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The objective of this study was to explore the functional anatomy of the globus pallidus internus (GPi) by studying the effects of unilateral pallidotomy on parkinsonian 'off' signs and levodopa-induced dyskinesias (LID). We found significant positive correlations between the preoperative levodopa responsiveness of motor signs and the levodopa responsiveness of scores in timed tests (Core Assessment Program for Intracerebral Transplantations) in the contralateral limbs and the improvement in these scores after surgery, whereas there was no correlation with the improvement in LID. We also found a highly significant correlation (P: < 0.0001, r = 0.8) between the volume of the ventral lesion in the GPi and the improvement in LID in the contralateral limbs, whereas there was no correlation between the ventral volume and the improvement in parkinsonian 'off' signs. The volumes of the total lesion cylinder and the dorsal lesion did not correlate with the outcome of either dyskinesias or parkinsonian 'off' signs. The differential predictive value of levodopa responsiveness for the outcome of parkinsonian 'off' signs and LID and the different correlations of ventral lesion volume with dyskinesias and parkinsonian 'off' signs indicate that different anatomical or pathophysiological substrates may be responsible for the generation of parkinsonian 'off' signs and dyskinesias. Whereas cells in a wider area of the GPi may be implicated in parkinsonism, the ventral GPi seems to be crucial for the manifestation of LID. We suggest that our observations are additional proof of the functional somatotopy of the systems within the GPi that mediate parkinsonism and dyskinesias, especially along the dorsoventral trajectory used in pallidotomy. The outcome of pallidotomy in which the lesion involves the ventral and dorsal GPi could be the net effect of alteration in the activity of pathways which mediate different symptoms, and hence could be variable. more...

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses. more...

Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo. more...

Tumors evade host immunity at both the induction and effector phases Most studies have focused on tumor evasion at the induction phase, and, due in part to poor antitumor CTL responses to most tumors, the mechanism for evasion of CTL effector function is less clear. Here we have taken advantage of the strong CTL responses to a costimulator B7-1-transfected tumor to study the mechanism for tumor evasion of preexisting host immunity. We have investigated six independent recurrent tumors isolated from mice that were challenged with and had rejected B7-1-transfected J558 (J558-B7) tumors. Because the mice had developed strong antitumor CTL responses, these recurrent tumors must have evaded preexisting antitumor CTLs. Indeed, whereas the parental J558-B7 cell line is efficiently lysed by the ex vivo tumor-infiltrating lymphocytes, all of the recurrent tumors are resistant to such lysis. Interestingly, the recurrent tumors can be divided into two groups. The group 1 tumors have vastly reduced levels of cell surface MHC class I with a concurrent reduction in the expression of multiple genes devoted to MHC class I antigen presentation. In contrast, the group 2 tumors have lost the expression of costimulatory molecule B7-1 while retaining cell surface MHC class I and expression of all antigen presentation genes studied. These results demonstrate that tumors can evade preexisting CTLs either by avoiding presentation of the tumor antigen or, surprisingly, by down-regulation of costimulatory molecules. The paradoxical requirements of both antigen and costimulatory molecules at the effector phase raised an interesting question on the nature of antitumor immunity. more...

Unmutated tumor antigens are chosen as primary candidates for tumor vaccine because of their expression on multiple lineages of tumors. A critical issue is whether unmutated tumor antigens are expressed in normal cells, and if so, whether such expression imposes special restrictions on cytotoxic T lymphocyte (CTL) responses. In this study, we use a transgenic approach to study the development and effector function of T cells specific for P1A, a prototypical unmutated tumor antigen. We report here that although P1A is expressed at low levels in normal tissues, including lymphoid tissues, the P1A-specific transgenic T cells develop normally and remain highly responsive to the P1A antigen. The fact that transgenic expression of P1A antigen in the thymus induces T cell clonal deletion demonstrates that normal hematopoietic cells can process and present the P1A antigen and that P1A-specific T cells are susceptible to clonal deletion. By inference, P1A-specific T cells must have escaped clonal deletion due to low expression of P1A in the thymus. Interestingly, despite the fact that an overwhelming majority of T cells in the T cell receptor for antigen (TCR)-transgenic mice are specific for P1A, these mice are no more resistant to a P1A-expressing plasmocytoma than nontransgenic littermates. Moreover, when the same TCR-transgenic mice were challenged simultaneously with B7-1(+) and B7-1(-) tumors, only B7-1(+) tumors were rejected. Therefore, even though P1A can be a tumor rejection antigen, the effector function of P1A-specific CTL is restrained in vivo. These results have important implications for the strategy of tumor immunotherapy. more...

