Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

BACKGROUNDMonoclonal antibodies (mAbs) such as DD3, raised against progastrin-releasing peptide(31-98) (ProGRP (31-98)) antigen, have been used to target small cell lung cancer (SCLC). However, as an intact mAb, DD3 is cleared slowly from the body, with an optimal radioimmunoimaging time of 72 hours. More recently, a single-chain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research. Thereby, it potentially increases the radioimmunoimaging efficacy. However, there have been few studies with this antibody fragment. The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of (131)I-anti-ProGRP(31-98) scFv in nude mice bearing SCLC xenografts.

METHODSAnti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry. (131)I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated. Similarly, the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated. After injection of (131)I-anti-ProGRP(31-98) scFv, treated mice were imaged at 1, 24, and 30 hours. Then the tumor/base ratios were calculated.

RESULTSProGRP was highly expressed in NCI-H446 cells and xenograft tissue. The metabolism of (131)I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes, respectively. The %ID/g of (131)I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection, reaching a maximum of (5.38±0.92) %ID/g at 24 hours. Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours, with 24 hours proving optimal.

CONCLUSION(131)I-anti-ProGRP(31-98) scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance, indicating that it could be a promising agent for SCLC radioimmunoimaging.

Animals ; Female ; Flow Cytometry ; Humans ; Immunoglobulin Fragments ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Peptide Fragments ; immunology ; Radioimmunodetection ; methods ; Recombinant Proteins ; immunology ; Small Cell Lung Carcinoma ; diagnostic imaging ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo evaluate the association of major histocompatibility complex class I chain related gene A (MICA) antibodies with acute rejection (AR), chronic rejection (CR) and renal function after renal transplantation.

METHODSSerum MICA antibodies were detected with ELISA before and after transplantation with also examinations of panel reactive antibodies (PRA), serum creatinine, urine, graft ultrasound, lymphocyte subsets and the pathology of graft biopsy. The study was carried out in two parts to monitor MICA antibodies in acute and chronic rejections after renal transplantation.

RESULTSIn the first part of the study 18 of the 41 recipients experienced episodes of acute rejection, and the incidence rate was markedly higher in MICA(+) group than in MICA(-) group (P<0.05). Compared with the recipients with stable renal functions, the patients with acute graft rejection showed a significantly higher positivity rate of MICA antibodies. Postoperative MICA antibody monitoring showed that MICA antibody level increased gradually 2-3 days after the occurrence of acute rejection; anti-rejection treatment lowered serum creatinine to a normal level but MICA antibodies remained positive. In the second part, 21 of 40 patients had chronic graft rejection and showed significantly higher positivity rate of MICA than the patients with stable renal functions (P<0.05). In patients with chronic rejections, the serum creatinine levels were significantly higher in MICA(+) than in MICA(-) cases (P<0.05). Graft biopsy of all MICA(+) cases showed C4d deposition.

CONCLUSIONThe status of MICA antibodies can predict the occurrence and treatment outcomes of acute rejection, and also as one of the major causes of chronic graft rejection, they affect the long-term survival of the renal grafts.

Adolescent ; Adult ; Antibodies ; blood ; immunology ; Complement C4b ; metabolism ; Creatinine ; blood ; Follow-Up Studies ; Graft Rejection ; blood ; immunology ; pathology ; HLA Antigens ; immunology ; Histocompatibility Antigens Class I ; immunology ; Humans ; Kidney ; metabolism ; physiopathology ; Kidney Transplantation ; Peptide Fragments ; metabolism ; Young Adult

BACKGROUNDWe previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice.

METHODSD-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g).

RESULTSThe labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle.

CONCLUSIONS(99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; metabolism ; Female ; Male ; Mice ; Mice, Inbred ICR ; Peptide Fragments ; immunology ; Recombinant Proteins ; immunology ; Technetium ; chemistry

Calreticulin (CRT) is a multifunctional molecule in both intracellular and extracellular environment. We have previously found that a recombinant CRT fragment (rCRT/39-272) could modulate T cell-mediated immunity in mice via activation and expansion of CD1d(hi)CD5⁺ B cells as well as induction of CRT-specific regulatory antibodies. Antibody secreting cells (ASCs) are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regulation. In this study, we demonstrate that rCRT/39-272 differentiates murine CD1d(hi)CD5⁺ B cells into ASCs marked by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro. Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1d(hi)CD5⁺ B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis. Thus, we propose that ASC differentiation and subsequent antibody production of CD1d(hi)CD5⁺ B cells are key steps in CRT-mediated immunoregulation on inflammatory T cell responses.
Animals ; Antigens, CD1d ; metabolism ; Autoantibodies ; biosynthesis ; B-Lymphocytes ; cytology ; drug effects ; immunology ; metabolism ; CD5 Antigens ; metabolism ; Calreticulin ; chemistry ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; Humans ; Mice ; Peptide Fragments ; chemistry ; pharmacology ; Solubility

OBJECTIVETo explore the clinical significance of cytokeratin 19 fragments test in the diagnosis of nasopharyngeal carcinoma.

METHODSThe study included 102 cases of nasopharyngeal carcinoma, 90 cases of nasal polyp/nasopharyngitis, and 150 healthy individuals. RT-PCR was used to detect CK19 mRNA expression and Western blot to detect CK19 fragment protein expression in tissues of nasopharyngeal carcinoma. Expression of CK19-2G2 was examined by immunohistochemistry. Chemiluminescence analysis was used to detect the serum levels of CK19-2G2, and ELISA to detect that of EB-VCA IgA.

