The trabecular bone score is associated with bone mineral density, markers of bone turnover and prevalent fracture in patients with end stage kidney disease.

Fracture risk increases in end-stage kidney disease (ESKD), but bone mineral density (BMD) measurement is less predictive of risk than in the general population. In this study of patients with ESKD, a lower trabecular bone score (TBS), indicative of microarchitectural deterioration, was associated with higher bone turnover markers and prevalent non-vertebral fracture. INTRODUCTION: Declining renal function carries increased fracture risks, but BMD is less predictive of fracture for dialysis patients than the general population. The TBS, obtained from lumbar spine dual-energy X-ray absorptiometry (DXA) images, provides information on microarchitectural integrity not captured by BMD. The aim of this study was to assess associations of the TBS to clinical, DXA, radiological, and laboratory measures in patients with ESKD undergoing kidney and simultaneous pancreas kidney (SPK) transplantation. METHODS: A total of 147 patients with ESKD underwent pre-transplant laboratory testing, DXA, lateral spine X-ray, and structured history within 4 weeks of transplantation. Associations of the TBS to demographic data, prevalent fracture, BMD, and laboratory variables were assessed. RESULTS: Of 147 patients (60% male, mean age 48 ± 13 years), 36% had diabetes mellitus (DM) and 54 patients had fractures: 21 prevalent vertebral fractures only, 22 non-vertebral fractures only, and 11 had both. The mean TBS (1.345 ± 0.125) was lower in patients undergoing SPK than kidney-only transplants (1.292 vs. 1.364, p = 0.001). The TBS correlated to spine and total hip BMD, body mass index and inversely to parathyroid hormone, alkaline phosphatase and procollagen 1 N-propeptide. By multivariable logistic regression, lower TBS was significantly associated to prior non-vertebral fracture (p = 0.026). CONCLUSIONS: A lower TBS, suggestive of increased microarchitectural damage, was associated with type 1 DM, markers of higher bone turnover, and prevalent fracture. These data support the need for prospective studies to evaluate whether TBS inclusion improves fracture prediction in patients with ESKD.

Worsening of soft tissue dystrophic calcification in an osteoporotic patient treated with teriparatide.

Teriparatide, used for treatment of osteoporosis in patients at high risk of fracture risk, sometimes results in mild and transient hypercalcemia. There have been two recent reports of worsening dystrophic calcification in patients with autoimmune disorders following teriparatide treatment. We report a patient with severe osteoporosis and without a pre-existing autoimmune disorder, who developed symptomatic worsening of dystrophic calcification 4 months after teriparatide was initiated. Symptoms resolved within 1 week of teriparatide cessation.

Multidrug-resistant surgical site infections in a humanitarian surgery project.

The epidemiology of surgical site infections (SSIs) in surgical programmes in sub-Saharan Africa is inadequately described. We reviewed deep and organ-space SSIs occurring within a trauma project that had a high-quality microbiology partnership and active follow-up. Included patients underwent orthopaedic surgery in Teme Hospital (Port Harcourt, Nigeria) for trauma and subsequently developed a SSI requiring debridement and microbiological sampling. Data were collected from structured chart reviews and programmatic databases for 103 patients with suspected SSI [79% male, median age 30 years, interquartile range (IQR) 24-37]. SSIs were commonly detected post-discharge with 58% presenting >28 days after surgery. The most common pathogens were: Staphylococcus aureus (34%), Pseudomonas aeruginosa (16%) and Enterobacter cloacae (11%). Thirty-three (32%) of infections were caused by a multidrug-resistant (MDR) pathogen, including 15 patients with methicillin-resistant S. aureus. Antibiotics were initiated empirically for 43% of patients and after culture and sensitivity report in 32%. The median number of additional surgeries performed in patients with SSI was 5 (IQR 2-6), one patient died (1%), and amputation was performed or recommended in three patients. Our findings suggest the need for active long-term monitoring of SSIs, particularly those associated with MDR organisms, resulting in increased costs for readmission surgery and treatment with late-generation antibiotics.

A case report of disabling bone pain after long-term kidney transplantation.

