Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody.

Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy.

Sensitivity, Specificity, and Safety of a Novel ESAT6-CFP10 Skin Test for Tuberculosis Infection in China: 2 Randomized, Self-Controlled, Parallel-Group Phase 2b Trials.

Background: Diagnostics to identify tuberculosis infection are limited. We aimed to assess the diagnostic accuracy and safety of ESAT6-CFP10 (EC) skin test for tuberculosis infection in Chinese adults.

Methods: We conducted 2 randomized, parallel-group clinical trials in healthy participants and tuberculosis patients.

A Computational Method for the Identification of Endolysins and Autolysins.

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious.

Ag85b/ESAT6-CFP10 adjuvanted with aluminum/poly-IC effectively protects guinea pigs from latent mycobacterium tuberculosis infection.

The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models.

Recombinant tuberculosis vaccine AEC/BC02 induces antigen-specific cellular responses in mice and protects guinea pigs in a model of latent infection.

Purpose: To preliminarily evaluate the immunogenicity and efficacy of the recombinant tuberculosis vaccine AEC/BC02 in which Ag85b and fusion protein ESAT6-CFP10 were combined with bacillus Calmette-Guérin CpG and an aluminum salt-based adjuvant system.

Methods: Groups of BALB/c mice were immunized intramuscularly three times at 10-day intervals with AEC/BC02 or the adjuvant alone and the vaccine-induced cell-mediated immune responses were evaluated. The efficacy of AEC/BC02 was evaluated in two guinea pig models, one a model of prevention and the other a model of latent infection.

Efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis: a phase II trial.

Background: This study aimed to determine the efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis (TB).

Material And Methods: A phase II trial was performed in 158 patients with pulmonary TB (145 initially-treated and 13 re-treated) and 133 healthy subjects. Skin testing was carried out by injecting purified protein derivative (PPD) (on left forearm) or recombinant ESAT-6 protein at a dosage of 2, 5, or 10 μg/mL (on the right forearm) in each subject.

Preclinical study and phase I clinical safety evaluation of recombinant Mycobacterium tuberculosis ESAT6 protein.

Background: To investigate the ability of rESAT6 to identify different mycobacteria-sensitized guinea pigs and its safety in preclinical and phase I clinical study.

Material And Methods: Guinea pigs were sensitized with different Mycobacteria. After sensitization, all animals were intradermally injected with rESAT6 and either PPD or PPD-B.

[The effect of various antigen compounds of tuberculosis composite antigen vaccine on the immune function in mice].

Objective: To study the immune function of mice immunized by different combinations of antigen 85b (Ag85b), fusion protein culture filtered protein 10 (CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille Calmette-Guerin (BCG) CpG and aluminum.

Methods: According to antigen combinations, 48 BALB/c mice were divided into 8 groups: (1) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; (3) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 + adjuvant; (7) group G: HspX + adjuvants; (8) control group: saline (6 mice per group). The mice were subcutaneously immunized 3 times.

[A guinea pig model of latent Mycobacterium tuberculosis H₃₇Rv infection].

Objective: To establish the guinea pig model of latent Mycobacterium tuberculosis (MTB) H₃₇Rv infection, and to study the multiplication dynamics of MTB in vivo, and the relationship between latent MTB infection and PPD skin test.

Methods: Sixty-two guinea pigs were randomly divided into the model group (n = 42) and the control group (n = 20), and the model group was subdivided into a 4 weeks group (n = 12), an 8 weeks group (n = 21) and a 12 weeks group (n = 9), challenged by 500 CFU H₃₇Rv with restored toxicity. After 2 weeks challenge, the model groups were treated with isoniazid (INH, 10 mg/kg) + pyrazinamidum aldinamide (PZA, 40 mg/kg) for 4 weeks, 8 weeks and 12 weeks respectively.

The development and preliminary evaluation of a new Mycobacterium tuberculosis vaccine comprising Ag85b, HspX and CFP-10:ESAT-6 fusion protein with CpG DNA and aluminum hydroxide adjuvants.

Ag85b and HspX of Mycobacterium tuberculosis (Mtb) (H37Rv) were expressed and purified in this study. These two proteins were combined with another fusion protein CFP-10:ESAT-6 (C/E) (Ag), then mixed with the adjuvants CpG DNA and aluminum hydroxide and used to vaccinate mice and guinea pigs challenged with Mtb (H37Rv). The number of spleen lymphocytes secreting Ag85b, HspX and C/E-specific interferon-gamma were significantly higher in the Ag+Al+CpG group than in the Ag and CpG groups.

