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Showing papers in "Nature Medicine in 1997"


Journal ArticleDOI
TL;DR: It is demonstrated that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) — termed the SCID leukemia-initiating cell, or SL-IC — possesses the differentiate and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell.
Abstract: On the subject of acute myeloid leukemia (AML), there is little consensus about the target cell within the hematopoietic stem cell hierarchy that is susceptible to leukemic transformation, or about the mechanism that underlies the phenotypic, genotypic and clinical heterogeneity. Here we demonstrate that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) - termed the SCID leukemia-initiating cell, or SL-IC - possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all subtypes of AML analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34++ CD38-, similar to the cell-surface phenotype of normal SCID-repopulating cells, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation. The SL-ICs were able to differentiate in vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy.

6,709 citations


Journal ArticleDOI
TL;DR: It is suggested that apoptosis inhibition may be a general feature of neoplasia and survivin is identified as a potential new target for apoptosis-based therapy in cancer and lymphoma.
Abstract: Inhibitors of programmed cell death (apoptosis) aberrantly prolonging cell viability may contribute to cancer by facilitating the insurgence of mutations and by promoting resistance to therapy Despite the identification of several new apoptosis inhibitors related to bcl-2 or to the baculovirus IAP gene, it is not clear whether apoptosis inhibition plays a general role in neoplasia Here, we describe a new human gene encoding a structurally unique IAP apoptosis inhibitor, designated survivin Survivin contains a single baculovirus IAP repeat and lacks a carboxyl-terminal RING finger Present during fetal development, survivin is undetectable in terminally differentiated adult tissues However, survivin becomes prominently expressed in transformed cell lines and in all the most common human cancers of lung, colon, pancreas, prostate and breast, in vivo Survivin is also found in approximately 50% of high-grade non-Hodgkin's lymphomas (centroblastic, immunoblastic), but not in low-grade lymphomas (lymphocytic) Recombinant expression of survivin counteracts apoptosis of B lymphocyte precursors deprived of interleukin 3 (IL-3) These findings suggest that apoptosis inhibition may be a general feature of neoplasia and identify survivin as a potential new target for apoptosis-based therapy in cancer and lymphoma

3,200 citations


Journal ArticleDOI
TL;DR: Tissue transglutaminase is identified as the unknown endomysial autoantigen of celiac disease, and gliadin is a preferred substrate for this enzyme, giving rise to novel antigenic epitopes.
Abstract: Celiac disease is characterized by small intestinal damage with loss of absorptive villi and hyperplasia of the crypts, typically leading to malabsorption. In addition to nutrient deficiencies, prolonged celiac disease is associated with an increased risk for malignancy, especially intestinal T-cell lymphoma. Celiac disease is precipitated by ingestion of the protein gliadin, a component of wheat gluten, and usually resolves on its withdrawal. Gliadin initiates mucosal damage which involves an immunological process in individuals with a genetic predisposition. However, the mechanism responsible for the small intestinal damage characteristic of celiac disease is still under debate. Small intestinal biopsy with the demonstration of a flat mucosa which is reversed on a gluten-free diet is considered the main approach for diagnosis of classical celiac disease. In addition, IgA antibodies against gliadin and endomysium, a structure of the smooth muscle connective tissue, are valuable tools for the detection of patients with celiac disease and for therapy control. Incidence rates of childhood celiac disease range from 1:300 in Western Ireland to 1:4700 in other European countries, and subclinical cases detected by serological screening revealed prevalences of 3.3 and 4 per 1000 in Italy and the USA, respectively. IgA antibodies to endomysium are particularly specific indicators of celiac disease, suggesting that this structure contains one or more target autoantigens that play a role in the pathogenesis of the disease. However, the identification of the endomysial autoantigen(s) has remained elusive. We identified tissue transglutaminase as the unknown endomysial autoantigen. Interestingly, gliadin is a preferred substrate for this enzyme, giving rise to novel antigenic epitopes.

