Xenogeneic bone marrow transplantation: II. Porcine-specific growth factors enhance porcine bone marrow engraftment in an in vitro primate microenvironment

Abstract: Establishment of mixed bone marrow chimerism in pig-to-primate transplantation, as a means of inducing specific immune tolerance, will require that both immune and nonimmune barriers be overcome. As a preliminary step in evaluating nonimmune barriers in this system, we have developed an in vitro model of engraftment in which long-term culture of porcine bone marrow-derived hematopoietic cells is supported on preformed primate bone marrow stromal layers. In the absence of cytokine supplementation, primate stromal cells were unable to support long-term porcine hematopoiesis in these cultures. Supplementation with porcine Steel Factor was required for long-term maintenance of hematopoietic progenitor cell content and total hematopoietic activity. Addition of porcine IL-3, in combination with porcine Steel Factor, increased long-term progenitor cell content and hematopoietic activity on primate stroma to levels comparable to that obtained in cultures on porcine stroma. The combination of porcine GM-CSF and Steel Factor increased progenitor cell content and hematopoietic activity early in the cultures, but had little effect in long-term cultures. The Steel Factor and IL-3 combination was species-specific in its action in these cultures, as the corresponding human cytokines were unable to effectively support long-term porcine hematopoiesis. Likewise, the combination of porcine cytokines had only minimal effects on long-term bone marrow culture of primate CD34+ cells I on primate stroma.     

Missense mutations in codon 225 of ornithine transcarbamylase (OTC) result in decreased amounts of OTC protein: A hypothesis on the molecular mechanism of the OTC deficiency

Mutations P225L and P225R were identified in codon 225 of the gene for ornithine transcarbamylase (OTC) in two patients with the neonatal form of OTC deficiency. The mutations occur at a CpG dinucleotide and eliminate a unique MspI restriction site in exon 7 of the OTC gene. They do not alter existing splice sites or create new sites, as judged from the nucleotide sequence. Both mutations are associated with undetectable levels of OTC antigen in liver homogenates, and with either complete lack of OTC activity (P225R mutation) or very small residual activity (0.15% of normal in the P225L mutation). The residual activity observed with P225L exhibits normal pH dependence, little or no increases in the Km values for ornithine and carbamoyl phosphate and normal stability at either 37°C or, in the presence of 0.66 mol/L urea, at 0°C. The latter conditions were used to examine whether the P225L mutation favours dissociation of the active OTC trimer. Given the normal stability and lack of tendency to dissociation of the mutant enzyme, it appears likely that the dramatic reduction in the level of OTC protein is due to inefficient conversion of the mutant OTC precursor polypeptide (pOTC) into the correctly localized, appropriately folded, mature enzyme trimer, suggesting degradation of pOTC in transit to the mitochondria.     

Inadequate dietary calcium and vitamin D intakes in renal-transplant recipients in Ireland.

OBJECTIVE: To quantify the dietary calcium and vitamin D intake in adult renal-transplant recipients attending at a large teaching hospital in Ireland for follow-up. SETTING: Outpatient renal-transplant follow-up clinic. SUBJECTS: Fifty-nine adult renal transplant recipients (58% male) with a mean age of 46 years, a median transplant duration of 6 years, and a mean estimated glomerular filtration rate (eGFR) of 50 mL/min per 1.73 m2. Fifty-three percent were at National Kidney Foundation stage 3 chronic kidney disease, and 14% had stage 4 chronic kidney disease. INTERVENTION: This cross-sectional, observational study used a tailored food frequency questionnaire specific for calcium and vitamin D intake in Irish adults, which was completed during a face-to-face interview with each subject. MAIN OUTCOME MEASURE: The main outcome measure was the average daily dietary and supplemented calcium and vitamin D intake. RESULTS: The median interquartile range (IQR) dietary calcium intake was 820 mg/day (range, 576-1,177 mg/day), and was similar in men and women (recommended intake > or = 1,000 mg/day in adult men and nonmenopausal adult women, > or = 1,500 mg/day in menopausal women). Five participants received calcium supplementation. Overall, 59% of men and 64% of women had total calcium intakes below the recommended amounts. The median IQR estimated dietary vitamin D intake was 5.2 microg/day (range, 2.4-6.4 microg/day) in women, and 4.6 microg/day (range, 2.2-6.6 microg/day) in men (recommended intake, > or = 10 microg/day). Six subjects received vitamin D supplementation. Total vitamin D intakes were suboptimal in 91% of men and 87% of women. Dietary calcium and vitamin D intakes significantly correlated with each other, but neither was significantly related to eGFR category, and was similarly low in both presumed menopausal women and in the initial year posttransplantation. CONCLUSION: These findings suggest that dietary and total calcium and vitamin D intakes in adult renal-transplant patients are in many cases inadequate.     

