Prentice's approach and the meta-analytic paradigm: a reflection on the role of statistics in the evaluation of surrogate endpoints

We put a perspective on the strengths and limitations of statistical methods for the evaluation of surrogate endpoints. Whereas using several trials overcomes some of the limitations of a single-trial framework (Prentice, 1989, Statistics in Medicine 8, 431-440), arguably the evaluation of surrogate endpoints can never be done using only statistical evidence but such evidence should be seen as but one component in a decision-making process that involves, among others, a number of clinical and biological considerations. We briefly present a hierarchical framework that incorporates ideas from Prentice's work and is uniformly applicable to different types of surrogate and true clinical outcomes.     

More than a monolayer: relating lung surfactant structure and mechanics to composition

Survanta, a clinically used bovine lung surfactant extract, in contact with surfactant in the subphase, shows a coexistence of discrete monolayer islands of solid phase coexisting with continuous multilayer "reservoirs" of fluid phase adjacent to the air-water interface. Exchange between the monolayer, the multilayer reservoir, and the subphase determines surfactant mechanical properties such as the monolayer collapse pressure and surface viscosity by regulating solid-fluid coexistence. Grazing incidence x-ray diffraction shows that the solid phase domains consist of two-dimensional crystals similar to those formed by mixtures of dipalmitoylphosphatidylcholine and palmitic acid. The condensed domains grow as the surface pressure is increased until they coalesce, trapping protrusions of liquid matrix. At approximately 40 mN/m, a plateau exists in the isotherm at which the solid phase fraction increases from approximately 60 to 90%, at which the surface viscosity diverges. The viscosity is driven by the percolation of the solid phase domains, which depends on the solid phase area fraction of the monolayer. The high viscosity may lead to high monolayer collapse pressures, help prevent atelectasis, and minimize the flow of lung surfactant out of the alveoli due to surface tension gradients.     

Recruitment of transferrin receptor to immunological synapse in response to TCR engagement

T cell receptor engagement by an APC induces the formation of a highly organized complex of surface receptors and intracellular signaling molecules, known as the immunological synapse, at the site of cell-cell contact. The transferrin receptor (TfR, CD71) is normally present in the plasma membrane and recycling endosomes. In this study, we show that, although the TfR is typically absent from lipid rafts at steady state, stimulation with a mitogenic mixture of anti-CD3 Abs of human Jurkat T cells leads to a rapid compartmentalization of the TfR into lipid rafts accompanying that of CD3epsilon and activated Lck. This change occurs very rapidly and is accompanied by an increase in the surface expression of the TfR, probably by translocation from an internal endosomal pool. TfR recruitment to lipid rafts was also observed in primary T cells treated with mitogenic anti-CD3 Abs and in Jurkat T cell-APC conjugates. The use of beads coated with Abs indicates that the surface and endosomal TfR pools redistribute to the contact site region in response to engagement of CD28 and CD3. In T cell-APC conjugates, the T cell TfR endosomal pool relocates beneath the contact site, whereas surface TfR localizes to the peripheral ring of the immunological synapse. In the presence of specific anti-TfR Abs, the total number of T cell-APC contacts and the percentage of conjugates with CD3 and Lck translocated to the contact site were reduced. Our results therefore suggest the involvement of the TfR in the formation of the immunological synapse.     

The minimal essential core of a cysteine-based protein-tyrosine phosphatase revealed by a novel 16-kDa VH1-like phosphatase, VHZ

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process.     

Protein tyrosine phosphatases in the human genome

Tyrosine phosphorylation is catalyzed by protein tyrosine kinases, which are represented by 90 genes in the human genome. Here, we present the set of 107 genes in the human genome that encode members of the four protein tyrosine phosphatase (PTP) families. The four families of PTPases, their substrates, structure, function, regulation, and the role of these enzymes in human disease will be discussed.     

Molecular bases of the treatment of Alzheimer's disease with antioxidants: prevention of oxidative stress

Alzheimer's disease is associated with a systemic oxidative stress situation which can be followed in vivo by determining biomarkers such as plasma lipoperoxides and TBARS levels and the oxidation degree of glutathione in red blood cells. It has been observed that Alzheimer's patients show an increased level of plasma TBARS, which indicates a higher free radical oxidation of plasma unsaturated phospholipids, and an increased oxidation of red blood cells glutathione, which indicates oxidative stress in peripheral cells. This latter, glutathione oxidation, was found to correlate statistically with the cognitive status of the patients. Treatment with vitamin E resulted in an improved cognitive performance only of those patients in which the tocopherol acted as an antioxidant, according to blood indicative markers of oxidative stress. Indeed, the effect of vitamin E on Alzheimer's disease patients showed considerable variations both in its antioxidant function and in its capacity to improve cognitive functions. An important conclusion from the reported results is that epidemiological or clinical studies that aim to test the effect of antioxidant supplementation on given functions should include the determination of the antioxidant status of the patients by the measurement of blood markers of oxidative stress.     

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.The two first authors participated equally in this workThis work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 02/3003 to M.V.T. Lobo)     

Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses

European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2) are widespread in Europe, although little is known of their evolutionary history. We undertook a comprehensive sequence analysis to infer the selection pressures, rates of nucleotide substitution, age of genetic diversity, geographical origin, and population growth rates of EBLV-1. Our study encompassed data from 12 countries collected over a time span of 35 years and focused on the glycoprotein (G) and nucleoprotein (N) genes. We show that although the two subtypes of EBLV-1--EBLV-1a and EBLV-1b--have both grown at a low exponential rate since their introduction into Europe, they have differing population structures and dispersal patterns. Furthermore, there were strong constraints against amino acid change in both EBLV-1 and EBLV-2, as reflected in a low ratio of nonsynonymous to synonymous substitutions per site, particularly in EBLV-1b. Our inferred rate of nucleotide substitution in EBLV-1, approximately 5 x 10(-5) substitutions per site per year, was also one of the lowest recorded for RNA viruses and implied that the current genetic diversity in the virus arose 500 to 750 years ago. We propose that the slow evolution of EBLVs reflects their distinctive epidemiology in bats, where they occupy a relatively stable fitness peak.