A 77-year-old man, who received a renal transplant 13 years before for IgA glomerulonephritis, was referred after he developed bilateral mid-tibial aching pain that did not improve with simple analgesia. He had recently been changed from low-dose cyclosporine to tacrolimus, but the pain did not improve when this was reversed. He had a history of focal prostatic adenocarcinoma, cryptococcal lung infection, osteoporosis treated with alendronate for 2 years and multiple squamous cell carcinomas, including one requiring left neck dissection and radiotherapy. Upon physical examination, he had gouty tophi and marked bilateral tibial tenderness but had no other clinical findings. Laboratory investigations included an elevated intact parathyroid hormone value of 7.9 pmol/L (1.6 to 6.9), bone specific alkaline phosphatase of 22 µg/L (3.7 to 20.9), urinary deoxypyridinoline/creatinine ratio of 7.2 nmol/mmol (2.5 to 5.4) and C-reactive protein. Chest X-ray and tibial X-rays were normal, but there was marrow oedema and a prominent periosteal reaction on magnetic resonance imaging. A radionuclide bone scan showed increased symmetrical, linear uptake in both tibiae and the left femur, and uptake was also noted in both clinically asymptomatic humeri. Tibial bone biopsy disclosed small deposits of poorly differentiated metastatic cancer and a follow-up chest CT revealed a lung lesion. It was concluded that the bone pain and periostitis was caused by primary lung cancer with metastatic disease to bone, and an associated hypertrophic osteoarthropathy.

Risk factors for cholera transmission in Haiti during inter-peak periods: insights to improve current control strategies from two case-control studies.

Two community-based density case-control studies were performed to assess risk factors for cholera transmission during inter-peak periods of the ongoing epidemic in two Haitian urban settings, Gonaives and Carrefour. The strongest associations were: close contact with cholera patients (sharing latrines, visiting cholera patients, helping someone with diarrhoea), eating food from street vendors and washing dishes with untreated water. Protective factors were: drinking chlorinated water, receiving prevention messages via television, church or training sessions, and high household socioeconomic level. These findings suggest that, in addition to contaminated water, factors related to direct and indirect inter-human contact play an important role in cholera transmission during inter-peak periods. In order to reduce cholera transmission in Haiti intensive preventive measures such as hygiene promotion and awareness campaigns should be implemented during inter-peak lulls, when prevention activities are typically scaled back.

Cooking, Shopping, and Eating Behaviors of African American and Hispanic Families: Implications for a Culturally Appropriate Meal Kit Intervention.

Families with low incomes face barriers to preparing healthy meals, including decreased food access and limited time, and may turn to fast, low-quality, and inexpensive foods. Affordable and accessible meal kits may reduce these barriers. The objective of this study was to explore the cooking, eating, and shopping behaviors of African American (AA) and Hispanic participants living in the United States with low incomes and determine the knowledge of and preferences for a culturally appropriate meal kit intervention. Trained researchers conducted focus groups using a semi-structured questionnaire with AA and Hispanic food preparers with low incomes. Participant cooking, eating, and shopping behaviors and knowledge of and preferences for a culturally appropriate meal kit intervention were evaluated using thematic analysis. AA participants (n = 16) reported cooking on average 2 to 3 days per week and more often on weekends. Hispanic participants (n = 15) reported cooking 5 days per week and more often during the week. Both groups identified cost as the number one consideration when shopping. Most were unfamiliar with meal kits but indicated they would try an affordable meal kit. AA and Hispanic participants differed in their cooking, eating, and shopping behaviors but were equally interested in trying meal kits if affordable and culturally appropriate.

Association between 25-hydroxyvitamin D, somatic muscle weakness and falls risk in end-stage renal failure.

