Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: a randomized controlled trial. Alzheimer's Disease Cooperative Study

CONTEXT: Several reports from small clinical trials have suggested that estrogen replacement therapy may be useful for the treatment of Alzheimer disease (AD) in women. OBJECTIVE: To determine whether estrogen replacement therapy affects global, cognitive, or functional decline in women with mild to moderate AD. DESIGN: The Alzheimer's Disease Cooperative Study, a randomized, double-blind, placebo-controlled clinical trial conducted between October 1995 and January 1999. SETTING: Thirty-two study sites in the United States. PARTICIPANTS: A total of 120 women with mild to moderate AD and a Mini-Mental State Examination score between 12 and 28 who had had a hysterectomy. INTERVENTIONS: Participants were randomized to estrogen, 0.625 mg/d (n = 42), or 1.25 mg/d (n = 39), or to identically appearing placebo (n = 39). One subject withdrew after randomization but before receiving medication; 97 subjects completed the trial. MAIN OUTCOME MEASURES: The primary outcome measure was change on the Clinical Global Impression of Change (CGIC) 7-point scale, analyzed by intent to treat; secondary outcome measures included other global measures as well as measures of mood, specific cognitive domains (memory, attention, and language), motor function, and activities of daily living; compared by the combined estrogen groups vs the placebo group at 2, 6, 12, and 15 months of follow-up. RESULTS: The CGIC score for estrogen vs placebo was 5.1 vs 5.0 (P = .43); 80% of participants taking estrogen vs 74% of participants taking placebo worsened (P = .48). Secondary outcome measures also showed no significant differences, with the exception of the Clinical Dementia Rating Scale, which suggested worsening among patients taking estrogen (mean posttreatment change in score for estrogen, 0.5 vs 0.2 for placebo; P = .01). CONCLUSIONS: Estrogen replacement therapy for 1 year did not slow disease progression nor did it improve global, cognitive, or functional outcomes in women with mild to moderate AD. The study does not support the role of estrogen for the treatment of this disease. The potential role of estrogen in the prevention of AD, however, requires further research.     

Morphological assessment of pancreatic islet hormone content following aerobic exercise training in rats with poorly controlled Type 1 diabetes mellitus

Regular exercise has been shown to improve many complications of Type 1 diabetes mellitus (T1DM) including enhanced glucose tolerance and increased cardiac function. While exercise training has been shown to increase insulin content in pancreatic islets of rats with T1DM, experimental models were severely hyperglycemic and not undergoing insulin treatment. Further, research to date has yet to determine how exercise training alters glucagon content in pancreatic islets. The purpose of the present investigation was to determine the impact of a 10-week aerobic training program on pancreatic islet composition in insulin-treated rats with T1DM. Second, it was determined whether the acute, exercise-mediated reduction in blood glucose experienced in rats with T1DM would become larger in magnitude following aerobic exercise training. Diabetes was induced in male Sprague-Dawley rats by multiple low dose injections of streptozotocin (20mg/kg i.p.) and moderate intensity aerobic exercise training was performed on a motorized treadmill for one hour per day for a total of 10 weeks. Rats with T1DM demonstrated significantly less islet insulin, and significantly more islet glucagon hormone content compared with non-T1DM rats, which did not significantly change following aerobic training. The reduction in blood glucose in response to a single exercise bout was similar across 10 weeks of training. Results also support the view that different subpopulations of islets exist, as small islets (<50 μm diameter) had significantly more insulin and glucagon in rats with and without T1DM.     

Beta 2-integrin and L-selectin are obligatory receptors in neutrophil aggregation [see comments]

We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2- integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates on the basis of size and color, as well as antibody binding to these two adhesive molecules. Formyl peptide-stimulated aggregate formation was measured for individual populations fluorescently labeled red (LDS-751) or green (CD44-FITC), and interpopulation red-green cell conjugates. Blocking either the beta 2-integrin or L-selectin adhesive epitope with monoclonal antibody on individual cell populations resulted in an approximately 50% reduction in two-color aggregation as compared with that in unblocked samples. Shedding the L-selectin on a cell population by preincubation with complexes of lipopolysaccharide and its plasma membrane binding protein also decreased aggregation to a control population by approximately 50%. We examined the aggregation of neutrophils from patients genetically deficient in beta 2-integrin and clinically leukocyte adhesion deficient (LAD). LAD adhesion to normal neutrophils was dependent on the expression of L-selectin on LAD cells and beta 2-integrin on normal cells. Thus, the minimum requirement for adhesion between two mixed populations of neutrophils was that one population expressed the beta 2-integrin and the other expressed the L- selectin adhesive epitope.     

The predominant form of the amyloid beta-protein precursor in human brain is protease nexin 2.

The amyloid beta protein and the amyloid beta-protein precursor (APP) are major constituents of senile plaques and cerebrovascular deposits in patients with Alzheimer disease and Down syndrome. Most human tissues contain mRNA that encodes forms of APP that contain the Kunitz protease inhibitor (KPI+) domain. A major 120-kDa protein corresponding to this KPI+ mRNA is also found in these tissues. This protein is identical to the protease inhibitor protease nexin 2. Brain contains an additional mRNA species that encodes a form of APP that lacks the KPI domain (KPI-). This latter mRNA has been suggested to encode a 105-kDa KPI- form of APP protein also found in brain. Using protease inhibitory functional assays, we show that both the 105-kDa and 120-kDa APP proteins in normal and Alzheimer disease brain contain the KPI domain. Moreover, KPI domain-specific precipitation assays reveal that KPI- forms of APP protein represent less than 14% of total brain APP. Lastly, an enriched fraction from total brain homogenate contains proteolytic activity that can process the purified 120-kDa KPI+ form of APP into a 105-kDa form, resulting in a high-molecular-mass doublet identical to that seen in brain. These findings indicate that although KPI- APP mRNA is abundant in brain, little corresponding protein is present. Thus, KPI+ APP protein (equivalent to protease nexin 2) is the predominant form of APP in human brain.     

