Issue published February 1, 2004

< i>Helicobacter pylori are bacteria that have coevolved with humans to be transmitted from person to person and to persistently colonize the stomach. Their population structure is a model for the ecology of the indigenous microbiota. A well-choreographed equilibrium between bacterial effectors and host responses permits microbial persistence and health of the host but confers risk of serious diseases, including peptic ulceration and gastric neoplasia.

T he recent history of research on cholera illustrates the importance of establishing research and care facilities equipped with advanced technologies at locations where specific health problems exist. It is in such settings, where scientific research is often considered difficult due to poverty and the lack of essential infrastructure, that investigators from many countries are able to make important advances. On this, the 25th anniversary of the founding of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), this article seeks to recount the Centre’s demonstration of how high-quality research on important global health issues, including cholera, can be accomplished in conditions that may be considered by many as unsuitable for scientific research.

P latelet activation occurs in response to vessel injury and is important for the arrest of bleeding. Platelet activation during disease states leads to vascular occlusion and ischemic damage. The P2Y₁₂ receptor, activated by ADP, plays a central role in platelet activation and is the target of P2Y₁₂ receptor antagonists that have proven therapeutic value.

A study in this issue of the JCI shows that in response to autoantigens consisting of peptides from normal proteins, patients with diabetes mount a T cell response characterized by production of IFN-γ . However, in response to these same antigens, T cells from normal control subjects produce IL-10. The antigen-specific response characterized by release of a regulatory cytokine suggests a mechanism for the control of autoimmunity that is initiated at the time of antigen presentation.

M utations in lamins A and C, nuclear intermediate-filament proteins in nearly all somatic cells, cause a variety of diseases that primarily affect striated muscle, adipocytes, or peripheral nerves or cause features of premature aging. Two new studies use lamin A/C–deficient mice, which develop striated muscle disease, as a model to investigate pathogenic mechanisms. These reports provide evidence for a stepwise process in which mechanically stressed cells first develop chromatin and nuclear envelope damage and then develop secondary alterations in the transcriptional activation of genes in adaptive and protective pathways.

A shortcoming in the clinical use of organic nitrates is the development of tolerance. Recent data have suggested that the denitrification of organic nitrates is mediated by mitochondrial aldehyde dehydrogenase and that dysfunction of this enzyme is an important cause of tolerance. In this issue of the JCI, evidence in support of this hypothesis is presented in an in vivo model of nitrate tolerance.

G enetic disorders of amino acid and fatty acid metabolism can be detected with tandem mass spectrometry (MS/MS). MS/MS screening of mice subjected to chemical mutagenesis defined a new disorder of branched-chain amino acid metabolism resembling human maple syrup urine disease. This approach has general application to the discovery of gene function in developmental and metabolic disorders.

L aminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C–deficient (Lmna^–/–) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna^–/– myocytes show reduced shortening with normal baseline and peak amplitude of Ca²⁺ transients. Lmna^–/– LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna^–/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARγ expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna^–/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna^–/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.

M utations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C–deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna–/– cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-κB–regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna–/– cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.

T he gp130-dependent cytokines, which signal through at least two intracellular pathways, regulate osteoclast and osteoblast formation. To define their roles in regulating bone mass, we analyzed mice in which gp130 signaling via either the signal transducer and activator of transcription (STAT) 1/3 (gp130^{ΔSTAT/ΔSTAT}) or SHP2/ras/MAPK (gp130^Y757F/Y757F) pathway was attenuated. In gp130^{ΔSTAT/ΔSTAT} mice, trabecular bone volume (BV/TV) and turnover were normal, but bone length was reduced by premature growth plate closure, indicating an essential role for gp130-STAT1/3 signaling in chondrocyte differentiation. In contrast, while bone size was normal in gp130^Y757F/Y757F mice, BV/TV was reduced due to high bone turnover, indicated by high osteoclast surface/bone surface (OcS/BS) and osteoblast surface/bone surface (ObS/BS). Furthermore, generation of functional osteoclasts from bone marrow of gp130^Y757F/Y757F mice was elevated, revealing that while gp130 family cytokines stimulate osteoclastogenesis through the osteoblast lineage, gp130, via SHP2/Ras/MAPK, inhibits osteoclastogenesis in a cell lineage–autonomous manner. Genetic ablation of IL-6 in gp130^Y757F/Y757F mice exacerbated this osteopenia by reducing ObS/BS without affecting OcS/BS. Thus, while IL-6 is critical for high bone formation in gp130^Y757F/Y757F mice, it is not involved in the increased osteoclastogenesis. In conclusion, gp130 is essential for normal bone growth and trabecular bone mass, with balanced regulation depending on selective activation of STAT1/3 and SHP2/ras/MAPK, respectively. Furthermore, the latter pathway can directly inhibit osteoclastogenesis in vivo.

