Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells.

Adhering platelets on the cell surface can give misleading results when doing flow cytometry analysis of platelet/megakaryocyte-specific glycoprotein (GP) antigens to enumerate megakaryocytes (MK) in mobilized peripheral blood (PB), apheresis products, or normal bone marrow (BM). For adequate quantification and characterization of human MK, we examined samples with parallel flow cytometry and immunocytochemistry. MK expression of GP IIb/IIIa (CD41a), GP Ib (CD42b), GP IIIa (CD61), CD45, CD33, and CD11b, and their light scatter properties were evaluated. Fresh samples of low density mononuclear cells (MNC) or purified CD34+ cells contained 10-45% of platelet-coated cells. Platelet-coated cells decreased dramatically after several days of incubation in a serum-free medium supplemented with stem cell factor, IL-3, IL-6, and/or GM-CSF. Between d 9-12, flow cytometry detected a distinct CD41a+ MK population, 8.3 +/- 1.3% in BM CD34 cell cultures (n = 7) and 13.1 +/- 2.1% in PB CD34 cell cultures (n = 14), comparable to immunocytochemistry data (7.8 +/- 1.9% and 16.4 +/- 2.6%, respectively). CD41a stained a higher proportion of MK than CD42b or CD61, while CD42b+ or CD61+ cells contained more morphologically mature MK than CD41a+ cells in cultures containing aplastic serum. When fluorescence emission of CD41a was plotted against forward-light scatter (FSC), subpopulations of small and large MK were observed. Such subpopulations overlapped in CD41a intensity and side-light scatter (SSC) property. Most MK co-expressed CD45 (98.8% positive) but not CD33 (80.7% negative) or CD11b (88.9% negative). Our data indicate that flow cytometry can be used effectively to identify MK. However, caution should be taken with samples containing adherent platelets.

An aspartic acid at amino acid 108 is required to rescue infectious virus after transfection of a poliovirus cDNA containing a CGDD but not SGDD amino acid motif in 3Dpol.

The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme. Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C. D. Morrow, J. Virol. 67:373-381, 1993). The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase. On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase. To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326). The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed. Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108. In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type. The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation. Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed. Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus. Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus. The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus. RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.

Lipoprotein lipase association with lipoproteins involves protein-protein interaction with apolipoprotein B.

Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM). To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins.

Molecular basis for prokaryotic specificity of magainin-induced lysis.

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

Perceived personal and institutional influences on child and adolescent psychiatry research careers.

To assess possible influences on the professional success of child and adolescent psychiatry investigators, all 187 physician-first authors of research posters presented at 4 annual meetings of the American Academy of Child and Adolescent Psychiatry were asked to complete a questionnaire. Responses numbered 147 (79%), with 80% (117/147) spontaneously naming one or more workplace factors, such as excessive clinical, administrative, or teaching demands, as hindrances to their success as researchers. The most important hindrances andfacilitatingfactors are discussed and suggestions are made to promote the development of research investigators in child and adolescent psychiatry.

Characterization of poliovirus replicons encoding carcinoembryonic antigen.

Recombinant vaccines hold great promise for the prevention and therapy of infections diseases and cancer. We have explored the use of poliovirus as a recombinant vector to deliver genes into cells for the purpose of vaccination. For our studies, we have chosen to express the gene-encoding carcinoembryonic antigen (CEA) using a novel poliovirus vector. We have constructed a recombinant CEA-poliovirus replicon in which the CEA gene was substituted for the poliovirus capsid gene. Following in vitro transcription, the RNA was transfected into cells to demonstrate CEA expression. We found that a genome in which the region encoding the signal sequence of the CEA protein (amino acids 1-34) was removed was replication competent (i.e., referred to as a replicon). We encapsidated the CEA-poliovirus replicon by transfecting this RNA into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid protein (P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with the encapsidated replicon containing the CEA-poliovirus genome resulted in expression of the CEA protein. To test immunogenicity, mice susceptible to poliovirus were given three doses of the encapsidated replicons via the i.m. route. By the third administration, a CEA-specific antibody response was detected. Potential future use of the poliovirus replicon system as both a parenteral and oral vaccine vector is discussed.

"Just right" perceptions associated with compulsive behavior in Tourette's syndrome.