Incidence of malnutrition in adult patients at Groote Schuur Hospital, 1984

In order to determine nutritional status and the incidence of malnutrition 700 consecutive adult medical (395) and surgical (305) patients admitted to Groote Schuur Hospital between January and November 1984 were studied. Significant depletion in body weight was observed in 20% of all patients, fat depletion in 30% and muscle depletion in 15%; 40% were hypo-albuminaemia. Intergroup analysis demonstrated that malnutrition was more common in medical than surgical patients, and in non-white patients 38% of coloureds and 42% of blacks were fat depleted. Since malnutrition is associated with enhanced mortality and morbidity rates from any disease state, the results indicate that, particularly in non-white patients, nutritional support should be given greater emphasis in overall patient care.     

Impact of a region-wide approach to improving systems for heart attack care: the West Midlands thrombolysis project.

OBJECTIVE: To describe changes in delay to administration of thrombolytic therapy associated with a region-wide audit. DESIGN: Observational study of patients admitted with suspected myocardial infarction (MI) based on continuous audit. SUBJECTS: 18877 patients admitted to 23 hospitals with suspected MI between April 1995 and March 1998. RESULTS: Of 11232 patients with a discharge diagnosis of definite MI, 8802 (46.6%) received thrombolytic therapy during hospitalisation, with 5155 patients eligible for treatment on admission to hospital on the basis of established indications. Call-to-needle time for those eligible for treatment on admission fell from median 105 min in the first year of the project to 85 min in year 3 (P<0.001), and door-to-needle time fell from 45 to 35 min (P<0.001). Forty percent of eligible patients were treated within the then current national standard of 90 min from time of call for help, with nearly 49% in the final year and 20% being treated within the new national standard of 60 min, by the third year. CONCLUSION: The proportion of eligible patients receiving thrombolysis within 1 h of the call for help doubled during the 3-year project but the majority of patients still wait longer than 60-min 'call-to-needle'. New systems to reduce delays to administration of thrombolysis to within 60 min of call for help are required, including consideration of pre-hospital treatment.     

RANK signaling is not required for TNFalpha-mediated increase in CD11(hi) osteoclast precursors but is essential for mature osteoclast formation in TNFalpha-mediated inflammatory arthritis.

To address the controversy of whether TNFalpha can compensate for RANKL in osteoclastogenesis in vivo, we used a TNFalpha-induced animal model of inflammatory arthritis and blocked RANKL/RANK signaling. TNFalpha increased osteoclast precursors available for RANK-dependent osteoclastogenesis. RANK signaling is not required for the TNFalpha-stimulated increase in CD11b(hi) osteoclast precursors but is essential for mature osteoclast formation. INTRODUCTION: Although critical roles of TNFalpha in inflammatory arthritis and RANKL in bone resorption have been firmly established, a central controversy remains about the extent to which TNFalpha can compensate for RANKL during osteoclastogenesis and the stage at which RANK signaling is required for osteoclastogenesis. Here, we used the human TNFalpha transgenic mouse model (TNF-Tg) of erosive arthritis to determine if there are both RANK-dependent and -independent stages of osteoclastogenesis in TNFalpha-induced erosive arthritis. MATERIALS AND METHODS: Osteoclastogenesis and osteoclast precursor (OCP) frequency were analyzed using histology, fluorescence-activated cell sorting (FACS), and cell culture from (1) TNF-Tg mice treated with the RANKL antagonist, RANK:Fc, or (2) TNF-Tg X RANK -/- mice generated by crossing TNF-Tg mice with RANK-/- mice. RESULTS: Treatment of TNF-Tg mice, which have increased OCPs in their spleens, with RANK:Fc dramatically reduced osteoclast numbers on the surface of their arthritic joints and within their bones, but did not decrease CD11b(hi) OCP numbers in their spleens. Long-term RANK:Fc administration alleviated joint erosion. Furthermore, TNF-Tg x RANK -/- mice had severe osteopetrosis, no osteoclasts, and no joint erosion, but increased CD11b(hi) precursor numbers that failed to form mature osteoclasts in vitro. CONCLUSION: RANK signaling is essential for mature osteoclast formation in TNFalpha-mediated inflammatory arthritis but not for the TNFalpha-induced increase in CD11b(hi) OCP that subsequently can differentiate into osteoclasts in inflamed joints.