1,931 citations


Journal ArticleDOI
TL;DR: The mechanism of action of Bcl-2 provides clues for therapeutic interventions and could help in the development of new drugs to treat central nervous system disorders.
Abstract: The mechanism of action of Bcl-2 provides clues for therapeutic interventions

1,684 citations


Journal ArticleDOI
TL;DR: The first combined, high-resolution measurements of interstitial pH and pO2 profiles between adjacent vessels in a human tumor xenograft are reported, using fluorescence ratio imaging and phosphorescence quenching microscopy.
Abstract: The partial pressure of oxygen (pO2) and pH play critical roles in tumor biology and therapy. We report here the first combined, high-resolution (≤10 μm) measurements of interstitial pH and pO2 profiles between adjacent vessels in a human tumor xenograft, using fluorescence ratio imaging and phosphorescence quenching microscopy. We found (1) heterogeneity in shapes of pH and pO2 profiles; (2) a discordant relation between local pH profiles and corresponding pO2 profiles, yet a strong correlation between mean pH and pO2 profiles; (3) no correlation between perivascular pH/pO2 and nearest vessel blood flow; and (4) well-perfused tumor vessels that were hypoxic and, consequently, large hypoxic areas in the surrounding interstitium. Such multiparameter measurements of the in vivo microenvironment provide unique insights into biological processes in tumors and their response to treatment.

1,579 citations


Journal ArticleDOI
TL;DR: The data demonstrate that the preseniiin mutations cause a dominant gain of function and may induce AD by enhancing Aβ42 production, thus promoting cerebral β-amyloidosis.
Abstract: The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.

1,361 citations


Journal ArticleDOI
TL;DR: Evidence is provided for leptin as a novel placenta-derived hormone in humans and the physiologic and pathophysiologic significance of leptin in normal pregnancy and gestational trophoblastic neoplasms is suggested.
Abstract: Leptin is a circulating hormone that is expressed abundantly and specifically in the adipose tissue. It is involved in the regulation of energy homeostasis, as well as the neuroendocrine and reproductive systems. Here, we demonstrate production of leptin by nonadipose tissue, namely, placental trophoblasts and amnion cells from uteri of pregnant women. We show that pregnant women secrete a considerable amount of leptin from the placenta into the maternal circulation as compared with nonpregnant obese women. Leptin production was also detected in a cultured human choriocarcinoma cell line, BeWo cells, and was augmented during the course of forskolin-induced differentiation of cytotrophoblasts into syncytiotrophoblasts. Plasma leptin levels were markedly elevated in patients with hydatidiform mole or choriocarcinoma and were reduced after surgical treatment or chemotherapy. Leptin is also produced by primary cultured human amnion cells and is secreted into the amniotic fluid. The present study provides evidence for leptin as a novel placenta-derived hormone in humans and suggests the physiologic and pathophysiologic significance of leptin in normal pregnancy and gestational trophoblastic neoplasms.

1,273 citations


Journal ArticleDOI
TL;DR: It is shown that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL.
Abstract: The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30–72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.

1,254 citations


Journal ArticleDOI
TL;DR: Evidence is reported for the presence of ALT in a subset of tumor-derived cell lines and tumors, presumably via one or more novel telomere-lengthening mechanisms that the authors refer to as ALT (alternative lengthening of telomeres)
Abstract: The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential1–3. Telomerase is an enzyme that restores telomeric DNA sequences4, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo5. Telomerase activity has been detected in 50–100% of tumors of different types, but not in most normal adult somatic tissues6,7. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date7. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization8–11 presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres)11. Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.

1,242 citations


Journal ArticleDOI
TL;DR: Six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope and two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9–12 years of epitope stability.
Abstract: The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability CTL escape may play an important role in the pathogenesis of HIV infection

1,192 citations


Journal ArticleDOI
TL;DR: Both secondary degeneration at the site of SCI and the chronic demyelination of tracts away from the injury appear to be due in part to apoptosis, and as cytokines have been shown to mediate oligodendrocyte death in vitro, it seems likely that chronic demYelination after CNS injury shares features with chronic degenerative disorders like multiple sclerosis.
Abstract: Apoptosis is a morphologically defined form of programmed cell death seen in a variety of circumstances, including immune cell selection, carcinogenesis and development. Apoptosis has very recently been seen after ischemic or traumatic injury to the central nervous system (CNS), suggesting that active cell death as well as passive necrosis may mediate damage after CNS injury. After spinal cord injury (SCI) in the rat, typical post-traumatic necrosis occurred, but in addition, apoptotic cells were found from 6 hours to 3 weeks after injury, especially in the spinal white matter. Apoptotic cells were positive for oligodendrocyte markers. After SCI in monkeys, apoptotic cells were found within remote degenerating fiber tracts. Both secondary degeneration at the site of SCI and the chronic demyelination of tracts away from the injury appear to be due in part to apoptosis. As cytokines have been shown to mediate oligodendrocyte death in vitro, it seems likely that chronic demyelination after CNS injury shares features with chronic degenerative disorders like multiple sclerosis.

