Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

Redirecting anti-viral CTL against cancer cells by surface targeting of monomeric MHC class I-viral peptide conjugated to antibody fragments.

To combine the advantage of both the tumor targeting capacity of high affinity monoclonal antibodies (mAbs) and the potent killing properties of cytotoxic T lymphocytes (CTL), we investigated the activity of conjugates made by coupling single Fab' fragments, from mAbs specific for tumor cell surface antigens, to monomeric HLA-A2 complexes containing the immunodominant influenza-matrix peptide 58-66. In solution, the monovalent 95 kDa Fab-HLA-A2/Flu conjugates did not activate influenza-specific CTL. However, when targeted to tumor cells expressing the relevant tumor-associated antigen, the conjugates induced CTL activation and efficient tumor cell lysis, as a result of MHC/peptide surface oligomerization. The highly specific and sensitive in vitro cytotoxicity results presented suggest that injection of Fab-MHC/peptide conjugates could represent a new form of immunotherapy, bridging antibody and T lymphocyte attack on cancer cells.

Sensitive headspace gas chromatography analysis of free and conjugated 1-methoxy-2-propanol in urine

Glycol ethers still continue to be a workplace hazard due to their important use on an industrial scale. Currently, chronic occupational exposures to low levels of xenobiotics become increasingly relevant. Thus, sensitive analytical methods for detecting biomarkers of exposure are of interest in the field of occupational exposure assessment. 1-Methoxy-2-propanol (1M2P) is one of the dominant glycol ethers and the unmetabolized urinary fraction has been identified to be a good biological indicator of exposure. An existing analytical method including a solid-phase extraction and derivatization before GC/FID analysis is available but presents some disadvantages. We present here an alternative method for the determination of urinary 1M2P based on the headspace gas chromatography technique. We determined the 1M2P values by the direct headspace method for 47 samples that had previously been assayed by the solid-phase extraction and derivatization gas chromatography procedure. An inter-method comparison based on a Bland-Altman analysis showed that both techniques can be used interchangeably. The alternative method showed a tenfold lower limit of detection (0.1 mg/L) as well as good accuracy and precision which were determined by several urinary 1M2P analyses carried out on a series of urine samples obtained from a human volunteer study. The within- and between-run precisions were generally about 10%, which corresponds to the usual injection variability. We observed that the differences between the results obtained with both methods are not clinically relevant in comparison to the current biological exposure index of urinary 1M2P. Accordingly, the headspace gas chromatography technique turned out to be a more sensitive, accurate, and simple method for the determination of urinary 1M2P.[Authors]

Long circulating half-life and high tumor selectivity of the photosensitizer meta-tetrahydroxyphenylchlorin conjugated to polyethylene glycol in nude mice grafted with a human colon carcinoma.

In a mode of nude mice bearing a human colon carcinoma xenograft, the biodistribution and tumor localization of metatetrahydroxyphenylchlorin (m-THPC) coupled to polyethylene glycol (PEG) were compared with those of the free form of this photosensitizer used in photodynamic therapy (PDT). At different times after i.v. injection of both forms of 125I-labeled photosensitizer, m-THPC-PEG gave on average a 2-fold higher tumor uptake than free m-THPC. In addition, at early times after injection, m-THPC-PEG showed a 2-fold longer blood circulating half-life and a 4-fold lower liver uptake than free m-THPC. The tumor to normal tissue ratios of radioactivity concentrations were always higher for m-THPC-PEG than for free m-THPC at any time point studied from 2 to 96 hr post-injection. Significant coefficients of correlation between direct fluorescence measurements and radioactivity counting were obtained within each organ tested. Fluorescence microscopy studies showed that m-THPC-PEG was preferentially localized near the tumor vessels, whereas m-THPC was more diffusely distributed inside the tumor tissue. To verify whether m-THPC-PEG conjugate remained phototoxic in vivo, PDT experiments were performed 72 hr after injection and showed that m-THPC-PEG was as potent as free m-THPC in the induction of tumor regression provided that the irradiation does for m-THPC-PEG conjugate was adapted to a well-tolerated 2-fold higher level. The overall results demonstrate first the possibility of improving the in vivo tumor localization of a hydrophobic dye used for PDT by coupling it to PEG and second that a photosensitizer conjugated to a macromolecule can remain phototoxic in vivo.

Impact of dietary betaine and conjugated linoleic acid on insulin sensitivity, protein and fat metabolism of obese pigs.

To determine possible mechanisms of action that might explain the nutrient partitioning effect of betaine and conjugated linoleic acid (CLA) in Iberian pigs and to address potential adverse effects, twenty gilts were restrictively fed from 20 to 50 kg BW Control, 0.5% betaine, 1% CLA or 0.5% betaine + 1% CLA diets. Serum hormones and metabolites profile were determined at 30 kg BW and an oral glucose test was performed before slaughter. Pigs were slaughtered at 50 kg BW and livers were obtained for chemical and histological analysis. Decreased serum urea in pigs fed betaine and betaine + CLA diets (11%; P = 0.0001) indicated a more efficient N utilization. The increase in serum triacylglycerol (58% and 28%, respectively; P = 0.0098) indicated that CLA and betaine + CLA could have reduced adipose tissue triacylglycerol synthesis from preformed fatty acids. Serum glucose, low-density lipoprotein (LDL) cholesterol and non-esterified fatty acids were unaffected. CLA and betaine + CLA altered serum lipids profile, although liver of pigs fed CLA diet presented no histopathological changes and triglyceride content was not different from Control pigs. Compared with controls, serum growth hormone decreased (20% to 23%; P = 0.0209) for all treatments. Although serum insulin increased in CLA, and especially in betaine + CLA pigs (28% and 83%; P = 0.0001), indices of insulin resistance were unaffected. In conclusion, CLA, and especially betaine + CLA, induced changes in biochemical parameters and hormones that may partially explain a nutrient partitioning effect in young pigs. Nevertheless, they exhibited weak, although detrimental, effects on blood lipids. Moreover, although livers were chemically and histologically normal, pigs fed CLA diet challenged with a glucose load had higher serum glucose than controls.

