Immunophenotypic analysis of the T cell receptor V beta repertoire in CD4+ and CD8+ lymphocytes from normal peripheral blood.

The antigenic specificity of the majority of T lymphocytes in the peripheral blood is determined by the combination of alpha and beta variable region chains present in the T cell receptor complex. Currently, the V beta chains are grouped into 25 families. Historically, determination of V beta usage has relied on detection of gene rearrangement on the nucleic acid level; however, with the increased availability of monoclonal antibodies to the product of these genomic rearrangements, immunophenotypic methods are rapidly becoming a reliable alternative method for studying the usage of V beta regions by T cells and T cell subsets. In the present study, multiparametric flow cytometry was used to determine the use of 10 V beta chains on CD4+ and CD8+ T cells in the peripheral blood of 28 normal donors. By obtaining absolute lymphocyte counts at the time blood was drawn, the absolute number of both CD4+ and CD8+ cells using particular V beta regions could be determined. Additionally, the intradonor consistency of V beta usage was examined by obtaining blood from 5 of the volunteers at an interval of approximately 1 year. The results of this study suggest a fairly homogeneous pattern of use for these V beta regions. The most striking longitudinal differences were observed in one individual who underwent a tonsillectomy midway between the T cell receptor V beta determinations.

Detection and monitoring of a concomitant atypical myeloproliferative disorder and chronic lymphocytic leukemia by flow-cytometric immunophenotyping.

OBJECTIVE: To illustrate the utility of a broad panel of monoclonal antibodies to detect secondary processes or unexpected characteristics of the primary blood dyscrasia. DESIGN: Case report and discussion. SETTING: Regional academic medical center. PATIENT: A 64-year-old man presenting with an apparent acute myeloid leukemia. INTERVENTIONS: Sequential immunophenotyping with a broad panel of monoclonal antibodies to monitor progression of disease and response to therapy. MAIN OUTCOME MEASURE: Identification and monitoring of the two atypical populations in this patient with correlation to the clinical status of the patient. RESULTS: Identification of an unsuspected mature lymphoid clone and characterization of the evolution of the myelomonocytic clone. CONCLUSION: The evolving mature lymphoid clone may have been overlooked in the context of a predominant atypical myeloproliferative process, particularly if a limited panel of monoclonal antibodies had been used for immunophenotyping. Sequential immunophenotyping was useful in monitoring the progression of each atypical process.

Congenital leukemia: report of two cases.

Congenital leukemia is a rare disease in which a leukemic process is present at birth or immediately thereafter. The majority of cases presented in the literature were reported prior to the availability of contemporary immunophenotyping methods, and lineage assignment was often made on the basis of morphology alone. Congenital leukemias may be of various lineages, although, historically, monocytic and myelomonocytic congenital leukemias appear to be the most prevalent. We present two cases of congenital leukemia with detailed immunophenotypic and cytochemical characterization. One case is of the lymphoid lineage, and the second is of myelomonocytic lineage. Neither patient displayed trisomy 21.

Alterations of T-cell receptor variable region expression in human immunodeficiency virus disease.

Since only a small percentage of CD4+ lymphocytes is infected at any one time during the course of human immunodeficiency virus (HIV) disease, a question central to the pathogenesis of HIV is whether or not the depletion of CD4+ lymphocytes is a random or selective event. The majority of peripheral blood T lymphocytes use alpha and beta variable chains as components of their T-cell receptor (TCR) complex. Depletion of CD4+ T lymphocytes from the peripheral blood may be dependent on the V beta chain expressed by the CD4+ cell, based on the hypothesis that HIV may encode a superantigen. Peripheral blood from normal controls and HIV+ patients was studied for alterations in the expression of various V beta chains of the TCR. Three-color flow cytometry was used to determine the expression of V beta 2, V beta 3, V beta 8, V beta 13, and V beta 19 on all lymphocytes and on both CD4+ and CD8+ lymphocytes independently. Alteration of the V beta chains in HIV+ disease was analyzed as a function of absolute CD4 count and Centers for Disease Control (CDC) stage of the patient. These data suggest that the loss of T helper (CD4) lymphocytes during the course of HIV disease may be a selective event. These data are consistent with the hypothesis that selective depletion of CD4+, V beta 19+ lymphocytes may be due to the encoding of a superantigen by HIV. Furthermore, using multicolor flow cytometry and stratifying patients by absolute CD4 counts (or stage of disease) may reveal immunologic changes that might otherwise be overlooked.

Accuracy and cost-effectiveness of a one-tube, three-color method for obtaining absolute CD4 counts and CD4:CD8 ratios.

An increasing number of assays to determine absolute CD4 counts are being performed as the acquired immunodeficiency syndrome epidemic continues. Simultaneously, there is increasing pressure to contain costs in the clinical laboratory. A rapid, one-tube, three-color method for obtaining CD4 counts has been evaluated for accuracy, compared with a more traditional two-color panel of markers. The data, compiled on 102 patients, including both pediatric and adult human immunodeficiency virus-infected patients, indicate a high degree of correlation between the one-tube method and a traditional panel. This approach should be considered for cost-effective and accurate CD4 determinations.

