Early Levels of CD4, Neopterin, and β2-Microglobulin Indicate Future Disease Progression

Reduced CD4 T cell level and increased serum neopterin and ₂-microglobulin levels, which reflect immunological activation and dysregulation, are three important markers of HIV disease. The aim in this study is to delineate more clearly the relation of activation to future CD4 values and disease progression. By analyzing a cohort of 198 seroconverters from the Multicenter AIDS Cohort Study with 9 years' follow-up, the dynamic changes and levels of these three markers and their interrelationships are explored. We observed that the levels of markers in the first year after seroconversion have a much stronger impact on the progression of the disease than the preseroconversion marker levels. The actual change during the year after seroconversion is not as important as the final level reached during that year. The early levels of markers after seroconversion appear to be good indicators of the subsequent course of disease as defined by CD4 level and slightly better than the quantitative changes following seroconversion or the changes in the period 1 to 2.5 years after seroconversion. To investigate the variation between subjects, the 198 seroconverters were stratified into three approximately equal-sized groups in 12 ways based on their pre- and postseroconversion levels and changes in the three markers. The group with the highest CD4 level within a year after seroconversion maintains the highest CD4 level 8 years after seroconversion. The group with the lowest level of neopterin or ₂-microglobulin in this period has much higher future CD4 counts than the other two groups. The level of markers during the first year after seroconversion has a high predictive power for AIDS onset. Substantial differences in the hazards of AIDS are found between the groups with the highest and lowest CD4 count, neopterin, and ₂-microglobulin following seroconversion. The three markers are generally correlated throughout the postseroconversion period but can provide distinct information. High current levels of neopterin or ₂-microglobulin tend to be associated with low future CD4 count, while current levels of CD4 count have less association with future neopterin and ₂-microglobulin levels.     

Electron Microscopic Characteristics of β2-Microglobulin Amyloid Deposits in Long-Term Haemodialysis

The electron microscopic features of β2-microglobulin amyloid, deposited in the synovial membrane, are presented and discussed. The patient, a 69-year-old woman underwent chronic hemodialysis for 3 years. Because of constant pain and destructive arthropathy, endoprosthesis of the hip joints were implanted. Extra- and intracellular filamentous–fibrillar amyloid deposits have been demonstrated in ultrathin sections. The extracellular amyloid deposits showed a loose, filamentous or fibrillar structure at the periphery and a dense central core. The loose, filamentous structure may represent an early stage of fresh, newly deposited β2-microglobulin amyloid, while the condensed and fragmented amyloid filaments may be an advanced “mature” stage of amyloid deposition.     

A Regional Blood Flow Model for ��2-Microglobulin Kinetics and for Simulating Intra-dialytic Exercise Effect

A kinetic model based on first principles, for β(2)-microglobulin, is presented to obtain precise parameter estimates for individual patient. To reduce the model complexity, the number of model parameters was reduced using a priori identifiability analysis. The model validity was confirmed with the clinical data of ten renal patients on post-dilution hemodiafiltration. The model fit resulted in toxin distribution volume (V(d)) of 14.22 ± 0.75 L, plasma fraction in extracellular compartment (f(P)) of 0.39 ± 0.03, and inter-compartmental clearance of 44 ± 4.1 mL min(-1). Parameter estimates suggest that V(d) and f(P) are much higher in hemodialysis patients than in normal subjects. The developed model predicts larger removed toxin mass than that predicted by the two-pool model. On the application front, the developed model was employed to explain the effect of intra-dialytic exercise on toxin removal. The presented simulations suggest that intra-dialytic exercise not only increases the blood flow to low flow region, but also decreases the inter-compartmental resistance. Combined, they lead to increased toxin removal during dialysis and reduced post-dialysis rebound. The developed model can assist in suggesting the improved dialysis dose based on β(2)-microglobulin, and also lead to quantitative inclusion of intra-dialytic exercise in the future.     

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 (Ctomp2) is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species . To purify the protein and to prepare monoclonal antibodies (mAbs) against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni2 -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1:40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans . It had high specifity. In comparison with gold standard test-cell culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein , but also to the establishment of the method for its clinical application, for it had not been reported before.     