Background: A previous retrospective study indicated that patch testing is cost-effective and well accepted by patients., Objective: The objective of this observational prospective study was to show the cost-effectiveness of patch testing in patients suspected of allergic contact dermatitis (ACD) and to determine the order in which different severity groups rank in terms of cost-effectiveness., Methods: This observational study was conducted in 567 patients from 10 investigator sites over a period of 1 year. All patients with a suspicion of contact allergy who exhibited at least moderate disease activity were included in the study and were stratified according to disease severity and whether or not they were patch tested. In each severity category, the cost-effectiveness of patch testing was evaluated. Patients who were ruled out for contact allergy by the first 6 months after admission were excluded. A validated dermatology-specific quality of life instrument was administered to all the patients at entrance into the study and at 6 and 12 months after that. The cost-effectiveness analysis is shown using a decision analysis model., Results: Patch testing was performed on 22% of patients with mild disease, 41% of patients with moderate disease, and 50% of patients with severe disease. As a result of changes made in their lifestyle, 66% in the patch-tested group and 51% in the non-patch-tested group reported 75% or more improvement in disease symptoms after 6 months. Early confirmation of diagnosis helped reduce the prediagnosis costs of treatment, which was mostly based on preliminary diagnosis. The greatest quality of life benefits from patch testing, relative to no patch testing, occurred in subjects with recurrent or chronic ACD., Conclusion: Patch testing is most cost-effective and reduces the cost of therapy in patients with severe ACD. more...

It is generally believed that CTLs mature in lymphoid organs and then migrate into target tissues to execute their effector functions. This notion, however, is based on studies using antigens that are readily localized in the lymphoid tissue, such as viruses and allogeneic transplants. The site for maturation of CTLs for nonmetastatic tumors has not been determined. Because nonmetastatic tumor cells are not localized in lymphoid tissues, it is questionable whether such tumors are efficient inducers of antitumor CTLs. Here, we report that a nonmetastatic B7+ plasmacytoma induces strong effector CTL response. Thus, it is possible to induce CTLs with strong ex vivo CTL activity in the absence of tumor metastasis. In addition, a detailed kinetic analysis of CD8 T cell recruitment and maturation of CTL activity suggests that antitumor CTLs mature within the tumor rather than in the lymphoid tissues. Interestingly, despite B7-1 expression on tumor cells, induction of effector CTLs also requires costimulation by B7 on host antigen-presenting cells. These findings have important implications for tumor gene therapy and for understanding the mechanism of CTL induction in vivo. more...

Objective: The objective of this observational prospective study was to demonstrate the cost effectiveness of patch testing in patients suspected of allergic contact dermatitis (ACD) and to determine the order in which different severity groups rank in terms of cost effectiveness., Design and Setting: This observational study was conducted on 567 patients from 10 investigator sites in the US over a period of 1 year. All patients with a suspicion of contact allergy who exhibited at least moderate disease activity were included in the study and were stratified according to disease severity and whether or not they were patch tested. In each severity category, the cost effectiveness of patch testing was evaluated. Patients who were ruled out for contact allergy (without the use of patch test) by the first 6 months after admission were excluded. A validated dermatology-specific quality of life (DSQL) instrument was administered to all patients at the start of the study, and at 6 and 12 months thereafter. The cost-effectiveness analysis is demonstrated using a decision-analytical model. Costs included office visits and prescription costs without generic substitution. The cost of patch testing was not included due to the large variation in price among commercially available products., Main Outcome Measures and Results: Patch testing was performed on 22% of patients with mild disease, 41% of patients with moderate disease and 50% of those with severe disease. There was a significant difference between the patch-tested and non-patch-tested groups in terms of the time to obtain a confirmed diagnosis (medians = 8 and 175 days, respectively) and a significant difference in the ratio of patients who had a confirmed diagnosis (88 and 69%, respectively). As a result of changes made in their lifestyle, 66% of patients in the patch-tested group and 51% in the non-patch-tested group reported 75% or more improvement in their disease symptoms after 6 months. Early confirmation of diagnosis helped reduce the prediagnosis cost of treatment which was mostly based on preliminary diagnosis. The greatest quality-of-life (QOL) benefits from patch testing relative to nonpatch testing occurred in patients with recurrent/chronic ACD., Conclusions: Patch testing is most cost effective and reduces the cost of therapy in patients with severe ACD. Greater improvements in quality of life were seen in patients with recurrent/chronic ACD who were patch tested than patients who were not patch tested within the same group. Results were not sensitive to changes due to the addition of indirect costs or costs using generic substitution. more...

