[Analysis of The Quality of Pneumoconiosis Network Direct Report in Sichuan during 2006-2016].

Objective: To evaluate the quality of Pneumoconiosis Network Direct Report in Sichuan Province in 2006-2016. Methods: download all the pneumoconiosis report cards from the Network Direct Report system. Screen out cards based on the diagnosis time that is between January 1st 2006 and December 31st 2016. Using R 3.4.0 software to analysis the number of missing or repeated cards, time-logical error rates, timeliness, reporting year, reporting intervals to evaluate the quality of Pneumoconiosis Network Direct Report and location distribution. Results: there are 38 855 pieces of Pneumoconiosis report card in total in 2006-2016. 352 pieces of cards were reported twice. 224 cards were missing. 229 cards have time-logical error. The rate of timely reporting for 2006-2016 years was 66.41% (2 5453/38 326) , 67.14% (24 658/36 726) for new cases, 58.87% (783/1 330) for promoting cases and 4.44% (12/270) for deaths. 87.38% (33 490/38 326) patients was reported in the same year. 10 days was needed to finish one report, confirming-filling cost much more time than filling-report (9.865/49.019) . Conclusion: the records of pneumoconiosis report cards are much more complete, logical errors are less, and the timeliness was a little bit higher than the average level in China. But it also should be improved. The death cases are difficult to report. It takes longer to diagnose and fill in cards. Improving the timeliness rate can significantly improve the quality of network direct reporting.

Analysis of the adult thymus in reconstitution of T lymphocytes in HIV-1 infection.

A key question in understanding the status of the immune system in HIV-1 infection is whether the adult thymus contributes to reconstitution of peripheral T lymphocytes. We analyzed the thymus in adult patients who died of HIV-1 infection. In addition, we studied the clinical course of HIV-1 infection in three patients thymectomized for myasthenia gravis and determined the effect of antiretroviral therapy on CD4(+) T cells. We found that five of seven patients had thymus tissue at autopsy and that all thymuses identified had inflammatory infiltrates surrounding lymphodepleted thymic epithelium. Two of seven patients also had areas of thymopoiesis; one of these patients had peripheral blood CD4(+) T-cell levels of <50/mm3 for 51 months prior to death. Of three thymectomized patients, one rapidly progressed to AIDS, one progressed to AIDS over seven years (normal progressor), whereas the third remains asymptomatic at least seven years after seroconversion. Both latter patients had rises in peripheral blood CD4(+) T cells after antiretroviral therapy. Most patients who died of complications of HIV-1 infection did not have functional thymus tissue, and when present, thymopoiesis did not prevent prolonged lymphopenia. Thymectomy before HIV-1 infection did not preclude either peripheral CD4(+) T-cell rises or clinical responses after antiretroviral therapy.

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Interactions of bovine mitochondrial phenylalanyl-tRNA with ribosomes and elongation factors from mitochondria and bacteria.

A homologous in vitro poly(U)-directed translation system has been established using animal mitochondrial ribosomes, elongation factors (EF) and phenylalanyl-tRNA(Phe). The rate of incorporation of phenylalanine into polyphenylalanine in the mitochondrial system is slower than that observed for the homologous Escherichia coli system. E. coli ribosomes can be used in place of mitochondrial ribosomes in this system with only a slight decrease in the efficiency of phenylalanine incorporation from mitochondrial Phe-tRNA. However, E. coli elongation factor Tu (EF-Tu) cannot replace the mitochondrial EF-Tu in promoting the use of mitochondrial Phe-tRNA. The interaction between EF-Tu and mitochondrial Phe-tRNA was investigated by using the ability of EF-Tu to protect the aminoacyl-tRNA bond from hydrolysis. These results showed that both mitochondrial and E. coli EF-Tus are capable of interacting with mitochondrial Phe-tRNA. However, ribosomal A-site binding assays demonstrated that efficient binding of the mitochondrial Phe-tRNA to the ribosomal A-site was only obtained with the homologous mitochondrial EF-Tu.

T cell receptor excision circle assessment of thymopoiesis in aging mice.

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in naïve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of naïve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 naïve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

HIV vaccine development at Duke University Medical Center.

With the AIDS epidemic continuing to spread throughout the world, development of a safe, practical, and effective HIV vaccine is a national priority. HIV vaccine research efforts are currently targeted towards design of HIV immunogens that induce both cellular and humoral immunity. This brief review summarizes ongoing work at the Duke University School of Medicine on HIV vaccine development.

Bovine mitochondrial initiation and elongation factors.

The procedures summarized above provide nearly homogeneous preparations of IF-2mt, EF-Tu. Tsmt, and EF-Gmt. The scheme developed for IF-2mr leads to a 24,000-fold purification of this factor with a 26% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that this factor functions as a monomer with a molecular weight of about 85,000. The scheme developed EF-Tu.Tsmt provides a 10,000-fold purification with an overall yield of about 10%. The EF-Tumt component in this complex has a molecular weight of about 46,000, whereas EF-Tsmt has a molecular weight of about 32,000 on SDS-polyacrylamide gel electrophoresis. The EF-Tu. Tsmt complex is tightly associated and appears to have a native molecular weight of about 70,000. The five-step purification procedure outlined above for EF-Gmt results in a 14,000-fold purification of EF-Gmt with a 2-5% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that EF-Gmt functions as a monomeric protein with an apparent molecular weight of about 80,000.

