Bone marrow transplantation in severe combined immunodeficiency from a sibling who had received a paternal bone marrow transplant.
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Bone Marrow↗
Biomedical subjects
R L Roberts
Publications and source records attributed to R L Roberts.
Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.
RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.
Animals↗
Perinatal steroid treatments alter alloparental and affiliative behavior in prairie voles.
This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12-20 or on Postnatal Days 1-6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.
Animals↗
Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight.
The effects of immediate versus delayed cell separation, storage temperature, presence of serum, and type of anticoagulation on the natural killer (NK) cytotoxicity of human mononuclear cells were assessed. The NK cytotoxicity of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMC) was tested in a 3-h chromium-51 release assay with K562 cells at various effector/target cell ratios. The NK activities of PBMC from blood anticoagulated with either heparin or EDTA and then immediately separated and assayed were not different (42.9 +/- 2.5% for heparin and 40.3 +/- 4.6% for EDTA). When these separated cells were cultured in medium with 10% fetal calf serum and stored at 4,25, or 37 degrees C for 18 h before the assay, there was a significant increase in cytotoxicity. PBMC from blood stored in heparin or EDTA for 18 h before separation had reduced NK cytotoxicity, particularly if they were kept at 37 degrees C. When separated PBMC were cultured in medium with 10% human AB serum, however, samples held at 25 and 37 degrees C decreased in cytotoxicity but samples held at 4 degrees C maintained the cytotoxicity demonstrated at the baseline level with fresh cells. We recommend that heparinized blood be used for NK assays and that the PBMC be isolated immediately and held overnight at 4 degrees C in medium with 10% AB serum if the assay must be delayed. The NK cytotoxicity under these storage conditions most closely matches the results obtained when the PBMC are isolated and tested on the same day. IF PBMC isolation must also be postponed, it is best to store the blood in heparinized tubes at 25 degrees C to prevent loss of cytotoxic function.
ABO Blood-Group System↗
Rab5 regulates the dynamics of early endosome fusion.
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Adenosine Triphosphate↗
Adrenocorticoid hormones and the development and expression of mammalian monogamy.
Based on research with prairie voles, we hypothesize that the unusual patterns of reproduction and social behavior associated with mammalian monogamy may arise as a consequence of normal developmental exposure to high levels of glucocorticoids and/or other hormones of the HPA axis. Increased HPA activity could functionally inhibit some of the masculinizing processes expected during the perinatal period. We hypothesize that the unique behavioral, physiological, and anatomical changes associated with monogamy may reflect the adaptive consequences of reduced exposure to the masculinizing actions of HPG hormones, such as testosterone. Reproductively naive, unpaired adult prairie voles also show unusual patterns of adrenal activity, including a marked decline in corticosterone levels within minutes following exposure to novel animals of the opposite sex. In females, this decline in corticosterone may contribute to pair bonding, since corticosterone injections inhibit, and adrenalectomy is associated with a facilitation of pair bond formation. In males, corticosterone injections facilitate pair bonding and adrenalectomy has the opposite effect. In animals from established social pairs corticosterone levels also fluctuate according to the social environment of the animal; the absence of a familiar partner is associated with increased corticosterone secretion, and in the presence of the familiar partner corticosterone levels tend to decline. Prairie voles may offer a valuable source of information regarding the behavioral, anatomical, and physiological consequences of long-term and short-term exposure to high levels of adrenal activity in the absence of pathology.
Adrenal Cortex Hormones↗
Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.
Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.
Androstadienes↗
N-acetylcysteine enhances antibody-dependent cellular cytotoxicity in neutrophils and mononuclear cells from healthy adults and human immunodeficiency virus-infected patients.
Patients with AIDS have decreased levels of the intracellular antioxidant, glutathione, in their circulating lymphocytes and plasma. N-acetylcysteine (NAC) increases intracellular stores of glutathione and has direct antioxidant properties. In this study, the effects of glutathione and NAC on the cytotoxicity of neutrophils and mononuclear cells were tested using cells from healthy controls and human immunodeficiency virus (HIV)-infected patients. NAC (1 and 5 mM) enhanced the antibody-dependent cellular cytotoxicity (ADCC) of neutrophils from healthy adult controls and HIV-infected adults and children. The antineoplastic drug, 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), which depletes intracellular glutathione, inhibited the ADCC of neutrophils; the addition of NAC partially reversed this inhibition. Similar effects of BCNU and NAC were seen when the cytotoxicity of mononuclear cells was tested using CEM tumor cells bearing the HIV gp120 antigen as targets. Thus, NAC enhances various forms of cytotoxicity and may be beneficial to AIDS patients whose defects in leukocyte cytotoxicity may be due to glutathione depletion.
