Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients.

Two coronaviruses were isolated from brain material obtained at autopsy from two multiple sclerosis patients. The viruses were neutralized by serum and spinal fluid from these patients. Although most of the population have antibody to these virus isolates, multiple sclerosis patients have slightly higher concentrations of serum antibody than controls. The results suggest that coronaviruses should be considered as one additional virus with a potential implication in the etiology of multiple sclerosis.

Osteopontin-deficient progenitor cells display enhanced differentiation to adipocytes.

OBJECTIVE: Osteopontin (OPN, Spp1) is a protein upregulated in white adipose tissue (WAT) of obese subjects. Deletion of OPN protects mice from high-fat diet-induced WAT inflammation and insulin resistance. However, the alterations mediated by loss of OPN in WAT before the obesogenic challenge have not yet been investigated. Therefore, we hypothesised that the lack of OPN might enhance the pro-adipogenic micro environment before obesity driven inflammation. METHODS: OPN deficiency was tested in visceral (V) and subcutaneous (SC) WAT from WT and Spp1-/- female mice. Gene expression for hypoxia, inflammation and adipogenesis was checked in WT vs. Spp1-/- mice (n=15). Adipocytes progenitor cells (APC) were isolated by fluorescence cell sorting and role of OPN deficiency in adipogenesis was investigated by cell images and RT-PCR. RESULTS: We show that Spp1-/- maintained normal body and fat-pad weights, although hypoxia and inflammation markers were significantly reduced. In contrast, expression of genes involved in adipogenesis was increased in WAT from Spp1-/- mice. Strikingly, APC from Spp1-/- were diminished but differentiated more efficiently to adipocytes than those from control mice. CONCLUSIONS: APC from SC-WAT of lean OPN-deficient mice display an enhanced capacity for differentiating to adipocytes. These alterations may explain the healthy expansion of WAT in the OPN-deficient model which is associated with reduced inflammation and insulin resistance.

Modulation of alpha-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3.

The Src homology 2 phosphotyrosyl phosphatase (SHP2) is a nonreceptor-type phosphatase that acts as a positive transducer of receptor Tyr kinase (RTK) signaling, particularly the Ras-REK and PI3K-Akt pathways. Recently, we have demonstrated that SHP2 is required for cell transformation induced by the constitutively active fibroblast growth factor receptor 3 (K/E-FR3) (Oncogene, 22, 6909-6918). In that study, we had detected a phosphotyrosyl protein of approximately 100 KDa (p100) in cells expressing dominant-negative SHP2 (R/E-SHP2), but its identity and relevance in SHP2-meditaed transformation was not known. Here, we report the identification of p100 as alpha-catenin, a vinculin-related protein involved in adherens junction-mediated intercellular adhesion. We show that alpha-catenin becomes Tyr phosphorylated in intercellular adhesion-dependent manner and this event is counteracted by SHP2. Substrate trapping in intact cells and immunocomplex phosphatse assays confirmed that alpha-catenin is in deed an SHP2 substrate. Tyr phosphorylation of alpha-catenin enhances its translocation to the plasma membrane and its interaction with beta-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Site-directed mutagenesis studies also suggested that Tyr phosphorylation of alpha-catenin enhances its inhibitory role on cell transformation. Based on our previous work and the current report, we demonstrate that mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. To our knowledge, this is the first report showing regulation of alpha-catenin function by Tyr phosphorylation and its inhibitory effect on cell transformation.

A randomized trial of intravenous heparin in conjunction with anistreplase (anisoylated plasminogen streptokinase activator complex) in acute myocardial infarction: the Duke University Clinical Cardiology Study (DUCCS) 1.

OBJECTIVES: We designed a randomized trial to evaluate the effects of heparin administration in conjunction with anistreplase (anisoylated plasminogen streptokinase activator complex [APSAC]) on arterial patency and clinical end points. BACKGROUND: The role of conjunctive intravenous heparin therapy with APSAC has not been tested despite the recommendations that intravenous heparin should be used. METHODS: Four hours after APSAC administration, 250 patients with acute myocardial infarction were randomly assigned to receive 325 mg of either aspirin alone or aspirin and a continuous infusion of heparin (15 IU/kg body weight per h). Clinical ischemic events and bleeding complications were monitored. On hospital day 5, coronary arteriography and left ventriculography were performed. RESULTS: The primary end point of the trial (the combined outcome of death, reinfarction, recurrent ischemia and occlusion of the infarct-related artery) occurred in 42% of the heparin-treated group versus 43% of the group treated without heparin (p = 0.94). A patent infarct-related artery was present in 80% of the patients treated with heparin and in 73% of those treated without heparin (p = 0.26). Left ventricular function, as measured by ejection fraction, was well preserved in both groups (52% vs. 50.5%, respectively, p = 0.29). The overall bleeding rate was higher in patients with (32%) than without (17.2%) heparin (p = 0.006). CONCLUSIONS: Weight-adjusted intravenous heparin therapy after APSAC in acute myocardial infarction does not reduce the combined incidence of death, reinfarction, recurrent ischemia and occlusion of the infarct-related artery. Furthermore, withholding intravenous heparin therapy is associated with a 46% reduction in bleeding complications. Our findings do not support the addition of intravenous heparin after APSAC therapy, as currently recommended, and suggest that a strategy of withholding heparin is simpler and safer and does not place the patient at increased risk for ischemic complications after myocardial infarction.

