Biomedical subjects
Publications and source records attributed to B Ernst.
Seven sLe(x) mimics where the -CH2OH group of the galactose moiety is replaced by -CH2NH3+, -CH2NHAc, -CH2NHBz, -CH2OSO3Na, -COONa and -CONH2 have been prepared and tested for their binding affinity to E-selectin.
Naive T cells undergo spontaneous slow proliferation on adoptive transfer into syngeneic T cell (T)-deficient hosts. Recent work has shown that such "homeostatic" T cell proliferation is driven by MHC molecules loaded with self-peptides rather than foreign peptides. Because naive T cells in normal T-sufficient hosts remain in interphase despite continuous contact with self-MHC/peptide ligands, T cells apparently inhibit homeostatic proliferation of neighboring T cells. To address this, we have investigated the requirements necessary for "bystander" T cells to inhibit homeostatic proliferation of other T cells. Three key findings are reported. First, homeostatic proliferation of T cells only occurs in specific microenvironments, namely the T cell compartment of the secondary lymphoid tissues. Second, direct entry into T cell compartments is also required for bystander inhibition of homeostatic proliferation. Third, bystander inhibition is mediated largely by naive rather than activated/memory T cells and does not require proliferation or TCR ligation. These findings suggest that homeostasis of naive T cells is unlikely to be regulated through competition for systemic soluble factors or for specific stimulatory self-MHC/peptide ligands. Rather, the data favor mechanisms that involve competition for local non-MHC stimulatory factors or direct cell-to-cell interactions between the T cells themselves within the T cell compartment.
Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus and cells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23 degrees S and 24 degrees S) in the Atacama Desert for up to 15 months. B. subtilis spores (survival approximately 15%) and A. niger conidia (survival approximately 30%) outlived the other species. The survival of the conidia and spores species was only slightly poorer than that of the corresponding laboratory controls. However, the Deinococcus radiodurans cells did not survive the desert exposure, because they are readily inactivated at relative humidities between 40 and 80% which typically occur during desert nights. Cellular monolayers of the dry spores and conidia have in addition been exposed to the full sun light for up to several hours. The solar fluences causing 63% loss in viability (F37-values) have been determined. These F37-values are compared with those determined at other global locations such as Punta Arenas (53 degrees S), Key Largo (25 degrees N) or Mainz (50 degrees N) during the same season. The solar UVB radiation kills even the most resistant microorganisms within a few hours due to DNA damages. The data are also discussed with respect to possible similarities between the climatic conditions of the recent Atacama Desert and the deserts of early Mars.
Positive selection has long been thought to be a devise for producing a repertoire of T cells that can efficiently recognize foreign peptides in the context of self-major histocompatibility complex (MHC) molecules. However, in the light of recent evidence that long-term survival of mature T cells requires continuous contact with self-MHC molecules, the possibility for an additional role for positive selection has emerged: to generate a repertoire of T cells that can be maintained in the periphery through contact with self-MHC/peptide ligands. In support of this idea, our recent work suggests that positive selection is highly peptide specific and, more important, that mature T cells require extrathymic contact with the same MHC/peptide ligands that initially induced positive selection in the thymus in order for prolonged survival and to undergo homeostatic proliferation in response to T cell deficiency.
Time-resolved small-angle light scattering and linear conservative dichroism measurements are presented for concentrated, sterically stabilized, aqueous latices under simple shear flow. At low stress levels, flow causes a mild distortion of the liquid-like structure in colloidally stable dispersions, which is quite well understood. In this paper flow-induced structures are investigated in concentrated dispersions when the system is brought far from equilibrium by means of hydrodynamic forces. At high stress levels various structural changes have been predicted by numerical simulation, among others string phases oriented in the flow direction. Here, experimental results are reported on a bundle-like ordering in very dense systems, which involves a length scale much larger than that of a single string of particles. Two latices, with different particle sizes and different thicknesses of the stabilizing layer, are compared. The occurrence of the bundle-like ordering is related to the rheological behavior: it causes a significant decrease in viscosity. It is shown that the presence of this phase results in a structural hysteresis, which explains a thixotropic behavior that is encountered in some stable colloidal suspensions. Also the relaxation behavior of the bundle-like phases has been studied. Interparticle forces are found to have a very strong effect on the relaxation time scales. Copyright 1999 Academic Press.
