Issue published September 1, 2003

T he development of targeted gene repair is under way and, despite some setbacks, shows promise as an alternative form of gene therapy. This approach uses synthetic DNA molecules to activate and direct the cell’s inherent DNA repair systems to correct inborn errors. The progress of this technique and its therapeutic potential are discussed in relation to the treatment of genetic diseases.

S mall DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, β-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

A novel circulation phosphaturic hormone is postulated to regulate systemic phosphate homeostasis. Two new studies reveal that the phosphaturic factor FGF-23 is increased in hypophosphatemic subjects with McCune-Albright syndrome and that secreted frizzled-related protein-4 (sFRP-4), a factor produced by tumors derived from subjects with tumor-induced osteomalacia, also has phosphaturic activity. It remains to be established whether FGF-23 and sFRP-4 represent two distinct phosphatonins or are somehow integrated in a novel phosphate-regulating bone-kidney axis.

C D1d, a nonclassical MHC class I–like molecule, is prominently expressed on intestinal epithelial cells and is thought to function in the regulation of intestinal intraepithelial lymphocyte activity. Hsp110, an abundant heat shock protein present in essentially all mammalian tissues, has now been shown to upregulate CD1d expression in colonic tissue culture cell lines. Might this abundant chaperone serve an autocrine function in the regulation of CD1d expression?

M ammals coexist in an overall symbiotic relationship with a complex array of commensal bacterial flora that colonizes the gastrointestinal tract. These intestinal bacteria interface with cells of the mucosal immune system, including DCs. Here we discuss mechanisms of interaction between intestinal bacteria and DCs and the role of localized gastrointestinal immune responses.

I t has long been noted that while patients with familial hypertrophic cardiomyopathy due to cardiac troponin T (cTnT) mutations often suffer sudden cardiac death, they do not develop significant ventricular hypertrophy, suggesting that a distinct cellular mechanism apart from alterations in myocardial contractility is responsible. A new study has revealed that a single missense mutation in cTnT causes a striking disruption to energy metabolism, leading to cardiomyopathy.

A new study demonstrates that angiotensin-induced hypertension results in a marked decrease in expression of the β subunit of the BK channel, suggesting a role for this critical subunit in the regulation of vascular tone.

B acillus anthracis, the causative agent of anthrax, is believed to induce disease and death in humans in an endotoxic shock–like manner. A comprehensive study of the effects of anthrax toxin in mice demonstrates that toxin-induced death is mediated not by cytokine release, as previously thought, but by hypoxia-induced liver failure. The study strongly suggests that the therapies developed for treatment of cytokine-mediated septic shock will not be appropriate for the treatment of anthrax.

G onadotropins induce ovarian follicle growth that is coincident with increased follicular vasculature, suggesting a role of angiogenesis in follicle development. Functional studies performed in nonhuman primates show that administration of substances that inactivate VEGF block the development and function of preovulatory follicles as demonstrated by histological analysis or hormone measurements. Blockage of function of VEGF receptor 2 (VEGFR-2) alters follicular hormone secretion, suggesting that the intraovarian effect of VEGF might be mediated by this receptor. The specific mechanism by which follicular development was blocked in these previous studies remains unclear, however. Here we characterize the intraovarian role of VEGFR-2 activity on follicular development by choosing a model in which active feedback is absent, the prepuberally hypophysectomized mouse. Hypophysectomy prevents advanced follicle growth and maturation; however, follicle development to the preovulatory stage can be stimulated by administration of gonadotropins. We report that exogenously administered gonadotropins are unable to drive follicle development to the preovulatory stage in the presence of antiangiogenic agent, VEGFR-2–neutralizing Ab’s. This inhibition of follicular development is caused by arrests to both angiogenesis and antrum formation. We conclude that the intraovarian VEGF/VEGFR-2 pathway is critical for gonadotropin-dependent angiogenesis and follicular development.

< i>Bacillus anthracis lethal toxin (LT) is the major virulence factor of anthrax and reproduces most of the laboratory manifestations of the disease in animals. We studied LT toxicity in BALB/cJ and C57BL/6J mice. BALB/cJ mice became terminally ill earlier and with higher frequency than C57BL/6J mice. Timed histopathological analysis identified bone marrow, spleen, and liver as major affected organs in both mouse strains. LT induced extensive hypoxia. Crisis was due to extensive liver necrosis accompanied by pleural edema. There was no evidence of disseminated intravascular coagulation or renal dysfunction. Instead, analyses revealed hepatic dysfunction, hypoalbuminemia, and vascular/oxygenation insufficiency. Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, eotaxin, FasL, and IL-1β. No changes in TNF-α occurred. The C57BL/6J mice did not mount a similar cytokine response. These factors were not induced in vitro by LT treatment of toxin-sensitive macrophages. The evidence presented shows that LT kills mice through a TNF-α–independent, FasL-independent, noninflammatory mechanism that involves hypoxic tissue injury but does not require macrophage sensitivity to toxin.