Journal ArticleDOI
TL;DR: Dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells, suggesting the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopolietic cells.
Abstract: We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

Journal ArticleDOI
TL;DR: Kidney, heart and spleen tissue obtained from domestic pigs contained multiple copies of integrated PERV genomes and expressed viral RNA, which could rescue a Moloney retroviral vector and acquired resistance to lysis by human complement.
Abstract: The possible use of pig organs and tissues as xenografts in humans is actively being considered in biomedical research. We therefore examined whether pig endogenous retrovirus (PERV) genomes can be infectiously transmitted to human cells in culture. Two pig kidney cell lines spontaneously produce C-type retrovirus particles. Cell-free retrovirus produced by the PK-15 kidney cell line (PERV-PK) infected pig, mink and human kidney 293 cell lines and co-cultivation of X-irradiated PK-15 cells with human cells resulted in a broader range of human cell infection, including human diploid fibroblasts and B- and T-cell lines. Kidney, heart and spleen tissue obtained from domestic pigs contained multiple copies of integrated PERV genomes and expressed viral RNA. Upon passage in human cells PERV-PK could rescue a Moloney retroviral vector and acquired resistance to lysis by human complement.

Journal ArticleDOI
TL;DR: Recovery of monocyte function resulted in clearance of sepsis in eight of nine patients and suggests that IFN-γ treatment in carefully selected septic patients is a novel therapeutic strategy worth pursuing.
Abstract: Neutralization of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1), decreases mortality in several animal models of sepsis. However, recent clinical trials did not show an unequivocal improvement in survival. In contrast to animals, which succumb to shock during the first 72 hours, we found that many patients die much later with signs of opportunistic infections accompanied by downregulation of their monocytic HLA-DR expression and reduced ability to produce lipopolysaccharide (LPS)-induced TNF-α in vitro1–3. This phenomenon of monocyte deactivation in septic patients with fatal outcome shows similarities to experimental monocytic refractoriness induced by LPS desensitization or by pretreatment with its endogenous mediators IL-10 and transforming growth factor-β (TGF-β)4. In order to strengthen their antimicrobial defense, here we tested whether interferon-γ (IFN-γ) can improve monocytic functions in these patients and in experimental monocytic deactivation. The considerably lowered in vitro levels of LPS-induced TNF-α in these situations were significantly enhanced by IFN-γ, but did not reach the extremely high levels of IFN-γ primed naive cells from healthy donors. Moreover, IFN-γ applied to septic patients with low monocytic HLA-DR expression restored the deficient HLA-DR expression and in vitro LPS-induced TNF-α secretion. Recovery of monocyte function resulted in clearance of sepsis in eight of nine patients. These data suggest that IFN-γ treatment in carefully selected septic patients is a novel therapeutic strategy worth pursuing.

Journal ArticleDOI
TL;DR: It was found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression.
Abstract: The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors1–4. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level5–7, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

Journal ArticleDOI
TL;DR: It is shown that genetic disruption ofPARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion, providing compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia.
Abstract: Nitric oxide (NO) and peroxynitrite, formed from NO and superoxide anion, have been implicated as mediators of neuronal damage following focal ischemia, but their molecular targets have not been defined. One candidate pathway is DNA damage leading to activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP), which catalyzes attachment of ADP ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP, which is consumed in regeneration of NAD, leading to cell death by energy depletion. We show that genetic disruption of PARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia and suggest that therapies designed towards inhibiting PARP may provide benefit in the treatment of cerebrovascular disease.

Journal ArticleDOI
TL;DR: Noncoding, ISS-enriched plasmid DMAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens and should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.
Abstract: An adjuvant role for certain short bacterial immunostimulatory DNA sequences (ISSs) has recently been proposed on the basis of their ability to stimulate T helper-1 (Th1) responses in gene-vaccinated animals. We report here that noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens. The ISS-DNAs suppress IgE synthesis, but promote IgG and interferon-gamma (IFN-gamma) production. They furthermore initiate the production of IFN-gamma, IFN-alpha, IFN-beta, and interleukins 12 and 18, all of which foster Th1 responses and enhance cell-mediated immunity. Consideration should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.

Journal ArticleDOI
TL;DR: Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity.
Abstract: Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

Journal ArticleDOI
TL;DR: It is shown that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma, and the data suggest that a similar approach may be efficacious for immunotherapy of human cancer.
Abstract: The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.

