Biomedical subjects
Publications and source records attributed to S Jiang.
A simple, rapid method for colony hybridization has been developed. The DNA probes were labelled by digoxigenin. Hybridization signal was detected by enzyme-linked immunoassay reaction. The results showed that this method is sensitive and reproducible, and it can be used for colony hybridization instead of isotopic methods.
A five-year-old boy with psychomotor retardation, microcephaly, bilateral cataracts, hearing impairment and hypospadia with microphallus was found to have multiple cell lines from peripheral blood: 46,XY/46,XY, -13,+r(13)/46, Xy, -13, +dic r(13) in the ratio of 35%/61%/4% by trypsin-Giemsa, and C-bandings. Using fluorescence in situ hybridization (FISH) with biotin-labeled alpha-satellite probe (D21Z1/D13Z1) and fluorescence staining (FITC), we confirmed that the ring originated from chromosome 13. To elucidate changes in the chromosome ends in the ring originated from chromosome 13. To elucidate changes in the chromosome ends in the ring formation, we used human telomere-specific probes for FISH study; it showed an absence of telomeres on the ring chromosome, although Ag-NOR staining was positive. These findings yielded different breaking points on the ends of both the short and long arms of chromosome 13 from those reported in the literature.
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The strong and long-lasting hematotoxic effect after benzene exposure in vivo (300 ppm, 6 hours/day, 5 days/week for 2 weeks) was assessed in mice with bone marrow cells grown in long-term bone marrow culture (LTBMC). Bone marrow cultures initiated 1 day after the last benzene exposure did not produce adequate numbers of hematopoietic cells over 3 weeks and, in most cases, no erythroid or myeloid clonogenic cells could be recovered. The adherent cell layer of these cultures had a lower capacity for supporting in vitro hematopoiesis after the second seeding with normal bone marrow cells compared with control cultures. Two weeks after the last benzene exposure, body weight, hematocrit, bone marrow cellularity, and committed hematopoietic progenitor content (BFU-E and CFU-GM) were regenerated to normal or subnormal values, whereas hematopoiesis in LTBMC was very poor. Over 8 weeks, little or no significant committed progenitor production was observed. Treatment of mice exposed to benzene with hemin (three doses of 3 micrograms/gm body wt, iv, over 2-week for a total dose 9 micrograms/gm) partially overcame the toxic effect of benzene on the hematopoietic system as measured by the LTBMC method. Cultures from mice treated with hemin had modest recovery of BFU-E and CFU-GM clonogenic potential after 5 to 6 weeks in LTBMC. In contrast, little or no recovery was obtained for the adherent cell layer clonogenic capacity, even after hemin treatment. These results clearly indicate a strong, long-lasting toxic effect on the bone marrow stroma and a limited recovery of hematopoietic potential by clonogenic cells of the nonadherent population after in vivo hemin treatment.
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Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.
Cytolytic lymphocytes have previously been reported to be resistant to the lytic effects of perforin. In this work, plasma membrane proteins from a CTL cell line were fractionated by HPLC, and the eluted fractions were collected based on their ability to inhibit perforin-mediated hemolysis. Three proteins with inhibitory activity were thus purified, the serine esterase MCSP-3/granzyme F and the histones H2B and H3. A commercial source of H2B was able to potently inhibit perforin-mediated lysis, and it was confirmed by FACS analysis that H2B is in fact present on the surface of cytolytic cells. However, H2B was also found on the surface of perforin-susceptible tumor cell lines, indicating that the histones may partially inhibit perforin-mediated lysis in vitro, but that they do not represent the factor conferring specific resistance on cytolytic lymphocytes. The origin of the surface histones and the possible role of the surface MCSP-3/granzyme F are discussed.