An unforeseen complication of home parenteral antibiotic therapy.

Increasing pressure to cut the length of hospital stay has resulted in a large number of patients receiving home parenteral antibiotic therapy. We present a case of an immediate allergic reaction in a penicillin-sensitive spouse of a patient receiving parenteral mezlocillin sodium therapy. A seminal level of 42 micrograms/mL of mezlocillin was documented by bioassay.

Evidence for preferential effects of parathyroid hormone, calcitonin and adenosine on bone and periosteum.

In order to explore the distribution of hormone-responsive cells in skeletal tissues, we have examined the effects of synthetic bovine parathyroid hormone N-terminal peptide (bPTH 1-34) and salmon calcitonin (sCT) on cyclic AMP levels in periosteum-free rat calvaria, segments of periosteum, and in isolated cells dispersed from each tissue by collagenase digestion. Synthetic bovine PTH increased cyclic AMP levels to a greater degree in calvaria and in isolated bone cells than in the periosteal segments and cells, whereas sCT was more effective in the periosteal than in the bone systems. Primary cultures prepared from bone and periosteal cell populations exhibited progressive increases in their responsiveness to bPTH (1-34) and progressive decreases in responsiveness to sCT. After six days in the culture, bone cells failed to respond to sCT, and sCT did not modify their response simultaneously added bPTH (1-34). Six-day periosteal cell cultures exhibited residual sCT responsivity and an additive response upon simultaneous exposure to high concentrations of bPTH (1-34) and sCT suggesting separate sites of hormone action. Adenosine, a known stimulator of bone cell adenylyl cyclase, caused a greater increase in periosteal cell than in bone cell cyclic AMP. bPTH (1-34)-responsive cells which enrich periosteum-free bone may be osteoblasts, in view of their histological prominence in this tissue and in the bone cell isolates. Periosteal cells which responded to sCT and to adenosine preferentially are unidentified. Although periosteal segments contained numerous fibroblast-like cells, skin fibroblasts cultured from the same fetuses were sCT-insensitive. Growth in primary culture appears to alter the number of hormone-responsive cells or responsiveness of existing cells to each hormone, or both.

An unusual case of encephalomyeloradiculoneuropathy in a young woman.

Fulminant and progressive CNS disease developed in a young woman and was associated with bilateral deafness and sharply elevated CSF protein levels. Two months after onset, a severe sensorimotor neuropathy developed. Clinical improvement occurred with corticosteroid therapy.

Uptake and release of putative neurotransmitters. Measurements in regions of the normal and Newcastle disease virus-infected mouse brain.

The capacity of various regions of the mouse brain to accumulate a series of putative neurotransmitter compounds has been studied in cerebral homogenates. This uptake is selective, sodium dependent, energy dependent, and exhibits characteristics of high affinity transport. Calcium-stimulated release under depolarizing conditions of accumulated radioactive compounds was also examined. Large regional variations of uptake and release capacity existed. No clear relation between intensity of uptake and releasability of transported compounds was seen. The effect of infection of mice with Newcastle disease virus on these processes was investigated. No significant differences were seen in infected mice despite their depressed metabolic rate.

Antibodies to vaccinia and measles viruses in multiple sclerosis patients.

A virologic comparison was made of 144 patients with multiple sclerosis, 34 of their healthy siblings, and 40 patients with other neurologic diseases (OND). Antibodies in serum and cerebrospinal fluid (CSF) to vaccinia and measles viruses were measured, and these were correlated in the multiple sclerosis patients with the clinical characteristics of their disease. The CSF antibody to vaccinia virus was more frequent and at a higher titer in multiple sclerosis patients than in either control group. Moreover, a statistically significant increase was found in both frequency and titer of CSF vaccinia antibody in patients with the progressive form of the disease as compared with those classified as relapsing-remitting. Statistically significant differences between multiple sclerosis patients and their siblings were not observed for CSF or serum measles virus antibody, although both groups had significantly higher serum antibody titers than patients with OND.