Distribution pattern of matrix metalloproteinases 1, 2, 3, and 9, tissue inhibitors of matrix metalloproteinases 1 and 2, and α2-macroglobulin in cases of generalized AA- and AL amyloidosis

Matrix metalloproteinases (MMPs) degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix which is disrupted by the deposition of amyloid. This immunohistochemical study investigated the distribution pattern of matrix metalloproteinases (MMP-1, -2, -3, and -9) and their inhibitors [alpha 2-macroglobulin (alpha 2-M), tissue inhibitors of MMPs (TIMP)-1, and TIMP-2] in human AA- and AL amyloid deposits. Specimens of liver, kidney, and spleen from 22 autopsy cases were investigated. Nine patients had suffered from generalized AA amyloidosis, eight from generalized AL amyloidosis, and five from rheumatoid arthritis or tuberculosis with no histological evidence of amyloid. In all amyloidotic and non-amyloidotic patients, each protease and protease inhibitor was detected in almost every organ investigated. In the amyloidotic cases, there was no indication that a specific protease or protease inhibitor was absent or expressed, but a difference was observed in their spatial distribution patterns. The most noticeable difference was found in immunostaining of amyloid. Only MMP-1, -2, and -3, and alpha 2-M were present in AA amyloid deposits, and only TIMP-1 and TIMP-2 were found in deposits of AL amyloid. This is the first study to show that MMP-1, -2, and -3 are present in AA amyloid deposits. They may be involved in tissue remodeling or in proteolysis of the precursor and fibril proteins.     

Hepatic levels of prostaglandins F2α and I2 (prostacyclin) and of thromboxane A2 in dogs developing hemorrhagic shock

Professorial Department of Surgery, L'vov Medical Institute. L'vov Branch, Kiev Research Institute of Hematology and Blood Transfusion. (Presented by Academician of the Academy of Medical Sciences of the USSR V. S. Savel'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 2, pp. 134–135, February, 1992.     

Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.     

Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.     

Axenfeld�CRieger syndrome and spectrum of PITX2 and FOXC1 mutations

Axenfeld–Rieger syndrome (ARS) is a rare autosomal dominant disorder, which encompasses a range of congential malformations affecting the anterior segment of the eye. ARS shows genetic heterogeneity and mutations of the two genes, PITX2 and FOXC1, are known to be associated with the pathogenesis. There are several excellent reviews dealing with the complexity of the phenotype and genotype of ARS. In this study, we will attempt to give a brief review of the clinical features and the relevant diagnostic approaches, together with a detailed review of published PITX2 and FOXC1 mutations.     

Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes

Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.     

Study of DNA damage induction and repair capacity of fresh and cryopreserved lymphocytes exposed to H2O2 and γ-irradiation with the alkaline comet assay

The alkaline SCGE assay was evaluated for use with cryopreserved lymphocytes in order to obtain results similar to the freshly isolated ones. The induction of DNA damage as well as the repair capacity of γ-rays and H₂O₂ exposed cryopreserved human lymphocytes was found to be the same to that of the freshly isolated. Human lymphocytes (fresh or cryopreserved) responded differently to the effects of γ-irradiation if compared to the H₂O₂ treatment. The distribution of DNA damage among γ-irradiated lymphocytes was more homogeneous compared to H₂O₂, both in freshly isolated and in cryopreserved cells. 2.4 μg/ml phytohemagglutinin at the start of a 2-h incubation in RPMI of cryopreserved samples gave similar DNA repair and distribution patterns to the 2-h post-exposure incubation of freshly isolated lymphocytes. H₂O₂-induced DNA damage was not repaired completely. However, the repair of γ-rays-induced DNA damage was more efficient. These findings confirm the different mode of action of the two agents on the induction of DNA damage, as well as, the different response of the lymphocytes' DNA repair system.     

Transmission heterogeneities,kinetics, and controllability of SARS-CoV-2

A long-standing question in infectious disease dynamics concerns the role of transmission heterogeneities,which are driven by demography,behavior,and interventions.On the basis of detailed patient and contact-tracing data in Hunan,China,we find that 80% of secondary infections traced back to 15% of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)primary infections,which indicates substantial transmission heterogeneities.Transmission risk scales positively with the duration of exposure and the closeness of social interactions and is modulated by demographic and clinical factors.     

Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI,and EUCAST methods,and detection of FKS1 and FKS2 mutations

Candida glabrata has emerged as a major pathogen in invasive candidiasis in recent years. Currently, guidelines for invasive candidiasis treatment recommend fluconazole or an echinocandin as the first-line therapy. Nevertheless, the resistance of Candida glabrata to echinocandin is an emerging problem and has been partly associated with mutations in the FKS1 and FKS2 genes. The Etest® is an appropriate method for determining antifungal susceptibility in emergency routine diagnosis. In this work, we evaluated the reliability of the Etest® in comparison with the two reference broth microdilution methods, Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), to assess the caspofungin resistance of 193 isolates of Candida glabrata. The interpretation of minimum inhibitory concentration (MIC) values was also discussed according to different breakpoints. Moreover, FKS1 and FKS2 mutations were investigated for isolates with high MICs. Our results showed that the MIC₅₀ value was similar to the MIC₉₀ value for each method. The Etest® method showed the lowest MIC values, whereas EUCAST presented the highest. Categorical agreement between the Etest® and CLSI methods was 100 % and 36 % using the breakpoints proposed by Arendrup et al. (Antimicrob Agents Chemother 56(7):3965–3968, 2012) and Pfaller et al. (Int J Antimicrob Agents 38(1):65–69, 2011), respectively. Two isolates showed high MIC values with the three methods and both presented FKS2 mutations. A novel FKS2 mutation was also reported for one isolate. Future epidemiological studies should also evaluate the reliability of the Etest® to detect echinocandin resistance, as it remains a routine method.     

Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin''s ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)₂] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)₂ fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.Approximately 20,000 cases of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, in which the O157:H7 serotype is the most prevalent serotype, are reported annually in the United States (for recent reviews, see references 6, 9, 10, 23, and 31). Transmission of E. coli O157:H7 is most frequently associated with the consumption of contaminated food (e.g., ground beef or spinach) or drinking unpasteurized dairy products. Infections can also be acquired through person-to-person contact. Infected individuals typically develop abdominal pain and bloody diarrhea 2 to 5 days following exposure. STEC infections are self-limiting and usually resolve in 7 to 10 days. However, in 10 to 15% of children under the age of 5 or in the elderly, E. coli O157:H7 infections can develop into diarrhea-associated hemolytic uremic syndrome (HUS), a serious, life-threatening complication (22, 26, 28, 31). HUS is associated with hemolytic anemia and thrombocytopenia as a result of the destruction of red blood cells and platelets, followed by acute renal failure. There are no effective therapies against HUS, and supportive therapies include dialysis and kidney transplantation. Thus, the best treatment for HUS is prevention or amelioration of the E. coli O157:H7 infection, as no protective therapies are presently available. Antibiotic therapy for treatment of E. coli O157:H7 infections does not shorten the infection period and, in fact, may increase the risk of developing HUS (34).The primary virulence factor for HUS is Shiga toxin 2 (Stx2), which is one of two antigenically distinct toxins produced by STEC. Stx2, like Stx1, consists of a single A subunit (32 kDa) linked to a ring of five B subunits (7 kDa) (18). The A subunit possesses RNA N-glycosidase activity, which cleaves a specific adenine residue in the 28S rRNA, resulting in the inhibition of protein synthesis. The B subunits are responsible for binding to the host cell receptor, globotriaosyl ceramide (Gb3; Galα[1-4]-Galβ[1-4]-Glcβ1-ceramide). The A subunit is cleaved as it is internalized through clathrin-dependent or independent endocytosis and translocated via the retrograde pathway to the cytosol where it inhibits protein synthesis (3, 4, 19).Therapies that inhibit cell binding, interfere with the intracellular transport of Stx2, or inhibit enzymatic activity are under development by several research groups. One of the most promising therapies is the use of Stx2-neutralizing human monoclonal antibodies, particularly those directed against the A subunit (13, 14). One of these antibodies, 5C12, was shown to protect mice and gnotobiotic piglets from the fatal complications of Stx2 (13, 24, 25). A recombinant 5C12 antibody also demonstrated similar protective activity in these two animal models (1). We have recently shown that 5C12 neutralizes the toxicity of Stx2 for HeLa cells by blocking the retrograde transport of the toxin to the cytosol where the A subunit inhibits protein synthesis (11). In the present study, we investigated the contribution of the Fc portion of 5C12 to its in vitro and in vivo toxin-neutralizing activity by evaluating the efficacies of the Fabs and F(ab′)₂ fragments of 5C12 in the HeLa cell and mouse toxicity assays. Smaller antibody fragments are advantageous for clinical use because of their lower immunogenicity and production costs. A comparison of a human monoclonal antibody against the B subunit of Stx2 (5H8) and its Fab fragment was performed to determine if similar results are obtained. We also investigated the contribution of the Fc functions by comparing the in vitro and in vivo neutralizing activities of the recombinant 5C12 isotype variants (e.g., IgG1, IgG2, IgG3, and IgG4).     

