Screening for activating EGFR mutations in surgically resected nonsmall cell lung cancer.

The clinical applicability of screening surgically resected nonsmall cell lung cancer (NSCLC) tumour tissue and serum for activating epidermal growth factor receptor (EGFR) mutation is unknown. Furthermore, the comparative accuracy of inexpensive EGFR mutation tests, mutant-enriched (ME)-PCR and high-resolution melt (HRM) has not been determined. Lung tumour DNA from 522 surgically resected stage I-IV NSCLC and matched serum DNA from a subset of 64 subjects was analysed for EGFR mutations in exons 19 and 21 using ME-PCR and HRM. Additionally, 97 subjects had previous EGFR DNA sequencing data available for comparison. ME-PCR and HRM detected EGFR mutations in 5% (27 out of 522) of tumour samples. Compared to DNA sequencing, ME-PCR had a sensitivity of 100% and specificity of 99%, while HRM had 100% sensitivity and specificity. Six subjects with EGFR mutation tumours had matched serum, where ME-PCR detected mutations in three samples and HRM in two samples. In the cohort of never-smoker subjects, those with EGFR mutated tumours had worse survival compared with wild-type tumours (30 versus 49 months; p=0.017). ME-PCR and HRM have similar accuracy in detecting EGFR mutations but the prognostic implications of the mutations in resected NSCLC warrants further study.

Nephropathy in model combining genetic hypertension with experimental diabetes. Enalapril versus hydralazine and metoprolol therapy.

We compared the effects of the angiotensin-converting enzyme inhibitor enalapril and a conventional antihypertensive regimen (hydralazine and metoprolol) on kidney function, albuminuria, and glomerular ultrastructure in hypertensive diabetic and nondiabetic rats. Diabetes was induced with streptozocin at 8 wk of age in spontaneously hypertensive (SHR) rats. Antihypertensive drugs were administered in drinking water from the time of induction of diabetes in all groups. Blood pressure reduction was equal in the diabetic and nondiabetic SHR rats receiving either enalapril or hydralazine plus metoprolol. In diabetic SHR rats, there was a rise in serum creatinine after 32 wk, which did not occur in diabetic rats treated with either antihypertensive regimen or in nondiabetic rats. Both drug regimens reduced albuminuria in diabetic and nondiabetic SHR rats to a similar degree. Enalapril and the combination of hydralazine and metoprolol were associated with decreased glomerular basement membrane thickness and glomerular volume in diabetic and nondiabetic SHR rats without significant effect on fractional mesangial volume. Thus, antihypertensive therapy retards the development of albuminuria, glomerular basement membrane thickening, and glomerular hypertrophy in the rat in the presence or absence of diabetes. No specific benefit of angiotensin-converting enzyme inhibition was observed in these hypertensive models of nephropathy. Human studies comparing the effects of different classes of antihypertensive drugs on kidney function, proteinuria, and glomerular morphology are warranted.

Synthesis and secretion of a functional antibody in a vaccinia virus expression system.

A humanized rat monoclonal antibody (Campath 1H) has been expressed in HeLa cells using recombinant vaccinia viruses. Heavy and light chain recombinant viruses were constructed separately and when grown independently produced proteins of the expected molecular weights. Expressed heavy chain was entirely intracellular but light chain was mainly excreted and processed. When cells were infected at high multiplicity with both heavy and light chain recombinants a proportion of the heavy chain was then found in the extracellular medium. This secreted heavy chain was shown to be associated with light chain as judged by co-electrophoresis in non-reducing SDS polyacrylamide gels and by co-purification on protein-A sepharose. The secreted heavy and light chain complexes were functionally active as an antibody, with activity comparable to authentic Campath 1H antibody as assessed by ELISA, T-cell binding and antigen binding assays. Production of antibody in this system was achieved in the absence of serum, which is an important consideration in the production of monoclonal antibodies (MAbs). The amount of antibody produced was 0.2-0.4 micrograms/10(6) cells without optimization of expression levels. The wide host cell range of vaccinia virus together with the recently developed methods for increasing expression levels make this an attractive candidate as a flexible general vehicle for producing MAbs.

The nucleotide sequence of the structural-protein-coding region of foot-and-mouth disease virus serotype SAT3.

The nucleotide sequence coding for the structural proteins and nonstructural protein P2A has been determined for a foot-and-mouth disease virus (FMDV) isolated in Africa. This virus, serotypically designated SAT3 (South African Territories type 3), shows about 60% homology at the nucleotide level to prototype viruses from the O, A and C serotypes of FMDV. The highest region of variability was shown in structural protein VP1, presumably a consequence of its position on the surface of the virus and its exposure to selection pressure by neutralising antibody. Within this region amino acids (aa) 141-160, which have been shown to represent an immunodominant region in other FMDV serotypes, showed hypervariability as well as the insertion of 5 or 9 additional aa relative to the O1 and C1 serotypes, respectively. In contrast, the sequence of nonstructural protein P2A was completely conserved indicating a probable important role in virus replication.

