An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

Reproducibility of histomorphologic diagnoses with special reference to the kappa statistic

Systems for classification and grading used in pathology should ideally be biologically meaningful and at least be reproducible from one pathologist to another. A statistical method to evaluate reproducibility (non-chance agreement) for several observers using nominal or ordinal categories has been developed and refined over the past few decades--the kappa statistic. A high level of observed agreement among different pathologists can either signify a high level of reproducibility, if agreement by chance is low, or express a low level of reproducibility, if agreement by chance is almost as high as the observed agreement. Therefore, the observed agreement says nothing in itself, unless it is low. The kappa value, however, indicates how much better the observers are compared to a throw of the dice, and therefore gives the real credit to the agreement which was found. We have developed a user-friendly computer program for calculating inter- and intra-observer agreement of 2 or more observers. By calculating associations between different categories and different observers, the statistic furthermore obtains a function close to the parameter of accuracy. We recommend the use of the above method before a set of nominal or rank scale parameters are used for deciding prognosis and treatment of patients. By submitting a diskette the computer program will be available at no cost.     

Mucosal/submucosal blood flow in the small intestine in pigs determined by local washout of 133Xe and microsphere techniques

In 11 anaesthetized pigs a laparotomy was performed and the mucosal and submucosal blood flow rate in the small intestine of the pig was determined by a local application of 133Xe and by 6.5-microns radioactive microspheres. The 133Xe washout plotted in a semilogarithmic diagram showed a multiexponential configuration. As localization studies of 133Xe in the intestinal mucosa showed a constant high concentration of 133Xe in the luminal part of the mucosa due to shunting by diffusion, the initial slope of the 133Xe washout was used for blood flow determination in the mucosa/submucosa. There was a good relationship between blood flow determined by the two techniques. The correlation coefficient, R, between the two techniques was 0.89.     

The P-selectin gene is highly polymorphic: reduced frequency of the Pro715 allele carriers in patients with myocardial infarction

P-selectin is an adhesion molecule, expressed at the surface of activated cells, that mediates the interaction of activated endothelial cells or platelets with leukocytes. P-selectin expression is increased in atherosclerotic plaques, and high plasma levels of this molecule have been observed in patients with unstable angina. We investigated the P-selectin gene as a possible candidate for myocardial infarction (MI). The P-selectin gene is situated on chromosome 1q21-q24, spans >50 kb and contains 17 exons. The sequences of the 5'-flanking region and exons of 40 alleles from patients with MI were screened for polymorphisms using polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) and sequencing. Thirteen polymorphisms were identified: five in the 5'-flanking and eight in the exonic sequences. Four polymorphisms (Ser290Asn, Asn562Asp, Leu599Val and Thr715Pro) predicted a change in the amino acid sequence of the P- selectin protein. All P-selectin polymorphisms as well as a common E- selectin polymorphism, Ser128Arg which has been reported as being associated with an increased risk of premature coronary heart disease (CHD), and is in tight linkage disequilibrium with several P-selectin polymorphisms, were investigated in 647 patients with MI and 758 control subjects from four regions of France and Northern Ireland (the ECTIM study). The entire set of P-selectin polymorphisms provided a heterozygosity of 91%. The polymorphisms were tightly associated with one another and displayed patterns of linkage disequilibrium suggesting the existence of highly conserved ancestral haplotypes. The five polymorphisms in the 5'-flanking region of the gene were unrelated to MI or any relevant phenotype measured in the ECTIM study. We inferred that the four missense variants identified in the coding region predicted eight common forms of the P-selectin protein. The Pro715 allele which characterizes one of these forms was less frequent in France than in Northern Ireland ( P < 0.002) and in cases than in controls ( P < 0.002; P < 0.02 after correction for the number of tests). We conclude that the P-selectin gene is highly polymorphic and hypothesize that the Pro715 variant may be protective for MI. Whether this variant affects the properties of the P-selectin protein in a way which is compatible with this hypothesis needs to be checked experimentally.      

