Phagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII-C1, to type II collagen

Antibody screening of phage-displayed random peptide libraries to identify mimotopes of conformational epitopes is promising. However, because interpretations can be difficult, an exemplary system has been used in the present study to investigate whether variation in the peptide sequences of selected phagotopes corresponded with variation in immunoreactivity. The phagotopes, derived using a well-characterized monoclonal antibody, CII-C1, to a known conformational epitope on type II collagen, C1, were tested by direct and inhibition ELISA for reactivity with CII-C1. A multiple sequence alignment algorithm, PILEUP, was used to sort the peptides expressed by the phagotopes into clusters. A model was prepared of the C1 epitope on type II collagen. The 12 selected phagotopes reacted with CII-C1 by both direct ELISA (titres from < 100-11 200) and inhibition ELISA (20-100% inhibition); the reactivity varied according to the peptide sequence and assay format. The differences in reactivity between the phagotopes were mostly in accord with the alignment, by PILEUP, of the peptide sequences. The finding that the phagotopes functionally mimicked the C1 epitope on collagen was validated in that amino acids RRL at the amino terminal of many of the peptides were topographically demonstrable on the model of the C1 epitope. Notably, one phagotope that expressed the widely divergent peptide C-IAPKRHNSA-C also mimicked the C1 epitope, as judged by reactivity in each of the assays used: these included cross-inhibition of CII-C1 reactivity with each of the other phagotopes and inhibition by a synthetic peptide corresponding to that expressed by the most frequently selected phagotope, RRLPFGSQM. Thus, it has been demonstrated that multiple phage-displayed peptides can mimic the same epitope and that observed immunoreactivity of selected phagotopes with the selecting mAb can depend on the primary sequence of the expressed peptide and also on the assay format used.

Molecular mimicry: Can epitope mimicry induce autoimmune disease?

Mimicry of host antigens by infectious agents may induce cross-reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. Epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post-viral myocarditis or Chagas disease, but for many other diseases in which it has been implicated, such as insulin-dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. Even if an epitope mimic can support a cross-reactive T or B cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of T cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the MHC-peptide complex with the T cell receptor. B cell presentation of mimicking foreign antigen to T cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. In this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory.

A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Solubility of ibuprofen in aqueous ethanol at low ethanol contents

The solubility of ibuprofen was measured in water (W) - ethanol (E) mixtures from 0 to 50% w/w ethanol at 10, 25 and 40 °C by the dissolution method using UV spectrophotometry to determine the ibuprofen concentrations. The UV calibration for ibuprofen in different water - ethanol mixtures showed Beer - Lambert linearity, however the slopes differed, which indicated the structure of the drug is influenced by the solvent system i.e. the water - ethanol ratio. The ibuprofen solubility in water (zero ethanol) is low (~ 50 ppm) but increases near exponentially with increasing ethanol content. At 40 °C, there is phase separation between 34% and 63% w/w E/(E+W). The solubility data will be used to select precipitation crystallizer conditions to directly produce free flowing ibuprofen particles (<5 m) for developing a dry powder inhaler (DPI) formulation for lung delivery.

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.