G iven our recent discovery that it is possible to separate human epidermal stem cells of the skin from their more committed progeny (i.e., transit-amplifying cells and early differentiating cells) using FACS techniques, we sought to determine the comparative tissue regeneration ability of these keratinocyte progenitors. We demonstrate that the ability to regenerate a fully stratified epidermis with appropriate spatial and temporal expression of differentiation markers in a short-term in vitro organotypic culture system is an intrinsic characteristic of both epidermal stem and transit-amplifying cells, although the stem cell fraction is most capable of achieving homeostasis. Early differentiating keratinocytes exhibited limited short-term tissue regeneration under specific experimental conditions in this assay, although significant improvement was obtained by manipulating microenvironmental factors, that is, coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11. Importantly, transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem, transit-amplifying, and early differentiating keratinocytes for up to 10 weeks. These data illustrate that significant proliferative and tissue-regenerative capacity resides not only in keratinocyte stem cells as expected, but also in their more committed progeny, including early differentiating cells.

T NF-α has long been regarded as a proimmune cytokine involved in antimicrobial type 1 immunity. However, the precise role of TNF-α in antimicrobial type 1 immunity remains poorly understood. We found that TNF-α–deficient (TNF^–/–) mice quickly succumbed to respiratory failure following lung infection with replication-competent mycobacteria, because of apoptosis and necrosis of granuloma and lung structure. Tissue destruction was a result of an uncontrolled type 1 immune syndrome characterized by expansion of activated CD4 and CD8 T cells, increased frequency of antigen-specific T cells, and overproduction of IFN-γ and IL-12. Depletion of CD4 and CD8 T cells decreased IFN-γ levels, prevented granuloma and tissue necrosis, and prolonged the survival of TNF^–/– hosts. Early reconstitution of TNF-α by gene transfer reduced the frequency of antigen-specific T cells and improved survival. TNF-α controlled type 1 immune activation at least in part by suppressing T cell proliferation, and this suppression involved both TNF receptor p55 and TNF receptor p75. Heightened type 1 immune activation also occurred in TNF^–/– mice treated with dead mycobacteria, live replication-deficient mycobacteria, or mycobacterial cell wall components. Our study thus identifies TNF-α as a type 1 immunoregulatory cytokine whose primary role, different from those of other type 1 cytokines, is to keep an otherwise detrimental type 1 immune response in check.

L ipodystrophy is characterized by the complete or partial absence of adipose tissue, insulin resistance, hepatic steatosis, and leptin deficiency. Here, we show that low-dose central leptin corrects the insulin resistance and fatty liver of lipodystrophic aP2-nSREBP-1c mice, while the same dose given peripherally does not. Central leptin also repressed stearoyl-CoA desaturase-1 (SCD-1) RNA and enzymatic activity, which were increased in livers of lipodystrophic mice. aP2-nSREBP-1c mice homozygous for an SCD-1 deletion had markedly reduced hepatic steatosis, increased saturated fatty acids, decreased acetyl-CoA carboxylase activity, and decreased malonyl-CoA levels in the liver. Despite the reduction in hepatic steatosis, these mice remained diabetic. A leptin dose-response curve showed that subcutaneous leptin improved hyperglycemia and hyperinsulinemia in aP2-nSREBP-1c mice at doses that did not substantially alter hepatic steatosis or hepatic SCD enzymatic activity. Leptin treatment at this dose improved insulin-stimulated insulin receptor and insulin receptor substrate 2 (IRS-2) phosphorylation, IRS-2–associated PI3K activity, and Akt activity in liver. Together, these data suggest that CNS-mediated repression of SCD-1 contributes to leptin’s antisteatotic actions. Intracerebroventricular leptin improves glucose homeostasis by improving insulin signal transduction in liver, but in this case the effect appears to be independent of SCD-1.

