Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n?=?16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24?h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3?K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2?h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.     

Chromosome studies in newborn infants following exchange transfusion using countersexual blood]

The chromosomes of 6 (5 females, 1 male) newborns have been examined before and after exchange transfusion. The highest percentage of donor lymphocytes has been found immediately after the exchange transfusion (9%, range 5 to 16%). The percentage of donor lymphocytes had decreased to 4% after 24 hours and to 1% after 3 weeks. There was no hint of a clastogenic effect of phototherapy and exchange transfusion.     

Determining blood pressure in infants: use of the flush technique

The flush technique that recently has come into use is probably the most satisfactory method available for measuring blood pressures in infants and is of great aid in diagnosis of diseases associated with hypertension or hypotension. The method is practical and highly accurate. Readings obtained by this technique are not influenced by the width of the cuff, are usually higher at the wrist than at the ankle, and approximate the mean rather than the systolic blood pressure.     

Copper,zinc and iron levels in premature infants following red blood cell transfusion

This study aimed to investigate effect of erythrocyte suspension (ES) transfusion on Cu, Zn, and Fe levels. It was conducted on 53 premature infants who were admitted to Hacettepe Hospital and received EST for first time. Blood samples were drawn before and 96 h after ES transfusion to determine Cu, Zn, and Fe levels in plasma and/or erythrocytes. The mean plasma Cu levels were 99 ± 3 μg/dl and 113 ± 3 μg/dl; Zn levels were 105 ± 2 μg/dl and 115 ± 23 μg/dl; mean plasma Fe level was 58.1 ± 19.4 and 75.2 ± 25.4 μg/dl and mean erythrocyte Fe level was 4182 ± 2314 μg/ml and 7009 ± 5228 μg/ml, before and after ES transfusion. The differences between before and after ES transfusion in Cu, Zn and Fe levels were significant. Correlation between plasma and erythrocyte Fe levels was significant both before and after ES transfusion. Though Fe overload is a major cause of morbidity/mortality after ES transfusion, alterations in trace elements should also be considered when transfusing blood to infants and children.     

Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin

Physicochemical and biological properties of recombinant human erythropoietin (rhEPO) were compared with human urinary erythropoietin (uEPO). uEPO and rhEPO were purified to apparent homogeneity from the urine of patients with aplastic anemia and from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human EPO, respectively. The microheterogeneous nature of both factors, observed on isoelectric focusing, is derived from the difference of the number of terminal sialic acid residues bound to the carbohydrate chains of the EPO molecule. The primary structure of rhEPO, consisting of 165 amino acid residues, was determined, and the C-terminal arginine predicted from the cDNA sequence was confirmed to be missing, as described previously (Recny et al. (1987) J. Biol. Chem. 262, 17156). Three N-glycosylation and one O-glycosylation sites of both factors were determined as Asn24, Asn38, and Asn83 and Ser126, respectively. Two disulfide linkages are located between Cys7 and Cys161, and between Cys29 and Cys33, in both EPOs. Hematogenic potencies of rhEPO and uEPO compared in normal and in partially nephrectomized rats were approximately the same. Both factors also stimulated the colony formation of CFU-E, BFU-E, and CFU-Meg in a dose-dependent manner. From these results, it is concluded that rhEPO produced in CHO cells transfected with cDNA clone for human EPO is indistinguishable from uEPO physicochemically and biologically, and is valuable for further research and for clinical use.     

The effect of recombinant erythropoietin on murine megakaryocyte colony formation

We investigated the effect of a recombinant human erythropoietin preparation (recombinant Epo) on murine megakaryocyte (MK) colony formation in serum-free and serum-containing culture systems, in order to study the relationship between Epo and megakaryopoiesis. Pokeweed mitogen spleen-conditioned medium (PWM-SCM), a standard source of MK colony stimulator, dose-dependently stimulated MK colony formation in the two culture systems. The plating efficiency of serum-free cultures was almost equal to that of cultures containing serum. Recombinant Epo also dose dependently stimulated MK colony formation in serum-containing cultures. However, in serum-free cultures recombinant Epo alone did not stimulate the growth of MK colonies; with the addition of fetal calf serum (FCS) to the serum-free cultures, recombinant Epo induced the growth of MK colonies. Furthermore, recombinant Epo enhanced MK colony formation through the stimulation of PWM-SCM or murine interleukin 3 (IL-3) in serum-free cultures. Our data show that Epo can act as a stimulator of megakaryopoiesis in collaboration with a factor in serum, or with an MK colony stimulator such as IL-3.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.