An SPR based sensor for allergens detection.

A simple, sensitive and label-free optical sensor method was developed for allergens analysis using α-casein as the biomarker for cow's milk detection, to be used directly in final rinse samples of cleaning in place systems (CIP) of food manufacturers. A Surface Plasmon Resonance (SPR) sensor chip consisting of four sensing arrays enabling the measurement of samples and control binding events simultaneously on the sensor surface was employed in this work. SPR offers several advantages in terms of label free detection, real time measurements and superior sensitivity when compared to ELISA based techniques. The gold sensor chip was used to immobilise α-casein-polyclonal antibody using EDC/NHS coupling procedure. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions giving a detection limit of 58ngmL-1 as a direct binding assay. The assay sensitivity can be further improved by using sandwich assay format and amplified with nanoparticles. However, at this stage this is not required as the detection limit achieved exceeded the required allergens detection levels of 2µgmL-1 for α-S1-casein. The sensor demonstrated good selectivity towards the α-casein as the target analyte and adequate recoveries from CIP final rinse wash samples. The sensor would be useful tool for monitoring allergen levels after cleaning procedures, providing additional data that may better inform upon wider food allergen risk management decision(s) that are made by food manufacturer. In particular, this sensor could potentially help validate or optimise cleaning practices for a given food manufacturing process.

Ethylene oxide allergy in patients on hemodialysis waiting for kidney transplantation: logistical nightmare or challenge? A case report.

Ethylene oxide (EO) is widely used as a sterilization gas for heat-sensitive devices. In EO-sensitized patients, this type of sterilization can cause rare but major allergic reactions such as hives, rash, asthma, or anaphylactic shock. Hemodialysis patients in particular are at risk of developing hypersensitivity to EO. In these patients, surgical interventions should be planned far in advance allowing a thorough EO-free preparation of all equipment needed for the surgery as well as for the pre-, peri-, and postoperative care. In contrast to elective surgery, kidney transplantation with allografts from deceased donors cannot be planned; exact timing is unpredictable. Furthermore, transplantation may take place years after patients have been put on the waiting list. Listing of patients sensitive for EO is therefore a logistical and medical challenge for all health care professionals involved in the patient's care (eg, surgeons, nephrologists, anesthetists, nurses, pharmacists, and sterilization specialists). This case report describes a patient with chronic kidney disease stage V who developed EO allergy during hemodialysis while waiting for a kidney transplantation. Diagnosis was made based on clinical signs and confirmed biochemically (including a positive radioallergosorbent test). Because the only treatment is avoidance of contact with EO-sterilized materials, a strict EO-free protocol was developed to allow an uneventful transplantation thereafter. Subsequently, 4 newly diagnosed EO-sensitive patients on the active kidney transplantation waiting list were diagnosed, and 1 of these patients has been transplanted successfully. EO allergy in patients on the waiting list for kidney transplantation is a unique challenging situation which, to the best of our knowledge, has not been reported yet for kidney transplantation. This report further highlights the logistical preparation of a renal transplantation, including anesthesiologic, surgical, and postoperative care.

Role of CD4+ and CD8+ T cells in the rejection of heart or islet xenografts in recipients with xenotolerance in the innate immune compartment.

UNLABELLED: To further study the interactions between innate and adaptive immunity in xenotransplantation, we explored the relative contribution of T-cell subsets in vascularized (heart) and cellular (islets) xenografts in a model with established xeno-non-reactivity of the innate system. MATERIALS: Specific innate xenotolerance was induced in xenoheart (hamster) recipients (nude rats) by a tolerizing regimen (TR), consisting of donor antigen infusion, temporary natural killer (NK)-cell depletion and a 4-week administration of leflunomide. Hamster pancreatic islets were transplanted either 1 week after heart transplantation or alone and syngeneic T-cell adoptive transfer was performed 10 days later. Purified CD3(+), CD4(+), and CD8(+) T cells were given 2 weeks after withdrawal of all drugs. At the day of rejection, xenografts were removed for histology. Serum was taken and IgM and IgG xenoantibody titers were measured by flow cytometry. RESULTS: Both heart and islet grafts were rejected after CD4(+) reconstitution. After CD8(+) T-cell adoptive transfer, cellular grafts were not rejected but vascularized grafts were rejected, although only after several months. Rejection in CD4(+) reconstituted nude rats was accompanied by the generation of predominantly IgG xenoantibodies. CONCLUSION: CD4(+) T lymphocytes are able to rapidly initiate the rejection of islet xenografts in the presence of a xenotolerant innate immune system either by breaking the "innate tolerance" (e.g., by activating macrophages and NK-cells) or through a mechanism without any involvement of the innate tolerance (e.g., T-dependent IgG antibody production). In contrast, CD8(+) T cells provoke a late rejection of only xenoheart grafts.

