Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.     

Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.     

Functional and anatomical evidence for different dopamine dynamics in the core and shell of the nucleus accumbens in slices of rat brain

BACKGROUND: Chlorambucil (CLB), 2-chlorodeoxyadenosine (2-CDA) and fludarabine (FAMP) are among the most widely used drugs in chronic lymphocytic leukemia (CLL). Therefore we evaluated in vitro sensitivity to these drugs and cross-resistance of purine analogs. In addition, we correlated the in vitro data with the main clinico-hematological variables and surface markers. PATIENTS AND METHODS: Eighty CLL samples obtained from 63 untreated and 17 treated CLL patients were tested in vitro with the MTT assay. Lethal dose (LD)50 values were calculated to determine sensitivity to CLB, 2-CDA and FAMP. RESULTS: Samples were clustered either for a one-log increase of LD50 values or for LD50 threshold values of 3 microM for FAMP, 0.3 microM for 2-CDA and 7 microM for CLB, which correspond to the therapeutically achievable plasmatic levels of these drugs. A higher number of samples sensitive to 2-CDA were identified by the first approach; with the second method the relative order of sensitivity was FAMP > 2-CDA > CLB. Concerning 2-CDA and FAMP cross-resistance, out of 61 samples resistant to 2-CDA, 29.5% were sensitive to FAMP. Conversely, 13.9% out of 43 samples resistant to FAMP were sensitive to 2-CDA. No correlation was found between the main clinico-hematological features and the LD50 values of each drug either considering the whole series or only the untreated cases. In vitro drug sensitivity was also evaluated during the steady-state of the disease and at disease progression in six untreated cases. We observed a mean increase in the LD50 values of about 13, 38 and 22 times for CLB, FAMP and 2-CDA, respectively. Among the treated cases, the LD50 values of both purine analogs and CLB correlated with bone marrow histology. CLL cells expressing CD14, CD11c, CD11b, and FMC7 were more resistant in vitro to purine analogs but not to CLB. CONCLUSIONS: This study suggests that i) the purine analogs exert a greater cytotoxic effect on CLL cells; ii) 2-CDA and FAMP are not cross-resistant in vitro in a percentage of CLL samples, iii) a possible change in LD50 values may be related to modification of the disease status, and iv) the expression of certain surface markers, which are CLL-unrestricted, identifies samples with higher in vitro resistance to purine analogs.     

Quantitative and qualitative analysis of accessory gland secretory proteins in a few species of Drosophila immigrans group

Quantitative and qualitative analysis of accessory gland proteins in four species of D. immigrans group has been made. Quantitative estimation of these proteins during development of the adult male revealed that there is a gradual increase in the quantity of secretions up to 7 days. The secretions constitute 72, 70, 50 and 54.2% in D.n. nasuta, D.s. neonasuta, D. rubida and D. pararubida respectively. Mating experiments showed that more than 1/3 of this stored secretory protein is transferred into the female reproductive tract during first mating. SDS-PAGE analysis of the accessory gland secretions revealed remarkable simplicity in their patterns, including the absence of age dependent alterations. In all the 4 species analysed, only the low mol. wt. fractions are not glycosylated. It was further observed that the extent of similarity between D.n. nasuta and D.s. neonasuta is greater than between D. rubida and D. pararubida.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

Mutation of hMSH3 and hMSH6 mismatch repair genes in genetically unstable human colorectal and gastric carcinomas

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.     

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug

OBJECTIVE: The 65 kDa heat shock protein (HSP) chaperonin is a highly conserved intracellular protein. HSP are involved in the pathogenesis of arthritis, but are not able to induce experimental arthritis. T cell clones recognizing the 180-188 amino acid sequence of 65 kDa HSP are present in inflamed synovium of both adjuvant arthritis and rheumatoid arthritis (RA). Oral administration of bovine collagen II or co-chaperonin 10 kDa HSP has been shown to induce an immune tolerance state to collagen induced arthritis (CIA). We investigate the effect of oral gavage with 65 kDa HSP on CIA. METHODS: We immunized 6-8-week-old DBA1 male mice with bovine type II collagen. A group of 25 mice were given oral recombinant mycobacterial 65 kDa HSP before immunization (30 microg in 200 microl phosphate buffered saline (PBS) at Days -7, -5, -2) while PBS alone was administered in 27 controls. A 3rd group was fed 65 kDa HSP according to the same protocol but was not immunized with collagen II (n = 8). The clinical arthritis score was recorded 3 times/week until Day 60. Antibodies to collagen II were determined by ELISA. RESULTS: The incidence of arthritis was comparable in the 2 groups (72 vs 70%). The onset of arthritis was not delayed in mice fed HSP. However, the severity of arthritis was lower 10 days after arthritis onset in animals fed 65 kDa HSP (clinical score 1.83 +/- 0.79 vs 2.74 +/- 1.1; p < 0.0001). No animals in Group 3 had arthritis. Serum IgG anti-type II collagen levels were decreased in HSP treated mice (optical density 0.33 +/- 0.21 vs 0.46 +/- 0.21; p < 0.0001). However, the ratio of IgG1/IgG2a antitype II collagen antibody response remained unchanged in the mice fed 65 kDa HSP. CONCLUSION: These results suggest that oral administration of 65 kDa HSP may diminish collagen induced arthritis.     

Structural and electrical behavior of polyacrylonitrile-polypyrrole composite film

Polyacrylonitrile-polypyrrole composite films were prepared by a vapor phase technique. The films obtained by this method have high conductivity and good mechanical properties. Growth of polypyrrole on the surface of polyacrylonitrile was investigated by electron microscopic studies. Electrical properties of composite films were investigated as a function of FeCl₃ content as well as polypyrrole uptake. The current-voltage characteristic suggest an ohmic conduction. The conductivity of these composite films was measured as a function of temperature yielding an activation energy of 0.16 eV.