Trisomy 14 in two cases of granulosa cell tumor of the ovary.

Cytogenetic analysis of granulosa cell tumor of the ovary was performed in two patients. G-banding analysis of cells cultured 3-5 days showed that the karyotype of each tumor contained normal diploid cells as well as cells with identical aberration: trisomy 14. This is the first report of trisomy 14 in two cases of granulosa cell tumor of the ovary. Flow cytometric DNA content analysis was also performed.

The changing private practice environment: how to analyze opportunities.

The search by physicians for opportunities to improve the short-term performance and long-term value of their practices has resulted in the creation of a range of affiliation and group practice structures and changed the traditional private practice profile. The resulting ability to structure an environment that is optimal for each practitioner or group of practitioners can enhance the delivery of medical care and serve to attract an array of individuals to private practice. In order for this evolution to be successful, however, the entities must be developed with thoughtful and detailed analysis and should be flexible enough to adapt to the continually changing environment.

Neuropeptide modulation of apomorphine-induced stereotyped behavior.

The nucleus accumbens contains many neuropeptides whose functions are presently unknown. The purpose of this study was to determine the extent to which these neuropeptides act in conjunction with the mesolimbic dopamine system. Microinjections of cholecystokinin, neurotensin, met-enkephalin, somatostatin, bombesin, as well as glutamate and muscimol, were made into the medial nucleus accumbens after systemic injection of apomorphine. Cholecystokinin and neurotensin, in nanogram doses, potentiated apomorphine-induced stereotypy. Met-enkephalin reduced, while somatostatin and bombesin were without effect on, apomorphine-induced stereotypy. In addition, both glutamate and muscimol potentiated this effect. These results suggest that several neuropeptides and amino acids act in the nucleus accumbens to modulate apomorphine-induced stereotyped behaviors.

Cholecystokinin potentiates dopamine-mediated behaviors: evidence for modulation specific to a site of coexistence.

Cholecystokinin coexists with dopamine in mesolimbic neurons in mammalian brain. When injected directly into the nucleus accumbens, cholecystokinin (CCK) potentiated dopamine (DA)-induced hyperlocomotion and apomorphine-induced stereotypy. These effects were not mimicked by nonsulfated CCK, but were blocked by proglumide, a putative CCK antagonist, as well as by antisera raised against sulfated CCK. CCK alone had no effect on locomotion or sterotypy, indicating that this peptide acts primarily as a modulator of DA-mediated behaviors in the mesolimbic pathway. In addition, CCK did not potentiate DA-induced hyperlocomotion or apomorphine-induced stereotypy when injected into the caudate nucleus, where CCK and DA are localized in separate neurons in rats. Facilitation of DA-mediated behaviors by CCK may represent a functional interaction specific to the neuromodulator-neurotransmitter coexistence phenomenon.

PKU, learning, and models of mental retardation.

Experimental phenylketonuria was induced in male rats by daily injections of alpha-methylphenylalanine and phenylalanine on postnatal Days 3-31. Beginning at 8 weeks of age, the animals were subjected to a test of observational learning followed by a test of latent learning (two tests of "advantageous" learning). The animals subjected to the PKU treatment early in life showed significant learning deficits in both tests. The importance of these studies lies in the fact that unlike conventional tests of learning, tests of advantageous learning are sensitive to the kinds of biological insults which cause mental retardation in humans. This differential sensitivity evident in studies of animal models of cognitive pathology is analogized to the areas of dysfunction which characterize human mental retardation. Suggestions for the development of appropriate models of intellectual development are made.

Anxiolytic-like properties of fominoben.

Fominoben, a centrally acting antitussive, has been shown recently to bind to the brain benzodiazepine binding site and to antagonize pentylenetetrazol-induced seizures. The present study reports the ability of fominoben to produce anti-anxiety effects analogous to diazepam in a mouse exploratory model for anxiolytics. This action of fominoben was blocked by Ro15-1788, a benzodiazepine receptor antagonist, implicating a brain benzodiazepine binding site in this anxiolytic action of fominoben.

Further characterization of a simple, automated exploratory model for the anxiolytic effects of benzodiazepines.

Anxiolytics specifically increase the number of exploratory transitions in a two-chambered model system for anxiety in mice. Characterization of parameters to optimize and standardize this model required analysis of multiple use of test animals, intertrial interval, and circadian variability. Time of day did not affect exploratory activity in mice treated with vehicle or diazepam between 10 a.m. and 11 p.m. Intertrial intervals of 1, 3, 5, or 7 days were equally effective. The diazepam-induced increase in exploratory activity was significant over the first three uses of test animals. These data recommend reuse of mice to a maximum of three trials, throughout the daytime or evening hours of their lighting schedule.