Cryopreservation of human mononuclear cells for quality control in clinical immunology. I. Correlations in recovery of K- and NK-cell functions,surface markers,and morphology

Cryopreserved human peripheral blood mononuclear cells (PBMC) were tested for natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) and for high-affinity (29°C) and total (4°C) rosette formation with sheep erythrocytes. PBMC produced variable NK activity following freezing and thawing, but consistently reacted well in ADCC. A significant correlation was found between low NK activity and a decreased percentage of low-affinity rosette-forming cells. On the contrary, the number of large granular lymphocytes (LGL), among which NK cells are restricted, and the reactivity with the monoclonal antibody OKT10, which recognizes the majority of LGL in the peripheral blood, were not significantly altered by cryopreservation. Cryopreserved cells proved to be excellent controls for determining the day-to-day variability of the NK assay and for selecting optimum conditions for this test in the clinical immunology laboratory.     

Fine structure of microgametogenesis of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus

Summary The microgamogony of Eimeria ferrisi from experimentally infected mice was investigated with the electron microscope. Microgamonts were recognizable by the presence of peripherally arranged nuclei and the presence of single or paired centrioles between each nucleus and the limiting membrane of the parasite. Often an intranuclear centrocone directed toward the centriole was present. Differentiation of the microgamete began when elevations of the limiting membrane, which indicated the commencement of flagellar development, appeared above the centrioles. This event was accompanied by the segregation of nuclear content into a dense osmiophilic portion and an electron-pale portion. Then followed a gradual protrusion of the dense portion of the nucleus and developing flagella into the parasitophorous vacuole. A dense ring developed at the base of the differentiating microgamete, resulting in the formation of a stalk which was occupied by the residual portion of the nucleus. Fully developed microgametes became detached and occupied the parasitophorous vacuole along with the residual cytoplasm. Microgametes had an anterior perforatorium, a dense elongate nucleus, with an anteriorly positioned mitochondrion in a small groove of the nucleus. Usually two flagella were present but one microgamete appeared to have three. Polysaccharide first appeared when differentiation was in progress and increased until large numbers of granules were present in the microgamont cytoplasm.Abbreviations AM Amylopectin - B Basal Body of Flagellum - CC Centrocone - CE Centriole - DR Dense Ring - ER Endoplasmic Reticulum - F Flagellum - HC Host Cell - HN Host Cell Nucleus - MI Mitochondrion - MN Microneme - MP Micropore - MT Microtubule - N Nucleus - P Perforatorium - PL Osmiophilic Plate - PV Parasitophorous Vacuole - RN Residual Nucleus Supported in part by the Deutsche Forschungsgemeinschaft¹, the Alexander von Humboldt Foundation² and a Faculty Development Grant to Andrews University by the Merck Foundation, Rahway, New Jersey, USA     

The Clinical Health Economics System Simulation (CHESS): a teaching tool for systems- and practice-based learning.

Academic medical centers face barriers to training physicians in systems- and practice-based learning competencies needed to function in the changing health care environment. To address these problems, at the University of Virginia School of Medicine the authors developed the Clinical Health Economics System Simulation (CHESS), a computerized team-based quasi-competitive simulator to teach the principles and practical application of health economics. CHESS simulates treatment costs to patients and society as well as physician reimbursement. It is scenario based with residents grouped into three teams, each team playing CHESS using differing (fee-for-service or capitated) reimbursement models. Teams view scenarios and select from two or three treatment options that are medically justifiable yet have different potential cost implications. CHESS displays physician reimbursement and patient and societal costs for each scenario as well as costs and income summarized across all scenarios extrapolated to a physician's entire patient panel. The learners are asked to explain these findings and may change treatment options and other variables such as panel size and case mix to conduct sensitivity analyses in real time. Evaluations completed in 2003 by 68 (94%) CHESS resident and faculty participants at 19 U.S. residency programs preferred CHESS to a traditional lecture-and-discussion format to learn about medical decision making, physician reimbursement, patient costs, and societal costs. Ninety-eight percent reported increased knowledge of health economics after viewing the simulation. CHESS demonstrates the potential of computer simulation to teach health economics and other key elements of practice- and systems-based competencies.     

Apoptosis of ileal Peyer's patch B cells is increased by glucocorticoids or anti-immunoglobulin antibodies

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (<5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24h, 60 ± 1 % of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2–2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 ± 6%, compared with 2.4 ± 0.1 % in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 ± 2 % and 75 ± 3 %, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca²⁺, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.     

The gene for prolactin-inducible protein (PIP), uniquely expressed in exocrine organs,maps to chromosome 7

The hormonally responsive prolactin-inducible protein (PIP), gene is expressed in benign and malignant breast tumor tissues and in such normal exocrine organs as sweat, salivary, and lacrimal glands. In this communication we report the regional chromosome localization of the PIPgene locus to chromosome 7 by Southern hybridization to DNA from human-hamster somatic cell hybrids, and to 7q32–36 by in situ hybridization.     

Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems

High levels ofl-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-K^r, derived from the more severely affected colony, was analyzed in detail. In starved cells theV _max of lysine uptake in CH-K^r was half that of CHO whileK _m was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y⁺, was significantly lower in CH-K^r. However, transport and pools of other amino acids, which are not substrates of y⁺, were also reduced in CH-K^r, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-K^r is pleiotropic, affecting some general aspects of amino acid transport.     

The search for a marsupial XIC reveals a break with vertebrate synteny

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the ‘X-inactivation center’ (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.