M ost of the human tumor-associated antigens (TAAs) characterized thus far are derived from nonmutated “self”-proteins. Numerous strategies have been developed to break tolerance to TAAs, combining various forms of antigens with different vectors and adjuvants. However, no study has yet determined how to select epitopes within a given TAA to induce the highest antitumor effector response. We addressed this question by evaluating in HLA-A*0201-transgenic HHD mice the antitumor vaccination efficacy of high- and low-affinity epitopes from the naturally expressed murine telomerase reverse transcriptase (mTERT). Immunity against low-affinity epitopes was induced with heteroclitical variants. We show here that the CTL repertoire against high-affinity epitopes is partially tolerized, while that against low-affinity epitopes is composed of frequent CTLs with high avidity. The high-affinity p797 and p545 mTERT epitopes are not able to protect mice from a lethal challenge with the mTERT-expressing EL4-HHD tumor. In contrast, mice developing CTL responses against the p572 and p988 low-affinity epitopes exhibit potent antitumor immunity and no sign of autoimmune reactivity against TERT-expressing normal tissues. Our results strongly argue for new TAA epitope selection and modification strategies in antitumor immunotherapy applications in humans.

T andem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea–treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain α-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.

P rior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the G_q-coupled receptors for thrombin and thromboxane A₂. Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Gα_q but was relatively unaffected by deletion of Gα_i2, which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of G_q-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.

A ccording to the quality of response they mediate, autoreactive T cells recognizing islet β cell peptides could represent both disease effectors in the development of type 1 diabetes (T1DM) and directors of tolerance in nondiabetic individuals or those undergoing preventative immunotherapy. A combination of the rarity of these cells, inadequate technology, and poorly defined epitopes, however, has hampered examination of this paradigm. We have identified a panel of naturally processed islet epitopes by direct elution from APCs bearing HLA-DR4. Employing these epitopes in a sensitive, novel cytokine enzyme-linked immunosorbent spot assay, we show that the quality of autoreactive T cells in patients with T1DM exhibits extreme polarization toward a proinflammatory Th1 phenotype. Furthermore, we demonstrate that rather than being unresponsive, the majority of nondiabetic, HLA-matched control subjects also manifest a response against islet peptides, but one that shows extreme T regulatory cell (Treg, IL-10–secreting) bias. We conclude that development of T1DM depends on the balance of autoreactive Th1 and Treg cells, which may be open to favorable manipulation by immune intervention.

A n antibiotic efflux gene cluster that confers resistance to chloramphenicol, trimethoprim, and ciprofloxacin has been identified in Burkholderia cenocepacia (genomovar III), an important cystic fibrosis pathogen. Five open reading frames have been identified in the cluster. There is apparently a single transcriptional unit, with llpE encoding a lipase-like protein, ceoA encoding a putative periplasmic linker protein, ceoB encoding a putative cytoplasmic membrane protein, and opcM encoding a previously described outer membrane protein. A putative LysR-type transcriptional regulatory gene, ceoR, is divergently transcribed upstream of the structural gene cluster. Experiments using radiolabeled chloramphenicol and salicylate demonstrated active efflux of both compounds in the presence of the gene cluster. Salicylate is an important siderophore produced by B. cepacia complex isolates, and both extrinsic salicylate and iron starvation appear to upregulate ceoR promoter activity, as does chloramphenicol. These results suggest that salicylate is a natural substrate for the efflux pump in B. cenocepacia and imply that the environment of low iron concentration in the cystic fibrosis lung can induce efflux-mediated resistance, even in the absence of antibiotic selective pressure.

T he inhibitor of NF-κB (IκB) kinases (IKK1[α] and IKK2[β]), the catalytic subunits of the IKK complex, phosphorylate IκB proteins on serine residues, targeting them for degradation and thus activating the transcription factor NF-κB. More recently, IKK2 has been implicated in mediation of insulin resistance caused by obesity, lipid infusion, and TNF-α stimulation, since salicylate and aspirin, known inhibitors of IKK activity, can reverse insulin resistance in obese mouse models. To further genetically elucidate the role of IKK2 in obesity-mediated insulin resistance, we have conditionally inactivated the mouse IKK2 gene in adult myocytes by Cre-loxP–mediated recombination in vivo. We have investigated the development of obesity-induced insulin resistance in muscle-specific IKK2 knockout mice and mice exhibiting a 50% reduction of IKK2 expression in every tissue and have found that, after gold thioglucose treatment, wild-type and mutant mice developed obesity to a similar extent. Surprisingly, no difference in obesity-induced insulin resistance was detectable, either at a physiological or at a molecular level. Moreover, impaired glucose tolerance resulting from a high-fat diet occurred to the same degree in control and IKK2 mutant mice. These data argue against a substantial role for muscular IKK2 in mediating obesity-induced insulin resistance in these models in vivo.

R ecent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (ρ⁰ cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.