RESULTSAmong 102 cases of nasophryngeal carcinoma, 64 showed CK19 mRNA expression by RT-PCR, 60 showed CK19 protein fragments in tumor tissues by Western blot, and 66 showed expression of CK19-2G2 by immunohistochemistry in nasopharyngeal carcinoma, including strong positivity in 20 cases, moderate in 34 cases and weak in 12 cases. The sensitivity and specificity of CK19-2G2 in the diagnosis of nasopharyngeal carcinoma were 49.0% and 89.2%, and those of EB-VCA IgA were 52.9% and 85.4%, respectively. The combined detection of CK19-2G2 and EB-VCA IgA increased the sensitivity to 73.5% while the specificity remained at 80.0%.

CONCLUSIONSHigh levels of CK19-2G2 fragment expressed in tissue and serum are present in patients with nasopharyngeal carcinoma. The serum level of CK19-2G2 is helpful in the diagnosis of nasopharyngeal carcinoma. Furthermore, the combination of serum CK19-2G2 and EB-VCA IgA improves the detection sensitivity.

Adult ; Aged ; Antigens, Viral ; blood ; Blotting, Western ; Capsid Proteins ; blood ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; pathology ; Herpesvirus 4, Human ; immunology ; Humans ; Immunoglobulin A ; blood ; Immunohistochemistry ; Keratin-19 ; blood ; metabolism ; Middle Aged ; Nasopharyngeal Neoplasms ; diagnosis ; metabolism ; pathology ; Neoplasm Staging ; Peptide Fragments ; blood ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVE: To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ(15)-c, and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli. METHODS: The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The synthetic, double-strand Aβ(1-15) gene was inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-71- Aβ(15) was spliced to HBc88-144, yielding the recombinant gene c-Aβ(15)-c; that gene was subcloned into pET-28a(+). The fusion protein (CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The virus-like particle (VLP) formed by fusion protein CA15C was observed with transmission electric microscope (TEM). Four Kunming (KM) mice were given intraperitoneal injections of CA15C, and the anti-Aβ antibody elicited was detected by indirect ELISA. RESULTS: The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, the fusion protein was expressed, mainly in the sediment from the bacterial lysate. The expression level was 40% of total protein in the sediment. The CA15C could form VLP. After 5 rounds of immunization, the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000, while the anti-HBc antibody was undetectable. CONCLUSION Recombinant c-Aβ(15)-c gene can be expressed in E.coli. The expressed protein can form VLPs and has a strong immunogenicity.
Alzheimer Disease ; prevention & control ; Amyloid beta-Peptides ; genetics ; Animals ; Base Sequence ; Genetic Vectors ; genetics ; Hepatitis B Core Antigens ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, Virus-Like Particle ; genetics ; immunology ; metabolism

OBJECTIVETo study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and β-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated β-amyloid peptide (Aβ) in rats.

METHODSSD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aβ antibody was evaluated by ELISA. When the titers of the anti-Aβ antibody reached 1:3 000, aggregated Aβ was injected into the CA1 region of the rat hippocampus. Two weeks after Aβ injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining.

RESULTSThe titer of anti-Aβ antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aβ injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aβ injection exhibited obvious cell damages with Aβ deposits and glial infiltration, whereas in CAC-immunized rats, Aβ deposits were significantly reduced or even absent.

CONCLUSIONImmunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aβ injection.

Amyloid beta-Peptides ; administration & dosage ; biosynthesis ; genetics ; immunology ; Animals ; Antibodies ; blood ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hippocampus ; metabolism ; Immunization ; Injections ; Male ; Peptide Fragments ; administration & dosage ; immunology ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate the expressions of matrix metalloprotein-2 (MMP-2) and tissue inhibitor of metallopeptidase inhibitor-1 (TIMP-1) in the renal allografts of patients with chronic active antibody-mediated rejection (ABMR), and explore their role in the pathogenesis of ABMR.

METHODSImmunohistochemistry and computer-assisted image analysis were used to detect the expression of MMP-2 and TIMP-1 in the renal allografts of 46 patients with interstitial fibrosis and tubular atrophy (IF/TA), with 15 normal renal tissue specimens as the control. The association of MMP-2 and TIMP-1 with the pathological grade of IF/TA in ABMR was analyzed.

RESULTSThe expressions of MMP-2 and TIMP-1 significantly increased in the renal tissues of the patients as compared with the normal renal tissues (P<0.05). MMP-2 expression tended to decrease, while TIMP-1 and serum creatinine increased with the pathological grades of IF/TA (P<0.05). In IF/TA group, the expression of TIMP-1 was positively correlated to serum creatinine level (r=0.718, P=0.00<0.05).

CONCLUSIONAbnormal expressions of MMP-2 and TIMP-1 can promote the development of renal fibrosis in chronic ABMR.

Adult ; Antibody Formation ; Complement C4b ; metabolism ; Female ; Fibrosis ; etiology ; Graft Rejection ; immunology ; Humans ; Kidney ; metabolism ; Kidney Diseases ; pathology ; Kidney Transplantation ; adverse effects ; immunology ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Middle Aged ; Peptide Fragments ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism

We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0 x 10(6) colonies and the phage library titer was 2.65 x 10(12) TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.
AIDS Vaccines ; immunology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; genetics ; Humans ; Mutation ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Peptide Library ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; tat Gene Products, Human Immunodeficiency Virus ; biosynthesis ; genetics ; immunology