Journal ArticleDOI
TL;DR: It is reported that normal human cells were highly resistant to ONYX-015-mediated, replication-dependent cytolysis and a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences, were efficiently destroyed.
Abstract: The 55-kilodalton (kDa) protein from the E1B-region of adenovirus binds to and inactivates the p53 gene, which is mutated in half of human cancers. We have previously shown that the replication and cytopathogenicity of an E1B, 55-kDa gene-attenuated adenovirus, ONYX-015, is blocked by functional p53 in RKO and U20S carcinoma lines. We now report that normal human cells were highly resistant to ONYX-015-mediated, replication-dependent cytolysis. In contrast, a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5-9), were efficiently destroyed. Antitumoral efficacy was documented following intratumoral or intravenous administration of ONYX-015 to nude mouse-human tumor xenografts; efficacy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone.

Journal ArticleDOI
TL;DR: A new mouse osteopetrotic mutant is defined and NF-κB proteins in bone development are implicate, raising new directions in the treatment of bone disorders.
Abstract: The nfkb1 and nfkb2 genes encode closely related products regulating immune and inflammatory responses. Their role during development and differentiation remains unclear. The generation of nfkb1 null mice (p50-/-) resulted in altered immune responses, but had no effect on development. Similarly, nfkb2 knockout mice (p52-/-) did not show developmental defects (J.C. et al., manuscript submitted). We have investigated the potential for in vivo compensatory functions of these genes by generating double-knockout mice. The surprising result was that the animals developed osteopetrosis because of a defect in osteoclast differentiation, suggesting redundant functions of NF-kappaB1 and NF-kappaB2 proteins in the development of this cell lineage. The osteopetrotic phenotype was rescued by bone marrow transplantation, indicating that the hematopoietic component was impaired. These results define a new mouse osteopetrotic mutant and implicate NF-kappaB proteins in bone development, raising new directions in the treatment of bone disorders.

Journal ArticleDOI
TL;DR: Changes in p27 and cyclin E protein levels in tumors might be more common than previously anticipated and may be indicators of tumor behavior.
Abstract: Mutations in certain genes that regulate the cell cycle, such as p16 and p53, are frequently found in human cancers1. However, tumor-specific mutations are uncommon in genes encoding cyclin E and the CDK inhibitor p27Kip1, two cell-cycle regulators that are also thought to contribute to tumor progression2–8. It is now known that levels of both cyclin E and p27 can be controlled by posttranscriptional mechanisms, indicating that expression of these proteins can be altered by means other than simply mutation of their respective genes9,10. Thus, changes in p27 and cyclin E protein levels in tumors might be more common than previously anticipated and may be indicators of tumor behavior.

Journal ArticleDOI
TL;DR: Different apoptosis-triggering pathways employ distinct signal transduction pathways that will culminate in the Induction of permeability transition (PT) and/or associated phenomena, and this work has shown that Bcl-2 regulates this pathway.
Abstract: Nature Medicine 3, 614–620 (1997) Owing to a production error, Figure 3 was presented incorrectly. The correct presentation is provided below. Fig, 3 The apoptotic process and its regulation by Bcl-2. Different apoptosis-triggering pathways employ distinct (“private”) signal transduction pathways that will culminate in the Induction of permeability transition (PT) and/or associated phenomena.

Journal ArticleDOI
TL;DR: It is demonstrated by immunohistochemistry that p27Kip1 protein levels are reduced in primary breast cancers and that this is associated with tumor progression in both in situ and invasive lesions, and shown to be regulated posttranscriptionally by in situ hybridization.
Abstract: Breast cancer is the second leading cause of cancer death in North American women. There is considerable need for reliable prognostic markers to assist clinicians in making management decisions. Although a variety of factors have been tested, only tumor stage, grade, size, hormone receptor status, and S-phase fraction are used on a routine basis1. The cell cycle is governed by a family of cyclin-dependent kinases (cdks), which are regulated by associated cyclins and by phosphorylation2. p27Kip1, a cyclin-dependent kinase inhibitor, regulates progression from G1 into S phase by binding and inhibiting cyclin/cdks3,4. p27Kip1 protein levels and/or activity are upregulated by growth inhibitory cytokines including transforming growth factor-β (TGF-β) and, thus, provide an important link between extracellular regulators and the cell cycle5–7. Loss of p27Kip1, a negative cell-cycle regulator, may contribute to oncogenesis and tumor progression. However, p27Kip1 mutations in human tumors are extremely rare8,9. We have demonstrated by immunohistochemistry that p27Kip1 protein levels are reduced in primary breast cancers and that this is associated with tumor progression in both in situ and invasive lesions. This was confirmed by western analysis, reflected in increased G1/S-phase cyclin-dependent kinase activities and shown to be regulated posttranscriptionally by in situ hybridization. Furthermore, on multivariate analysis, low p27Kip1 is a predictor of reduced disease-free survival. This simple and reliable immunohistochemical assay may become a routine part of breast cancer evaluation and may influence patient management.