F GF-23, a novel member of the FGF family, is the product of the gene mutated in autosomal dominant hypophosphatemic rickets (ADHR). FGF-23 has been proposed as a circulating factor causing renal phosphate wasting not only in ADHR (as a result of inadequate degradation), but also in tumor-induced osteomalacia (as a result of excess synthesis by tumor cells). Renal phosphate wasting occurs in approximately 50% of patients with McCune-Albright syndrome (MAS) and fibrous dysplasia of bone (FD), which result from postzygotic mutations of the GNAS1 gene. We found that FGF-23 is produced by normal and FD osteoprogenitors and bone-forming cells in vivo and in vitro. In situ hybridization analysis of FGF-23 mRNA expression identified “fibrous” cells, osteogenic cells, and cells associated with microvascular walls as specific cellular sources of FGF-23 in FD. Serum levels of FGF-23 were increased in FD/MAS patients compared with normal age-matched controls and significantly higher in FD/MAS patients with renal phosphate wasting compared with those without, and correlated with disease burden bone turnover markers commonly used to assess disease activity. Production of FGF-23 by FD tissue may play an important role in the renal phosphate–wasting syndrome associated with FD/MAS.

I L-12 p40–related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses. Little is known, however, about the transcriptional regulation of the p40 gene in vivo. In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins. The cells constitutively producing IL-12 p40 were identified as CD8α and CD11b double-negative CD11c⁺ lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon. FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression. Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression. In addition, analysis of transgenic mice with a mutated NF-κB target site in the p40 promoter showed a critical role of NF-κB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-κB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

S ubsets of parasympathetic and enteric neurons require neurturin signaling via glial cell line–derived neurotrophic factor family receptor α2 (GFRα2) for development and target innervation. Why GFRα2-deficient (Gfra2^–/–) mice grow poorly has remained unclear. Here, we analyzed several factors that could contribute to the growth retardation. Neurturin mRNA was localized in the gut circular muscle. GFRα2 protein was expressed in most substance P–containing myenteric neurons, in most intrapancreatic neurons, and in surrounding glial cells. In the Gfra2^–/– mice, density of substance P–containing myenteric ganglion cells and nerve bundles in the myenteric ganglion cell layer was significantly reduced, and transit of test material through small intestine was 25% slower compared to wild-type mice. Importantly, the knockout mice had approximately 80% fewer intrapancreatic neurons, severely impaired cholinergic innervation of the exocrine but not the endocrine pancreas, and increased fecal fat content. Vagally mediated stimulation of pancreatic secretion by 2-deoxy-glucose in vivo was virtually abolished. Retarded growth of the Gfra2^–/– mice was accompanied by reduced fat mass and elevated basal metabolic rate. Moreover, the knockout mice drank more water than wild-type controls, and wet-mash feeding resulted in partial growth rescue. Taken together, the results suggest that the growth retardation in mice lacking GFRα2 is largely due to impaired salivary and pancreatic secretion and intestinal dysmotility.

H ypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca²⁺ release events through ryanodine receptors (Ca²⁺ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca²⁺-activated K⁺ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca²⁺ spark–BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the β1 subunit was decreased relative to the pore-forming α subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca²⁺ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the β1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

I ncreased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate–cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.

M eningitis occurs when blood-borne pathogens cross the blood-brain barrier (BBB) in a complex interplay between endothelial cells and microbial gene products. We sought to understand the initial response of the BBB to the human meningeal pathogen group B Streptococcus (GBS) and the organism’s major virulence factors, the exopolysaccharide capsule and the β-hemolysin/cytolysin toxin (β-h/c). Using oligonucleotide microarrays, we found that GBS infection of human brain microvascular endothelial cells (HBMEC) induced a highly specific and coordinate set of genes including IL-8, Groα, Groβ, IL-6, GM-CSF, myeloid cell leukemia sequence-1 (Mcl-1), and ICAM-1, which act to orchestrate neutrophil recruitment, activation, and enhanced survival. Most strikingly, infection with a GBS strain lacking β-h/c resulted in a marked reduction in expression of genes involved in the immune response, while the unencapsulated strain generally induced similar or greater expression levels for the same subset of genes. Cell-free bacterial supernatants containing β-h/c activity induced IL-8 release, identifying this toxin as a principal provocative factor for BBB activation. These findings were further substantiated in vitro and in vivo. Neutrophil migration across polar HBMEC monolayers was stimulated by GBS and its β-h/c through a process involving IL-8 and ICAM-1. In a murine model of hematogenous meningitis, mice infected with β-h/c mutants exhibited lower mortality and decreased brain bacterial counts compared with mice infected with the corresponding WT GBS strains.