OBJECTIVE: Tourette's syndrome is a chronic disorder of childhood onset that is characterized by motor and phonic tics and a broad range of associated behavior including obsessive-compulsive symptoms. Less well appreciated are the sensory and other perceptually tinged mental phenomena associated with both tic and compulsive behavior. This cross-sectional study evaluated the "just-right" phenomena commonly associated with compulsive behavior in subjects with tic disorders. METHOD: A total of 134 subjects with tic disorders, aged 9 to 71 years, completed a questionnaire concerning their current and past tic symptoms. Subjects were also asked to describe their "just right" perceptions. The Yale-Brown Obsessive Compulsive Scale was used to assess severity of current obsessive-compulsive symptoms. RESULTS: Eight-one percent of the subjects with both Tourette's syndrome and obsessive-compulsive disorder (N = 31) and 56% of the subjects with Tourette's syndrome and obsessive-compulsive symptoms (N = 61) reported being aware of a need to perform compulsions until they were "just right." This awareness most commonly referred to visual or tactile (as opposed to auditory) features of the compulsive act or its consequences. Three-quarters of the subjects with these perceptions reported that they usually were aware of them immediately before or concurrently with the initiation of compulsions. CONCLUSIONS: While epidemiological studies of tic disorders and obsessive-compulsive disorder have yet to incorporate questions concerning "just right" perceptions, these results suggest that such perceptions may be commonplace in adolescent and adult subjects with both obsessive-compulsive disorder and tic disorders. They also implicate brain regions involved in the processing of sensorimotor information in the pathobiology of tic disorders.

Videotape tic counts in the assessment of Tourette's syndrome: stability, reliability, and validity.

OBJECTIVE: We examined the short- and long-term temporal stability of tic counts to estimate the minimum length of videotape needed for a reliable index of overall tic activity and determined the interrater reliability and validity of tic counts based on prolonged videotape segments (> 10 minutes). METHOD: Motor and phonic tic counts and clinician ratings were performed on 43 patients with Tourette's syndrome (TS), aged 7 to 50 years. Short-term stability was estimated by determining the mean interval-to-interval correlation of sequential equal-length segments from 30-minute videotape recordings of 20 subjects. Long-term stability was determined by correlating tic counts at 1-week (N = 14) and 2-week intervals (N = 11). In addition, tic counts were correlated with the most widely used clinical ratings of TS. RESULTS: The short-term stability data indicated that estimates of motor and phonic tic frequencies should be based on videotape counts of at least 5 minutes' duration. Tic counts also were highly reliable and were significantly correlated with clinical ratings with the Yale Global Tic Severity Scale and the Clinical Global Impression Scale for Tourette Syndrome. CONCLUSIONS: Standardized videotape tic counts can provide highly reliable, stable measures of tic frequencies that are moderately correlated with selected global ratings of tic severity.

Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation.

Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 x 10(4) to 0.65 x 10(4)/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamide-mobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 +/- 16.9%, mean +/- 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA- CD71+ population containing 89 +/- 6.3% (n = 4) BFU-E and a CD45RA+ CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.

Determinants of academic survival: survey of AACAP poster authors.

OBJECTIVE: To assess personal and institutional factors that may influence the development and retention of child psychiatry researchers and to estimate academic survival rates of full-time child psychiatry researchers. METHOD: One hundred forty-seven (79%) of 187 physician-first authors of research posters presented at the annual meetings (1984 to 1990) of the American Academy of Child and Adolescent Psychiatrists were surveyed for demographic data, current academic status, level of research involvement, career path from 1984 to 1991, and factors facilitating or hindering research career success. RESULTS: Survival analysis of strictly defined full-time researchers (N = 46) revealed a 67% 7-year survival rate. Significantly higher survival rates were obtained for investigators with more than 2 years of research training or with affiliation with major child psychiatric research institutions when beginning a full-time research position. Both these factors were significant and independent predictors of academic survival. CONCLUSIONS: Research training and affiliation with a major child psychiatric research institution are associated with longer academic survival for full-time researchers.

Premonitory urges in Tourette's syndrome.

OBJECTIVE: Tourette's syndrome traditionally has been viewed as a hyperkinetic movement disorder characterized by involuntary motor and phonic tics. Many patients, however, describe their tics as a voluntary response to premonitory urges. This cross-sectional study evaluated premonitory urges and related phenomena in subjects with tic disorders. METHOD: A total of 135 subjects with tic disorders, aged 8 to 71 years, completed a questionnaire concerning their current and past tic symptoms. Subjects were asked to describe and, if possible, localize their premonitory urges. The Yale Global Tic Severity Scale was used to assess current tic severity. The method of case finding does not provide prevalence data for premonitory urges. RESULTS: Ninety-three percent of the subjects reported premonitory urges. Anatomical regions with the greatest density of urges were the palms, shoulders, midline abdomen, and throat. Eighty-four percent of the subjects reported that tics were associated with a feeling of relief. A substantial majority (92%) also indicated that their tics were either fully or partially a voluntary response to the premonitory urges. CONCLUSIONS: While epidemiological studies of tic disorders have yet to incorporate questions concerning premonitory urges, these results suggest that such urges may be commonplace in adolescent and adult subjects with tic disorders. These results challenge the conventional wisdom that tic behaviors are wholly involuntary in character. They also implicate brain regions involved in the processing of sensorimotor information in the pathobiology of tic disorders.

Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry.

Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

13-cis retinoic acid augments the production of macrophages in mouse bone marrow cultures stimulated with interleukin 3.

