Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization

Summary The present study utilized biochemical and immuno-and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific forN-acetyl-d-glucosamine,N-acetylneuraminic acid, andN-acetyl-d-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates.     

Controlling mechanism of ATP regeneration by glycolytic pathway in a yeast: Hansenula jadinii

Summary The regulatory mechanism of ATP regeneration by the glycolytic pathway in Hansenula jadinii cells was investigated by analyzing the initial stage of CDP-choline fermentation. As a result, the on-off of ATP regeneration was found to be determined by the ATP concentration overcoming the inhibitory effect of phosphate buffer on hexokinase activity. The concentration of ATP at the initial stage of fermentation was greatly influenced by the kinds and amounts of glycogen in cells. Based on these results, the regulatory mechanism of ATP regeneration by the glycolytic pathway is discussed in detail.     

Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model

Background

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.

Methods

We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10⁶ MSCs were implanted into each ischemic limb.

Results

Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N^G-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.

Conclusions

These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.     

Application of Plasmid to ATP Production by E. coli

A variety of immunomodulators were synthesized by combining biologically active derivatives of 1-thio-muramoyl dipeptide with 4-O-phosphono-d-glucosamine derivatives related to bacterial lipid A, and using the (succinoylamino)undecanoyl group as a spacer. Their immunoadjuvant activities in guinea-pigs were examined.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Dynamic Changes of CD44 Expression from Progenitors to Subpopulations of Astrocytes and Neurons in Developing Cerebellum

We previously reported that CD44-positive cells were candidates for astrocyte precursor cells in the developing cerebellum, because cells expressing high levels of CD44 selected by fluorescence-activated cell sorting (FACS) gave rise only to astrocytes in vitro. However, whether CD44 is a specific cell marker for cerebellar astrocyte precursor cells in vivo is unknown. In this study, we used immunohistochemistry, in situ hybridization, and FACS to analyze the spatial and temporal expression of CD44 and characterize the CD44-positive cells in the mouse cerebellum during development. CD44 expression was observed not only in astrocyte precursor cells but also in neural stem cells and oligodendrocyte precursor cells (OPCs) at early postnatal stages. CD44 expression in OPCs was shut off during oligodendrocyte differentiation. Interestingly, during development, CD44 expression was limited specifically to Bergmann glia and fibrous astrocytes among three types of astrocytes in cerebellum, and expression in astrocytes was shut off during postnatal development. CD44 expression was also detected in developing Purkinje and granule neurons but was limited to granule neurons in the adult cerebellum. Thus, at early developmental stages of the cerebellum, CD44 was widely expressed in several types of precursor cells, and over the course of development, the expression of CD44 became restricted to granule neurons in the adult.