Biodegradable amphiphilic polymer-drug conjugate micelles

The coupling of drugs to macromolecular carriers received an important impetus from Ringsdorf's notion of polymer-drug conjugates. Several water-soluble polymers, poly(ethylene glycol), poly[N-(2-hydroxypropyl) methacrylamidel, poly(L-glutamic acid) and dextran, are studied intensively and have been utilized successfully in clinical research. The promising results arising from clinical trials with polymer-drug conjugates (e.g., paclitaxel, doxorubicin, camptothecins) have provided a firm foundation for other synthetic polymers, especially biodegradable polymers, used as drug delivery vehicles. This review discusses biodegradable polymeric micelles as an alternative drug-conjugate system. Particular focus is on A-B or B-A-B type biodegradable amphiphilic block copolymer such as polylactide, morpholine-2,5-dione derivatives and cyclic carbonates, which can form a core-shell micellar structure, with the hydrophobic drug-binding segment forming the hydrophobic core and the hydrophilic segment as a hydrated outer shell. Polymeric micelles can be designed to avoid uptake by cells of reticuloendothelial system and thus enhance their blood lifetime via the enhanced permeability and retention effect.

Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate

A paclitaxel/MPEG-PLA block copolymer conjugate was prepared in three steps: (1) hydroxyl-terminated diblock copolymer of monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA) was synthesized by ring-opening polymerization of L-lactide using MPEG as a maroinitiator, (2) it was converted to carboxyl-terminated MPEG-PLA by reacting with mono-i-butyl ester of diglycolic acid and subsequent deprotecting the t-butyl group with TFA; (3) the latter was reacted with paclitaxel in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Structures of the polymers synthesized were confirmed by H-1 NMR, and their molecular weights were determined by gel permeation chromatography. The antitumor activity of the conjugate against human liver cancer H7402 cells was evaluated by MTT method. The results showed that paclitaxel can be released from the conjugate without losing cytotoxicity.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.

Characterization of the artificially covalent conjugate of B-phycoerythrin and R-phycocyanin and the phycobilisome from Porphyridium cruentum

B-phycoerythrin (BPE) and R-phycocyanin (RPC) were purified from Porphyridium cruentum by Sephadex G-200 chromatography, then the BPE was attached covalently to the RPC by reacting their amino groups to form the artificially covalent BPE-RPC conjugate in which the excitation energy can transfer from the BPE to the RPC with low efficiency. Meanwhile, the intact phycobilisome (PBS) consisting of BPE, RPC, APC and L-CM was isolated and purified from Porphyridium cruentum, and the purified PBS was found to keep intact if the solution contains sucrose. Comparison of spectroscopic properties between the purified PBS and the BPE-RPC conjugate suggests that the BPE-RPC conjugate is much more stable than the purified PBS. The construction of BPE-RPC conjugate with low efficiency of the excitation energy transfer may be useful for preparing phycobiliprotein probes. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their epsilon-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or L-cm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 degrees C for various times showed that the connection between the APC and the APC B or L-cm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS.

The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice

The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SE Delta waaL) or deep-defective (SE Delta gal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SE Delta waaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SE Delta waaL as non-live vaccine in the mouse model.

Optimal assessment of the ability of children with recurrent respiratory tract infections to produce anti-polysaccharide antibodies.

Specific anti-polysaccharide antibody deficiency (SPAD) is an immune disorder. Diagnostic criteria have not yet been defined clearly. One hundred and seventy-six children evaluated for recurrent respiratory tract infections were analysed retrospectively. For each subject, specific anti-pneumococcal antibodies had been measured with two enzyme-linked immunosorbent assays (ELISAs), one overall assay (OA) using the 23-valent pneumococcal polysaccharide vaccine (23-PPSV) as detecting antigen and the other purified pneumococcal polysaccharide serotypes (serotype-specific assay, SSA) (serotypes 14, 19F and 23F). Antibody levels were measured before (n = 176) and after (n = 93) immunization with the 23-PPSV. Before immunization, low titres were found for 138 of 176 patients (78%) with OA, compared to 20 of 176 patients (11%) with the SSA. We found a significant correlation between OA and SSA results. After immunization, 88% (71 of 81) of the patients considered as responders in the OA test were also responders in the SSA; 93% (71 of 76) of the patients classified as responders according to the SSA were also responders in the OA. SPAD was diagnosed in 8% (seven of 93) of patients on the basis of the absence of response in both tests. Thus, we propose to use OA as a screening test for SPAD before 23-PPSV immunization. After immunization, SSA should be used only in case of a low response in OA. Only the absence of or a very low antibody response detected by both tests should be used as a diagnostic criterion for SPAD.

