Use of Total Error concept in the validation of viral activity in cell cultures.

Due to the high variability inherent of experimental recipients, validating biological methods is often a complex exercise, and following ICH Q2R1 recommendations is not always feasible and/or meaningful. Linking systematic error and random error to obtain a unique criterion, as defined in ISO guideline, could be of interest to capture the total variability in biological assays. In this paper, the use of Total Error concept in the validation of biological assays was for the first time investigated and compared to a conventional interpretation of the ICH guideline. Both decision methodologies concluded that the assay was valid from 2.13 to 5.83 log(10)(CCID(50)/ml). However, only the Total Error approach using accuracy profile as decision tool allowed to guarantee that accurate and reliable results will be obtained during the future routine application of the assay. In addition, the risk to obtain out of acceptance limits results was estimated using this approach and was found out to be at the most 3.1% irrespective of the concentration level, thus demonstrating the reliability of the biological assay.

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.

Comparative study of poliovirus excretion after vaccination of infants with two oral polio vaccines prepared on Vero cells or on primary monkey kidney cells.

A comparative study was designed to assess the bioequivalence of 2 oral poliovaccines (OPV) produced on 2 different cell systems: primary monkey kidney (PMK) cells and the Vero cell line. The Vero cell line has been used to overcome the problem of obtaining a regular supply of high quality monkeys that are devoid of latent viruses. For this study, 9 children were vaccinated with PMK-OPV and 12 children with Vero-OPV. The comparison covered poliovirus excretion, reversion of polioviruses in the 5'-noncoding region, and immunogenicity. Major molecular markers in the 5'-noncoding region related to neurovirulence already had been identified at position 480 for type 1, position 481 for type 2, and position 472 for type 3 poliovirus. Two nucleic-acid based methods were designed for studying these positions: a RT-PCR followed by sequencing, which required preliminary culture and cloning; and a type-specific nested PCR followed by sequencing, which enabled direct detection and genotyping of polioviruses. Twenty-eight stool specimens were analyzed by this second method with no PCR inhibition problem. The use of Vero cell line did not modify the global pattern of poliovirus excretion, reversion frequency, or seroconversion. These results provide additional support for the use of the well-characterized Vero cell line in OPV manufacturing.

Calcium-dependent, slowly inactivating potassium currents in cultured neurons of rat neocortex.

Slowly inactivating outward currents were examined in neurons from rat anterior cortex dissociated at postnatal day 1 and recorded after 7-48 days in vitro by the use of whole-cell patch-clamp technique, in the presence of 0.5-0.8 microM tetrodotoxin (TTX). 50 microM carbachol and 1-5 mM CsCl2. Experiments were often carried out in the additional presence of 1-5 mM CsCl2, which blocks the anomalous, inwardly rectifying IQ, the fast Ca(2+)-dependent K+ current (IC), and 50 microM carbachol, which depresses the IM current. These currents were evoked by depolarizing steps to -40 +/- 5 mV from a conditioning hyperpolarization to -110 +/- 10 mV. Their sensitivity to elevation from 2.5 to 12.5 mM in extracellular K+ concentration, together with their sensitivity to 5-15 mM tetraethylammonium, suggests that they are mainly carried by K+ ions. Their activation and inactivation curves show that the threshold for activation is -65 mV, that their inactivation is achieved at -75 mV and that potentials more negative than -120 mV are needed to abolish it. The time-dependence of de-inactivation gives a maximal current amplitude for conditioning hyperpolarizations of 2 s and is best described by a monoexponential function with a time constant of 0.7 s. Slow transient K+ currents were depressed by low doses of 4-aminopyridine (30-100 microM), which indicates the occurrence of an ID-type component in the recorded K+ currents. No slowly declining K+ current was expressed when a recording solution containing 10 mM 1,2-bis (2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid (BAPTA), instead of 1-5 mM BAPTA, was used. When recorded without Ca2+ chelator in the pipette, slowly declining K+ currents were blocked by bath-applied 40-50 microM BAPTA-aminoethoxy, revealing a large-amplitude, rapidly inactivating outward current. This residual component is insensitive to 50 microM 4-aminopyridine and may include a current more related to the IA-type. Our data provide evidence that, in cultured cortical neurons from rat, the expression of an ID-like K+ current is highly dependent on internal Ca2+ concentration.

Lack of adjuvant effect of the pertussis component on IPV DTP-polio vaccine in children.

On day 0, four groups of children (3 to 6 months old) randomly received IPV alone or IPV + pertussis, or IPV + DP, or IPV + DTP. At days 28 and 56, all the children received the same IPV + DTP vaccine. Polio neutralizing and diphtheria antibodies were determined at days 0, 28 and 56. No adjuvant and even some inhibitory effect of pertussis was observed at days 28 and 56 on mean polio antibody titers. These results are compared to those observed with diphtheria.

Potency test of killed polio vaccines. Statistical analysis of test on chicken and preliminary results in combination with ELISA D-antigen determinations.

The authors resume a several years' experience of potency testing inactivated polio vaccines with an in vivo-vitro assay based on inoculation of 3 week-old chicken and measure of neutralizing antibodies at 5 days on Hep 2 cells in a microplate system. Critical points, specificity and reproducibility are successively outlined. Preliminary combining results with determination of D-antigen by ELISA method and human immunization are submitted in view of a future standardization.