9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine: a novel prodrug of 9-(2-phosphonylmethoxyethyl)guanine with improved antitumor efficacy and selectivity in choriocarcinoma-bearing rats.

The novel acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPr-PMEDAP) was shown in vitro to act as an intracellular prodrug of 9-(2-phosphonylmethoxyethyl)guanine (PMEG). We compared the in vivo antitumor efficacy and selectivity of cPr-PMEDAP, its progenitor PMEDAP, and PMEG in a rat choriocarcinoma tumor model. The rats, inoculated with rat choriocarcinoma (RCHO) cells under the renal capsule, were treated IP during 10 days. Macroscopical and histological examination of the RCHO-inoculated kidneys was performed at two time points (i.e., immediately after the end of treatment or after an additional drug-free period of 2 weeks). Complete inhibition of choriocarcinoma tumor development was achieved upon treatment with cPr-PMEDAP, PMEG, and PMEDAP at a daily dose of 10, 1, and 50 mg/kg, respectively. At these doses, all three compounds produced moderate to strong toxicity (evidenced by atrophy of lymphoid organs and reduced body weight gain). When compared at the maximum tolerated (sublethal) doses (i.e., 0.5, 10, and 50 mg/kg for PMEG, cPr-PMEDAP, and PMEDAP, respectively), cPr-PMEDAP proved superior to PMEG and PMEDAP in achieving a complete inhibition of tumor development. Also, whereas PMEG was unable to produce a prolonged antitumor effect, the animals treated with cPr-PMEDAP still showed prominent inhibition of tumor development when tumor size was evaluated at 2 weeks after end of treatment. Based on its efficacy and therapeutic safety, cPr-PMEDAP can be regarded as a promising antitumor agent, which merits further in vivo evaluation in additional tumor models for human neoplasms.

Induction of T-independent xenotolerance in a semi-discordant hamster-to-presensitized, nude rat model.

BACKGROUND: We have previously demonstrated that in a concordant hamster-to-nude rat cardiac transplant model, T-independent specific B-lymphocyte and natural killer (NK)-cell tolerance could be induced, leading to long-term xenograft (Xg) survival. Here, we investigated whether the same could be achieved in a clinically more relevant semi-discordant model involving hamster hearts transplanted into pre-sensitized, nude rats. METHODS: Sensitized, nude rats with high titers of anti-hamster immunoglobulin (Ig)M xenoantibodies (XAbs) were prepared by transplanting a first hamster heart without treatment. One week after rejection, a complete tolerizing regimen was given, including the following: a) an i.v. injection of hamster heart antigens; b) a 4-week administration of malononitriloamide; and c) a single injection of an anti-NK antiserum. Two weeks later, a second hamster heart was grafted. The isotype and level of XAb were examined by fluorescence-activated cell sorting. NK cytotoxicity was evaluated by a standard 4-hr 51Cr release assay. Hamster heart Xgs were examined by conventional histologic and immunohistochemical analysis. RESULTS: Untreated, presensitized, nude rats developing high titers of IgM XAb underwent hyperacute rejection within 4 hr (n=4) after transplantation of the second hamster heart. Immunohistochemical analysis showed intensive staining for IgM and C3 along the vascular endothelia in the rejected Xgs. In contrast, presensitized, nude rats receiving the complete tolerizing regimen had a rapid decrease in anti-hamster IgM XAb. The second hamster hearts were not rejected and showed long-term survival even after withdrawal of malononitriloamide (n=6). Moreover, tolerant rats showed specific B-lymphocyte tolerance and a specific continuous absence of anti-hamster NK-cell reactivity. CONCLUSION: T-independent B-lymphocyte and NK-cell xenotolerance can also be achieved in recipients with pre-existing IgM XAb.