The positions of all 9 intrachain disulfide bonds within the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) have been established recently. Peptides expected to mimic some of the disulfide-bonded domains [(120-133)-(203-221); (133-138)-(164-203); (224-254); (391-425) and (385-392)-(425-452)] were synthesized. All peptides, except (120-133)-(203-221), elicited in immunized rabbits relatively high levels of antibodies reacting with gp120 in enzyme-linked immunosorbent assay (ELISA) and/or Western immunoblot assays. However, these antibodies failed to neutralize the infectivity of HIV-1. Combined with earlier reports concerning other gp120 loop peptides, these results confirm the uniqueness of the V3 (303-338) loop in encompassing a principal determinant(s) involved in virus neutralization.
Immunization of primates or humans with human immunodeficiency virus type 1 (HIV-1) glycoproteins usually elicited moderate immune responses to the principal neutralizing determinant (PND) located within the V3 hypervariable loop of gp120. Since an antibody response to the PND appears to be protective, experiments were carried out to determine the responsiveness of chimpanzees to immunization with synthetic peptides corresponding to the full-length V3 loop. Seven chimpanzees (4 preimmunized with gp160, 2 preimmunized with HIV-1 antigens unrelated to gp160, and 1 unimmunized) were vaccinated with a mixture of full-length V3 loop peptides from 21 distinct HIV-1 isolates (clones) either in unconjugated form or linked to carrier proteins from HIV-1 nef and gag P18, respectively. Six chimpanzees developed high levels of antibodies to the peptides (dilution endpoints 1: greater than 25,000), and 5 had high levels of antibodies to gp120 from HIV-1IIIB (endpoint titers 1: greater than 500,000). Chimpanzees immunized with peptide-carrier conjugates (4) had antibodies to the carrier proteins nef and gag P18, respectively (endpoint titers 1: greater than or equal to 35,000). Virus-neutralizing (VN) antibodies were detected in sera of 5 of 7 chimpanzees, but were present at titers of 1: greater than or equal to 400 only in sera of 2 chimpanzees. One of these was challenged with HIV-1 and was protected against infection, as reported elsewhere. The antibodies were primarily specific for the HIV-1 isolate used for primary immunization before boosting with peptides. The relatively low dilution endpoints of VN antibodies as compared with endpoints determined by site-specific immunoassays probably can be ascribed to imperfect mimicry of conformational epitopes by synthetic peptides. Nevertheless, sequential or simultaneous immunization with recombinant envelope glycoproteins of HIV-1 and selected synthetic peptides offers an approach for eliciting protective immunity against HIV-1.
The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.
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Cine-coronary arteriography in 186 consecutive patients with coronary heart disease demonstrated 109 cases with transmural infarctions, among which 59 revealed ventricular aneurysm (true 57, false 2). Twenty-eight cases underwent aneurysmectomy and 1 autopsy was performed. The anterolateral wall and/or apex of the left ventricle were involved in all 57 true cases of ventricular aneurysm. Single, double and triple vessel diseases of the coronary arteries were found in 29/57 (50.9%), 11/57 (19.3%) and 13/57 (22.8%), respectively. The anterior descending artery (LAD) was involved in 53 of the 57 cases (92.9%). In two cases with false ventricular aneurysm, the postero-lateral wall of the left ventricle was involved in one and the right ventricular wall in the other. Single vessel disease of the right coronary artery and the left circumflex branch was found respectively in the two cases. The problems of angiographic diagnosis and differential diagnosis of the true and false ventricular aneurysm are discussed.
The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.
Six cases of mental complications after cataract extraction performed on 779 patients by the author when he worked at Do Ye Ha Hospital of Algeria were reported in this paper. The symptoms included confusion, agitation, schizophrenia, aberrant behavior and nervousness. The treatment consisted of sedative and psychotherapy. The patients were all recovered within 72 hours. The methods for preventing mental complications after operation are discussed.
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A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.
A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytolytic lymphocytes. In the complement (C) system, a C8-binding protein (C8bp) or homologous restriction factor (HRF) has been described that protects cells against lysis mediated by homologous C. C8bp/HRF is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C8bp/HRF as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C8bp/HRF and against a 65 kDa protein band in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in C8bp/HRF; and (iii) the three antibodies compete with each other for binding to C8bp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforin-mediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C8bp/HRF does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C8bp/HRF and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for C8bp/HRF.
The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.