Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.     

Sex differentiation and germ cell production in chimeric pigs produced by inner cell mass injection into blastocysts

This study aimed at collecting background knowledge for chimeric pig production. We analyzed the genetic sex of the chimeric pigs in relation to phenotypic sex as well as to functional germ cell formation. Chimeric pigs were produced by injecting Day 6 or Day 7 inner cell mass (ICM) cells into Day 6 blastocysts. Approximately 20% of the piglets born from the injected blastocysts showed overt coat color chimerism regardless of the embryonic stage of donor cells. The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICM cells, respectively, showing an obvious bias toward males. When XX donor cells were injected into XY blastocysts at the same embryonic stage, the phenotypic sex of the resulting chimera was male with no germ-line cells formed from the donor cell lineage. On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was male, and germ-line cells were derived only from the donor cells. The combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when it appeared in coat color. When the embryonic stage of the donor was advanced by 1 day in the XY-XY combination, 100% of the germ-line cells of the chimeras were derived from the donor cell lineage. These data showed that characteristics of sex differentiation and germ cell formation in chimeric pigs are similar to those in chimeric mice.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

Plastid genomes reveal recurrent formation of allopolyploid Fragaria

Premise of the Study

Recurrent formation of polyploid taxa is a common observation in many plant groups. Haploid, cytoplasmic genomes like the plastid genome can be used to overcome the problem of homeologous genes and recombination in polyploid taxa. Fragaria (Rosaceae) contains several octo‐ and decaploid species. We use plastome sequences to infer the plastid ancestry of these taxa with special focus on the decaploid Fragaria cascadensis.

Methods

We used genome skimming of 96 polyploid Fragaria samples on a single Illumina HiSeq 3000 lane to obtain whole plastome sequences. These sequences were used for phylogenetic reconstructions and dating analyses. Ploidy of all samples was inferred with flow cytometry, and plastid inheritance was examined in a controlled cross of F. cascadensis.

Key Results

The plastid genome phylogeny shows that only the octoploid F. chiloensis is monophyletic, all other polyploid taxa were supported to be para‐ or polyphyletic. The decaploid Fragaria cascadensis has biparental plastid inheritance and four different plastid donors. Diversification of the F. cascadensis clades occurred in the last 230,000 years. The southern part of its distribution range harbors considerably higher genetic diversity, suggestive of a potential refugium.

Conclusions

Fragaria cascadensis had at least four independent origins from parents with different plastomes. In contrast, para‐ and polyphyletic taxa of the octoploid Fragaria species are best explained by incomplete lineage sorting and/or hybridization. Biogeographic patterns in F. cascadensis are probably a result of range shift during the last glacial maximum.     

Elastic properties of cancellous bone: measurement by an ultrasonic technique

The high degree of porosity of cancellous bone makes elastic property measurement difficult by traditional mechanical testing methods. An ultrasonic technique is described with which mechanical properties of anisotropic, rigid, porous materials, such as cancellous bone, can be measured. The technique utilizes unique piezoelectric transducers operated in a continuous wave mode at a frequency of approximately 50 kHz. Both longitudinal and shear waves can be propagated and received with the transducers allowing both Young's moduli and shear moduli to be determined with the technique. A comparison between moduli measured with the ultrasonic technique and moduli measured with traditional mechanical testing shows the new method to be quite accurate in elastic property determination, (r2 = 0.935, Emech = 1.00E1dt + 23.3 MPa) (r2 = 0.656, Gmech = 1.08 Gult--3.3MPa).     

Mitogen-stimulated phospholipid synthesis in normal and immune-deficient human B cells

Eight patients with common variable panhypogammaglobulinemia were shown in the in vitro Ig biosynthesis assay to have defective B cell responses to pokeweed mitogen (PWM). Phospholipid synthesis was assessed in the B cell plus monocyte fraction (MB) and irradiated T cells (T*) of patients and paired normal controls. Cell populations were studied separately and in the four possible combinations (1:1), with and without PWM, to reveal the effect of cell interactions. At 16 to 20 hr the mean stimulation index (SI) +/- standard error for MB cells alone was 1.01 +/- 0.02 for eight patients and 0.99 +/- 0.02 for the paired normals; the T* cell SI was 1.25 +/- 0.04 for patients and 1.28 +/- 0.05 for normals. Combinations of normal MB cells with normal T* cells showed significantly higher SI when compared with the combinations of normal MB cells with patient T* cells (p less than 0.005). However, the combination of patient MB cells with patient T* cells and the combination of patient MB cells with normal T* cells were not significantly different in SI (0.05 less than p less than 0.1). Isolation of patient and normal B cells, T* cells, and monocytes after the choline pulse showed that patient B cells gave a higher SI with normal T* help than with patient T* help. Of greatest interest is the finding that patient B cells that were defective in PWM-stimulated Ig production nevertheless showed a phospholipid synthesis response to PWM in the normal range, suggesting that the maturation defect in these B cells occurs later than the phospholipid synthesis acceleration step, or on a different pathway.     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.