Potent antitumor activity of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine in choriocarcinoma-bearing rats.

The acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective antiretroviral agent which is currently evaluated in its oral prodrug form, bis(POM)PMEA (adefovir dipivoxil), in phase II and III clinical trials in human hepatitis B virus (HBV)- and human immunodeficiency virus (HIV)-infected individuals, respectively. We have now found that PMEA is also a potent inhibitor of growth of the highly aggressive choriocarcinoma tumor arising from rat choriocarcinoma RCHO cells grafted under the kidney capsule of syngeneic WKA/H rats. In untreated rats, massive invasive RCHO tumors, covering the whole surface of the kidney and resulting in a marked enlargement of the kidney, were observed at day 10 after tumor cell grafting. Daily treatment with PMEA at 25 mg/kg/day afforded a marked reduction in tumor size (i.e., smaller tumors and slight, if any, enlargement of the kidney). Increasing the PMEA dose to 50, 100 or 250 mg/kglday resulted in a gradual increase of the antitumor effect of the compound. At the highest dose tested, i.e., 250 mg/kg/day, PMEA completely suppressed tumor growth. The antitumor activity of PMEA persisted for at least 10 days after termination of drug treatment. In addition, delayed treatment with PMEA at a dose of 200 mg/kg/day, started at a time point where choriocarcinoma tumors had already developed, stopped further growth and even induced regression of the tumors. PMPA, a closely related structural analogue of PMEA, failed to inhibit choriocarcinoma tumor growth. This observation points to the specificity of PMEA as an antitumor agent. In view of our findings, the therapeutic potential of PMEA for the treatment of neoplastic diseases appears to merit further investigation.

Use of the methylxanthine derivative A802715 in transplantation immunology: II. In vivo experiments.

BACKGROUND: We have previously demonstrated in vitro that the methylxanthine derivative A802715 suppresses the cyclosporine (CsA)-resistant "signal two"-dependent pathway of T cell activation and hence acts synergistically with CsA. Here, this synergism was further investigated in vivo in rats. METHODS: Primary cardiac allografts were placed in the neck, and secondary grafts were transplanted intra-abdominally. A802715 was given orally for 30 days or by continuous intravenous infusion via a mini-osmotic pump for 2 weeks. CsA was given orally for up to 30 days. T cell responses were examined in vitro using mixed lymphocyte reaction, concanavalin A whole blood, and cell-mediated lympholysis assays. RESULTS: In a major histocompatibility complex incompatible WKAH-->PVG combination, neither oral CsA (7.5 mg/kg/day) nor oral A802715 (100 mg/kg/day) was able to prolong graft survival. However, a combination of both drugs, given at the same dose, sustained graft survival during treatment. A similar synergism was not obtained with pentoxifylline, another methylxanthine derivative. The synergism between A802715 and CsA could be further increased by using a continuous intravenous infusion of A802715, since (1) lower doses of A802715 (20 mg/kg/day) and CsA (5 mg/kg/day) could be used, and (2) six of seven grafts survived permanently. In a major histocompatibility complex compatible Wag/Rij-->R/A combination, similar synergistic effects and permanent graft survival could also be obtained by oral A802715 (100 mg/kg/day) in combination with a low dose of CsA (2.5 mg/kg/day). In both strain combinations, long-term survivors accepted donor-type but rejected third-party second grafts in the absence of immunosuppression. This specific tolerance was not related to clonal deletion nor anergy, as recipient lymphocytes proliferated normally in the anti-donor mixed lymphocyte reaction. Instead, a defect in generating specific cytotoxic T lymphocytes was involved. CONCLUSIONS: A802715 synergizes with CsA in vivo to induce specific transplantation tolerance and hence should be considered as a promising new immunosuppressant.