11Li Breakup on 208 at energies around the Coulomb barrier.

The inclusive breakup for the (11)Li + (208)Pb reaction at energies around the Coulomb barrier has been measured for the first time. A sizable yield of (9)Li following the (11)Li dissociation has been observed, even at energies well below the Coulomb barrier. Using the first-order semiclassical perturbation theory of Coulomb excitation it is shown that the breakup probability data measured at small angles can be used to extract effective breakup energy as well as the slope of B(E1) distribution close to the threshold. Four-body continuum-discretized coupled-channels calculations, including both nuclear and Coulomb couplings between the target and projectile to all orders, reproduce the measured inclusive breakup cross sections and support the presence of a dipole resonance in the (11)Li continuum at low excitation energy.

Measurement of the 18Ne(α,p0) 21Na reaction cross section in the burning energy region for x-ray bursts.

The 18Ne(α,p) 21Na reaction provides one of the main HCNO-breakout routes into the rp process in x-ray bursts. The 18Ne(α,p0) 21Na reaction cross section has been determined for the first time in the Gamow energy region for peak temperatures T∼2 GK by measuring its time-reversal reaction 21Na(p,α) 18Ne in inverse kinematics. The astrophysical rate for ground-state to ground-state transitions was found to be a factor of 2 lower than Hauser-Feshbach theoretical predictions. Our reduced rate will affect the physical conditions under which breakout from the HCNO cycles occurs via the 18Ne(α,p) 21Na reaction.

Do halo nuclei follow Rutherford elastic scattering at energies below the barrier? The case of 11Li.

The first measurement of the elastic scattering of the halo nucleus 11Li and its core 9Li on 208Pb at energies near the Coulomb barrier is presented. The 11Li+208Pb elastic scattering shows a strong reduction with respect to the Rutherford cross section, even at energies well below the barrier and down to very small scattering angles. This drastic change of the elastic differential cross section observed in 11Li+208Pb is the consequence of the halo structure of 11Li, as it is not observed in the elastic scattering of its core 9Li at the same energies. Four-body continuum-discretized coupled-channels calculations, based on a three-body model of the 11Li projectile, are found to explain the measured angular distributions and confirm that the observed reduction is mainly due to the strong Coulomb coupling to the dipole states in the low-lying continuum of 11Li. These calculations suggest the presence of a low-lying dipole resonance in 11Li close to the breakup threshold.

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the host's immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a 'mixed response', and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.

Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.

Interactions between beta 2-syntrophin and a family of microtubule-associated serine/threonine kinases.

A screen for proteins that interact with beta 2-syntrophin led to the isolation of MAST205 (microtubule-associated serine/threonine kinase-205 kD) and a newly identified homologue, SAST (syntrophin-associated serine/threonine kinase). Binding studies showed that beta 2-syntrophin and MAST205/SAST associated via a PDZ-PDZ domain interaction. MAST205 colocalized with beta 2-syntrophin and utrophin at neuromuscular junctions. SAST colocalized with syntrophin in cerebral vasculature, spermatic acrosomes and neuronal processes. SAST and syntrophin were highly associated with purified microtubules and microtubule-associated proteins, whereas utrophin and dystrophin were only partially associated with microtubules. Our data suggest that MAST205 and SAST link the dystrophin/utrophin network with microtubule filaments via the syntrophins.

T-cell epitope determination.

Synthetic approaches to T-cell epitope determination have recently been developed that complement the search for natural T-cell epitopes and the investigation of the preferences of the different MHC alleles for particular motifs in cognate peptide sequences. The combination of these different strategies opens new possibilities for basic, as well as for applied, immunology. The outlines of the strategies for determination of natural T-cell epitopes are well established. These strategies have contributed substantially to our understanding of the nature of T-cell epitopes and of many diseases. Positional scanning approaches with random synthetic peptide libraries allow comprehensive surveys of the sequence requirements for peptide selection by MHC molecules and for induction of T-cell responses. Synthetic T-cell epitopes can be determined independently of the knowledge of the natural T-cell antigen. This opens new perspectives for the development of synthetic vaccines, TCR antagonists and MHC blockers.

T cell receptor specificity and mimotopes.

Degeneracy rather than unique ligand specificity seems to guide T cell functions. This view has evolved from analyses of T cell development and responses in vivo, as well as studies with synthetic molecular libraries in vitro, and has opened new prospects both for understanding T cell biology and for applied immunology.

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

Nanoparticle-Based Mucosal Vaccines Targeting Tumor-Associated Antigens to Human Dendritic Cells.