Acetylcysteine↗
Detection of Epstein-Barr virus DNA in cerebrospinal fluid for diagnosis of AIDS-related central nervous system lymphoma.
The diagnostic utility of Epstein-Barr virus (EBV) DNA detection in cerebrospinal fluid for the diagnosis of central nervous system lymphoma was evaluated with two different PCR assays to test a collection of cerebrospinal fluid samples from 24 AIDS patients with central nervous system disorders. A PCR assay amplifying a fragment from the BamHI-W region had the highest clinical and analytic sensitivity. The BamHI-W PCR assay detected EBV DNA in cerebrospinal fluid from 83% (5 of 6) of patients with pathologically proven primary central nervous system lymphoma and 7% (1 of 16) of controls with autopsy-proven nonlymphomatous central nervous system disorders. EBV DNA was also detected in one patient with autopsy-proven systemic lymphoma involving the central nervous system and one patient with probable primary central nervous system lymphoma. EBV DNA was detected consistently when central nervous system lymphoma involved meningeal surfaces. PCR for EBV in cerebrospinal fluid appears to be useful for diagnosis of AIDS-related central nervous system lymphoma, but additional studies are required to better define the sensitivity of the assay and to understand the significance of a positive test in the absence of lymphoma.
Central Nervous System Neoplasms↗
Chondroid chordoma of the lateral skull base.
Clinical, radiological, and pathologic features of an intracranial chondroid chordoma in a 9-year-old boy are described. This is the first reported case of a chordoma, the center of which was laterally situated in the cranial base, lying in or near jugular foramen and carotid canal, but without midline involvement. Although cranial chordomas in childhood are extremely rare, and all previously reported cases appeared to have arisen in the clivus, this location should not be considered ectopic. Forking at the rostral end of the notochord has been demonstrated in embryos, and would be the presumed embryological source for this tumor. This is also the first reported case of a chondroid chordoma in a child with immunohistochemical documentation distinguishing it from a chondrosarcoma. This chondroid chordoma contained two populations of cells: neoplastic cartilage and chordoid tissue in a myxoid stroma. The distinction between chordoma and chondrosarcoma and the implications on treatment will be discussed.
Child↗
Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth.
Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from a colonial form of growth to a filamentous pseudohyphal form. This dimorphism requires a polar budding pattern and elements of the MAP kinase signal transduction pathway essential for mating pheromone response in haploids. We report here that haploid strains exhibit an invasive growth behavior with many similarities to pseudohyphal development, including filament formation and agar penetration. Haploid filament formation depends on a switch from an axial to a bipolar mode of bud site selection. Filament formation is distinct from agar penetration in both haploids and diploids. We find that the same components of the MAP kinase cascade necessary for diploid pseudohyphal development (STE20, STE11, STE7, and STE12) are also required for both filament formation and agar penetration in haploids. Thus, haploid yeast cells can enter either of two developmental pathways: mating or invasive growth, both of which depend on elements of a single MAP kinase cascade. Our results provide a novel developmental model to study the dynamics of signal transduction, with implications for higher eukaryotes.
Calcium-Calmodulin-Dependent Protein Kinases↗
Transport of transferrin across the blood-thymus barrier in young rats.
The interaction of transferrin-peroxidase (Tf-HRP) with the capillary endothelium of the rat thymus was analyzed by diaminobenzidine (DAB) cytochemistry and electron microscopy. The thoracic aortas of young rats were cannulated and the upper bodies perfused with the Tf-HRP conjugate. In the thymus, plasmalemmal vesicles of the endothelium mediated the endocytosis and transport of Tf-HRP. Transcytosis of Tf-HRP appeared to occur by micropinocytosis, without morphological evidence of involvement by endothelial endosomes. DAB reaction product was commonly present in the subendothelial interstitial space and, in addition, was often localized in clathrin coated pits and vesicles in epithelial reticular cells that surround the thymic capillaries. In perfusions done at 4 degrees C, no binding of Tf-HRP to the lumenal membranes of capillaries was detected. The transport of Tf-HRP across the capillary endothelium in the thymus was not inhibited by competition with excess native transferrin; however, the uptake of Tf-HRP by epithelial reticular cells was completely inhibited by excess transferrin. Transferrin receptors were localized in the thymus by indirect immunocytochemistry using OX-26, a mouse anti-rat transferrin receptor monoclonal antibody. No transferrin receptors were detected on the capillary endothelium but diffuse reaction product was localized on the subjacent epithelial reticular cells. These results indicate that transport of Tf-HRP across the thymus capillary wall is independent of transferrin receptors at the level of the endothelial cells but that subsequent uptake of Tf-HRP by epithelial reticular cells is a transferrin receptor dependent process.
