Florida Red Tide: Inhalation Toxicity of Karenia brevis Extract in Rats.

Brevetoxins are neurotoxins produced by the marine dinoflagellate Karenia brevis. Histopathologic examination of marine mammals dying following repeated exposure of brevetoxins during red tide events suggests that the respiratory tract, nervous, hematopoietic, and immune systems are potential targets for toxicity in repeatedly exposed individuals. The purpose of this experiment was to evaluate the effects of repeated inhalation of K. brevis extract on these potential target systems in rats. Male Sprague-Dawley rats were exposed four hours/day, five days/week for up to four weeks to target concentrations of 200 and 1000 µg/L K. brevis extract (approximately 50 and 200 µg/L brevetoxin-like compounds; positive neurotoxicity in a fish bioassay). Control rats were sham exposed to air. Immunohistochemical staining of pulmonary macrophages indicated deposition of brevetoxin-like compound within the lung. However, exposure resulted in no clinical signs of toxicity or behavioral changes. There were no adverse effects on hematology or serum chemistry. No histopathological changes were observed in the nose, lung, liver, kidneys, lymph nodes, spleen, or brain of exposed rats. Immune suppression was suggested by reduced responses of spleen cells in the IgM-specific antibody-forming plaque cell response assay and reduced responses of lymphocytes to mitogen stimulation in vitro. Differences between responses observed in rats in this study and those observed in manatees may be a function of dose or species differences in sensitivity.

Brevetoxin Depuration in Shellfish via Production of Non-toxic Metabolites: Consequences for Seafood Safety and the Environmental Fate of Biotoxins.

During blooms of the dinoflagellate Karenia brevis, filter-feeders such as oysters and clams bioaccumulate brevetoxins, often to levels that are toxic to humans. In controlled aquarium experiments, we exposed live oysters to bloom levels of toxic K. brevis, followed by 10 weeks of exposure to non-toxic microalgae. Oysters were harvested weekly and analyzed for brevetoxins and brevetoxin metabolites to quantify toxin bioaccumulation and depuration. All of the PbTx-2 concentrated by oysters was immediately converted to a mixture of polar metabolites that were then slowly eliminated from the oysters. However, 90% of measured PbTx-3 was eliminated within two weeks of toxic exposure but without apparent biotransformation. Extracts of oysters containing high levels of PbTx-3 were toxic to mice by intraperitoneal (IP) injection. Extracts of oysters harvested after PbTx-3 had been eliminated were non-toxic despite high concentrations of PbTx-2 metabolites. Oysters collected in Florida during and after a bloom of K. brevis contained polar metabolites of PbTx-2 as well as PbTx-3, but no PbTx-2. Again, PbTx-3 concentration was a good predictor of mouse toxicity. One hundred percent conversion of PbTx-2 to polar metabolites was also accomplished in vitro by spiking oyster or clam homogenate with PbTx-2, followed by a brief incubation at room temperature. These PbTx-2 metabolites did not kill mice, either orally or by intraperitoneal injection, even at concentrations 30 times greater than toxic PbTx-3 levels.

Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium.

Minute amount of Brevetoxin PbTx-3 (400 microg; 0.446 micromol) was converted into an hemisuccinate derivative (PbTx-3 HS) then covalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) in a reversed micellar medium. According to the efficient cyclic synthetic procedure described, the epitope density of the conjugates was around 10 and 20 for OVA and BSA carriers, respectively. The kinetics of antibody production in sequential sera harvested from a single BALB/c mouse immunised by multiple intraperitoneal (i.p.) injections of PbTx-3-BSA conjugate was performed by enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies (MAbs) against PbTx-3 were selected from fusion of the mouse immune splenocytes with the P3-X63-Ag 8.653 myeloma cells. In competitive inhibition ELISA experiments, both polyclonal antibodies and MAbs exhibited strong cross-reactivity (> or = 100%) to other PbTx-2-type toxins (PbTx-2 and -9) but low or moderate cross-reactivity (6-15%) to a PbTx-1-type toxin (PbTx-1). Moreover, using these two MAbs, a low cross-reactivity with okadaic acid (3%) was noticed but no significant cross-reactivity was observed with two ciguatoxins (CTX-1B and CTX-3C) over the concentration range studied. The apparent dissociation constant (K(D)) for the interaction of these MAbs with free PbTx-2-type toxins was in the 10(-6)-10(-7)M range. The performance of this MAb-based assay (limit of detection approximately 5ng/well; working range=8-150ng/well) coupled with adequate extraction methods would provide an alternative assay to the mouse i.p. bioassay for routine shellfish monitoring. This production and characterisation of MAbs using small amount of polyether toxins in a reversed micellar medium appear most valuable for the development of immunoassays to other highly potent but poorly available marine polyether toxins like ciguatoxins (CTXs).

Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins.

As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of JKLM ring fragment, a carboxylic derivative of the right-hand tetracyclic terminus portion of CTX-1B (the most potent CTX) with two carrier proteins have been synthesized. Two procedures using different amount of hapten were evaluated: (i) a bulk technique (3-5 mg) via the N-hydroxysuccinimide ester of the carboxylic fragment in the presence of a water-soluble carbodiimide according to the standard method in aqueous buffer, or (ii) a micro-scale technique (300 microg) via the mixed anhydride method performed in a reversed micellar medium. In both cases, bovine serum albumin and ovalbumin were respectively used for immunization of BALB/c mice and antibody screening by a solid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedule appeared to be more efficient to specifically trigger the mice immune system. These antisera titers determined in an end-point titration standard ELISA format were found around 1/128,000 as compared to 1/16,000 obtained in the short-term protocol (immunogen prepared via the bulk procedure). In competitive inhibition ELISA experiments, both types of antisera did not significantly cross-react with a brevetoxin congener (PbTx-3), okadaic acid (OA), monensin or other polyether compounds, but only sera from the short-term protocol did show high cross-reactivity to CTX-1B (133%). With sera from the long-term protocol, a lower detection limit for JKLM (1.23 x 10(-9) M) was achieved by implementation of a biotin-avidin amplification system rather than by miniaturization of the assay in Terasaki plates. This study confirms the feasibility of the immunological approach for CTXs assay in fish tissues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen, (ii) the immunization schedule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs.

Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay.

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.

An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens.

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.

Intragraft cytokine expression in tolerant rat renal allografts with rapamycin and cyclosporin immunosuppression.

The Th-1/Th-2 paradigm proposes clonal expansion of Th-2 lymphocytes as the basis of tolerance towards allografts. Intragraft cytokine expression was evaluated in a highly stringent model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. Group A (n = 8) received a single dose of rapamycin and cyclosporin 12 h prior to engraftment, followed by 7 d of cyclosporin post-operatively. Isografts (Group B, n = 5) and control allografts (Group C, n = 4) received no immunosuppression. Sacrifice was performed after 120 d. Intragraft expression of IL-10, IL-4, and IFN-gamma was determined using qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). All groups had functionally normal grafts at sacrifice, with 50% histological tolerance among Group A animals. No isografts showed evidence of cellular infiltrate, and all control allografts showed severe rejection. IL-10 was only detected in the tolerant animals (p < 0.001). Similarly, IL-4 was detected predominantly in the tolerant allografts (p < 0.05). IFN-gamma was only isolated in rejected allografts, whether treated or untreated (p < 0.001). We conclude that the expansion of Th-2 cells is associated with tolerance, while the expansion of Th-1 cell is associated with acute cellular rejection.

Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels.

Ciguatoxins (CTXs) and brevetoxins (PbTxs) modify the activation and inactivation processes of voltage-sensitive sodium channels (VSSC). In this study, the specific binding to rat brain synaptosomes of two commercial PbTxs, five purified CTXs and their derivatives was evaluated in competition with various concentrations of radiolabelled brevetoxin ([3H]PbTx-3). The results indicate that all CTXs bind specifically and with high affinity to sodium channels. Statistical analysis of the calculated inhibition constants identified two classes of toxins: the PbTxs and the less polar CTXs, and a group of CTXs of very high affinity. Relatively small chemical differences between the CTXs gave rise to significant differences in their affinity to the rat brain sodium channels. Cytotoxic effects associated with sodium channel activation were evaluated for the two classes of toxins on murine neuroblastoma cells, and their acute toxicity was determined in mice. CTXs have shown high affinities to VSSC of rat brain membranes and strong cytotoxic effects on neuroblastoma cells which correlate with their very low LD50 in mice. For PbTxs, it is different. Although binding with high affinity to VSSC and giving rise to significant cytotoxic effects, they are known to be poorly toxic intraperitoneally to mice. Furthermore, within the CTXs family, even though the most toxic compound (CTX-1B) has the highest affinity and the less toxic one (CTX-4B) the lowest affinity, a detailed analysis of the data pointed out a complex situation: (i) high affinity and toxicity seem to be related to the hydroxylation of the molecule on the A-ring rather than to the backbone type, (ii) acute toxicity in mice does not follow exactly the sodium-dependent cytotoxicity on neuroblastoma cells. These data suggest that the high toxicity of CTXs is related to sodium-dependent disturbances of the excitable membranes but might also involve other cellular mechanisms.

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate.