The induction of effective T cell-mediated immune responses is the main objective of vaccination against cancer. T cell responses are initiated by dendritic cells (DCs) as the most potent antigen-presenting cells. Designing vaccines for efficient delivery of tumor antigens to these cells in immunogenic fashion is, therefore, a major task in tumor immunology. In this human-based in vitro study we investigated the suitability of different polymeric nanoparticles (NPs) for delivering the tumor-associated antigen Her2/neu to DCs for induction of T cell responses by mucosal vaccination. The natural polymer chitosan and novel functionalized PLGA-based polymers were used for NP production. All NPs were efficiently taken up by DCs. Her2/neu delivered by NPs was more efficiently processed and presented by DCs than the soluble protein and induced more vigorous CD4+ and CD8+ T cell proliferation, and cytotoxic T cells. Testing the suitability of this platform for mucosal vaccination, NPs were applied to the apical side of an intestinal epithelium model and found to be efficiently transported across the epithelial layer to become available to basolateral DCs. Thus, chitosan and PLGA-based NPs are efficient carriers for delivery of antigens to DCs for induction of T cell-based immunity, and suitable for mucosal vaccine formulations.

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines.

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.

Evidence that the mammary fat pad mediates the action of growth hormone in mammary gland development.

Recent evidence from our laboratory suggests that GH and insulin-like growth factor I (IGF-I) mediate glandular mammary development together with estrogen. It has also been well established that both stromal and epithelial elements must interact for mammary glandular development to occur. To determine whether the effect of GH is mediated by the stromal or epithelial tissue, we set up the following experiment. Bovine GH (bGH; 100 microg) or BSA (as a control), without or with estradiol (E2), was injected i.p. into sexually immature female rats that were hypophysectomized and oophorectomized. Mammary glands and subscapular fat pads were removed from the animals. The mammary glands were divided into two parts: a gland-free fat pad and remaining glandular tissue. The end point of bGH activity was induction of IGF-I messenger RNA (mRNA). This was determined quantitatively by solution hybridization and also by RT-PCR. We found that the effects of GH on stimulation of IGF-I mRNA in the gland-free mammary fat pad and the remainder of the mammary gland were similar (3.6- vs. 3.9-fold, respectively; P < 0.001). In both sorts of mammary tissue, bGH was found to synergize with E2 in the induction of IGF-I mRNA (5.8- vs. 5.3-fold; P < 0.001). There was also an increase in IGF-I mRNA in subscapular fat pads in response to 100 microg bGH (5.3-fold; P < 0.001); however, no synergism between bGH and E2 was found. These data indicate that bGH works as well on mammary stromal tissue as on tissue with glands and suggests that GH acts on the stromal compartment of the mammary gland to induce IGF-I mRNA and possibly IGF-I itself, which, in turn, causes differentiation of epithelial ducts into terminal end buds. These data also might explain why mammary epithelium is also able to differentiate in nonmammary fat pads when transplanted there.

Efficacy of synthetic vaccines in the induction of cytotoxic T lymphocytes. Comparison of the costimulating support provided by helper T cells and lipoamino acid.

Synthetic vaccines that specifically induce active immunity mediated by cytotoxic T lymphocytes (CTL) are of great interest considering the central role of these cells in immune responses against intracellular antigens. The influence of specific T helper (Th) cell recruitment and of the potent immunostimulating lipoamino acid tripalmitoyl-S-glycerylcysteine (P3C) on CTL mediated immunity induced by CTL epitopes was analysed and compared. Synthetic peptides that represent CTL epitopes were found to be inefficient for CTL priming. However, when combined with peptides that contain Th cell epitopes, with proteins that carry multiple Th cell epitopes or with P3C, efficient priming of CTL was obtained. The costimulating support by P3C and proteins resulted in high cytolytic activities already after 9 days whereas, in the case of single helper epitopes, incubation periods of about 4 weeks were required. The effects of P3C and helper epitopes were additive.

Determination of T cell epitopes with random peptide libraries.

A new approach to T cell epitope determination is presented. Critical amino acids for the induction of cytotoxic T cell responses were identified using synthetic peptide libraries with single defined sequence positions combined with randomized sequence positions. Sequences for potential T cell epitopes were deduced from scan profiles using combinations of the active amino acids. Highly potent epitopes for cytotoxic T lymphocytes were obtained. Epitopes defined by this approach are, as shown in this communication, not necessarily the natural epitopes and, therefore, were named synthetic epitopes. They can serve effectively for the development of vaccines or for the determination of T cell receptor antagonists.