In H2-M- mice, the presence of a single peptide, CLIP, bound to MHC class II molecules generates a diverse repertoire of CD4+ cells. In these mice, typical self-peptides are not bound to class II molecules, with the result that a very high proportion of H2-M- CD4+ cells are responsive to the various peptides displayed on normal MHC-compatible APC. We show here, however, that such "self" reactivity is controlled by low-affinity CD4+ cells. These cells give spectacularly high proliferative responses but are virtually unreactive in certain other assays, e.g., skin graft rejection; responses to MHC alloantigens, by contrast, are intense in all assays. Possible explanations for why thymic selection directed to a single peptide curtails self specificity without affecting alloreactivity are discussed.
Positive selection to self-MHC/peptide complexes has long been viewed as a device for skewing the T cell repertoire toward recognition of foreign peptides presented by self-MHC molecules. Here, we provide evidence for an alternative possibility, namely, that the self-peptides controlling positive selection in the thymus serve to maintain the longevity of mature T cells in the periphery. Surprisingly, when total T cell numbers are reduced, these self-ligands become overtly stimulatory and cause naive T cells to proliferate and undergo homeostatic expansion.
It has been shown that all selectins recognize the carbohydrate epitopes sialyl Lewis(x) and sialyl Lewis(a). For the establishment of the structure-activity relationship, the efficient synthesis of these tetrasaccharides and derivatives is therefore of vital interest. The glycosyl transferase-mediated approach is summarized with emphasis on the use of modified acceptors and modified sugar-nucleotide donors. A survey of the involved enzymes: beta(1-3) and beta(1-4)galactosyl transferases, alpha(2-3)sialyl transferase, FucT III and FucT VI reveals that the enzymatic synthesis is highly efficient for the rapid preparation of sialyl Lewis(x)- and sialyl Lewis(a)-derivatives.
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Monocyclic and bicyclic lactam units representing beta-turn surrogates were incorporated into a sialyl Le(X) structure by replacement of the natural sugar components. Low micromolar activity was found in a new P-selectin binding assay.
Application of the Ugi four-component condensation to rapidly synthesize a library of glycopeptide mimics of the tetrasaccharide SLe(x) as inhibitors of E- and P-selectin, and to study the effect of varied functionality in mimics on the inhibition is described.
Leukocyte rolling is the earliest observable even in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewis(x) (sLe(x)) is required for function. Despite findings that sLe(x) inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin-dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNF alpha)-stimulated mouse cremaster is increased following treatment with either sLe(x) or the sLe(x)-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti-P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin-dependent) rolling is unaffected by either sLe(x) or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.
The conformation of the sialyl Lewis(x) tetrasaccharide bound to E-selectin was previously determined from transfer NOE (trNOE) experiments in conjunction with a distance-geometry analysis. However, the orientation of the tetrasaccharide ligand in the binding site of E-selectin is still unknown. It can be predicted that the accurate quantitative analysis of all trNOEs, including those originating from spin diffusion, is one key to analyze the orientation of sialyl Lewis(x) in the binding pocket of E-selectin. Therefore, we applied homonuclear 3D NMR experiments and 1D analogs to obtain trNOEs that could not unambiguously be assigned from previous 2D trNOESY spectra, due to severe resonance-signal overlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a 1D trNOESY-TOCSY experiment of the sialyl Lewis(x)/E-selectin complex furnished new interglycosidic trNOEs and provided additional information for the interpretation of trNOEs that have been described before. A 2D trROESY spectrum of the sialyl Lewis(x)/E-selectin complex allowed one to identify the amount of spin-diffusion contributions to trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysis of spin-diffusion pathways, was obtained, creating a basis for a quantitative analysis of trNOEs in the sialyl Lewis(x)/E-selectin complex.
A 32 amino acid peptide called histatin-3 (H3; 22% His) and its N-terminal 24 amino acid fragment histatin-5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His-rich peptides have been synthesized by Fmoc-based solid-phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9-24 segment of H3 with all the His at positions 15,18,19,21 replaced by Tyr was also prepared (delta 1-8 H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PC1, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PC1, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR decrease SN). On prolonged incubation some minor cleavage was also observed C-terminal to the first LysArg6 pairs of basic amino acids namely at: HAKR decrease HH, which contains a P4 as well as P'1 and P'2 His residues. The second potential site YKRK12-FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PC1 only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR decrease HH. As expected, neither the D-amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all. In contrast to the above findings for hPC1, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin-mediated cleavage of the pentapeptide pGlu-Arg-Thr-Lys-Arg-MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 microM for H3 and 2.98 microM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 microM. The partial sequence identity of H3 to the inhibitory pro-peptide of furin and PC7 provides a rationale for our observation.