Age Factors↗
Disseminated measles infection after vaccination in a child with a congenital immunodeficiency.
An infant boy with a congenital immunodeficiency had fatal disseminated measles after administration of a live attenuated measles vaccine. This rare complication was confirmed with molecular virologic techniques. Although efforts to expand availability of vaccinations are critically important, caution is warranted in children with potentially severe immunologic dysfunction.
Failure to Thrive↗
Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines.
We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.
Adult↗
Deferoxamine posttreatment reduces ischemic brain injury in neonatal rats.
BACKGROUND AND PURPOSE: Iron catalyzes the formation of damaging reactive species during cerebral reperfusion. Brain iron concentration is highest at birth, so the brain of the asphyxiated newborn may be at increased risk of iron-dependent injury. We investigated whether the ferric iron chelator deferoxamine could reduce hypoxic-ischemic brain injury in neonatal rats. Because deferoxamine has concentration-dependent activities other than iron chelation, we measured brain deferoxamine levels and calculated deferoxamine pharmacokinetic parameters. METHODS: We produced hypoxic-ischemic injury to the right cerebral hemisphere of 7-day-old rats by right common carotid artery ligation followed by 2.25 hours of hypoxia in 8% oxygen. At 5 minutes of recovery from hypoxia the rats received 100 mg/kg deferoxamine mesylate or saline subcutaneously. Rats (saline, n = 33; deferoxamine, n = 38) were killed at 42 hours of recovery to assess early acute edema by measurement of hemispheric water content. Other rats (saline, n = 31; deferoxamine, n = 32) were killed at 30 days of age for morphometric determination of right hemisphere atrophy. In still other rats, we measured deferoxamine levels in blood and brain after hypoxia-ischemia. RESULTS: Deferoxamine significantly reduced right hemisphere injury as measured by early water content (P < .01) and later atrophy (P = .019). Deferoxamine brain levels peaked between 100 and 200 mumol/L at 40 to 60 minutes after injection and exceeded serum levels by +/- 70%. CONCLUSIONS: Deferoxamine administered after induction of cerebral hypoxia-ischemia reduces injury in 7-day-old rats. Deferoxamine concentrates in the brain at levels between 100 and 200 mumol/L. At the concentrations achieved, deferoxamine might protect the brain through mechanisms unrelated to its ability to chelate iron.
Animals↗
Antiviral properties of neonatal and adult human neutrophils.
The antiviral properties of neonatal and adult human neutrophils were investigated by their ability to inhibit the formation of herpes simplex virus (HSV) plaques using an extrinsic viral resistance (EVR) assay. The EVR assay was performed by incubating neutrophils or mononuclear cells (MNC) with HSV-infected Vero or CEM tumor cells for 48 h. The cells were then frozen and viable HSV was quantitated by the ability of the lysate to form viral plaques. Activation of neutrophils from normal adults with phorbol myristate acetate increased their ability to inhibit HSV plaque formation more than 10-fold. The EVR response of neutrophils from newborn infants was much lower, and no significant inhibition occurred using activated neutrophils from patients with chronic granulomatous disease. The presence of rabbit anti-HSV antiserum slightly increased the EVR response of neutrophils but produced a greater increase in the response of the MNC in both adults and newborns. However, the combination of antiserum plus cytokines (granulocyte-macrophage colony-stimulating factor, IL-2, and interferon-gamma) greatly augmented the neutrophil EVR response to the level of the MNC response. Thus, neutrophils are capable of exerting a strong antiviral response comparable to that of MNC that may be important as a second line of defense in the immunocompromised patient.
Adolescent↗
Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease.
The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.
Adult↗
Phagocytic processing of bacterial antigens for class I MHC presentation to T cells.
Class I major histocompatibility complex (MHC) molecules present antigens that are produced within the presenting cell or penetrate from the vacuolar system into the cytosol for processing. Most studies of exogenous antigen processing have used soluble antigens, which are not efficiently presented by class I MHC molecules and do not elicit CD8 T-cell responses in vivo. But particulate antigen preparations with no known mechanism for cytosolic penetration can also elicit CD8 T-cell responses in vivo. We report here that phagocytosis of bacteria with no mechanism for cytosolic penetration also results in presentation of bacterial antigens by class I MHC molecules. Moreover, this mechanism is resistant to cycloheximide and Brefeldin A, which block the classical class I processing pathway. These results suggest a novel vacuolar class I processing pathway for exogenous phagocytic antigens.
Amino Acid Sequence↗