Mitogenic activation of human prostate-derived fibromuscular stromal cells by bradykinin.

Biologically active kinin peptides are released from precursor kininogens by kallikreins. Kinins act on kinin receptors to mediate diverse biological functions including smooth muscle contraction, inflammation, pain and mitogenicity. All components of the kallikrein-kinin system exist in human male genital secretions suggesting that these molecules participate in physiological and pathophysiological genitourinary function. The objective of this study was to assess the consequences of kinin action on prostate cells. Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from human prostate tissue. Transcripts encoding both the human B1 and B2 bradykinin receptor subtypes were detected in human prostate transition-zone tissue and in cultured cells by RT-PCR. In receptor binding assays, the B1 subtype predominated on PE cell membranes and the B2 subtype predominated on PS cell membranes. In PS cells, but not in PE cells, BK induced significant inositol phosphate accumulation and [3H]-thymidine uptake. These responses were mediated through the B2 receptor subtype. The use of signal transduction inhibitors indicated that mitogenic activation by BK occurred through both protein kinase C (PKC) and protein tyrosine kinase dependent mechanisms. PMA (phorbol 12-myristate 13-acetate) produced maximal [3H]-thymidine uptake by PS cells, resulted in cell elongation and caused the alpha-actin fibres present in PS smooth muscle cells to became organized into parallel arrays along the length of the elongated cells. In summary, the prostate contains a functional kallikrein-kinin system, which could be significant in physiological and pathophysiological prostate function.

The alpha-adrenoceptor antagonist properties of the enantiomers of doxazosin in the human prostate.

The alpha-adrenoceptor antagonist properties of doxazosin and its enantiomers were characterized using human prostate tissue and cell membranes isolated from rat-1 fibroblast expressing each of the cloned human alpha 1-adrenoceptor subtypes. In the alpha 1-adrenoceptor-binding studies on the human prostate with [3H]doxazosin and 2-{[beta-(3-[125I],4-hydroxyphenyl)ethyl]aminomethyl}-l-tetralone ([125I]HEAT), no significant differences were observed between racemic doxazosin, R-doxazosin and S-doxazosin (mean -log Ki (pKi) values were 8.60-8.63, 8.47-8.55 and 8.61-8.65, respectively), whereas the alpha 2-adrenoceptor-binding studies with [3H]rauwolscine and [3H]clonidine revealed that the alpha 2-adrenoceptor-binding affinity of S-doxazosin (pKi = 5.91-5.94) was slightly (3- or 4-fold), but significantly lower than that of R-doxazosin (pKi = 6.47-6.54). Studies in phenylephrine-contracted prostatic tissue showed no significant difference in alpha 1-adrenoceptor antagonist potency between racemic doxazosin, R-doxazosin and S-doxazosin (pA2 values were 8.43 +/- 0.28, 8.64 +/- 0.56 and 8.75 +/- 0.38, respectively). In the binding studies with cloned alpha 1-adrenoceptor subtypes using [3H]prazosin and [125I]HEAT, racemic doxazosin, R-doxazosin and S-doxazosin showed no selectivity for the alpha 1-adrenoceptor subtypes. The present study demonstrated that doxazosin and its enantiomers are highly selective alpha 1-adrenoceptor antagonists and that there is no evidence suggesting differential alpha 1-adrenoceptor antagonist effects of doxazosin and its enantiomers in the human prostate. Doxazosin, therefore, could be described as displaying balanced activity across all three alpha 1-adrenoceptor subtypes.

Development of a lymphocytic lymphoma during immunosuppressive therapy with azathioprine for systemic lupus erythematosus with renal involvement induced by phenylbutazone.

A case of systemic lupus erythematosis (SLE) with renal involvement after the administration of phenylbutazone is described. The patient subsequently developed a lymphocytic lymphoma following two years treatment with azathioprine and prednisone. Although the association between SLE, immunosuppressive therapy and the development of lymphomas is well documented, the increased incidence of malignant disease in uremic patients is less well recognized. The varied factors that may have contributed to the development of the lymphoma in this case are discussed, together with a review of the literature.

Reticulosarcoma occurring during long-term hemodialysis.

Reticulosarcoma occuring during long-term hemodialysis. A case of chronic renal failure due to polycystic disease of the kidneys with development of a reticulosarcoma with cerebral involvement during maintenance hemodialysis is described. Immunosuppression from uremia could have played an important part in tumor induction in this case and might also be a significant factor in the high incidence of de-novo malignancies in renal transplant recipients.