HLA (A*0201) mimicry by anti-idiotypic monoclonal antibodies

Soluble MHC Ags and anti-Id (anti-anti-MHC) Abs have both been shown to inhibit MHC alloantigen-specific B cell responses in vivo. We hypothesized that some anti-idiotypic Abs function as divalent molecular mimics of soluble HLA alloantigen. To test this idea, we studied two well-defined anti-idiotypic mAbs, T10-505 and T10-938, elicited in syngeneic BALB/c mice by immunization with CRll-351, an HLA-A2,24,28-specific mAb. Each anti-Id induced "Ab-3" Abs in rabbits that cross-reacted with HLA-A2 but not with HLA-B Ags. Furthermore, each anti-Id could bind to and block Ag recognition by Ha5C2.A2, a human homologue of mAb CRll-351. Both anti-Id mAb displayed weak reactivity with the human mAb SN66E3, which recognized an overlapping but distinct determinant of HLA-A2 Ags; neither reacted with human mAb MBW1, which recognized a nonoverlapping HLA-A2 determinant. Amino acid sequence comparison of mAb CRII-351 heavy and light chain variable region complementarity-determining regions (CDRs) with those of mAb Ha5C2.A2 and SN66E3 revealed short regions of homology with both human mAb; a large insert in the light chain CDR1 of mAb SN66E3 distinguished it from both CRll-351 and Ha5C2.A2. The amino acid sequences of mAb T10-505 and T10-938, which differed markedly from each other, revealed no homology to the alpha2 domain sequence of HLA-A*0201 that contains the CRll-351 mAb-defined epitope. We conclude that structurally different anti-Id Abs can mimic a polymorphic conformational epitope of an HLA Ag. In the case of T10-505 and T10-938 mimicry was not based on exact replication of the epitope by the hypervariable loops of the anti-Id mAb.     

Reactivity of twenty-two cytotoxic human monoclonal HLA antibodies towards soluble HLA class I in an enzyme-linked immunosorbent assay (PRA-STAT)

An ELISA, PRA-STAT was recently introduced for the detection of HLA class I specific antibodies of IgG isotype in patients' sera. We studied the antigenicity of the soluble HLA (sHLA) preparations that are used in this ELISA as the detection matrix, with the aid of a panel of complement binding human HLA monoclonal antibodies (HuMAbs). A total of 22 HuMAbs, including both IgG and IgM were used. CDC and PRA-STAT ELISA were in complete agreement on 9 of the mAbs tested, with 16 HLA-A and 16 HLA-B locus antigens or their splits identified identically on CDC and PRA-STAT. In 7 of the remaining 13 HuMAbs, there was a difference of one antigen in the specificity pattern of the two techniques three times a specificity call not made by CDC, and four times a call not made by PRA-STAT. For the remaining 6 HuMAbs the differences involve 2 antigens (4 HuMAbs), and 3 or 4 antigens (1 HuMAb each). This study shows the validity of PRA-STAT for detection of HLA-class I antibodies, irrespective of isotype, in serum. The immunological integrity of the sHLA preparations used in PRA-STAT is also confirmed, albeit with some slight discrepancies in antibody specificity seen between PRA-STAT and CDC.     

Effect of heparin on the interaction between platelets with collagen

An effect of a standard heparin preparation on the interaction between blood platelets and collagen has been investigated. The experiments have shown, that the addition of heparin in the concentration of 0.3; 0.6 or 0.9 IU/mL did not change the interaction between blood platelets and collagen. Such interaction increased, when heparin concentration was 1,2 IU/mL, and remained unchanged despite the further increase in heparin concentration. The authors suggest that such a course of this interaction results from the stimulating action of heparin added in adequate concentration on protein release from platelet alpha granulations--which bound GP II b/III a complex with collagen.     

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT "DNA-unwinding motif," which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.     

Specific interaction of Golgi coatomer protein alpha-COP with phosphatidylinositol 3,4,5-trisphosphate

The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the alpha-COP subunit was observed with a p-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6 probe labeled six of the seven subunits, with only beta, beta', delta, and epsilon-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only alpha-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.     