Sperm quality in Hodgkin's disease versus non-Hodgkin's lymphoma

The study was conducted to determine the deleterious effect of lymphoma disease on spermatogenesis and to evaluate the possibility that the disease is mediated primarily by inherent mechanisms in Hodgkin's disease and non-Hodgkin's lymphoma patients. A total of 89 patients with lymphoma disease (Hodgkin's and non-Hodgkin's) were referred for sperm preservation prior to adjuvant treatments. A comparison was made of pre- and post-thaw sperm quality between lymphoma patients and healthy volunteers who applied for sperm donation. This was followed by further assessment of the differences between patients with Hodgkin's disease and non-Hodgkin's lymphoma in terms of sperm variables, clinical parameters and blood hormone concentrations. It was found that patients with lymphoma disease had significantly impaired pre-freeze and post-thaw sperm quality compared with that of healthy volunteers. Patients with non-Hodgkin's lymphoma had spermatozoa of higher quality than patients with Hodgkin's disease. No differences were found in the clinical or hormonal parameters between these two groups. As expected, reduced testicular size and abnormal testicular consistency were correlated with decreased sperm quality. The mere presence of cancer disease has a direct negative effect on spermatogenesis, which is probably not related to incidental side-effects. A variable degree of impairment should be expected with different categories of cancer.      

Using infertile patients in epidemiologic studies on subfecundity and embryonal loss

Subfecundity is a frequent and serious problem that may sometimes be preventable, but we need to know more about its determinants. Different epidemiologic designs are available. The best of these use prospectively collected data from the population, but they are time consuming, expensive and often hampered by low-participation rates. Most patients undergoing infertility treatment are closely monitored for clinical reasons, making it feasible to use secondary data to study the period from conception to implantation and pregnancy. In spite that infertility patients are highly selected, there are specific exposure-effect relations that can be studied in cohorts of infertility patients. These patients offer a potentially useful setting for studying exposures that operate late in fertilization, whereas the designs may be inadequate to identify exposures that cause reduced sperm counts, anovulation and total occlusion. The clinical sampling and the treatment set limitations for what can be studied. In certain situations, infertile patients can, however, provide useful epidemiologic evidence for learning about the causes of subfecundity.     

Phosphate glasses for tissue engineering: Part 2. Processing and characterisation of a ternary-based P2O5-CaO-Na2O glass fibre system

This paper presents the results of a study of the thermal properties, solubility and dimensions of a range of phosphate-based glass fibres (PB-GFs). The glass compositions were limited by fixing the P2O5 content to 45, 50 and 55 mol%, and varying the CaO mol% at 30, 35 and 40. PB-GFs were obtained from the 50 and 55 mol% P2O5 compositions; however, we were unable to obtain fibres from the 45 mol% compositions. This was linked to the cross-linked density, network connectivity and average chain length of the compositions studied. With regards to thermal parameters investigated, initial data showed an increase of the Tg and crystallisation temperatures with increasing CaO mol% at each fixed phosphate content. A decrease in Tg temperatures was also observed with increasing P2O5 content to 55 mol%. The crystallisation temperatures obtained for compositions with fixed phosphate at 55 mol%, showed a reverse pattern, with a decrease in values as compared to the fixed 50 mol% phosphate compositions. The diameters of the fibres all decreased with increasing RPMs as expected, and the solubility also increased with increasing RPMs. This was related to the increased surface area of the higher RPM fibres. There was also a decrease seen in solubility with increasing CaO mol%.     

Haemophilus ducreyi Outer membrane determinants, including DsrA, define two clonal populations

The Haemophilus ducreyi outer membrane component DsrA (for ducreyi serum resistance A) is necessary for complete resistance to normal human serum (NHS). When DsrA expression in 19 temporally and geographically diverse clinical isolates of H. ducreyi was examined by Western blotting, 5 of the strains expressed a different immunotype of the DsrA protein (DsrA(II)) than the well-characterized prototypical strain 35000HP (DsrA(I)). The predicted DsrA proteins expressed by the DsrA(II) strains were 100% identical to each other but only 48% identical to that of strain 35000HP. In addition to the DsrA(II) protein, class II strains also expressed variant forms of other outer membrane proteins (OMPs) including NcaA (necessary for collagen adhesion A), DltA (ducreyi lectin A), Hlp (H. ducreyi lipoprotein), major OMP, and/or OmpA2 (for OMP A2) and synthesized a distinct, faster-migrating lipooligosaccharide. Based on these data, strains expressing DsrA(I) were termed class I, and those expressing DsrA(II) were termed class II. Expression of dsrA(II) from strain CIP 542 ATCC in the class I dsrA(I) mutant FX517 (35000HP background), which does not express a DsrA protein, rendered this strain resistant to 50% NHS. This demonstrates that DsrA(II) protein is also critical to serum resistance. Taken together, these results indicate that there are two clonal populations of H. ducreyi. The implications of two classes of H. ducreyi strains differing in important antigenic outer membrane components are discussed.