A comparison of dementia in Alzheimer's disease and multiple sclerosis.

We compared results of comprehensive neuropsychological testing in 42 patients with clinically diagnosed Alzheimer's disease (AD) and in an equal number of patients with clinically definite chronic-progressive multiple sclerosis. Age, sex, and education were controlled using demographically corrected T scores based on a large normal sample. Both groups showed significant impairment on the test battery, but the degree of dementia was more severe in the patients with AD. A deviation score analysis, controlling for overall level of cognitive impairment, revealed significant differences between the groups. Alzheimer's disease was associated with relatively greater impairment of learning, memory, and verbal skills, whereas the MS group showed greater relative impairment of attention, incidental memory, and psychomotor functions. These data suggest that both the degree and pattern of mental impairement differ in patients with AD and patients with multiple sclerosis. Our results support a distinction between "gray matter" and "white matter" dementia, and may help clarify the issue of "cortical" vs "subcortical" dementia by demonstrating neuropsychological differences based on secure neuropathologic distinctions.

Antigenic assessment of coronaviruses isolated from patients with multiple sclerosis.

Many studies have either supported or discounted the role of coronaviruses as etiologic agents in multiple sclerosis (MS). Two new approaches were applied to investigate this controversy. First, monoclonal antibodies specific for either murine coronaviruses (mouse hepatitis viruses) or human coronaviruses were used to characterize the antigenic features of MS-derived coronaviruses SK and SD. Both isolates were found to have a mouse hepatitis virus-type profile. Second, serum and cerebrospinal fluid antibodies to different coronaviruses, including SD, were measured in MS and control groups. No significant difference in antibody level to coronaviruses was found between MS and control samples. The results of these antigenic studies do not support a specific association between MS and coronaviruses.

Pathogenesis of coronavirus SD in mice. I. Prominent demyelination in the absence of infectious virus production.

Following intracerebral inoculation of 3- to 4-week-old C57 B16/J mice with coronavirus SD, 23% exhibited neurologic signs within the first week. However, only 6% died. Within the first week after inoculation (AI), we noted a panencephalitis. Prominent demyelination detected in the spinal cord on day 6 continued through day 29 AI. Demyelinated lesions in the spinal cord were either subpial with few inflammatory cells except for macrophages or perivascular with prominent accumulation of lymphocytes, plasma cells, and macrophages. Beginning on day 6 AI, IgG was detected in the lesions. Although an infectious virus was detectable in the CNS only through day 12 AI, viral antigen expression continued through day 24. We concluded that coronavirus SD persists in a nonrecoverable form throughout the initial phase of demyelination, day 6 to day 24 AI.

Acute encephalopathy caused by defective virus infection. I. Studies of Newcastle disease virus infections in newborn and adult mice.

An acute encephalopathy caused by a defective paramyxovirus infection was studied. Newcastle disease virus (ndv), given intracerebrally, caused neurologic disease and death in mice. Infected newborn mice died by the fourth day after inoculation, and abundant amounts of virus were recovered from their brains. Infected 4-week-old mice died by the eighth day, but only minimal amounts of virus, if any, were recovered. The brains of many moribund 4-week-old mice were histologically normal and contained no NDV antigen on fluorescent antibody staining. No serum antibody to NDV was detected. These features make this infection difficult to distinguish from a metabolic encephalopathy.

The effects of pregnancy in multiple sclerosis: a retrospective study.

We reviewed the medical records of 178 women with multiple sclerosis to evaluate the number of completed pregnancies, current disability status, and relationship of pregnancy to onset of MS symptoms. We found no differences in the long-term disability of women with no pregnancies, one pregnancy, or two or more pregnancies. Women who had initial symptom onset in pregnancy experienced less subsequent disability than women whose symptoms began before or after pregnancy. Therefore, pregnancy per se or number of pregnancies has no effect on subsequent disability.

Management of patients receiving interferon beta-1b for multiple sclerosis: report of a consensus conference.

Results of a double-blind, placebo-controlled study in ambulatory patients with relapsing-remitting MS showed that interferon beta-1b reduced the rate of exacerbations by one-third compared with placebo and limited new disease activity in the brain as evidenced by MRI. Interferon beta-1b, administered subcutaneously at a dosage of 0.25 mg (8 million IU) every other day is indicated for the treatment of ambulatory patients with relapsing-remitting MS. Interferon beta-1b may help a wider range of patients, but it should be prescribed only for patients with a diagnosis of clinically definite or laboratory-supported definite MS. The decision to treat a patient with interferon beta-1b should be individualized; that is, based on each patient's clinical presentation and course of MS. The most common adverse effects include (1) injection-site reactions and (2) flu-like symptoms, which are generally manageable and usually abate after the first few months of treatment. Spasticity may increase. Patients with severe depression or suicidal ideation should be monitored carefully, and symptomatic treatment should be pursued. Interferon beta-1b is contraindicated in pregnant and nursing women. Interferon beta-1b is effective in reducing the progression of total disease burden as seen on MRI in patients with MS. Its use is relatively straightforward and generally does not require alteration in the symptomatic treatment of MS. Patient education and support remain the mainstays of maintaining compliance through the early phases of therapy.