OBJECTIVE: Suboptimal levels of 25-hydroxyvitamin D (25OHD) are common in haemodialysis patients (Chronic Kidney disease-5D: CKD-5D) and may be associated with reduced muscle strength and increased falls risk. We tested the hypothesis that 25OHD levels may be independently associated with falls risk in CKD-5D. BACKGROUND: Supplementation with calcium and cholecalciferol reduces hip and other nonvertebral fractures in elderly individuals, and this effect may in part be attributable to reduction in falls frequency. The relationship between 25OHD and falls risk has not been investigated in CKD-5D. DESIGN AND PATIENTS: This is a cross-sectional study of 25 CKD-5D patients with predialysis 25OHD, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and intact parathyroid hormone (iPTH) measurement. Falls risk was assessed by quadriceps muscle strength, FallsScreen((c)) test (FST), Berg Balance Scale (BBS), timed 'up and go' (TUG) test, Modified Barthel Index (MBI) and Falls Efficacy Scale (FES). RESULTS: Mean age was 69.8 +/- 12.1 years, and median time on dialysis was 3.1 years. Median 25OHD level was 55.3 nmol/l (range 20.8-125.8 nmol/l). Muscle strength was significantly positively correlated with 25OHD (P = 0.024) but not with 1,25(OH)(2)D (P = 0.477) or PTH (P = 0.461). Statistically significant correlation between 25OHD levels and FST (P = 0.028) plus MBI (P = 0.0046) was noted. No significant correlation was detected between falls risk and 1,25(OH)(2)D or PTH. CONCLUSIONS: Suboptimal levels of 25OHD in CKD-5D are associated with reduced quadriceps muscle strength and increased falls risk. 25OHD may be more important than the active renal metabolite 1,25(OH)(2)D for muscle strength with implications for vitamin D choice and goals of supplementation. Further investigation is required to examine effectiveness of calciferol supplementation on the incidence of falls in CKD-5D.

Molecular epidemiology of erythropoietic protoporphyria in the U.K.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a cutaneous porphyria caused by mutations in the ferrochelatase (FECH) or, less frequently, the delta-aminolaevulinate synthase 2 (ALAS2) gene. Predictive genetic counselling requires accurate molecular diagnosis and knowledge of patterns of inheritance. OBJECTIVES: To investigate the molecular epidemiology of EPP in the U.K. METHODS: DNA samples from 191 unrelated patients resident in the U.K. were analysed for mutations in the FECH and ALAS2 genes and for the FECH IVS3-48 dimorphism. RESULTS: Mutations were identified in 179 (94%) patients. Most (169; 94%) had a FECH mutation on one allele and were classified as having pseudodominant EPP (psdEPP); seven (4%) patients had FECH mutations on both alleles (autosomal recessive EPP) and three (2%) patients had ALAS2 mutations (X-linked dominant protoporphyria). The FECH IVS3-48C allele was strongly associated with psdEPP and with the absence of mutations at the FECH or ALAS2 loci. Fifty-six FECH mutations were identified, 19 being previously unreported. Missense mutations were predominant in autosomal recessive EPP (82%) but not in psdEPP (32%). One mutation (c.314 + 2T>G) was present in 41 (24%) of EPP families, most of whom appeared to be descended from a common ancestor resident in the north of England. CONCLUSIONS: These data define the prevalence and molecular epidemiology of each type of EPP in the U.K.

Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M-null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M-null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.

Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits.

Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.

Requirement of heavy neurofilament subunit in the development of axons with large calibers.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H-null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H-null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content.

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.

The molecular genetics of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a syndrome in which accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver leads to acute photosensitivity and, in about 2% of patients, liver disease. More than 95% of unrelated patients have ferrochelatase (FECH) deficiency (MIM 177000) while about 2% have X-linked dominant protoporphyria (XLDPP) (MIM 300752) caused by gain-of-function mutations in the ALAS2 gene. Most FECH-deficient patients are compound heterozygotes for a hypomorphic allele (FECH IVS3-48C) and a deleterious FECH mutation that together lower FECH activity to around 30% of normal. The frequency of the IVS3-48C allele varies between populations, ranging from less than 1% to 45%. About 4% of unrelated FECH-deficient patients are compound heterozygotes or homozygotes for rare FECH mutations and have lower enzyme activities. Acquired somatic mutation of FECH secondary to myeloid disease may rarely cause EPP. The risk of liver disease is increased in XLDPP and in FECH-deficient patients who are hetero- or homoallelic for rare FECH mutations. Inherited FECH-deficient EPP is an autosomal recessive disorder with some families showing pseudodominant inheritance; the proportion of such families being determined by the population frequency of the IVS3-48C allele.