Identification and characterization of the cytoplasmic antiproteinase (CAP) in human platelets. Evidence for the interaction of CAP with endogenous platelet proteins

To define the presence and potential role of platelet-associated protease inhibitors, we initiated a study designed to characterize the platelet components that are responsible for the formation of two SDS-stable complexes of approximately 58 and 70 kDa initially observed following the incubation of 125I-thrombin and human platelets. We demonstrate that thermal-mediated unfolding of the 58-kDa complex between 125I-thrombin and a nonsecreted platelet protein leads to an apparent molecular mass of 70 kDa. This platelet component is functionally and immunologically indistinguishable from the cytoplasmic antiproteinase (CAP), also known as placental thrombin inhibitor, a recently cloned member of the ovalbumin family of intracellular serpins (serine proteinase inhibitors). CAP-specific mRNA and antigen were detected in human platelets, suggesting that CAP synthesis occurs concurrent with platelet development. Utilizing quantitative immunoblotting, CAP antigen was estimated at 1.014 +/- 0.181 microg/10(9) nonstimulated platelets. After platelet activation with the calcium ionophore A23187, CAP antigen was detected in released microparticles at approximately 0. 195 +/- 0.031 microg/10(9) platelets and a fraction of platelet CAP was proteolytically modified. We provide evidence that these lower molecular mass species arise by cleavage of CAP at or near the reactive site loop. Most importantly, molecular sieving chromatography indicates the presence of an approximately 68-kDa SDS-labile complex between cleaved CAP and a cellular component in A23187-stimulated platelets, suggesting a physiological target of this intracellular serpin and a potential role for this inhibitor in regulating proteolytic activity that may be formed during platelet activation.     

Ultrastructural observation of platelets from patients with progressive systemic sclerosis (PSS)

We observed the ultrastructure of platelets from patients with PSS (7 cases; 48.2 +/- 12.3 y-old; M:F = 1:6_ and healthy controls (HC) (5 cases; 44.8 +/- 8.0 y-old; M:F = 1:4) by using transmission (TEM) and freeze-fracture electron microscopy (FEM). The open canalicular system (OCS) connected with the plasma membrane (PM) formed pinhole-like invaginations (50 nm in diameter) in the cleaved face (P-face) of the plasma membrane seen from the outside of the platelets and sharply elevated structures in the cleaved face (E-face) of PM seen from the inside of the platelets by FEM. The density of OCS on the surface of the platelets from PSS patients was 3 +/- 1/micron 2, which was higher than that from HC (1 +/- 0.5/micron 2) (p < 0.02). Dome-shaped structures, which clearly differ from OCS and were 80-150 nm in diameter without intramembranous particles, were seen in the P-face, and the complementary depressed structures were seen in the E-face. These structures were thought to be vesicles fused onto the PM of the platelets. The total volume of platelets (7.62 +/- 0.11 micron 3), total volume of granules (0.79 +/- 0.01 micron 3) and vacuoles including OCS (0.78 +/- 0.05 micron 3), and the total surface area of platelets (17.25 +/- 1.30 micron 2) from four PSS patients calculated by the morphometrical method were similar to those from four HC (7.32 +/- 0.25 micron 3, 0.76 +/- 0.03 micron 3, 0.80 +/- 0.05 micron 3, 18.75 +/- 0.35 micron 2, respectively); there were no statistical significances between the data from PSS patients and HC. The total volumes of vacuoles in platelets from both PSS patients and HC significantly decreased after a 2 min-vibration stress of the hands (p < 0.02) and the total volume of granules in platelets from PSS patients decreased significantly after the same stress (p < 0.002), although that from HC showed no similar significant change. However, there were no statistically significant differences in total volume or total surface of platelets from PSS patients and HC after the stress. These data may suggest that depletion of granules occurred due to activation of platelets from PSS patients following a secretion of their proteins, because their plasma protein levels were elevated after the stress (Jpn J Dermatol, 98; 1205, 1988). Higher density of OCS on the surface of the platelets from PSS patients may play an important role in secretion of their proteins, although the detailed mechanism of secretion of specific proteins derived from platelet granules is still unknown. These ultrastructural abnormalities of platelets may correlate with some involvement of a platelet disorder and with a possible role for the activation of platelets from PSS patients.     