A new and improved method for 3'-end labelling DNA using [alpha-32P]ddATP.

We have synthesised dideoxyadenosine-5'-[alpha-32P]triphosphate [( alpha-32P] ddATP ) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5'-[alpha-32P]triphosphate [( alpha-32P] KTP ) as a means to 3'-end label DNA. The [alpha-32P] ddATP was found to be three to five times more efficient than [alpha-32P] KTP . Blunt and 3'-protruding ends were labelled more efficiently with [alpha-32P] ddATP using terminal transferase than were the 5'-ends with [gamma-32P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3'-end labelling of DNA to become a realistic alternative to 5'-end labelling. We have also compared [alpha-32P] ddATP - and [alpha-32P] KTP -labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.

The sequence of foot-and-mouth disease virus RNA to the 5' side of the poly(C) tract.

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O1 compared with 362 nt for A10, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.

Synthetic peptides mimic subtype specificity of foot-and-mouth disease virus.

The major immunogen of foot-and-mouth disease virus (FMDV) is located between amino acids 141-160 of the capsid protein VP1. Synthetic peptides corresponding to the major immunogenic region give good neutralising antibody responses and protection in guinea pigs. To define more precisely the immunogenic site of the virus, we have examined serological differences between subtypes of the A serotype using synthetic peptides covering the 141-160 region. We show that these synthetic peptides carry determinants which mimic the subtype specificity of the virus. The correlation between these results and predictive structural models, based on the amino acid sequence, is discussed.

Benign fibrous histiocytoma of bone.

The clinical, radiologic, and pathological features of eight cases of fibrohistiocytic bone lesions histologically identical to the nonossifying fibroma of childhood are presented. They differed from the childhood lesion in their clinical and radiological features. They occurred in adults, and were frequently associated with pain in the absence of complicating fracture. They were not confined to the metaphysis of long bones. When metaphyseal, the lesions also frequently showed a tendency to involve the epiphysis. Others occurred in the diaphysis of long bones, in the pelvis, and in a rib. Three recurred locally, but none has metastasized. Other fibrohistiocytic and fibroblastic tumours of bone, including malignant fibrous histiocytoma, giant cell tumour, fibrosarcoma, and desmoplastic fibroma can be differentiated on radiological and histological features, and hyperparathyroidism may need to be excluded by biochemical investigations.

Transesophageal echocardiographic abnormalities in a case of cardiac sarcoidosis.

Sarcoidosis is a granulomatous disease that may involve multiple organ systems, including the heart. Manifestations include atrial and ventricular arrhythmias, conduction abnormalities, congestive cardiac failure, pericarditis, and sudden death. Whereas cardiac involvement is a relatively common finding at autopsy, antemortem diagnosis is often missed because the clinical manifestations are nonspecific, and the sensitivity and specificity of investigations are low. We report a case of a 62-year-old woman who had clinically significant cardiac sarcoidosis associated with echocardiographic abnormalities that had not been reported previously in association with this condition.

Gelatinous transformation of the bone marrow.

A case of gross macroscopic gelatinous transformation of bone marrow associated with prolonged malabsorption due to celiac disease is reported. Histochemical studies support the view that the extracellular myxoid tissue is composed predominantly of acid mucopolysaccharides, and electron microscopy shows extracellular fine fibrillar and granular material. The replacement of marrow in this condition is histochemically different from serous fat atrophy occurring in epicardial fat, and it is suggested that it results from an increase in normal ground substance. It must be differentiated from metastatic signet ring adenocarcinoma.

Macrophages and glomerular crescent formation. Studies with rat nephrotoxic nephritis.

A model of crescentic glomerulonephritis in the rat was developed, based upon an augmented form of nephrotoxic nephritis. The glomerular lesions were relatively mild, permitting an analysis of the morphologic events in crescent formation to an extent not possible in models in other species in which the sequence of changes is obscured by the severity of the inflammatory process. Monocytes and macrophages accumulated in glomerular capillary lumens and walls. Crescents were composed of cells with ultrastructural features indistinguishable from those of the intracapillary mononuclear cells. The findings support the view that the crescent cells are predominantly composed of macrophages derived from circulating monocytes.

Production of replication-deficient adenovirus recombinants.

In essence, replication-deficient (RD) adenovirus (Ad) vectors can be considered to function as an extremely efficient DNA transfection system capable of providing transgene expression in up to 100% of cells both in vitro or in vivo. As researchers continue to realize the full potential of this vector system, discover novel applications, and further develop and enhance systems, the use of this vector system has increased exponentially. The exploitation of Ad recombinants in HCV research is encouraged by demonstration that the virus will efficiently infect and express transgenes in hepatocytes and that following iv inoculation, transgene expression can readily be detected in hepatocytes in the liver (1-5). A number of HCV proteins have now been expressed in Ad recombinants (6-8) whereas these vectors have also been used to deliver antisense and ribozyme molecules as prototype HCV therapeutics (9).