Molecular cloning of genes flhA and flhB_2 for flagellar biosynthesis of Leptospira interrogans and functional prediction of the prokaryotic expressing products

Leptospirosis is a worldwide zooanthroponosis thatis common in waterlogging areas and rice paddies[1] .Chinais one of the widelyspreadarea of thisdisease.Leptospiraincludes two species :Lepto-spira interrogansandLeptospira biflexa. Theformer is pathogenic for humans and animals andthe latteris saprophytic which usually existsinthesurface of waters and soils .Sofar ,no anytypicalexotoxins ofLeptospira interroganshas beenfound,and the content and toxicity of lipopolys-sachride fromL.interrogan…     

Immunochemical identification and physicochemical characteristics of the specificα 2-globulin of human granulocytes

Research Institute of Transplantology and Artificial Organs, Ministry of Health of the USSR. Department of Biochemistry, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR T. T. Berezov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 4, pp. 353–355, April, 1990.     

Expression of FGF10 and FGFR2Ⅲb in development of mouse kindey

目的 观察FGF10及其受体FGFR2Ⅲb在小鼠肾脏发生发育中的表达和分布,探讨其在肾脏发生发育中的作用.方法 选取胚龄12、14、16及18 d和出生后1、7、14、21及42 d小鼠肾组织,用免疫组织化学和Western blot,检测FGF10和FGFR2Ⅲb的表达.结果 胚龄12 d组FGF10及FGFR2Ⅲb开始在输尿管芽微弱表达;随着肾脏发育成熟,FGF10及FGFR2Ⅲb主要表达于远端小管和集合管;FGF10在近曲小管表达,近直小管无表达;FGFR 2Ⅲb在近端小管无阳性表达;生后肾组织及发育各阶段肾小体未见FGF10及FGFR2Ⅲb表达.Western blot显示随着胚(日)龄的增加,FGF10及FGFR2Ⅲb在肾脏的表达量先增后减.结论 FGF10和FGFR2Ⅲb在肾脏发生发育过程中发挥重要作用.     

Sulforaphane Inhibits IL-1β-Induced Proliferation of Rheumatoid Arthritis Synovial Fibroblasts and the Production of MMPs,COX-2, and PGE2

This study was performed to define the effects of sulforaphane on interleukin-1β (IL-1β)-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), the expression of matrix metalloproteinases (MMPs) and cyclooxygenase (COX), and the production of prostaglandin E2 (PGE2) by RASFs. The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1β with/without sulforaphane. The expression of MMPs, tissue inhibitor of metalloproteinase-1, COXs, intracellular mitogen-activated protein kinase signalings, including p-ERK, p-p38, p-JNK, and nuclear factor-kappaB (NF-kB), and the production of PGE2 were examined by Western blotting or semi-quantitative RT-PCR and ELISA. Sulforaphane inhibits unstimulated and IL-1β-induced proliferation of RASFs; the expression of MMP-1, MMP-3, and COX-2 mRNA and protein; and the PGE2 production induced by IL-1β. Sulforaphane also inhibits the phosphorylation of ERK-1/2, p-38, and JNK and activation of NF-kB by IL-1β. These results indicate that sulforaphane inhibits the proliferation of synovial fibroblasts, the expression of MMPs and COX-2, and the production of PGE2, which are involved in synovitis and destruction of RA, and suggest that sulforaphane might be a new therapeutic agent for RA.