Preoperative blood tests in elective general surgery: cost and clinical implications.

A retrospective observational study was performed in our trust in October 2010 that examined compliance, and the financial and clinical implications of performing inappropriate preoperative blood tests on adult patients prior to elective surgery, against the 2003 NICE guidelines. An unacceptable proportion of inappropriate tests (31.3%) were being performed. None were associated with adverse outcome or changes in management. Based on our results, we estimate that an extrapolated cost of pound 11.2 million is being spent on inappropriate blood tests in NHS England and Wales.

Dimeric beta-turn peptidomimetics as ligands for the neurotrophin receptor TrkC.

Twelve dimeric peptidomimetics 1 were prepared via a divergent-convergent strategy. These peptidomimetics incorporated the same amino acids as i +1 and i + 2 residues in key beta-turns of the neurotrophin NT-3. Cytosensor microphysiometry was used to gauge the effects of the dimers 1 on cells that overexpress the NT-3 receptor, TrkC. Increases in extracellular acidification rates were observed for some monomers 3, but the active dimers gave greater effects.

Cellular responses of NG108-15 and SK-N-MC lines to sweet and bitter tastants as measured by extracellular acidification rates.

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of detecting small changes in cellular metabolism in response to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). By measuring the ECAR we were able to detect responses of tissue culture cell lines to a variety of sweet- and bitter-tasting compounds. We examined in detail the responses of the NG108-15 (mouse neuroblastoma x rat glioma hybrid) and SK-N-MC (human neuroepithelioma) cell lines. We determined that NG108-15 cells were consistently very responsive to several potent sweeteners and bitter compounds, such as sodium saccharin, guanidino- sweeteners, denatonium benzoate, quinine, and ranitidine. These compounds could evoke changes in cellular metabolism (measured as ECAR) that were rapid in onset, saturable with respect to ligand concentration, and sensitive to several inhibitors of G-protein-coupled receptor signaling pathways. In sharp contrast, the neuroepithelioma SK-N-MC did not respond to any of the sweet or bitter compounds. Rapid changes in ECAR were easily detectable in both cell lines with the calcium ionophore A23187. Bradykinin elicited changes in the ECAR only in the NG108-15 cell line, which is known to express the B2 receptor. The changes in ECAR of the NG108-15 cell line in response to sweet and bitter taste compounds suggest these cells may expresses a receptor(s) specific for small sapid molecules.

Vasoactive amine responses in murine cerebrovascular endothelial cells as measured by extracellular acidification rates.

The Cytosensortrade mark microphysiometer device is capable detecting cellular responses to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). Using microphysiometry, we were able to determine that cerebovascular endothelial cells derived from SJL/J mice were more sensitive to serotonin (maximal ECAR response at 100 nM), whereas BALB/c-derived cerebrovascular endothelial cells (CVE) were relatively insensitive (maximal ECAR response at 30 microM). Serotonin (5HT)(1) and 5HT(2) receptor antagonists inhibited the serotonin-mediated increases in ECAR. The cells' responses to histamine in both strains were similar (maximal ECAR required 100 microM of histamine). H(1) and H(3) receptor subtype antagonists specifically inhibited the histamine responses. Bradykinin also revealed sensitivity differences in that maximal ECAR changes for CVE from SJL/J mice could be observed with 1 microM, as compared to the ECAR responses from BALB/c mice CVE, which required 10 microM. Exposure to bradykinin antagonists revealed that the response was due to the stimulation of B(2) receptors. These microphysiometry results reveal that the cerebrovascular endothelial cells of SJL/J mice tend to be more sensitive to vasoactive substances than those of BALB/c mice.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

Bioactive fragment of human interleukin-1beta (163-171) modulates the immune response to synthetic peptides of RESA of Plasmodium falciparum.

In this study a bioactive fragment, residues 163-171 of human interleukin-1beta (hu IL-1beta), that mimics the functions of the whole IL-1 molecule was chemically linked to two peptide sequences of the ring erythrocyte surface antigen (RESA), an asexual blood stage antigen of Plasmodium falciparum. The immunogenicity of different formulations was studied in different haplotypes of inbred mice. The peptide-IL-1beta conjugates delivered in liposomes showed the maximal antibody production. Antigen entrapped in liposomes at a dose of 5 microg showed antibody levels comparable to that of peptide conjugate in alum. The IgG subclass profile with different RESA peptides and its conjugates at various doses in liposomes induced predominantly IgG2a/2b isotypes while alum-delivered formulations showed both IgG1 and IgG2a/2b isotypes. In vitro studies of merozoite reinvasion showed varying degree (50-85%) of inhibition, maximum being with the peptide conjugates in liposomes (80-87%). Thus, this approach suggests that linking of hu IL-1beta is instrumental in augmenting the immune response against otherwise poorly immunogenic synthetic peptides.

Construction of chimeric immunogens: Bioactive fragment of human IL-1ß or polytuftsin (PT) capable of eliciting immune responses to HIV peptides.

In this study, we have examined the effect of linking of bloactive fragment of human IL-1ß (163-171) or polytuftsin (PT, a synthetic polymer of natural immunomodulator "tuftsin") with synthetic peptides of HIV on the induction of immune response to the synthetic peptides. A panel of synthetic peptides representing defined region of gp41, gp120 and gag were used as antigens. Immunomodulators linked peptides (i.e. peptide-IL-1ß or peptide-PT) or peptide dimers were employed for immunization in Balb/c mice. Mice immunized with the peptide-immunomodulator develop effective T-cell proliferation,in vitro cytokine release and higher antibody production, but not with peptide dimers. We also found that peptide-immunomodulators induced high level of IgG2a antibody production. Furthermore, there was a positive correlation between the levels of cytokine (IL-2 & IFN-γ) and IgG isotype production. Thus it would appear that incorporation of IL-1ß fragment or PT selectively enhances the Th1 type response to these peptides and may therefore be important for virus neutralization and clearance. However, the effect of IL-1ß fragment was found to be more pronounced than polytuftsin. Such an approach may provide effective vaccination against other infectious diseases.

Modification of delivery system enhances MHC nonrestricted immunogenicity of V3 loop region of HIV-1 gp120.

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2b, H-2d, H-2k and H-2s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P < 0.05 to P < 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.

Advantage of dimeric peptide antigens in serodiagnosis of HIV-1 infection.

The reactivity of antibodies with dimeric and monomeric peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV-1 representing defined regions, 598-609 and 524-533 (fusion domain) of gp41 and 306-320 of gp120, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV-1 infected individuals and 53 HIV-1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV-1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V3 loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4-fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV-1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV-1 infection.

Bioactive fragment of human IL-1beta [163-171] modulates the immune response to synthetic peptides of HIV.

The activation of T helper cells specific for viral antigens is critical for antibody production and the generation of cytotoxic T cells during retroviral infection. In this study, we examined the effect of linking HIV peptides with a bioactive fragment of human interleukin-1beta (IL-1beta) (163-171) on the induction of immune response to the peptides. A panel of highly purified synthetic peptides representing defined regions of gp41, Gag and gp120 were used as antigens. Mouse spleen cells primed with the peptide conjugates produced greater proliferation on in vitro stimulation than spleen cells primed with peptide alone. In addition, antibody production as assessed by ELISA was observed after immunization with conjugated peptides but not with peptide alone, indicating B-cell activation. We also found that a high level of IgG2a antibody production correlated with a high level of IFN-gamma production. These findings favor the notion that IL-1beta plays an important role in immune responses. These observations support the formulation and design of synthetic vaccines against HIV using synthetic HIV peptides conjugated with immunomodulators. Such an approach may provide an effective vaccination against other infectious agents.