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (ABA) and two other closely related small chromophoric haptens. The amide bond-generating mixed anhydride method of Erlanger was modified to promote conjugation to various proteins (bovine serum albumin, ovalbumin, casein and hemocyanin) or to a synthetic homopolymer (Poly-DL-lysine). The key process in this synthesis is the use of a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement at characteristic hapten absorption peaks. This coupling procedure is applicable to as little hapten material as 0.2 micromol and is disclosed to be most valuable for other rare lipid haptens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple intraperitoneal injections of (ABA)23-BSA conjugate as revealed by indirect and competitive ELISA. Calculated KD for the interaction of the antibodies with free ABA was found to be 5 X 10(-5)M.

Flow cytometric analysis of chimerism in the rat tolerant to a renal allograft.

BACKGROUND: Chimerism, produced by the two-way migration of cells between graft and host, is a proposed mechanism by which tolerance occurs. The appearance of donor/recipient chimeras in tolerant ACI to Lewis rat heterotopic renal transplants was assessed in peripheral blood leukocytes using flow cytometry after staining with monoclonal antibodies. MATERIALS AND METHODS: ACI and Lewis rats were used as donor and recipient, respectively, after Rapamycin and Cyclosporin immunosuppression with or without donor blood or bone marrow transfusion. ACI and Lewis animals were also used for isograft and single-kidney controls. Animals were sacrificed at various time points after initial operation. Flow cytometry was performed on isolated peripheral blood leukocytes at sacrifice. Histologic and functional data were also obtained. The monoclonal antibody panel included RT1(a) (ACI, MHC I) combined with CD2, CD4, CD8, CD16, and CD25 or RT1(a,c) (bone marrow chimeras). RESULTS: RT1(a)+, CD8+ cells were transiently present in the peripheral blood leukocytes of Lewis recipients with the exception of allogeneic bone marrow recipients. No significant number of RT1(a)+, CD16+ ("dendritic" cell-line) chimeras was seen. Veto cells (RT1(a,c)+) were transiently present in the bone marrow recipients, but they did not lead to improved outcome. Furthermore, no correlation was made between histologic tolerance and any of these donor-derived cells. CONCLUSION: Donor/recipient chimerism, and the veto cell phenomenon are not operational tolerance mechanisms in this stringent model of ACI to Lewis rat renal transplantation.

The immunologic role of IFN-gamma in ACI to Lewis kidney transplantation.

BACKGROUND: One of the proposed mechanisms of tolerance induction is the Th-1/Th-2 paradigm. The Th-1 cell is proinflammatory, secreting IFN-gamma and IL-2. Conversely, the Th-2 cell is anti-inflammatory, secreting IL-4 and IL-10. In our earlier studies a shift toward Th-2 dominance was required for tolerance induction in this model. MATERIALS AND METHODS: ACI and Lewis rats were used as donors and recipients, respectively. Twelve hours prior to engraftment, rapamycin 1.5 mg/kg po and cyclosporin 10 mg/kg sc were given, followed by 5 mg/kg sc postop (days 1-7). Lewis rats were used as isografts. Functional allograft tolerance was induced consistently in 100% of the recipients with 50% of the allografts exhibiting normal histology beyond 120 days. Qualitative RT-PCR was performed on the grafts to determine IFN-gamma expression with beta-actin housekeeping gene as control. RESULTS: IFN-gamma was expressed in all untreated allografts (5/5) and all treated, yet rejecting, allografts (4/4). None of the isografts (0/5) or histologically tolerant allografts (0/4) expressed IFN-gamma. This distribution was statistically significant (P < 0.001, Fischer's exact test). CONCLUSION: Our findings support a shift from Th-2 to Th-1 predominance as the corollary mechanism responsible for preventing histologic tolerance.

Paragangliomas del cuerpo carotídeo / Paraganglioma of the carotid body

Los tumores del cuerpo carotideo (paragangliomas, quimiodectomas), se originan en el tejido paraganglionico de la bifurcaci6n carotidea; son lesiones de lenta evolución que aumentan gradualmente en el curso de los años y pueden comprimir las estructuras neurovasculares del cuello; su transformación maligna es rara. La lesión, a menudo asintomatica, puede aparecer en la tercera o cuarta decadas de la vida y es mas frecuente en personas que viven a grandes alturas, y en pacientes con enfermedad pulmonar obstructiva crónica (EPOC). El propósito de este estudio es enfatizar los beneficios de nuestra tecnica quirurgica, ya que con ella hemos obtenido excelentes resultados. Vale destacar la utilidad de la angiografia carotidea bilateral en el estudio y tratamiento de esta patologia. En nuestros pacientes tratados quirurgicamente no se presentó ninguna complicacion definitiva, y la unica morbilidad fue una paresia de la rama mandibular del nervio facial y otra del hipogloso; tampoco hubo mortalidad.