Specific interaction of wild-type and truncated mouse N-methylpurine-DNA glycosylase with ethenoadenine-containing DNA

BACKGROUND: The present study was designed to test the biocompatibility of a new vitamin E-modified multi-layer membrane (CL-E filter), as well as its ability to protect against oxygen free radicals during hemodialysis (HD). METHODS: We investigated, both in vitro and in vivo, the bioreactivity of the filter with respect to the blood antioxidants and its ability to prevent lipoperoxidation. The effects on the leukocyte respiratory burst were also studied. Cuprammonium rayon was used as a comparison material (CL-S filter). RESULTS: The in vitro results demonstrated that, under controlled conditions, CL-E is able to preserve blood antioxidants, and particularly vitamin E, from the spontaneous consumption observed in the incubation with CL-S filters and in control incubations. In accordance with this observation, the rate of the oxidative demolition of lipids either in plasma and red blood cells (RBC) or from rat brain homogenate decreased after the exposure to CL-E filters in comparison with the CL-S filter. Moreover, in the absence of any significant cytotoxic effects due to both the types of material studied, the production of oxygen free radicals and nitric oxide (NO) by leukocytes was higher after their in vitro exposure to CL-S, but was quite similar to that of the control leukocytes after exposure to CL-E. In vivo, a one-month treatment with the CL-E filter increased plasma vitamin E by 84.3% with respect to treatment with CL-S; this gain slightly decreased to 68.9% when CL-E treatment was prolonged to three months. In the RBC, vitamin E was found to have increased by 76.7% and 113.4% at one and three months, respectively. Plasma glutathione (GSH) levels determined at three months were significantly increased from 0.10 +/- 0.02 to 0.33 +/- 0.12 mumol/ml, while the erythrocyte GSH was only slightly increased. The leukocyte function estimated as responsiveness to soluble chemical stimuli in CL-S-treated patients was significantly improved both qualitatively and quantitatively after CL-E treatment. The presence of an increased number of mononuclear cells undergoing programmed cell death (apoptosis) in CL-S-treated patients (18.8 +/- 1.7% vs. a control value of 6.5 +/- 2.3%) as well as the apoptogenic effect of their plasma in vitro on U937 cells was significantly corrected after CL-E treatment (mean decrease in apoptotic mononuclear cells at 24 hours of culture, 25.5% and 27.1% at 1 and 3 months, respectively). The anti-apoptogenic effect of CL-E treatment showed a close dependence on the increase in vitamin E in the blood cell compartment. CONCLUSIONS: This study suggests that this vitamin E-modified membrane can be considered a highly biocompatible material, the antioxidant properties of which can exert a site-specific and timely scavenging function against oxygen free radicals in synergy with a hypostimulatory action on the PMN respiratory burst.     

Demonstration of complimentarity between monoclonal antibodies (MAbs) to human chorionic gonadotropin (hCG) and polyclonal antibodies to luteinizing hormone/hCG receptor (LH-R) and their use in better understanding hormone-receptor interaction

The concurrent influence of multiple neurotransmitter systems in mediating cocaine-induced convulsions is predicted by the results of previous receptor binding studies. The present results demonstrate that pharmacological manipulations of these predicted neurotransmitter systems alters the occurrence of cocaine-induced convulsions. The 5-HT reuptake inhibitor fluoxetine enhanced the occurrence and severity of convulsions produced by 100 mg/kg (-) cocaine, while the 5-HT2 receptor antagonists cinanserin, ketanserin and pirenperone antagonized cocaine-induced convulsions in a dose-dependent manner. Further, the M1 receptor antagonist pirenzepine antagonized cocaine-induced convulsions, but atropine did not. In addition, both the (+) and (-) stereoisomers of the sigma ligand SKF 10047 significantly attenuated cocaine-induced convulsions. (+)SKF 10047 was more potent than (-)SKF 10047 in this effect, suggesting a stereoselective effect at sigma receptor sites. In constrast, DA and NE neurotransmission do not appear to modulate the proconvulsant effects of cocaine in a specific, dose-dependent manner. Thus, of the CNS binding sites with which cocaine is known to interact, the results are consistent with the conclusion that 5-HT transporters and 5-HT2 receptor sites appear to be direct and primary sites related to cocaine-induced convulsions, while M1 and sigma binding sites appear to play important but secondary and modulatory roles in this response.     

