Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice.

A PCR-based assay has been devised for the detection and semiquantitation of cells originating from a few donor hematopoietic stem cells (HSCs) in a background of recipient cells. Upon sequencing a segment of murine Y chromosome contained in the plasmid pY2, oligonucleotide primers were designed for specific amplification of the Y chromosome-restricted segment. The HSCs were isolated from the bone marrow of mice on day 4 following a single i.v. injection of 5-fluorouracil and were readily distinguished from other bone marrow elements by the characteristics of low density, absence of lineage-specific surface markers, lack of expression of transferrin receptor, and a high expression of major histocompatibility complex class I antigen. Injection of as few as four such HSCs was shown to produce donor-derived cells (including lymphoid cells) for at least 8 months after transplantation into syngeneic female recipients. Retransplantation, employing 10(6) bone marrow cells from the initial recipients, also yielded clear evidence of repopulation with detectable levels of male donor cells. On statistical grounds, it is clear that long-term repopulation in vivo may result from even a single HSC having the characteristics defined herein.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Ciliary muscle in avian is derived from mesenchymal and epithelial cells

It has long been maintained that the ciliary muscle derives from mesenchymal cells. The embryonic development of the avian ciliary muscle was studied in chick embryos from stage 25 HH to the time of hatching. Serial sections of the eye were stained routinely or immunocytochemically using the monoclonal antibody 13F4, which recognizes a cytoplasmic antigen specific for all types of muscle cells. We found that the mesenchymal immunoreactive cells, at stage 37 HH, are arranged in two distinct orientations forming the anterior and posterior portions of the ciliary muscle. At stages 38 and 39 HH the pigmented epithelium contained 13F4 positive cells, which detach from the epithelium and apparently migrate into stroma. These epithelial cells may differentiate into muscle cells. Within this same time period a progressive accumulation of myoblasts was detected between the pigmented epithelium and the ciliary muscle. Some myoblasts containing melanin were also observed. At stage 40 HH the internal portion of the ciliary muscle was visible. These findings indicate that the immunopositive epithelial cells participate in the formation of the internal portion of the muscle. We conclude that the ciliary muscle derives not only from the mesenchymal cells but also from the pigmented epithelium.

The Christopher Foster family history, 1603-1953; a history of the descendants of Christopher Foster and Frances Stevens, his wife, who came to America in 1635 and lived in Southampton, Long Island.

Includes indexes.

CMRF-44 antibody-mediated depletion of activated human dendridic cells: A potential means for improving allograft survival

Background. Activated dendritic cells (DC) initiate immune responses by presenting antigen, including alloantigen from tissue grafts, to T lymphocytes. The potential to deplete or inactivate differentiated-activated DC during allogeneic transplantation represents a new approach to immunosuppression. Methods. The authors investigated the potential of the monoclonal antibody CMRF-44, which has specificity for a DC-associated differentiation-activation antigen, to induce complement-mediated lysis of activated human DC. Peripheral blood mononuclear cells (PBMC), or purified DC preparations, were cultured overnight to activate endogenous DC, resulting in the expression of CNW-44 antigen and CD83. These were then treated with CMRF-44 and complement. Depletion of activated DC was monitored by flow cytometry. Results. Eighty-nine percent of activated (CD83(+)) DC in cultured PBMC were depleted by treatment with CMRF-44 and autologous serum (AS) (complement source; mean percentage of CD83(+)-CD14(-)-CD19(-) cells=0.06%; cf 0.50% for heat-inactivated AS controls, P

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal β-cells

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro:simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swift recognition and removal of apoptotic cells is important for normal tissue homeostasis and failure in the underlying clearance mechanisms has pathological consequences associated with inflammatory and auto-immune diseases. Cell cultures in vitro usually lack the capacity for removal of non-viable cells because of the absence of phagocytes and, as such, fail to emulate the healthy in vivo micro-environment from which dead cells are absent. While a key objective in cell culture is to maintain viability at maximal levels, cell death is unavoidable and non-viable cells frequently contaminate cultures in significant numbers. Here we show that the presence of apoptotic cells in monoclonal antibody-producing hybridoma cultures has markedly detrimental effects on antibody productivity. Removal of apoptotic hybridoma cells by macrophages at the time of seeding resulted in 100% improved antibody productivity that was, surprisingly to us, most pronounced late on in the cultures. Furthermore, we were able to recapitulate this effect using novel super-paramagnetic Dead-Cert Nanoparticles to remove non-viable cells simply and effectively at culture seeding. These results (1) provide direct evidence that apoptotic cells have a profound influence on their non-phagocytic neighbors in culture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy for improving antibody manufacture in vitro.

Acute and long-term effects of peroxisome proliferator-activated receptor-γ activation on the function and insulin secretory responsiveness of clonal beta-cells

Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 M) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 M) enhanced (p

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

The hormone glucagon-like peptide-1(7-36)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the E-amino group in the side chain of the LyS26 residue and to combine this modification with substitutions of the Ala 8 residue, namely Val or amino-butyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal) 26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys-(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal β-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal) 26]GLP-1 and [Val8,Lys(pal) 26]GLP-1 did not demonstrate acute glucose-lowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability. Copyright © by Walter de Gruyter.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (β)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.

Cross-Cultural Cuisine: Long-Term Trend or Short-Lived Fad

Defining a consumer interest as a long-term trend or short-lived fad has significant implicatiosn for restauranteurs' management decisions. The terms "trend" and "fad" can be operationally defined for the food service industry. The authors examine today's popular cross-cultural cuisine to determine its trend or fad status and discuss the catalysts that promoted or hindered its trend/fad status, as well as implications for the food service industry.