Expression and characterization of HCV NS3 protease.

The overexpression of a gene in a heterologous system is often the prelude to or the prerequisite of the elucidation and characterization of a given protein, in particular where the protein is difficult to obtain in sufficient quantity from natural sources. Prokaryotic expression systems, in particular Escherichia coli have been exploited successfully for a number of viral proteins. A large number of E. coli expression systems are currently commercially available that offer the investigator a large number of choices with regard to promoter choice, site of expression, fusions, and so forth. Since the variety and number of expression systems available are extensive, it is not within the scope of this chapter to discuss them fully, and the final choice of expression system used, is often arrived at empirically and often reflects the investigator's "favored system."

Expression and characterization of the HCV NS3 helicase domain.

The hepatitis C virus (HCV) NS3 protein has two distinct biochemical domains. The N-terminal 20 kDa has serine protease activity (see Chapter 31 ) and the C-terminal 50 kDa has both nucleoside triphosphatase (NTPase) and helicase activities (1-4).

Microvascular complications in cystic fibrosis-related diabetes mellitus: a case report.

CONTEXT, The prevalence of cystic fibrosis-related diabetes mellitus is increasing and is associated with increased survival from cystic fibrosis. CASE REPORT, This study describes a case of the premature onset of disabling and widespread microvascular complications resulting from cystic fibrosis-related diabetes mellitus. Previously asymptomatic retinopathy was diagnosed on recognition of diabetic nephropathy. CONCLUSIONS, The treatment of pulmonary exacerbations has become more complex due to the nephrotoxic potential of intravenous aminoglycoside drugs which are frequently used to control chronic Pseudomonas infection in cystic fibrosis.

Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties.

Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

Gene expression of lung squamous cell carcinoma reflects mode of lymph node involvement.

Tumour, node, metastasis staging is essential for lung cancer management. However, similarly staged cancers may have markedly different prognoses, indicating that stage cannot completely explain tumour behaviour. While ipsilateral hilar node involvement is designated N1, the current authors hypothesised that primary tumours involving nodes by direct extension are biologically distinct from those involving nodes through lymphatic metastasis. Microarrays were used to investigate the gene expression profiles of 59 primary lung squamous cell carcinomas, comparing N0 tumours (n = 35), N1 tumours by direct extension (N1d; n = 8), and N1/N2 tumours by lymphatic metastasis (N1/N2m; n = 16). Hierarchical clustering using 125 genes differentially expressed between N0 and N1/N2m tumours found N1d tumours clustered with N0 tumours. Class prediction modelling found the expression profiles of all eight N1d tumours were more similar to N0 than to N1/N2m tumours. The present study demonstrates for the first time that N1 tumours directly invading hilar nodes are genomically different to those that metastasise via lymphatics. Independent reports suggest that tumours with direct, rather than metastatic node involvement have better outcomes. Consequently, the data suggest that there is a need to re-evaluate the N1 staging definition in lung cancer. This is relevant for prognosis prediction and also for clinical management, particularly in selecting those patients most likely to benefit from adjuvant chemotherapy.

Approaches to the development of novel inhibitors of hepatitis C virus replication.

Chronic infection with the hepatitis C virus is a major health problem. Conventional therapy, with interferon-alpha is effective in only a minority of patients. The failure of current treatments has led to a major initiative to identify new antiviral agents. In the absence of a tissue culture model for hepatitis C infection the pharmaceutical industry has been obliged to investigate the basic biology of hepatitis C viral replication. Such studies have identified novel translational regulatory elements and new proteolytic enzymes which may serve as targets for new antiviral drugs.

New drugs for hepatitis C virus (HCV).

Lack of efficacy and significant side effects have severely limited the use of interferon-alpha (IFN-alpha) as the standard therapy for non-A non-B hepatitis (NANBH) caused by hepatitis C virus (HCV) and alternative, improved therapies are urgently sought. Attempts have been made to improve the potency and tolerability of IFN-alpha by adjusting dosing regimens, methods of delivery and length of treatment. Furthermore, a number of different agents have been used in combination wit IFN-alpha and, from these studies, therapeutic options have been galvanized by the synergistic effects of IFN-alpha and ribavirin. Nevertheless, the majority of patients with HCV still do not sustain lasting therapeutic benefit from this combination and continuing research is required to identify new therapeutic candidates that will have more potent antiviral activity and less severe side effects. This review focuses on the progress that has been made in this area and the prospects for new effective therapies in the near future.