C D1d is expressed on the surface of professional and nonprofessional APCs, including intestinal epithelial cells (IECs), for a role in the presentation of glycolipid-based antigens to subsets of T cells. The mechanisms that regulate CD1d expression in any cell type are unknown. To investigate the possibility that expression of CD1d is influenced by exogenous factors present within the intestinal lumen, CD1d expression was analyzed in several IEC lines after culturing in the presence of lumenal contents (LC) of the normal human intestine. Exposure of the colon-derived cell lines T84, HT-29, and Caco-2 to soluble LC resulted in a marked induction of CD1d expression as determined by RT-PCR, confocal microscopy, cell surface ELISA, and Western blot analysis. Similarly, exposure of human IECs to LC isolated from mice bred in both specific pathogen–free and germfree conditions also resulted in the induction of CD1d expression, with the maximum CD1d-inducing activity observed in the small intestine. Biochemical and biophysical characterization of the human CD1d-inducing activity identified heat shock protein 110 (Hsp110) as a major functional component of the LC that contributes to CD1d surface regulation, and immunolocalization studies revealed Hsp110 expression in subsets of human IECs in vivo. These data support the presence of a novel autocrine pathway of CD1d regulation by Hsp110.

I n studies using genetically deficient mice, a role for the lymphotoxin (LT) system in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has remained controversial. Here, we have reassessed this conclusion by using a fusion protein decoy that blocks the LT pathway in vivo without evoking the developmental defects inherent in LT-deficient mice. We have found that inhibition of the LT pathway prevented disease in two models of EAE that do not rely on the administration of pertussis toxin. Surprisingly, disease attenuation was due to specific blockade of LTαβ binding rather than the binding of LIGHT to its receptors. In a third system that requires pertussis toxin, LT inhibition did not affect disease, as was observed when the same model was used with LT-deficient mice. Disease prevention in pertussis toxin–free models was associated with defects in T cell responses and migration. When the DO11.10 T cell transgenic system was used, inhibition of the LT pathway was shown to uncouple T cell priming from T cell recall responses. Therefore, it is hypothesized that the LT pathway and its ability to maintain lymphoid microenvironments is critical for sustaining late-phase T cell responses in multiple sclerosis.

T he thin filament protein cardiac troponin T (cTnT) is an important regulator of myofilament activation. Here we report a significant change in cardiac energetics in transgenic mice bearing the missense mutation R92Q within the tropomyosin-binding domain of cTnT, a mutation associated with a clinically severe form of familial hypertrophic cardiomyopathy. This functional domain of cTnT has recently been shown to be a crucial modulator of contractile function despite the fact that it does not directly interact with the ATP hydrolysis site in the myosin head. Simultaneous measurements of cardiac energetics using ³¹P NMR spectroscopy and contractile performance of the intact beating heart revealed both a decrease in the free energy of ATP hydrolysis available to support contractile work and a marked inability to increase contractile performance upon acute inotropic challenge in hearts from R92Q mice. These results show that alterations in thin filament protein structure and function can lead to significant defects in myocardial energetics and contractile reserve.

T cell receptor engagement with CD28 costimulation is generally required for naive T cell activation, whereas reactivation of memory cells is less dependent on CD28 costimulation. We studied this process in chronic beryllium disease, in which the frequency of antigen-specific CD4⁺ T cells in the lung is large and circulating antigen-specific cells are also detectable. In the lung, a large fraction of CD4⁺ T cells stopped expressing CD28 mRNA and protein, and this change in phenotype correlated with lung inflammation. In the presence of concentrations of CTLA-4Ig that inhibited the CD28-B7 interaction, beryllium-specific CD4⁺ T cells in lung were still able to proliferate and secrete IFN-γ in response to beryllium in culture. This functional independence of CD28 costimulation included lung CD28⁺ effector cells. Although lung CD4⁺CD28^– cells retained the ability to secrete Th1-type cytokines in response to beryllium, they showed less proliferative capacity and were more susceptible to cell death compared with CD28⁺ T cells. In contrast to lung cells, inhibition of the CD28-B7 interaction markedly reduced responses of beryllium-specific T cells in blood. Taken together, these findings suggest transition within memory CD4⁺ T cells from CD28 dependence in central memory cells to functional independence and then loss of CD28 expression in effector cells.

T umors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FE_Pi) from 14% ± 2% to 34% ± 5% (mean ± SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FE_Pi from 0.7% ± 0.2% to 3.8% ± 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FE_Pi, decreased serum phosphate (1.95 ± 0.1 to 1.53 ± 0.09 mmol/l, P < 0.05) but did not alter serum 1α, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1α-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal β-catenin and increased phosphorylated β-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1α, 25-dihydroxyvitamin D.

A llogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade–based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8⁺ T cells and nascent intrathymic alloreactive CD8⁺ T cells. In the absence of large numbers of host alloreactive CD8⁺ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.