibition of JNK activation decreased As₂O₃-induced RXRα phosphorylation and blocked the effects of As₂O₃ on RXRα-mediated transcription. A mutational analysis indicated that phosphorylation of a specific serine residue, S32, was primarily responsible for inhibition of RXRα-mediated transcription. These data may provide some insight into the rational development of chemotherapeutic combinations involving As₂O₃ as well as into molecular mechanisms of arsenic-induced carcinogenesis resulting from environmental exposure.

L ysosomal proteases generate peptides presented by class II MHC molecules to CD4⁺ T cells. To determine whether specific lysosomal proteases might influence the outcome of a CD4⁺ T cell–dependent autoimmune response, we generated mice that lack cathepsin L (Cat L) on the autoimmune diabetes-prone NOD inbred background. The absence of Cat L affords strong protection from disease at the stage of pancreatic infiltration. The numbers of I-A^g7–restricted CD4⁺ T cells are diminished in Cat L–deficient mice, although a potentially diabetogenic T cell repertoire persists. Within the CD4⁺ T cell compartments of Cat L–deficient mice, there is an increased proportion of regulatory T cells compared with that in Cat L–sufficient littermates. We suggest that it is this displaced balance of regulatory versus aggressive CD4⁺ T cells that protects Cat L–deficient mice from autoimmune disease. Our results identify Cat L as an enzyme whose activity is essential for the development of type I diabetes in the NOD mouse.