Embryonic mosaic deletion of APP results in displaced Reelin-expressing cells in the cerebral cortex.

It is widely accepted that amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease. In addition, APP has been proposed to have functions in numerous biological processes including neuronal proliferation, differentiation, migration, axon guidance, and neurite outgrowth, as well as in synapse formation and function. However, germline knockout of APP yields relatively subtle phenotypes, and brain development appears grossly normal. This is thought to be due in part to functional compensation by APP family members and other type I transmembrane proteins. Here, we have generated a conditional mouse knockout for APP that is controlled temporally using CreER and tamoxifen administration. We show that total cortical expression of APP is reduced following tamoxifen administration during embryonic time points critical for cortical lamination, and that this results in displacement of Reelin-positive cells below the cortical plate with a concurrent elevation in Reelin protein levels. These results support a role for APP in cortical lamination and demonstrate the utility of a conditional knockout approach in which APP can be deleted with temporal control in vivo. This new tool should be useful for many different applications in the study of APP function across the mammalian life span.

Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets.

Lymphoma is a common tumor in ferrets, but anatomic distribution, histomorphology, immunophenotype, laboratory abnormalities, and response to chemotherapy are incompletely defined. In this study, lymphoma was diagnosed by histopathology of tumor tissue in 29 ferrets ranging in age from 0.8 to 8.5 years, including 12 males and 17 females. Tumors involved the viscera of the abdominal cavity (n = 11), thoracic cavity (n = 1), or abdominal and thoracic cavities (n = 7); the skin (n = 2); or the viscera of both body cavities plus other sites (n = 8). Microscopically, all tumors had diffuse architecture. Assessment by histomorphology and immunophenotype classified tumors as peripheral T-cell lymphoma (n = 17), anaplastic large T-cell lymphoma (n = 5), anaplastic large B-cell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 1), and Hodgkin-like lymphoma (n = 2). Cytologic evaluation of tumor tissue was diagnostic in 11 of 13 cases. Twenty-two of 27 ferrets had anemia, 2 had leukemia, and 5 were neutropenic. Common comorbid disorders were adrenal disease (n = 27) and insulinoma (n = 6). Tumors most frequently involved mesenteric lymph nodes, while enlargement of peripheral lymph nodes was uncommon (n = 3). Ferrets with Hodgkin-like lymphoma had massive enlargement of single lymph nodes. Mean survival of ferrets not immediately euthanized was 5.0 months (T-cell lymphoma) and 8.4 months (B-cell lymphoma). Ferrets treated with chemotherapy survived an average of 4.3 months (T-cell lymphoma, n = 9) or 8.8 months (B-cell lymphoma, n = 4). Results indicate that lymphomas in ferrets most commonly affect abdominal viscera, may be amenable to cytologic diagnosis, are frequently associated with anemia and, in some cases, may be chemosensitive, resulting in relatively long survival times.

Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys.

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.

Role of mucosal IgA in the resistance to Acanthamoeba keratitis.

PURPOSE: To determine whether oral immunization with Acanthamoeba castellanii antigens elicits mucosal antibodies of the IgA isotype and whether mucosal antibodies affect parasite adhesion to the corneal epithelium. METHODS: Chinese hamsters were immunized with 100 microg aqueous Acanthamoeba antigen mixed with cholera toxin (Ac-CT) and subsequently challenged with parasite-laden contact lenses that were applied to abraded corneal surfaces. Tears and stool samples were examined for the presence of Acanthamoeba-specific IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The effect of mucosal antibody on trophozoite binding to corneal epithelium and viability of trophozoites was examined in vitro. RESULTS: Hamsters immunized orally with Ac-CT showed significantly lower infection rates than did control groups (21.4% versus 72.6%). ELISA analysis of mucosal specimens showed the presence of parasite-specific IgA in stool samples and tears from hamsters orally immunized with Ac-CT, but not in control animals. In vitro assays showed that anti-Acanthamoeba IgA did not affect parasite viability. However, mucosal anti-Acanthamoeba IgA profoundly inhibited (>75%) the binding of parasites to corneal epithelial cells in vitro. CONCLUSIONS: Oral immunization with Ac-CT induces the production of parasite-specific IgA in mucosal secretions and prevents corneal infection. Mucosal antibody does not affect the viability of Acanthamoeba trophozoites but seems to prevent infection by inhibiting parasite binding to the corneal epithelium.