Large decrease of anti-tetanus anatoxin and anti-pneumococcal antibodies at one year after renal transplantation

info:eu-repo/semantics/published

Evaluation of a WHO-Validated Serotype-Specific Serological Assay for the Diagnosis of Pneumococcal Etiology in Children with Community-Acquired Pneumonia.

BACKGROUND: The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. METHODS: In this study, pneumococcal aetiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalised in Belgium with X-ray-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of Streptococcus pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for nine serotypes using World Health Organization validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with non proven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal aetiology in 55% of patients with non-proven P-CAP. Serotypes 1 (51.6%), 7F (19%), and 5 (15.7%) were the most frequent according to IgG ELISA. CONCLUSIONS: In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.

Large decrease of anti-tetanus anatoxin and anti-pneumococcal antibodies at one year after renal transplantation.

Aims: In kidney transplant recipients (KTR), antibody (Ab) synthesis is hampered by AZA and CsA. We here report in a prospective cohort study, the effects of mycophenolate mofetil (MMF) associated to a calcineurin inhibitor on plasma levels of anti-tetanus anatoxin Ab (TAnAb) and anti-pneumococcal Ab (PnPsAb). Methods: Serum titers of the TAnAb and the PnPsAb against serotypes 14, 19F and 23F were measured in 94 KTR on Day 0 (T0) and 1 year (T12) after renal transplantation and in 49 healthy controls. Results: 1) At T0, TAnAb were detected in only 71% of patients vs. 98% of controls (p < 0.0001) and the titers were significantly lower in KTR (1.46 UI/ml vs. 2.74 in controls, p = 0.01); they further decreased between T0 and T12 (1.46 UI/ml to 0.31, p < 0.0001). The calculated half-life (t1/2) of TAnAb was 7.7 months, as compared to more than 10 years in a normal population. 2) In KTR, PnPsAb titers decreased significantly between T0 and T12 (p < 0.005); the t1/2 of the different PnPsAb ranged from 9.2 to 11.9 months. Conclusions: In KTR treated by MMF and CNI, the TAnAbs and PnPsAbs titers decrease significantly and profoundly during the first year. Immunization pre-transplantation should be encouraged to maintain adequate post-transplant Abs levels.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

Pneumococcal etiology in hospitalised children with community-acquired penumonia in Belgium :evaluation by culture, RT-PCR and serotype-specific IgG and IgA serology.

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How a different look at latency can help to develop novel diagnostics and vaccines against tuberculosis.

Mycobacterium tuberculosis is one of the most successful human pathogens. It kills every year approximately 1.5 - 2 million people, and at present a third of the human population is estimated to be infected. Fortunately, only a relatively small proportion of the infected individuals will progress to active disease, and most will maintain a latent infection. Although a latent infection is clinically silent and not contagious, it can reactivate to cause highly contagious pulmonary tuberculosis, the most prevalent form of the disease in adults. Therefore, a thorough understanding of latency and reactivation may help to develop novel control strategies against tuberculosis. The most widely held view is that the mycobacteria are imprisoned in granulomatous structures during latency, where they can survive in a non-replicating, dormant form until reactivation occurs. However, there is no hard data to sustain that the reactivating mycobacteria are indeed those that laid dormant within the granulomas. In this review an alternative model, based on evidence from early studies, as well as recent reports is presented, in which the latent mycobacteria reside outside granulomas, within non-macrophage cell types throughout the infected body. Potential implications for new diagnostic and vaccine design are discussed.