Identification of a synthetic peptide that mimics an HIV glycoprotein 120 envelope conformational determinant exposed following ligation of glycoprotein 120 by CD4.

CD4 ligation of HIV envelope gp120 results in conformational changes in gp120 that lead to exposure of the gp41 fusogenic domain and fusion with the host cell membrane. One determinant at or near the CD4-binding site exposed on gp120 subsequent to CD4 binding is defined by two human MAbs termed 17b and 48d. These MAbs do not block CD4 binding to gp120; rather, their binding to gp120 is upregulated following CD4 binding. To determine if synthetic peptide mimetopes could be found that reflect conformational determinants on the surface of gp120, synthetic gp120 peptides from 10 divergent HIV isolates were screened for their ability to bind to 17b and 48d in ELISAs. Although MAb 48d binds to HIV IIIB recombinant gp120 protein, in our studies 48d selectively bound only to the HIV Can0A V3 peptide and not to HIV IIIB V3 peptide, whereas MAb 17b bound none of the peptides tested. Monoclonal antibody 48d bound to the HIV Can0A V3 peptide both in solid-phase ELISA and in solution in a competitive ELISA, but could not bind to HIV Can0A V3 peptide bound to human T cells. The HIV Can0A V3 peptide induced anti-HIV antibodies in rhesus monkeys that neutralized the laboratory-adapted HIV MN strain but did not induce antibodies that neutralized HIV IIIB/LAI, HIV SF-2, or HIV RF isolates, or that neutralized HIV primary isolates. These data suggested that the primary sequence of the HIV Can0A V3 loop exists in a conformer that mimicks a non-V3 determinant of native gp120 exposed subsequent to CD4 binding on the surface of gp120 of laboratory-adapted HIV strains. Structural studies of the Can0A V3 peptide and/or the 48d MAb may provide important information regarding the nature of gp120 conformational changes that occur following gp120 ligation by CD4.

Role of adhesion molecules in the pathogenesis of rheumatoid arthritis.

The pathogenesis of rheumatoid arthritis centers on as yet unknown initiating events in the synovium that result in synovial vessel proliferation, and upregulation of endothelial cell ligands for leukocyte adhesion molecules. Ligation of adhesion molecules on synovial microenvironment cells and immune cells probably regulates synovial and immune cell inflammatory cytokine production. Interruption of adhesion molecule function and interruption of inflammatory cytokine production are promising new sites of therapeutic inhibition of synovial inflammation.

Effects of length and mRNA secondary structure on the interaction of bovine mitochondrial ribosomes with messenger RNA.

The mRNA for cytochrome oxidase subunit II (CoII) from bovine mitochondria binds to the small subunit of the mitochondrial ribosome in the absence of auxiliary factors. The synthetic polymer poly(U) is effective in competing with CoII mRNA for binding, although the polymer poly(A,U,G) competes very weakly. The effects of mRNA length on the interaction between the 28 S ribosomal subunit and mRNA have been examined using truncated derivatives of CoII mRNA. These results indicate that there is a minimum length of approximately 400 nucleotides required for the efficient binding of the mRNA to the small subunit. Shorter mRNAs will bind, but do so with much lower association constants. mRNAs of various lengths but with reduced secondary structure were prepared by substituting ITP for GTP during in vitro transcription reactions. These derivatives show the same effects of length as do the normal mRNA, indicating that RNA secondary structure is not a critical factor in subunit-mRNA interaction. The binding of the mRNA to the 28 S subunit is not influenced by the presence of guanine nucleotides or by the presence of a triphosphate at the 5' end of the RNA.

Initiation of protein synthesis in animal mitochondria. Purification and characterization of translational initiation factor 2.

Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity. The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity. SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa. IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative. IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes. IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide. It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly. The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP. DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP. No activity is observed with ATP, CTP, or UTP. Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria.

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.

Interaction of bovine mitochondrial ribosomes with messenger RNA.

The gene for subunit II of cytochrome oxidase (CoII) from bovine mitochondria has been cloned behind a T7 promoter and the corresponding mRNA synthesized in vitro. The RNA transcribed from this vector has a single nucleotide 5' to the start AUG and, thus, corresponds closely to the native mRNA. It binds to the small 28 S ribosomal subunit of bovine mitochondria but not to the large (39 S) subunit or to 55 S ribosomes. The binding occurs readily in the absence of auxiliary initiation factors or initiator tRNA. The complex formed appears to contain 1 mRNA/28 S subunit. The observed binding is specific for mRNA since neither tRNA nor ribosomal RNA can act as competitive inhibitors. The interaction of the mRNA with the 28 S subunit does not require an AUG codon near the 5' end and constructs containing 5' leaders of more than 100 nucleotides still bind efficiently. About 5% of the bound mRNA is protected from digestion by T1 RNase. The protected fragments do not arise from a specific region of the mRNA since they hybridize to several restriction fragments of the cloned CoII gene.

N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding.

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.