Acute lymphoblastic leukemia of childhood: identification of two distinct regions of deletion on the short arm of chromosome 12 in the region of TEL and KIP1

Cytogenetic analysis of acute lymphoblastic leukemia (ALL) of childhood identified nonrandom chromosomal abnormalities of the short arm of chromosome 12. The alterations include deletions that are thought to be indicative of the presence of a tumor suppressor gene that is mutated on the remaining allele. To refine further the chromosomal localization of this gene, we analyzed the loss of heterozygosity (LOH) of chromosome 12 in 100 primary ALL samples using 22 polymorphic markers and identified two distinct smallest common deleted regions on chromosome 12p13. One region is flanked by D12S77 and D12S98 and has a size of 4 cM. Twenty-six percent of informative patients showed LOH in this region. This region may contain the TEL gene. The other region is flanked by D12S269 and D12S308 including the KIP1 gene. Forty-four percent of informative patients showed LOH in this second region. Mutational analysis of KIP1 using polymerase chain reaction-single- strand conformation polymorphism analysis and Southern blot analysis showed no homozygous deletions and point mutations suggesting that the altered gene in this second region is not the KIP1. Clinical data showed that LOH of 12p was demonstrated more frequently in precursor-B ALLs (32 of 80; 40%) than in T-ALLs (1 of 20; 5%) (P = .0027). Furthermore, patients with 12p LOH were younger (P = .013), with a lower DNA index (P = .046), but they had the same survival rates at 3 years. In summary, these data suggest that two different tumor suppressor genes are on chromosome arm 12p, which act separately in the development of childhood precursor-B ALLs. One of the tumor suppressor genes is in the region the KIP1 gene, but our data suggest this gene is not abnormal. The other target is in the region of the TEL gene; and this candidate deserves further study.     

Exploring the utility of whole‐exome sequencing as a diagnostic tool in a child with atypical episodic muscle weakness

The advent of whole‐exome next‐generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½‐year old female patient with a 2‐year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work‐up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di‐deoxy sequencing. WES revealed a de novo non‐synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.     

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL- 2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2- activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL- 2 may be of clinical use to promote hematopoietic reconstitution after BMT.     

Effectiveness of methyl-GAG (methylglyoxal-bis[guanylhydrazone]) in patients with advanced malignant lymphoma

We treated 51 patients with advanced malignant lymphoma refractory to conventional therapy with methyl-glyoxal-bis(guanylhydrazone) (methyl- GAG) at doses ranging from 400 to 800 mg/sq m. Therapy was started on a weekly schedule and was switched to every other week in responding patients at the onset of toxicity. Partial responses were observed in 6 of 13 evaluable patients with Hodgkin's disease (46%), 5 of 10 patients with diffuse poorly differentiated lymphocytic lymphoma (50%), 2 of 4 patients with nodular poorly differentiated lymphocytic lymphoma (50%), and 3 of 13 patients with diffuse histiocytic lymphoma (23%). Two of six patients with mycosis fungoides showed objective improvement in cutaneous disease. Toxicity was generally mild and included muscular weakness, myalgia, mucositis, and diarrhea; two patients developed bronchospasm following drug infusions. We conclude that methyl-GAG has major antitumor activity when administered on this schedule to patients with advanced malignant lymphoma. The low degree of toxicity, unique mechanism of action, and minimal myelosuppressive effects suggest that methyl-GAG will prove useful in future trials of combination chemotherapy regimens for the treatment of lymphoma.     

Age and distraction are determinants of performance on a novel visual search task in aged Beagle dogs

Aging has been shown to disrupt performance on tasks that require intact visual search and discrimination abilities in human studies. The goal of the present study was to determine if canines show age-related decline in their ability to perform a novel simultaneous visual search task. Three groups of canines were included: a young group (N = 10; 3 to 4.5 years), an old group (N = 10; 8 to 9.5 years), and a senior group (N = 8; 11 to 15.3 years). Subjects were first tested for their ability to learn a simple two-choice discrimination task, followed by the visual search task. Attentional demands in the task were manipulated by varying the number of distracter items; dogs received an equal number of trials with either zero, one, two, or three distracters. Performance on the two-choice discrimination task varied with age, with senior canines making significantly more errors than the young. Performance accuracy on the visual search task also varied with age; senior animals were significantly impaired compared to both the young and old, and old canines were intermediate in performance between young and senior. Accuracy decreased significantly with added distracters in all age groups. These results suggest that aging impairs the ability of canines to discriminate between task-relevant and -irrelevant stimuli. This is likely to be derived from impairments in cognitive domains such as visual memory and learning and selective attention.     

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1‐EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1‐EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild‐type mice. The fractured bones of Col1‐EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild‐type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1‐EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU‐F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild‐type control mice. Furthermore, Col1‐EphB4 mice had significantly lower numbers of TRAP‐positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones. © 2013 American Society for Bone and Mineral Research.