Journal ArticleDOI
TL;DR: The specific production of the critical Aβ isoform in the ER of neurons links this compartment with the generation of Aβ and explains why primarily ER localized (mutant) proteins such as the presenilins3 could induce AD.
Abstract: The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

Journal ArticleDOI
TL;DR: The unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV) is described, where the rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers.
Abstract: Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Journal ArticleDOI
TL;DR: A consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection is document.
Abstract: Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines.

Journal ArticleDOI
TL;DR: Intratumoral implantation of murine cells modified to produce retroviral vectors containing the HSV-TK gene induces regression of experimental brain tumors in rodents after ganciclovir administration, suggesting that indirect, “bystander,” mechanisms provide local antitumor activity in human tumors.
Abstract: Intratumoral implantation of murine cells modified to produce retroviral vectors containing the herpes simplex virus-thymidine kinase (HSV-TK) gene induces regression of experimental brain tumors in rodents after ganciclovir (GCV) administration. We evaluated this approach in 15 patients with progressive growth of recurrent malignant brain tumors. Antitumor activity was detected in five of the smaller tumors (1.4 +/- 0.5 ml). In situ hybridization for HSV-TK demonstrated survival of vector-producing cells (VPCs) at 7 days but indicated limited gene transfer to tumors, suggesting that indirect, "bystander," mechanisms provide local antitumor activity in human tumors. However, the response of only very small tumors in which a high density of vector-producing cells had been placed suggests that techniques to improve delivery and distribution of the therapeutic gene will need to be developed if clinical utility is to be achieved with this approach.

Journal ArticleDOI
TL;DR: High leptin levels in the obese subject were due solely to increased leptin pulse height; all concentration-independent pulsatility parameters were almost identical in the two women, suggesting that leptin pulsatility therefore can be preserved in the obesity.
Abstract: Leptin communicates nutritional status to regulatory centers in the brain1,2. Because peripheral leptin influences the activity of the highly pulsatile adrenal and gonadal axes3,4, we sought to determine whether leptin levels in the blood are pulsatile. We measured circulating leptin levels every 7 minutes for 24 hours, in six healthy men, and found that total circulating leptin levels exhibited a pattern indicative of pulsatile release, with 32.0 ± 1.5 pulses every 24 hours and a pulse duration of 32.8 ± 1.6 minutes. We also show an inverse relation between rapid fluctuations in plasma levels of leptin and those of adrenocorticotropic hormone (ACTH) and cortisol that could not be accounted for on the basis of glucocorticoid suppression of leptin. As leptin levels are pulsatile, we propose that a key function of the CNS is regulated by a peripheral pulsatile signal. In a separate pilot study we compared leptin pulsatility in 414 plasma samples collected every 7 minutes for 24 hours from one obese woman and one normal-weight woman. We found that high leptin levels in the obese subject were due solely to increased leptin pulse height; all concentration-independent pulsatility parameters were almost identical in the two women. Leptin pulsatility therefore can be preserved in the obese.

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TL;DR: The first successful in vivo transfer of NFκB decoy oligodeoxynucleotides to reduce the extent of myocardial infarction following reperfusion is reported, providing a new therapeutic strategy for myocardia infarctions.
Abstract: The transcriptional factor nuclear factor-kappaB (NFkappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes that might be involved in myocardial damage after ischemia and reperfusion. Therefore, we hypothesized that synthetic double-stranded DNA with high affinity for NFkappaB could be introduced in vivo as "decoy" cis elements to bind the transcriptional factor and to block the activation of genes mediating myocardial infarction, thus providing effective therapy for myocardial infarction. Treatment before and after infarction by transfection of NFkappaB decoy, but not scrambled decoy, oligodeoxynucleotides before coronary artery occlusion or immediately after reperfusion had a significant inhibitory effect on the area of infarction. Here, we report the first successful in vivo transfer of NFkappaB decoy oligodeoxynucleotides to reduce the extent of myocardial infarction following reperfusion, providing a new therapeutic strategy for myocardial infarction.