Induction of an antigen specific humoral immune response by immunization with the aggregate‐free human TGFα‐P64k fusion protein

Transforming growth factor alpha (TGFα) is an important epidermal growth factor receptor (EGFR) ligand. Over‐expression of both molecules in epithelial tumors has been correlated with poor prognosis and disease progression. Due to the importance of TGFα in tumorigenesis, this molecule has great potential for cancer immunotherapy. We previously designed a TGFα‐based vaccine consisting of a fusion protein between human TGFα (hTGFα) and P64k protein from Neisseria meningitidis expressed in Escherichia coli. However, this protein was obtained highly aggregated, which hampered its introduction into clinical use. In this study, we demonstrate that this aggregation state is not a consequence of IMAC purification, but is formed after bacterial disruption. To obtain this protein as a monomer, we designed a procedure that included an unfolding/refolding step at the end of purification. We verified that hTGFα in the refolded fusion protein (hTGFα‐P64k‐r) is immunogenic in mice. The latter was capable of inducing a humoral immune response against hTGFα identical to that generated with the aggregated fusion protein, demonstrating that the aggregation level has no influence on hTGFα immunogenicity. We also showed that TGFα‐directed antibodies induced apoptosis in A431 cells. The present results also validated the potential use of this vaccine in cancer patients with tumors overexpressing TGFα. Drug Dev Res 69 2008. © 2008 Wiley‐Liss, Inc.     

McLeod syndrome resulting from a novel XK mutation

McLeod Syndrome (MLS) is a rare X-linked disorder characterized by haemopoietic abnormalities and late-onset neurological and muscular defects. The McLeod blood group phenotype is typically associated with erythrocyte acanthocytosis, absence of the Kx antigen and reduced expression of Kell system antigens. MLS is caused by hemizygosity for mutations in the XK gene. We describe a patient with MLS who first showed symptoms in 1989 (aged 51 years). As the disease progressed, the patient developed a slight dementia, aggressive behaviour and choreatic movements. A cardiomyopathy was also diagnosed. An electroneuromyography showed neuropathic and myopathic changes. Liver enzymes were elevated and a blood smear showed acanthocytes. MLS was confirmed by serological analysis of the Kell antigens. Analysis of red blood cells by flow cytometry revealed the patient and his grandson to have reduced Kell antigen expression. The patient's daughters had two populations of red cells, consistent with them being heterozygous for an XK0 allele. The molecular basis of MLS in this family is a novel mutation consisting of a 7453-bp deletion that includes exon 2 of the XK gene. This confirms that the patient's 7-year-old grandson, who is currently asymptomatic, also has the XK0 allele and is therefore likely to develop MLS.     

ALT-946 and aminoguanidine,inhibitors of advanced glycation,improve severe nephropathy in the diabetic transgenic (mREN-2)27 rat

The severe diabetic nephropathy that develops in the hypertensive transgenic (mRen-2)27 rat with streptozotocin (STZ) diabetes has previously been considered angiotensin II-dependent. Because metabolic pathways are also activated in the diabetic kidney, the present study aimed to determine whether renoprotection could be afforded with inhibitors of advanced glycation end products (AGEs), ALT-946, and aminoguanidine (AG). At 6 weeks of age, nondiabetic control and STZ diabetic Ren-2 rats were randomized to receive vehicle, ALT-946 (1 g/l), or AG (1 g/l) and were studied for 12 weeks. Systolic blood pressure was unchanged with diabetes, ALT-946, or AG. Both kidney weight and glomerular filtration rate were increased with diabetes and unchanged with ALT-946 or AG. ALT-946 and AG equally ameliorated glomerulosclerosis and medullary pathology; however, ALT-946 did reduce cortical tubular degeneration to a greater extent than AG. Albumin excretion rate, which was elevated with diabetes, was reduced with ALT-946 but not AG. AGE immunolabeling was increased in glomeruli and reduced with ALT-946 and AG. These findings indicate that even in the context of renal injury presumed to be primarily blood pressure- and/or angiotensin II-dependent, approaches that interfere with metabolic pathways such as inhibitors of AGE formation can confer renal protection in experimental diabetes.     

Cognitive deficits in neurofibromatosis 1

Cognitive deficits and academic learning difficulties are the most common neurologic "complication" of neurofibromatosis 1 in childhood and can be responsible for significant lifetime morbidity. There is a slight increase in the frequency of mental retardation (Wechsler Full-Scale IQ < 70) in children with neurofibromatosis 1, but the mean Full-Scale IQ for the patient group is within 1 SD of the population mean. Academic difficulties are common, as are specific deficits in visuospatial ability, executive function, expressive and receptive language, and attentional skills. Behavioral and psychosocial problems have a major impact on quality of life, although there are few objective studies in this area Current research is focusing on the pathogenesis of the disorder. Clinical studies have identified possible radiologic and pathologic markers for cognitive deficits in neurofibromatosis 1, which can now be explored in animal models.     

The relationship between pre-registration house officers and their consultants

CONTEXT: Previous studies have drawn attention to the importance of the trainee/trainer relationship in determining job satisfaction and motivation to learn. OBJECTIVES: To study the relationship between pre-registration house officers and their consultants through exploring an interpersonal exchange and the emotional context in which the exchange took place. To consider any association between the type of relationship implied and the trainee's attitude to their career. DESIGN: Postal questionnaire covering a wide range of issues. This study focused on an open question about a significant or interesting exchange, followed by supplementary questions exploring the emotional context of the exchange. SETTING: 336 hospitals throughout the United Kingdom. SUBJECTS: A cohort of doctors were followed from the time of their application to medical school, and studied towards the end of their pre-registration year (n=2456). RESULTS: The response rate to the questionnaire was 58.4%. Responses were categorised as Support and supervision; Unreasonable behaviour; Consultant fallibility; Fair criticism and No exchange. Over half the responses described an interaction that made them feel positive. Trainees particularly appreciated positive feedback, clinical support, teaching, career advice, patronage, or social interaction. The importance of formal appraisal or review sessions in providing the setting for a positive exchange was confirmed. Positive interactions were associated with a positive view of medicine as a career. A minority described an interaction that was negative, involving unreasonable demands, criticism (whether perceived as fair or unfair), humiliation, or sexism. These were associated with a more negative view of medicine as a career, and of themselves as doctors.     

Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each ≈100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.