Poly(2-aminoadenylic acid): interaction with poly(uridylic acid)

Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.     

Human leucocytic antigens (HLA) in breast cancer

HLA frequencies of fifty (50) female breast cancer patients were compared with 200 age matched female controls. A total of 20 HLA-A locus, 35 HLA-B locus and 8 HLA-C locus antigens were studied. The HLA-A2, A11, Aw19 and A30; HLA-B8, B14 and HLA Cw6 were found significantly higher than the controls. The HLA-A11, HLA-Aw19 and HLA-B8 were found protective whereas, HLA-A2, HLA-B14 and HLA-Cw6 were a risk for breast cancer. The prective antigens' probable involvement through immunogenic mechanism in breast cancer is emphasized in this article.     

Molecular genetics (HLA) of Beh?et's disease

Beh?et's disease (BD) has been known to be strongly associated with the human leukocyte antigen (HLA) B51. This B51 association has been confirmed in many different ethnic groups between the Middle East and Japan, and it has been proposed that BD is prevalent in those ethnic groups along the old Silk Route. The hypothesis could be made that B51 molecules are primarily involved in BD development through specific antigen presentation. However, polymorphic analyses of the TNFB gene and Tau-a microsatellite between the HLA-B and TNF genes indicate that the pathogenic gene of BD is not the HLA-B51 gene itself but another gene located around the HLA-B gene. HLA-C genotyping by the PCR-SSP method also suggests that the BD pathogenic gene is not the HLA-C gene itself but other gene located near the HLA-B gene. Recently we sequenced a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C and identified 8 novel genes designated NOB1-8 (NOB: new organization associated with HLA-B). During the course of the genomic sequence analysis we clarified the genetic structure of the MICA (MHC class I chain-related gene A) gene and found a triplet repeat microsatellite polymorphism of (GCT/AGC)n in the transmebrane (TM) region. Furthermore, the microsatellite allele consisting of 6 repetitions of GCT/AGC (MICA A6 allele) was present at a significantly higher frequency in the BD patient group than in the control group and a significant fraction of B51-negative patients were positive for this MICA A6 allele. These results suggest the possibility of a primary association of BD with MICA rather than HLA-B.     

Human platelets possess tyrosylprotein sulfotransferase (TPST) activity

Phosphorylation of myosin light chain kinase by a Ca(2+)-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTP gamma S and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca(2+)-dependent phosphorylation of these two contractile proteins in smooth muscle.     

Autoimmune hepatitis with antibodies against liver cytosol (anti-LC1)

Antibody against liver cytosol (anti-LC1) was proposed in 1988 as a new and very specific immunoserologic marker of autoimmune hepatitis of childhood and young age. In adults, anti-LC1 might be masked by the presence in serum of anti-LKM1, usually associated with antibodies to hepatitis-C virus. We report the case of a 60-year-old woman who had active chronic hepatitis not related to infection by hepatitis C virus, with autoantibody reacting against liver cytosol as the unique marker of autoimmune hepatitis. Treated with corticosteroids (prednisone, 1 mg/kg per day), coagulation disorder, bilirubin, transaminase activity and immunoglobulins normalized in the following months. A year and a half later, autoantibodies anti-LC1 and p-ANCAs were no longer detected. We have only found one report of a young patient with anti-LC1 autoimmune hepatitis who had cirrhosis and portal hypertension, in national publications.     

Specific 3H-imipramine binding in human platelets. Influence of age and sex

Human platelets have been shown to possess high-affinity binding sites for 3H-imipramine. These binding sites have a similar affinity and drug specificity to those already described in rat brain. The platelets from healthy volunteers show no difference in 3H-imipramine binding between the sexes but there is a decrease in maximal 3H-imipramine binding with increasing age of the donor.