[Preliminary study on rapid identification the species of Mycobacteria by PCR-restriction fragment length polymorphism based upon hsp65 gene].

Objective: To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level.

Methods: hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae III and Bstp I, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed.

Isolated congenital heart disease is associated with the 22q11 deletion even though it is rare.

It is well known that a deletion within chromosome 22q11.2 has been identified in most cases of congenital heart disease (CHD) with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). Whether the 22q11.

[Effect of Mycobacterium smegmatis vaccine on the level of nitric oxide produced by peritoneal macrophages in mice].

Objective: To study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice.

Methods: Balb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro.

[Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice].

Objective: To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.

Methods: Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme.

[Identification of Mycobacterium species using reversed-phase high performance liquid chromatographic analysis of mycolic acid].

A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4 degrees C.

[Analysis of the genotype of intraspecies of common Mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences].

Objective: To analyze the genotypes of intraspecies of common mycobacteria.

Methods: The genotypes of 94 strains of mycobacteria from DSMZ, as compared with 12 reference strains, were studied by 16S-23S rRNA internal transcription space (ITS) sequence analysis.

Results: The sequencing of 16S-23S rRNA ITS of the 106 strains of common mycobacteria were completed.

[Differentiation of infection with Mycobacterium tuberculosis by recombinant Mycobacterium tuberculosis 11000 protein].

Objective: To study the differentiation effect of recombinant Mycobacterium tuberculosis 11000 protein on infection of Mycobacterium tuberculosis.

Methods: Guinea pigs were immunized with different strains of mycobacterium, and then all guinea pigs were given intradermal injections with recombinant Mycobacterium tuberculosis 11000 protein and purified protein derivative of tuberculin (PPD) or purified protein derived from M. intracellulare (PPD-B).

[The animal experimental study on rifampicin-dependent Mycobacterium tuberculosis].

Objective: To investigate the presence of rifampicin-dependent Mycobacterium tuberculosis strains by use of a guinea pig model of tuberculosis of rifampicin-dependent Mycobacterium tuberculosis.

Methods: Guinea pigs were randomly divided into groups of infection by rifampicin-dependent Mycobacterium tuberculosis strains (1130 strain, 1219 strain, b858 strain), rifampicin-resistant Mycobacterium tuberculosis strain (1290 strain) and ATCC 35810 strain and each group was further divided into an experimental group and a control group. The guinea pigs were challenged with 1130 strain, 1219 strain, b858 strain, 1290 strain and ATCC 35810 strain to establish the tuberculosis model.

[Effects of Mycobacterium smegmatis vaccine on cytokines production and Th1/Th2 responses in mice].

Objective: To validate the immunogenicity of Mycobacterium smegmatis and to study the immune modulatory function of Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis by analyzing the effects of the vaccine on immune responses in mice.

Methods: Spleen cells and peritoneal macrophages from BALB/c mice which were randomized into a control group and Mycobacterium smegmatis vaccine groups (low, middle, and high doses) were cultured in vitro. Then the supernatants were collected and the concentrations of IL-2, IL-4, IL-12, and IFN-gamma were analyzed through ELISA.

Association of 22q11 deletion with isolated congenital heart disease in three Chinese ethnic groups.

Background: Congenital heart disease (CHD) is the most common type of heart disease among children. About 75% of DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) includes CHD. A deletion within chromosome 22q11.

[The in vitro killing of intracellular Mycobacterium smegmatis by Mycobacteriophage].

Objective: To study the effect of Mycobacteriophage on the lysis of intracellular Mycobacterium smegmatis.

Methods: Peritoneal macrophages from BALB/C mice were incubated with Mycobacterium smegmatis for 4 h, and the extracellular bacteria were removed. Then the infected macrophages were treated for 2 h with normal saline, or different doses of Mycobacteriophages (2.

[Clinical analysis of primary biliary cirrhosis: a report of 42 cases].

Objective: To study the clinical features of primary biliary cirrhosis (PBC) in order to facilitate cognition of the disease.

Methods: Clinical data of 42 patients clinically and/or histologically diagnosed with PBC were reviewed. Anti mitochondrial antibody (AMA) negative/positive patients as well as the patients who were/were not associated with Sjogren Syndrome (SS) were compared in terms of clinical, biochemical and immunological features.