The full length cloning of a novel porcine gene CFL2b and its influence on the MyHC expression.

Porcine CFL2b gene play an important role in the muscle development and myofibrillar formation in pig. To explore whether CFL2b expression affects muscle fiber trait, the porcine CFL2b full-length cDNA was amplified using homology based cDNA cloning and SMART RACE. Then the full length cDNA of porcine CFL2b was inserted into pEGFP-N1 and transfected into C2C12 cells. The cells stably expressing CFL2b were selected by G418. We examined the expression of MyHC 2x, MyHC 2b and MyHC1/slow in C2C12 cells stably expressing CFL2b. The results showed that the level of MyHC 2x and MyHC 2b mRNA were dramatically increased compared with control cells, while the level of MyHC1/slow mRNA is not changed. To identify the transcription events of CFL2b, the porcine CFL2b mRNA was detected by Northern blotting, two transcripts, long transcript (3,012 bp) and short transcript (1,466 bp) were found in porcine skeletal muscles. The nucleotide sequence of CFL2b shares 88.1 and 74.9% homology with the CFL2b gene in human and mouse. The deduced amino acid sequence of CFL2b (166 amino acids) in pig shares 100, 99.1% identity with the CFL2b in human and mouse, respectively. Taken together, our research revealed that porcine CFL2b may be involved in the regulation muscle fiber trait by affecting the expression of MyHC.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures.

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.

Platelet-derived growth factor enhances ex vivo expansion of megakaryocytic progenitors from human cord blood.

Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34(+) cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1 beta (IL-1 beta), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and Flt-3L. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61(+) population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N--32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1 beta, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34(+) cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications.

Plasma PTH and PTH-rP levels and clodronate therapy in cancer patients with hypercalcemia.

To investigate the roles of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTH-rP) in hypercalcemic patients with various malignancies, we measured the plasma levels of intact PTH and intact PTH-rP in five hypercalcemic patients with hematologic malignancies and 29 hypercalcemic patients with solid cancers. Ten eucalcemic cancer patients with either hepatocellular carcinoma or squamous cancer of the lung served as controls. The results showed a suppressed PTH level in all of the 34 hypercalcemic cancer patients. The PTH-rP levels were within normal limits (< 2.5 pg/mL) for all of the eucalcemic cancer controls, all of the five hematologic cancer patients with hypercalcemia, and only two out of the 29 hypercalcemic patients with solid cancers. The PTH-rP level was elevated in nearly all (27/29) hypercalcemic patients with or without bony metastasis. There was a good correlation between the corrected total serum calcium level and the natural log of PTH-rP level in 39 patients (10 eucalcemic and 29 hypercalcemic) with solid cancers (r = 0.75, p < 0.001). These results suggest that PTH-rP plays an important role in the hypercalcemia of patients with solid cancers, but this was not the case in our patients with hematologic malignancies. True ectopic secretion of PTH is unlikely in most of these patients. The mean serum total calcium levels of 17 hypercalcemic patients (13 solid cancers, four hematologic malignancies) dropped significantly after the third day of treatment with a new-generation bisphosphonate-clodronate product. This treatment consisted of 300 mg daily intravenous infusion for seven days, followed by oral administration (800 mg, bid) for the following three weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial azurophilic granule deficiency. Report of 2 cases with ultrastructural and light-and-electron-microscopic cytochemical observation.

The neutrophils in 2 patients with atopic disease were studied. Ultrastructurally, a number of abnormal azurophilic granules (AG) with low electron-density (Case 1) and secondary lysosomes with "myelinoid membranes" figures (Case 2) were found. Myeloperoxidase (MPO) and acid phosphatase activity were detected by light cytochemical techniques, and showed significantly low values. The deficiency of MPO and abnormal distribution of AG were also demonstrated by electron microscopic cytochemical technique. The neutrophils from the parents revealed changes similar to the patients. This study suggests that since genetic partial deficiency of neutrophil AG enzymes existed, the phagocytosed substances were only partially degraded, leading to accumulation of substances with antigenicity, and became trigger event of atopic disease.

Identification of autoantigen recognized by autoimmune ophthalmopathy sera with immunoblotting correlated with orbital computed tomography.

We have demonstrated that there is an antibody related to extraocular muscle enlargement in autoimmune ophthalmopathy (Graves' ophthalmopathy, thyroid-associated correlated with orbital computed tomography (CT). This study was designed to identify the autoantigen and to determine whether there are common antigens among the extraocular muscle, the lacrimal gland, and the thyroid. We prepared a 100,000g sediment fraction of porcine extraocular muscle, lacrimal gland, thyroid, and human thyroid, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with sera from patients with Graves' disease, with or without ophthalmopathy, classified by symptoms and signs combined with orbital CT and normal controls. The results showed there was an approximately 55-kDa protein band which was recognized by the sera in 32.1% (9/28) of patients with autoimmune ophthalmopathy and in 47.3% (9/19) of patients with extraocular muscle enlargement demonstrated by orbital CT. It was significantly higher than the positive rates in patients without autoimmune ophthalmopathy and normal controls (15.8 and 11.1%, respectively, P < 0.025). However, there was no common antigen among the extraocular muscles, the lacrimal gland, and the thyroid. To further confirm this eye muscle-specific antigen, the approximately 55-kDa protein band was cut and solubilized from the nitrocellulose paper after SDS-PAGE, and electrophoretically transferred and used as an antigen in enzyme-linked immunosorbent assay. The absorbance was significantly higher in patients with autoimmune ophthalmopathy than patients without ophthalmopathy (P < 0.005), and normal controls (P < 0.01). Our findings suggest that an approximately 55-kDa protein may be a possible antigen in the eye muscle related to autoimmune ophthalmopathy.

Bedtime intermediate-acting insulin in the treatment of secondary failure to oral hypoglycemic agents.

Low-dose bedtime insulin therapy in combination with oral hypoglycemic agents (OHAs) has become an alternative treatment for NIDDM subjects with secondary failure to OHA. To assess its clinical efficacy, patient compliance, and its possible side effects, 33 patients with secondary OHA failure were recruited in this study. All of the subjects had experienced poor glycemic control for at least six months on their maximal OHAs before the institution of the bedtime insulin injection. Monotard HM (human insulin zinc suspension) was given at an initial dose of 0.15-0.2 U/kg body weight and was adjusted thereafter. As a whole, low-dose bedtime insulin with OHAs improved glycemic control. According to the clinical response, 10 patients (30.3%) were graded as responders, 12 (36.4%) were partial responders, 10 (30.3%) were non-responders, and one (3%) discontinued insulin therapy. There was no difference in demographic features among these three groups of patients. During this period, eight (25%) cases experienced mild hypoglycemic symptoms. In conclusion, combination of OHAs with a low-dose bedtime insulin injection is an alternative therapy for NIDDM patients with OHA failure.