During most clinically relevant infections with cytopathic viruses, neutralizing antibodies are generated early, i.e., within the first week of infection. As early as 4 days after immunization of mice with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, hybridomas could be isolated that secreted virus-neutralizing IgGs. Such antibodies were devoid of somatic mutations, showed high binding avidities (approximately 10(9) M-1), and used V gene fragments predominantly belonging to the VHQ52 and VK19-28 families. In contrast, most secondary and hyperimmune response IgGs isolated 12 and 150 days after infection used several additional V gene combinations. These, which used the VHQ52/VK19-28 combination of early IgGs, were point mutated but showed only marginally enhanced binding avidities. Since all VHQ52/ VK19-28-positive IgGs bound to one subsite within the major antigenic site of VSV-G irrespective of the presence or absence of somatic point mutations, fine specificity diversification of secondary and hyperimmune responses was achieved by newly appearing V gene combinations.
OBJECTIVE: Oxipurinol has been shown to be sufficiently absorbed after oral administration as a rapid release preparation of oxipurinol sodium. We compared the uric acid lowering affect of allopurinol and oxipurinol. METHODS: In a multicenter, randomized, double blind crossover trial in 99 hyperuricemic patients with normal renal function we investigated the uric acid lowering effect of oxipurinol sodium (O) in daily amounts equimolar to 300 mg allopurinol (A). Mean pretreatment plasma uric acid concentrations in groups A/O and O/A were 8.3 +/- 1.4 and 8.7 + /- 1.4 mg/dl, respectively. RESULTS: In group A/O the mean plasma uric acid decreased to 5.4 +/- 1.2 mg/dl with allopurinol treatment, and increased slightly to 5.7 + /- 1.3 mg/dl during the consecutive oxipurinol period. In group O/A plasma uric acid declined to 6.0 +/- 1.4 mg/dl with oxipurinol and was 5.6 + /- 1.3 mg/dl at the end of the allopurinol period. The overall average reduction compared to baseline was 3.0 mg/dl with allopurinol and 2.6 mg/dl with oxipurinol. The difference between the 2 treatments was small but significant (multiple p=0.027,2 tailed). The corresponding mean plasma oxipurinol concentrations were 9.24 mu g/dl at the end of the allopurinol period and 9.9 mu g/dl after treatment with oxipurinol (NS). CONCLUSION: Oxipurinol is well absorbed and sufficiently effective in hyperuricemic patients when administered as a rapid release preparation of oxipurinol sodium. Oxipurinol sodium could be a substitute for allopurinol in hyperuricemic patients and possibly also with new uses for allopurinol.
Cell division during thymic selection was studied with a system in which purified populations of T cell antigen receptor (TCR)- CD4+8+ (double-positive [DP]) cells and fetal thymic epithelial cells (TEC) were reaggregated in tissue culture. In this system, immature DP cells differentiate into mature single-positive (SP) CD4+8- and CD4-8+ TCRhi cells within 3-4 d, indicative of positive selection. By adding the DNA precursor, bromodeoxyuridine, to the cultures and staining cells for bromodeoxyuridine incorporation, T cell division in reaggregation cultures was found to be high on day 1, low on day 2, and high on days 4-5. Cell separation studies established that cell division on day 1 was restricted to DP blast cells. In the absence of blast cells, small DP cells failed to proliferate and differentiated into SP cells without cell division, thus indicating that proliferation is not an essential component of positive selection. This applied to SP cells generated within the first 2-3 d. Surprisingly, the SP cells generated later in culture showed a high rate of cell division; the proliferating SP cells were TCRhi and included both CD4+8- and CD4-8+ cells. Turnover of TCRhi SP cells was also prominent in the normal neonatal thymus and in TEC reaggregation cultures prepared with adult lymph node T cells. We speculate that division of mature SP cells in the perinatal thymic microenvironment is driven by stimulatory cytokines released from TEC. Such proliferation could be a device to expand the mature T cell repertoire before export to the periphery.