Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs

BACKGROUND: Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. METHODS: Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. RESULTS: Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. CONCLUSIONS: The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Potent innate immune response to pathogenic leptospira in human whole blood.

BACKGROUND Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600–800 μCi of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm3) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay

Neisseria gonorrhoeae has well-characterized oxidative stress defense systems that protect against oxidative killing in in vitro assays. In contrast, mutant strains of N. gonorrhoeae lacking oxidative stress defenses are identical to the wild type when tested in an ex vivo survival assay using human polymorphonuclear leukocytes.

New insights into the interactions of serum proteins with bis(maltolato)oxovanadium(IV): Transport and biotransformation of insulin-enhancing vanadium pharmaceuticals

Significant new insights into the interactions of the potent insulin-enhancing compound bis(maltolato)oxovanadium(IV) (BMOV) with the serum proteins, apo-transferrin and albumin, are presented. Identical reaction products are observed by electron paramagnetic resonance (EPR) with either BMOV or vanadyl sulfate (VOSO4) in solutions of human serum apo-transferrin. Further detailed study rules out the presence of a ternary ligand-vanadyl-transferrin complex proposed previously. By contrast, differences in reaction products are observed for the interactions of BMOV and VOSO4 with human serum albumin (HSA), wherein adduct formation between albumin and BMOV is detected. In BMOV-albumin solutions, vanadyl ions are bound in a unique manner not observed in comparable solutions Of VOSO4 and albumin. Presentation of chelated vanadyl ions precludes binding at the numerous nonspecific sites and produces a unique EPR spectrum which is assigned to a BMOV-HSA adduct. The adduct species cannot be produced, however, from a solution Of VOSO4 and HSA titrated with maltol. Addition of maltol to a VOSO4-HSA solution instead results in formation of a different end product which has been assigned as a ternary complex, VO(ma)(HSA). Furthermore, analysis of solution equilibria using a model system of BMOV with 1-methylimidazole (formation constant log K = 4.5(1), by difference electronic absorption spectroscopy) lends support to an adduct binding mode (VO(ma)(2)-HSA) proposed herein for BMOV and HSA. This detailed report of an in vitro reactivity difference between VOSO4 and BMOV may have bearing on the form of active vanadium metabolites delivered to target tissues. Albumin binding of vanadium chelates is seen to have a potentially dramatic effect on pharmacokinetics, transport, and efficacy of these antidiabetic chelates.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Levels of dioxin-like PCBs in low-volume serum samples of male patients attending fertility clinics

An accurate and easy method for the extraction, cleanup, and HRGC-HRMS analysis of dioxin-like PCBs (DL-PCBs) in low-volume serum samples (1 mL) was developed. Serum samples were extracted several times using n-hexane and purified by acid washing. Recovery rates of labeled congeners ranged from 70 to 110 % and the limits of detection were below 1 pg/g on lipid basis. Although human studies are limited and contradictory, several studies have shown that DL-PCBs can have adverse effects on the male reproductive system. In this way, the present method was applied to 21 serum samples of male patients attending fertility clinics. The total levels obtained for the patients ranged from 6.90 to 84.1 pg WHO-TEQ/g lipid, with a mean value of 20.3 pg WHO-TEQ/g lipid. The predominant PCBs (the sum of PCB 118, 156, and 105) contributed 67 % to the mean concentration of total DL-PCBs in the samples analyzed.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Medida da capacidade antioxidante de fluidos biologicos por voltametria de pulso diferencial associada ao uso de surfactante como resposta as adaptações geradas pelo exercicio

Universidade Estadual de Campinas . Faculdade de Educação Física

Avaliação cintilográfica de diferentes dosagens de Tecnécio-99m na padronização da perfusão pulmonar em cães da raça Rottweiller

A avaliação da função pulmonar em cães pode ser obtida mediante exames cintilográficos, os quais incluem a injeção de radiofármacos, seguida de radiografias. Neste estudo, foram utilizados 10 cães machos hígidos da raça Rottweiler e radiograficamente normais e soronegativos para dirofilariose, com administração de doses variadas de macroagregado de albumina sérica humana marcados com tecnécio-99m ([99mTc](MAA)). Os resultados foram avaliados qualitativa e quantitativamente, considerando o índice de perfusão pulmonar e sua homogeneidade, sendo dessa maneira visualizado um aumento na homogeneidade da imagem diretamente relacionado ao número de partículas de MAA injetado. Este estudo define os critérios de normalidade e de dosagem na perfusão pulmonar em cães hígidos da raça Rottweiller estabelecidos com a técnica de cintilografia.

Validação de um radioimunoensaio para a quantificação de metabólitos fecais de testosterona em hamster Sírio (Mesocricetus auratus)

Modelo para o estudo de diversas doenças humanas, o hamster tem sido objeto de numerosos estudos comportamentais e envolvendo estresse e/ou comportamento agonístico que supõem, muitas vezes, o monitoramento das flutuações hormonais nos indivíduos envolvidos. O objetivo do presente trabalho foi confirmar a adequação de um conjunto comercial para dosagem de testosterona em sangue humano para a quantificação de metabólitos fecais de testosterona (MFT) em hamsters Sírios machos e fêmeas. Dez machos foram submetidos a um desafio com um agonista de GnRH para estimular a atividade testicular, elevando os níveis circulantes de testosterona. Cinco fêmeas receberam uma injeção de testosterona enquanto cinco outras receberam uma injeção de solução salina. Amostras de fezes coletadas antes e depois dos procedimentos, assim como amostras fecais de 20 fêmeas gestantes coletadas ao longo da gestação foram analisadas com um conjunto comercial para radioimunoensaio. Um pico de MFT 12h após a injeção seguido de uma queda abaixo do nível basal comprovou que, nos machos, as alterações nos níveis de MFT refletem as alterações da concentração de testosterona no sangue. Nestes observou-se um ciclo circadiano das concentrações de MFT com acrofase no início do período claro correspondendo ao ciclo descrito para as concentrações sanguíneas na literatura. Nas fêmeas a administração de testosterona exógena provocou uma elevação importante dos níveis de MFT, mas as concentrações medidas ao longo da gestação não refletiram o padrão dos níveis sanguíneos do hormônio endógeno. O conjunto para radioimunoensaio para testosterona em sangue humano mostrou-se adequado para o monitoramento da função testicular no hamster macho, mas um ensaio mais específico seria necessário para as fêmeas.

Hantavirus Pulmonary Syndrome, Central Plateau, Southeastern, and Southern Brazil

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HIPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent lung tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.