Autoradiographic detection of IgG and viral antigens.

Autoradiographic methods can be used as an alternative to indirect immunofluorescence to detect viral antigen expression or the presence of IgG in tissue sections. Iodinated protein A isolated from Staphylococcus aureus detects an influx of IgG into the central nervous system of mice inoculated with the coronavirus SD. Antispecies antibody that has been iodinated detects coronavirus antigen expression for 24 days post-inoculation while it is only detectable for 10 days by immunofluorescence. A direct comparison of indirect fluorescence and autoradiographic methods indicates that the autoradiographic techniques are considerably more sensitive. This increased sensitivity is sufficient to permit the detection of viral antigen in formalin fixed paraffin embedded tissue sections.

Prognostic implications of ventricular arrhythmias during 24 hour ambulatory monitoring in patients undergoing cardiac catheterization for coronary artery disease.

The prognostic importance of ventricular arrhythmias detected during 24 hour ambulatory monitoring was evaluated in 395 patients with and 260 patients without significant coronary artery disease. Ventricular arrhythmias were found to be strongly related to abnormal left ventricular function. A modification of the Lown grading system (ventricular arrhythmia score) was the most useful scheme for classifying ventricular arrhythmias according to prognostic importance. When only noninvasive characteristics were considered, the score contributed independent prognostic information, and the complexity of ventricular arrhythmias as measured by this score was inversely related to survival. However, when invasive measurements were included, the ventricular arrhythmia score did not contribute independent prognostic information. Furthermore, ejection fraction was more useful than the ventricular arrhythmia score in identifying patients at high risk of sudden death.

A randomized factorial trial of reperfusion strategies and aspirin dosing in acute myocardial infarction. The DUCCS-II Investigators.

The focus of new research efforts to improve the morbidity and mortality associated with acute myocardial infarction (AMI) has turned to adjuvant agents that show promise of improving outcomes following coronary thrombolysis. We enrolled 162 patients with AMI in a randomized trial comparing front-loaded tissue-plasminogen activator (t-PA) plus weight-adjusted heparin with anisoylated plasminogen streptokinase activator complex (APSAC) without heparin as well as standard-dose (325 mg) and low-dose (81 mg) aspirin. The primary end point was an in-hospital morbidity profile; secondary end points were clinical and angiographic potency and hemorrhagic events. Selected sites performed an electrocardiographic substudy to determine the time to 50% ST-segment recovery and the time to steady state. Although the trial was terminated when the Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries-I trial showed that t-PA had a significant mortality advantage over streptokinase, important trends were evident. Patients given t-PA and heparin were better anticoagulated (p = 0.001), yet AP-SAC-treated patients had more bleeding complications. The primary end point favored t-PA (25.4% vs 31.3%), and the secondary end points were similar in both groups. In the electrocardiographic substudy, the t-PA group achieved both 50% ST-segment recovery and steady-state recovery sooner than the APSAC group. Patients taking low-dose aspirin had lower in-hospital mortality and less recurrent ischemia but more strokes than the standard-dose aspirin group. Thus, this trial demonstrated trends favoring front-loaded t-PA with weight-adjusted heparin over APSAC without heparin in the treatment of AMI. The use of low-dose aspirin did not appear to impose a loss of protection from adverse events, nor did standard-dose aspirin increase serious bleeding.

Referral bias in multiple sclerosis research.

Referral bias is a significant problem affecting the generalizability of clinical studies conducted in a university setting. To examine referral bias in our university-based multiple sclerosis referral center, we analyzed the characteristics of referral center patients compared to the population-based group of multiple sclerosis patients from which the referral center patients originated. The referral center patient group differed from those that remained in the population-based group in the following important ways: (1) they were younger, (2) they had more mobility impairment for their age, (3) disabled females were overrepresented compared to disabled males, (4) they more often reported recent disease worsening, (5) they had a higher frequency of early diagnosis supported by laboratory tests, and (6) they more often relied on neurologists and therapists for routine care of their disease. The multiple sclerosis referral center setting would appear to be ideal for the conduct of intervention trials, but inadequate for collecting representative natural history data.