Serum 25-hydroxyvitamin D in erythropoietic protoporphyria.

BACKGROUND: Vitamin D, produced by the action of sunlight on skin, is an important hormone for calcium homeostasis and has been implicated as tumour-protective agent. Some previous studies of photosensitive patients who actively avoid sunlight have failed to show convincing evidence of vitamin D insufficiency. OBJECTIVES: The aim of this study was to characterize the vitamin D status of a large cohort of patients with erythropoietic protoporphyria (EPP). METHODS: U.K. patients with EPP were recruited prospectively and seen locally by a single study investigator. A blood sample was taken for vitamin D assay. All blood analyses were performed in the same laboratory. RESULTS: A cohort of 201 patients with known EPP was seen over a 7-month period between January and July. Thirty-four patients (17%) were deficient in vitamin D and 126 (63%) had insufficient vitamin D. Both insufficiency and deficiency were significantly associated with the total erythrocyte protoporphyrin concentration and inversely with the time in minutes to the onset of symptoms following sunlight exposure. CONCLUSIONS: This is the first report of significant levels of vitamin D deficiency and insufficiency in a large cohort of patients with a photodermatosis. Such individuals are at risk of associated adverse events. In future, clinicians should consider monitoring 25-hydroxyvitamin D levels and instigating oral supplementation or dietary advice if appropriate.

Iron and the liver: acute effects of iron-loading on hepatic heme synthesis of rats. Role of decreased activity of 5-aminolevulinate dehydrase.

Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed by addition of dithiothreitol, Zn2+ or chelators of Fe2+ and Fe3+. Addition of FeSO4, ferric citrate, or ferritin to homogenates of control liver had no effect of activity of ALA dehydrase. The decrease in activity of ALA dehydrase, caused by iron-dextran, was mirrored by a reciprocal increase in ALA synthase. Iron-dextran potentiated the induction of ALA synthase by allylisopropylacetamide. However, this potentiation could be dissociated from the decrease in ALA dehydrase caused by iron loading.

Calcimimetics for secondary hyperparathyroidism in chronic kidney disease patients.

BACKGROUND: Calcimimetic agents have recently been evaluated in the treatment of secondary hyperparathyroidism (SHPT) as add-on therapy to calcitriol and vitamin D analogues and dietary phosphate binders. OBJECTIVES: To evaluate the benefits and harms of calcimimetics for the prevention of secondary hyperparathyroid bone disease (including osteitis fibrosa cystica and adynamic bone disease) in dialysis patients with chronic kidney disease (CKD). SEARCH STRATEGY: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials and conference proceedings were searched for randomised controlled trials (RCTs) evaluating any calcimimetic against placebo or another agent in pre-dialysis or dialysis patients with CKD. SELECTION CRITERIA: We included all RCTs of any calcimimetic agent, cinacalcet HCl (AMG-073, Sensipar), NPS R-467 or NPS R-568 administered to patients with CKD for the treatment of SHPT. DATA COLLECTION AND ANALYSIS: Data were extracted on all relevant patient-centred and surrogate outcomes. Analysis was by a random effects model and results expressed as relative risk (RR) or weighted mean difference (MD) with 95% confidence intervals. MAIN RESULTS: Eight studies (1429 patients) were identified, which compared a calcimimetic agent plus standard therapy to placebo plus standard therapy. The end of treatment values of parathyroid hormone (pg/mL) (MD -290.79, 95% CI -360.23 to -221.34), serum calcium (mg/dL) (MD -0.85, 95% CI -1.14 to -0.56), serum phosphorus (mg/dL) (MD -0.29, 95% CI -0.50 to -0.08) and the calcium by phosphorus product (mg(2)/dL(2))(MD -7.90, 95% CI -10.25 to -5.54) were significantly lower with calcimimetics compared to placebo. No significant effects on patient-based endpoints were demonstrated except for the risk of hypotension which was significantly reduced with calcimimetics compared to placebo (RR 0.53, 95%CI 0.36 to 0.79). AUTHORS' CONCLUSIONS: Calcimimetic treatment of SHPT leads to significant improvements in biochemical parameters that observational studies have shown to be associated with increased mortality, cardiovascular risk and osteitis fibrosa, but patient-based benefits have not yet been demonstrated in trials. For patients with SHPT, the benefits of calcimimetics over standard therapy remain uncertain until further RCTs become available.