Distribution of free and conjugated cannabinoids in human bile samples.

The metabolism of Δ(9)-tetrahydrocannabinol (THC) is relatively complex, and over 80 metabolites have been identified. However, much less is known about the formation and fate of cannabinoid conjugates. Bile excretion is known to be an important route for the elimination of phase II metabolites. A liquid chromatography-tandem mass spectrometry LC-MS/MS procedure for measuring cannabinoids in oral fluid was adapted, validated and applied to 10 bile samples. THC, 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), Δ(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc) and Δ(9)-tetrahydrocannabinol glucuronide (THC-gluc) were determined following solid-phase extraction and LC-MS/MS. High concentrations of THCCOOH-gluc were found in bile samples (range: 139-21,275 ng/mL). Relatively high levels of THCCOOH (7.7-1548 ng/mL) and THC-gluc (38-1366 ng/mL) were also measured. THC-A, the plant precursor of THC, was the only cannabinoid that was not detected. These results show that biliary excretion is an important route of elimination for cannabinoids conjugates and that their enterohepatic recirculation is a significant factor to consider when analyzing blood elimination profiles of cannabinoids. Furthermore, we suggest that the bile is the matrix of choice for the screening of phase II cannabinoid metabolites.

Anharmonicity contributions to the vibrational second hyperpolarizability of conjugated oligomers

Restricted Hartree-Fock 6-31G calculations of electrical and mechanical anharmonicity contributions to the longitudinal vibrational second hyperpolarizability have been carried out for eight homologous series of conjugated oligomers - polyacetylene, polyyne, polydiacetylene, polybutatriene, polycumulene, polysilane, polymethineimine, and polypyrrole. To draw conclusions about the limiting infinite polymer behavior, chains containing up to 12 heavy atoms along the conjugated backbone were considered. In general, the vibrational hyperpolarizabilities are substantial in comparison with their static electronic counterparts for the dc-Kerr and degenerate four-wave mixing processes (as well as for static fields) but not for electric field-induced second harmonic generation or third harmonic generation. Anharmonicity terms due to nuclear relaxation are important for the dc-Kerr effect (and for the static hyperpolarizability) in the σ-conjugated polymer, polysilane, as well as the nonplanar π systems polymethineimine and polypyrrole. Restricting polypyrrole to be planar, as it is in the crystal phase, causes these anharmonic terms to become negligible. When the same restriction is applied to polymethineimine the effect is reduced but remains quantitatively significant due to the first-order contribution. We conclude that anharmonicity associated with nuclear relaxation can be ignored, for semiquantitative purposes, in planar π-conjugated polymers. The role of zero-point vibrational averaging remains to be evaluated

Basis set and electron correlation effects on initial convergence for vibrational nonlinear optical properties of conjugated organic molecules

The level of ab initio theory which is necessary to compute reliable values for the static and dynamic (hyper)polarizabilities of three medium size π-conjugated organic nonlinear optical (NLO) molecules is investigated. With the employment of field-induced coordinates in combination with a finite field procedure, the calculations were made possible. It is stated that to obtain reasonable values for the various individual contributions to the (hyper)polarizability, it is necessary to include electron correlation. Based on the results, the convergence of the usual perturbation treatment for vibrational anharmonicity was examined

Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

PURPOSE: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats. METHODS: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy. RESULTS: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP. CONCLUSIONS: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Spin-Peierls vs Peierls distortions in a family of conjugated polymers

Distortions in a family of conjugated polymers are studied using two complementary approaches: within a many-body valence bond approach using a transfer-matrix technique to treat the Heisenberg model of the systems, and also in terms of the tight-binding band-theoretic model with interactions limited to nearest neighbors. The computations indicate that both methods predict the presence or absence of the same distortions in most of the polymers studied.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.

Crystallization and properties of poly(ethylene terephtalate) copolymers containing 5-tert-butyl isophtalic units

The influence of incorporating 5-tert-butyl isophthalic units (tBI) in the polymer chain of poly(ethylene terephthalate) (PET) on the crystallization behavior, crystal structure, and tensile and gas transport properties of this polyester was evaluated. Random poly(ethyleneterephthalate-co-5-tert-butyl isophthalate) copolyesters (PETtBI) containing between 5 and 40 mol% of tBI units were examined. Isothermal crystallization studies were performed on amorphous glassy films at 120 8C and on molten samples at 200 8C by means of differential scanning calorimetry. Furthermore, the non-isothermal crystallization behavior of the copolyesters was investigated. It was observed that both crystallinity and crystallization rate of the PETtBI copolyesters tend to decrease largely with the comonomeric content, except for the copolymer containing 5 mol% of tBI units, which crystallized faster than PET. Fiber X-ray diffraction patterns of the semicrystalline PETtBI copolyesters proved that they adopt the same triclinic crystal structure as PET with the comonomeric units being excluded from the crystalline phase. Although PETtBI copolyesters became brittle for higher contents in tBI, the tensile modulus and strength of PET were barely affected by copolymerization. The ncorporation of tBI units slightly increased the permeability of PET, but copolymers containing up to 20 mol% of the comonomeric units were still able to present barrier properties.