Sequential flow cytometry and fluorescence in situ hybridization for the study of formalin-fixed, paraffin-embedded breast cancer cells.

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.

Catheter-tract infections in rhesus macaques (Macaca mulatta) with indwelling intravenous catheters.

Development of catheter-tract infections in experimental animals can have devastating consequences on animal health and the functional lifespan of surgical implants. To measure the incidence of catheter-tract infections in animals with exteriorized intravenous catheters in this facility and assess the effects of these infections on mean catheter lifespan, health records of 31 Macaca mulatta with catheters were reviewed. Records spanned the interval of January 1, 1996 through October 1, 1997. Catheter-tract infections in 16 of 53 (30.2%) monkeys with catheters were diagnosed based on a combination of clinical signs of infection and results of bacterial culture. Segmental catheter-tract infections reduced mean catheter lifespan to 147 days, compared with 354 days for uninfected catheters. Exit-wound, local tunnel, and surgical-site infections did not significantly reduce catheter lifespan. Bacterial culture reports documented 31 isolates; 41.9% (13 of 31) were coagulase-negative staphylococci, and 22.6% (7 of 31) were Staphylococcus aureus. Of 20 isolates tested, 15 (75%) were resistant to methicillin/oxacillin in vitro. Antimicrobial susceptibility testing of methicillin-resistant and methicillin-susceptible isolates indicated that, compared with methicillin-sensitive isolates, methicillin-resistant isolates had a pattern of multiple antibiotic resistance. Catheter-tract infections were common in this colony of rhesus macaques, and clinically severe infections caused a drastic reduction in catheter lifespan. Approximately half (48%) the bacterial isolates were methicillin-resistant gram-positive bacteria.

Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency.

Hereditary spherocytosis (HS) is a common hemolytic anemia of variable clinical expression. Pathogenesis of HS has been associated with defects of several red cell membrane proteins including erythroid band 3. We have studied erythrocyte membrane proteins in 166 families with autosomal dominant HS. We have detected relative deficiency of band 3 in 38 kindred (23%). Band 3 deficiency was invariably associated with mild autosomal dominant spherocytosis and with the presence of pincered red cells in the peripheral blood smears of unsplenectomized patients. We hypothesized that this phenotype is caused by band 3 gene defects. Therefore, we screened band 3 DNA from these 38 kindred for single strand conformational polymorphisms (SSCP). In addition to five mutations detected previously by SSCP screening of cDNA, we detected 13 new band 3 gene mutations in 14 kindred coinherited with HS. These novel mutations consisted of two distinct subsets. The first subset included seven nonsense and frameshift mutations that were all associated with the absence of the mutant mRNA allele from reticulocyte RNA, implicating decreased production and/or stability of mutant mRNA as the cause of decreased band 3 synthesis. The second group included five substitutions of highly conserved amino acids and one in-frame deletion. These six mutations were associated with the presence of comparable levels of normal and mutant band 3 mRNA. We suggest that these mutations interfere with band 3 biosynthesis leading thus to the decreased accumulation of the mutant band 3 allele in the plasma membrane.

The role of macrophages in Acanthamoeba keratitis.