European porphyria initiative (EPI): a platform to develop a common approach to the management of porphyrias and to promote research in the field.

Porphyrias are uncommon inherited diseases of haem biosynthesis for which the diagnosis and treatment varies in individual countries. Despite the existence of guidelines recommended by porphyria experts concerning the diagnosis and management of the acute porphyrias, and of specialist centres in most European countries, many clinicians still do not apply these guidelines. The European Porphyia Initiative (EPI) network was formed in 2001 in order to compare experience among countries to attempt to develop a common approach to the management of the porphyrias, particularly concerning recommendation of safe and unsafe drugs, and to facilitate international collaborative clinical and biological research. The main achievements of EPI during this period have been: * Drafting and agreeing to consensus protocols for the diagnosis and management of acute hepatic porphyrias. * Creation of a multilingual website, particularly focusing on guidelines for common prescribing problems in acute porphyria and on providing information for patients that is now available in 10 languages: (www.porphyria-europe.org). EPI's current objectives are to develop the EPI platform, expand to new countries, extend to non-acute porphyrias and design European research and clinical trials in porphyria. The project will focus on: 1. Setting up a European laboratory external quality assurance scheme (EQAS) for biochemical and molecular investigations and their interpretation 2. Establishing a consensus drug list in collaboration with the Nordic porphyria network 3. Improving patient counseling 4. Developing large multi-centre, multi-national research projects. Due to the rarity of the porphyrias, it would be very difficult for any one country to provide this data with a sufficient number of patients and within a reasonable timescale. The progress achieved will facilitate improvements in the treatment and development of new therapeutic strategies. It will set a pattern for establishing, and subsequently harmonising, between countries best clinical practice for a rare but important group of diseases, and will help to develop the optimal therapy and ensure its cost effectiveness.

Alternative splicing and tissue-specific transcription of human and rodent ubiquitous 5-aminolevulinate synthase (ALAS1) genes.

The rate of haem synthesis in non-erythroid mammalian tissues is controlled by the ubiquitous isoform of 5-aminolevulinate synthase (ALAS1). In order to explore the regulation of mammalian ALAS1 genes, we have investigated the transcription of the human and rat genes. The 17 kb human gene differs from the rat gene in containing an additional untranslated exon that is alternatively spliced to produce a longer, minor mRNA transcript. Relative amounts of the two transcripts were similar in all tissues examined. Analysis of mRNA transcripts in human and rat tissues revealed tissue-specific differences in the use of transcription start sites by closely similar core promoters. In brain, initiation was from sites within and upstream from the TATA box, including an initiator-like element. In liver, initiation was TATA-driven from a single downstream site that appeared to be used exclusively for induction by drugs. Intermediate patterns were observed in other tissues and cell lines. Mutation of the TATA box did not impair transcription in transfected HeLa cells but activated upstream start sites, recapitulating the brain pattern. Our findings indicate that the conformation of the core ALAS1 promoter that directs assembly of the transcription pre-initiation complex may vary between tissues and have implications for understanding the tissue-specific regulated expression of this gene.

Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli.

Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined. Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EPO was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive. The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis. Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins. Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.

Effect of recombinant human erythropoietin on anemia caused by a murine mammary carcinoma.

CA NT is a transplantable murine mammary carcinoma that causes progressive anemia accompanied by granulocytosis and splenomegaly. Serum erythropoietin (Epo) levels, as measured by RIA, did not become elevated in anemic tumor-bearing mice; there was no correlation between hematocrit and serum Epo levels. Treatment with recombinant human (rHu) Epo prevented anemia in tumor-bearing mice when given in large doses, commencing on days 3-5 of tumor growth. Recombinant human Epo-treated mice had smaller spleens than controls. When treatment commenced on day 7, the development of anemia was retarded but not completely prevented. Treatment commenced on day 14 was less effective. This study demonstrates that treatment with rHu Epo can markedly influence the course of tumor-induced anemia.