On the basis of a comparison of high-resolution solution structures calculated for both equilibrium forms of rat ferrocytochrome b5, differences in reduction potential and thermodyanmic stability have been characterized in terms of significant structural and dynamic differences between the two forms. The dominant difference between A and B conformations has long been known to be due to a 180 degrees rotation of the heme in the binding pocket about an axis defined by the alpha- and gamma-meso carbons, however, the B form has not been structurally characterized until now. The most significant differences observed between the two forms were the presence of a hydrogen bond between the 7-propionate and the S64 amide in the A form but not the B form and surprisingly a displacement of the heme out of the binding pocket by 0.9 A in the B form relative to the A form. The magnitude of other factors which could contribute to the known difference in reduction potentials in the bovine protein [Walker, F. A., Emrick, D., Rivera, J. E., Hanquet, B. J., and Buttlaire, D. H. (1988) J. Am. Chem. Soc. 110, 6234-6240], such as differences in the orientation of the axial imidazoles and differences in hydrogen bond strength to the imidazoles, have been evaluated. The dominant effector of the reduction potential would appear to be the lack of the hydrogen bond to the S64 amide in the B form which frees up the propionate to charge stabilize the iron in the oxidized state and thus lower the reduction potential of the B form. The structure we report for the A form, based on heteronuclear NMR restraints, involving a total of 1288 restraints strongly resembles both the X-ray crystal structure of the bovine protein and a recently reported structure for the A form of the rat protein based on homonuclear data alone [Banci, L., Bertini, I., Ferroni, F., and Rosato, A. (1997) Eur. J. Biochem. 249, 270-279]. The rmsd for the backbone atoms of the A form is 0.54 A (0.92 A for all non-hydrogens). The rmsd for the backbone of the B form is 0.51 A (0. 90 A for all non-hydrogen atoms). An analysis of backbone dynamics based on a model-free analysis of 15N relaxation data, which incorporated axially symmetric diffusion tensor modeling of the cytochrome, indicates that the protein is more rigid in the reduced state relative to the oxidized state, based on a comparison with order parameters reported for the bovine protein in the oxidized state [Kelly, G. P., Muskett, F. W., and Whitford, D. (1997) Eur. J. Biochem. 245, 349-354]. more...

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation. more...

Mutational studies directed at evaluating the effect of the axial ligand plane orientation on electrochemical properties of cytochrome b5 have been performed. As described in the previous paper, structural consequences of one of these mutations, the A67V mutation, have been evaluated using NMR solution methods. The lack of large shifts relative to the wild-type protein in both the imidazole Ndelta nitrogen and proton resonances of the H63 imidazole ring indicates that the hydrogen bond between the carbonyl of F58 and the imidazole ring of H63 remains intact in this mutant. Effects of the imidazole plane reorientation on the Fe d-orbitals were evaluated on the basis of interpretation of EPR spectra, near-infrared bands associated with ligand-to-metal charge transfer transitions, reorientation of the anisotropy of the paramagnetic center determined by calculation of pseudocontact shifts, and the temperature dependence of the contact-shifted resonances. The dominant effect of the imidazole reorientation appears to have been a destabilization of the d(xz) orbital energy and a reorientation of the d(pi) orbitals. This is surprising in light of the -20 mV shift in the reduction potential of the mutant relative to the wild-type protein and indicates that a destabilization of d(yz)-orbital energy level of the reduced state dictates the observed change in reduction potential. Measured values for the reorganizational energy and heterogeneous electron transfer rates were indistinguishable for wild-type and mutant proteins. This is perhaps surprising, given significant differences in the pattern of electron delocalization into the porphyrin ring observed as significantly altered contact shift patterns. Mutational studies perturbing the H39 imidazole were also performed but with more limited success. more...

Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain 13C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b5. The assignment of backbone 13C resonances was used to confirm previous 1H and 15N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329-8340]. On the basis of short- and medium-range NOEs and backbone 13C chemical shifts, the solution secondary structure of rat cytochrome b5 has been determined. The striking similarity of backbone 13C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the 13C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts. more...