PURPOSE: Macrophages are thought to be the first line of defense in many infectious diseases and are present in high numbers in corneas with Acanthamoeba keratitis. Conjunctival macrophage depletion was performed in an animal model of Acanthamoeba infection to determine the importance of macrophages in this disease. METHODS: Selective elimination of macrophages was achieved by repeated subconjunctival injection of liposomes containing dichloromethylene diphosphonate in a Chinese hamster model of Acanthamoeba keratitis. RESULTS: Macrophage depletion affected the incidence, severity, and chronicity of keratitis. The incidence of infection in normal animals was approximately 60% but rose to 100% on day 4 in animals treated with liposomes containing dichloromethylene diphosphonate (C12MDP-LIP). Moreover, the clinical appearance of the keratitis in the C12MDP-LIP group was much more severe than in animals treated with liposomes containing phosphate-buffered saline at all time points. There was also a major change in the chronicity of keratitis, with an earlier onset and a prolonged and chronic course in the C12MDP-LIP treated hamsters. CONCLUSIONS: The profound exacerbation of Acanthamoeba keratitis in hamsters treated with C12MDP-LIP strongly suggests that macrophages play an important role in corneal infection with Acanthamoeba trophozoites, probably by acting as a first line of defense and eliminating significant numbers of Acanthamoeba trophozoites.

In vitro characterization of Acanthamoeba castellanii cytopathic effect.

This study examined the mechanism of the cytopathic effect (CPE) of Acanthamoeba castellanii on human target cells. Pathogenic Acanthamoeba trophozoites were incubated with human ocular melanoma (OCM1) cells for 30 min, 1 hr, and 3 hr. The amoebae were treated with a calcium ionophore (A23187), phorbol myristate ester (PMA), calcium channel blocker (Bepridil), cytochalasin D, and L-leucyl-L-leucine methyl ester (leu-leu-OMe). Cytolysis was quantified using a spectrophotometric assay. Cocultures of amoeba and cells were also observed by transmission electron microscopy at 1, 2, and 3 hr. Results show that trophozoites formed pseudopodia that made intimate contact with the target cell membrane. Neither amebostomes nor phagocytosis was seen. The calcium ionophore A23187 increased the cytopathic effect of the trophozoites on the cultured OCM1. In contrast, cytochalasin D, Bepridil, and PMA reduced the cytopathic effect. Leu-leu-OMe did not result in killing of Acanthamoeba trophozoites. The results suggest that the cytopathic effect of Acanthamoeba trophozoites involves calcium channels and cytoskeletal elements. There was no evidence of trogocytosis or phagocytosis as sometimes occurs in cytolysis by other free-living amoeba. Although Acanthamoeba-mediated CPE in some ways resembles CPE produced by cytotoxic lymphocytes, the mechanisms are not identical.

The structure and organization of the human erythroid anion exchanger (AE1) gene.

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans approximately 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3' noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.

Thirty-second sampling of plasma growth hormone in man: correlation with sleep stages.

Both fasting and sleep increase the secretion of human GH and, therefore, might explain its predominantly nocturnal release. To study the precise temporal relationship between GH secretory episodes and cortical activity, GH measurements and electroencephalogram sleep stage recordings were performed every 30 s for 8 h in six young male volunteers fasted for 24 h. GH was measured in two drops of whole blood, which were directly sampled into the assay tube using a continuous blood withdrawal pump and a fraction collector. Concomitant serum sampling during a GH-releasing hormone test (n = 4) revealed a high correlation (r = 0.98) between GH measurements in serum and whole blood. GH pulses were objectively identified with Cluster analysis, and GH secretion rates were calculated with a waveform-independent deconvolution algorithm. When data were analyzed as replicates with 1-min intervals, the nocturnal pulse frequency was 1.2 pulses/h. Elimination of data points demonstrated 43% and 64% reductions in the number of GH pulses detected for 5- and 20-min sampling intervals, respectively. Mean GH concentrations and secretory rates were significantly higher during stage 3 and 4 sleep compared to stage 1, 2, and rapid eye movement sleep. GH secretory rates and peripheral GH concentrations were maximally correlated with sleep stage, with lags of 4.5 and 16 min, respectively, suggesting that maximal GH release occurs within minutes of the onset of stage 3 or 4 sleep. This temporal coincidence between pituitary GH secretion and delta sleep is consistent with cortical control over hypothalamic-pituitary function.