Mouse bone marrow cells in liquid culture with interleukin 3 generate nonadherent granulocytes, mast cells, and macrophages. The addition of 13-cis retinoic acid (13cRA) (10(-8)-10(-6) M) enhanced proliferation of the nonadherent cells, and concentrations greater than 5 x 10(-7) M stimulated a sixfold increase in adherent macrophages. Four-color flow cytometry was used to identify the lineages present using the following antibodies: MAC1 (granulocytes and macrophages), F4/80 (macrophages), B54.2 (mast cells), and H12 (anti-Thy1.2 to identify myeloid precursors). This analysis demonstrated a twofold increase in MAC1+ F4/80+ cells, which were sorted and identified morphologically as macrophages. 13cRA also increased by 60%-95% the numbers of colony-forming cells responsive to interleukin 3 (IL-3) and macrophage colony-stimulating factor (M-CSF) but did not significantly change the colony-forming cells responsive to granulocyte-macrophage colony-stimulating factor (GM-CSF). These data suggest that 13cRA increases the production of macrophages by modulating the commitment of IL-3-expanded progenitor cells to the macrophage lineage.

Functional and phenotypical studies of the Leu-4 (CD3)+, Leu-1 (CD5)- T lymphocyte.

A small T cell subpopulation expressing the phenotype Leu-5(CD2)+, Leu-4(CD3)+, Leu-1 (CD5)- can be found in peripheral blood and bone marrow of normal individuals. When these cells were sorted out by three colour immunofluorescence cell sorting and tested in limiting dilution assays, they were found to have lower frequencies of proliferating (9.0 +/- 5.6 times, n = 7) and of IL-2 producing cells (11.5 +/- 5.0 times n = 5), and a higher frequency of cytotoxic cells (3.1 +/- 2.6 times, n = 2) than T lymphocytes expressing the three markers. In peripheral blood lymphocytes, 1/3 of the CD3+, CD5- cells were positive for Leu-2a (CD8) while virtually all were negative for Leu-3a (CD4). Four colour flow cytometric analysis revealed a small subset of T cells positive for CD3 and negative for CD5, CD4 and CD8. Approximately 75% of the CD3+, CD5- cells were negative for Leu-7 and CD16 simultaneously. These results shed a light on the phenotype of T cells that escape killing by CD5 and complement in T cell depleted bone marrow and may explain why fewer residual T cells in the depleted marrow are detected by limiting dilution assays than by flow cytometric analysis.

Prediction of recovery for closed-head-injured adults: an evaluation of the MMPI, the Adaptive Behavior Scale, and a "Quality of Life" Rating Scale.

Twenty-five closed-head-injured adults (24 males, 1 female; M age = 28.8 years) were classified as "recovered" if they had returned to work, school, or a sheltered workshop for which pay was received and "non-recovered" if they did not meet these criteria. Current status was compared with MMPI, Adaptive Behavior Scale (ABS), and "Quality of Life" Rating Scale (QLRS) scores at time of entry into a rehabilitation program. The "non-recovered" group was significantly higher on the PD (Psychopathic deviate) scale. No differences were found between groups on the Sc (Schizophrenia) or K (Validity) scales. The "recovered" group was significantly higher on the ABS Economic Activity domain and significantly lower in the Violent & Destructive, Antisocial, Rebelliousness, Untrustworthiness, Stereotyped Behavior & Odd Mannerisms, and Psychological Disturbance behavior domains. The self-ratings (QLRS) of the "nonrecovered" subjects were significantly more distinct from the ratings made by their relatives or significant others than were those of the "recovered" subjects.

Similarity of central and peripheral alpha-1 adrenoceptors in rat and rabbit.

In order to examine species and tissue differences in alpha 1 adrenoceptors, binding experiments were performed using 3H-prazosin and membrane homogenates of central nervous and peripheral tissues of rabbit (cortex and spleen), and rat (cortex, spleen, and liver). Saturation studies indicated one binding site for 3H-prazosin, with apparent log molar dissociation constants (pKD) ranging from 9.43 to 10.20. The rank orders of affinities of three competing antagonists (prazosin much greater than idazoxan greater than rauwolscine) and five agonists (cirazoline greater than clonidine approximately equal to (-)-norepinephrine greater than (-)-phenylephrine greater than (+)-norepinephrine) were typical of alpha 1 receptors in all tissues. There were small but significant differences in the mean affinities of rauwolscine, idazoxan and cirazoline among the five tissues. No significant differences in pseudo-Hill coefficients were observed among tissues, although agonist binding curves were shallow (.7 to .85) and prazosin competition curves were significantly steeper (greater than .85). Guanine nucleotide did not affect the position or slope of the (-)-norepinephrine competition profile in rat cortex. These results demonstrate a qualitative similarity among central and peripheral alpha 1 receptors of the rat and rabbit, with small differences observed between central and peripheral sites in both species.