The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 A from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. This absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations. more...

Whether tumors of different lineages share common Ags is a critical issue for understanding anti-tumor immune responses and for designing Ag-specific tumor immunotherapy. Because of lack of cross-protection among individually derived tumors, it has been proposed that tumor Ags are specific for individual tumors. Here we show that lack of cross-protection is not due to lack of a shared tumor Ag. Thus, a plasmocytoma J558 transfected with the costimulatory molecule B7 activates a cross-reactive CTL response in vivo. The major Ag recognized by the cross-reactive CTL is P1A, which is expressed in mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Surprisingly, no significant cross-protection can be detected among P1A-expressing tumors after immunization with either P1A-expressing or B7-transfected P815 cells. Our results demonstrate that multiple lineages of tumors are not cross-protected even though they share a tumor Ag that can be recognized by CTL. These results have important implications for tumor immunotherapy. more...

Background: In a continuing effort to better serve the low vision community, The Center for the Partially Sighted has developed an in-home low vision aids training (LVAT) program. Of interest to anyone working in the field of low vision is a description of our training materials and methodology, and a discussion of the efficacy of this program., Methods: The previous literature on low vision was evaluated and organized into an orderly program geared toward the visual rehabilitation of low vision patients., Results: Implementation of LVAT in the home began as a pilot project directed toward patients with macular degeneration who have begun reading as a primary goal of rehabilitation., Conclusions: Successful vision rehabilitation with low vision devices can be accomplished with proper training. Typically the exam does not allow ample opportunity to ensure that a patient is proficient in the use of a device. Follow-up training in the home environment may provide the necessary intervention to maximize patients' comfort and efficiency with low vision devices and their ability to accomplish near point tasks. more...

Chloroform extract of the aerial parts ofEupatorium adenophorum Spreng was fractionated and examined for growth inhibition. Bioassay-directed fractions of the plant materials afforded three known cadinenes and β-sitosterol. The effects of different fractions as well as isolated cadinenes were determined usingAllium cepa, Raphanus sativus, andCucumis sativus seeds. Three-day exposure to these cadinenes significantly inhibited germination and seedling growth of all three assay seeds. The degree of inhibition was dependent upon seed species and the concentrations of the compounds tested. Cadinene(1) was found to be more inhibitory to the seeds tested and the activity of the cadinene(3) was less than that of(1) and(4). more...

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees. Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112, 5258-5263]. more...

This cost-benefit analysis attempts to translate the clinical findings of the Helsinki Heart Study (HHS) and published results regarding additional cardiovascular conditions into economic terms meaningful to US managed care providers. The study has the following 3 key objectives: to define the cost effectiveness of gemfibrozil in the prevention of coronary heart disease (CHD); to assess the net impact of gemfibrozil on total treatment costs for CHD; and to identify those patient groups for whom gemfibrozil therapy is most cost effective. In order to reach these findings a cost-effectiveness model was constructed based on original clinical data provided by the HHS, published findings for CHD risk and cost of treatment in the US, expert opinion and extension of HHS to other cardiovascular conditions, and documented costs and treatment protocols of US Medicaid and privately managed health care programmes. The model was applied to the California Medicaid (Medi-Cal) programme to estimate costs of hyperlipidaemia therapy using gemfibrozil. In parallel, savings to Medi-Cal from averted coronary events were estimated. From these data, the net expected savings to Medi-Cal were calculated. The probability of experiencing CHD varies with cholesterol level, age, sex and risk factors such as smoking, hypertension and diabetes. Therefore, it is possible to use risk-factor profiles to define groups of individuals with low, moderate or high risk of experiencing acute myocardial infarction (AMI) or sudden cardiac death. The probability of a cardiac event within 5 years ranges from 1.1% in a 45-year-old low risk male to over 36% in a 55-year-old high risk male. The average total cost of CHD care was found to be US$22,271 within 5 years. Using the probability of a CHD event to calculate the expected 5-year cost of CHD care produces a range from US$242 in the 45-year-old low risk male to US$8084 in the 55-year-old high risk male. Treatment with gemfibrozil reduces the probability of AMI and sudden cardiac death events by 34%, as demonstrated in the HHS. Therefore, the corresponding probability ranges are reduced to 0.7% in the 45-year-old low risk male and 27.3% in the 55-year-old high risk male after treatment with gemfibrozil. The expected cost of a coronary event is reduced by US$82 and US$1997, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) more...