Effect of fructose on phenylephrine-induced glucose output in perfused rat liver.

Glucose output induced by phenylephrine in perfused livers of fed rats was decreased by 34% during an infusion of fructose, but was increased by 30% (compared to controls) following cessation of fructose infusion. Corresponding changes in hepatic inorganic phosphate (Pi) were also observed with a 40% decrease and a 48% increase in Pi concentration being measured during and following fructose infusion, respectively. The data suggest that the glycogenolytic response to phenylephrine is dependent on the hepatic Pi concentration. It is also suggested that enhanced hepatic Pi concentrations and glycogenolytic responses observed following fructose infusion may antagonize insulin-induced suppression of hepatic glucose output and thus play a role in the glucose intolerance and insulin resistance associated with sucrose or fructose feeding.

On the stimulation of respiration by alpha-adrenergic agonists in perfused rat liver.

Interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48-h-fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitrophenol were still evident. Phenylephrine-induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48-h-fasted animals. These data indicate that phenylephrine-induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.

The stimulation of tricarboxylic acid-cycle flux by alpha-adrenergic agonists in perfused rat liver.

Output of 14CO2 from 1-14C-labelled glutamate, 2-oxoglutarate or octanoate and from 4-methyl-2-oxo[2-14C]pentanoate was increased by more than 100% after infusion of phenylephrine into perfused livers of fed rats. Infusion of ethanol or sorbitol raised 3-hydroxybutyrate/acetoacetate ratios and decreased the output of 14CO2. Increases in 14CO2 output induced by phenylephrine were observed in the presence or absence of ethanol or sorbitol and were accompanied by elevated 3-hydroxybutyrate/acetoacetate ratios under all conditions examined. Phenylephrine had no effect on total tissue ATP/ADP ratios in livers from fed or starved rats. The data suggest that phenylephrine-induced increases in tricarboxylic acid-cycle flux do not arise from lowered matrix NADH/NAD+ or ATP/ADP ratios.

Effect of depolarizing concentrations of potassium on calcium uptake and metabolism in rat liver.

Exposure of perfused livers of fed rats to 60 mM K+ induces rapid responses in the Ca2+-sensitive metabolic events, glycogenolysis, cytoplasmic and mitochondrial NADH/NAD ratios and octanoate oxidation. All increase within 45 s of K+ addition. Metabolic responses were not observed following K+ addition to livers perfused in the absence of added Ca2+. Movements of Ca2+ into the liver were suggested from experiments in which 45Ca2+ uptake was measured. The Ca2+ antagonists verapamil, diltiazem and Ni2+ essentially abolished changes to tissue metabolism and Ca2+ fluxes induced by K+ addition. K+-induced changes were consistent with Ca2+ channel activation.

The contribution of both extracellular and intracellular calcium to the action of alpha-adrenergic agonists in perfused rat liver.

The role of both intracellular and extracellular Ca2+ pools in the expression of alpha-adrenergic-agonist-mediated responses was examined in perfused rat liver. Responses studied included glycogenolysis, respiration, lactate and pyruvate formation, ketone-body production, changes in the cytoplasmic and mitochondrial redox ratio and cellular K+ fluxes. Each of these was shown to be dependent on the mobilization of intracellular Ca2+ and can be grouped into one of two response types. Transient responses (ion fluxes and the redox ratios) are obligatorily dependent on the mobilization of intracellular Ca2+ and occur irrespective of the extracellular Ca2+ concentration. Sustained responses, on the other hand, initially require intracellular Ca2+ and, subsequently, extracellular Ca2+. The data indicate that alpha-adrenergic agonists mobilize extracellular Ca2+ as well as intracellular Ca2+ and that both pools are required for the full expression of hormone-induced responses in rat liver.

The action of alpha-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver.

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.