Manijeh Phillips

A Whole Blood Bactericidal Assay for Tuberculosis

The bactericidal activity of orally administered antituberculosis (anti-TB) drugs was determined in a whole blood culture model of intracellular infection in which microbial killing reflects the combined effects of drug and immune mechanisms. Rifampin (Rif) was the most active compound studied and reduced the number of viable bacilli by >4 logs. Isoniazid (INH), 2 quinolones, and pyrazinamide (PZA) showed intermediate levels of activity. Ethambutol exerted only a bacteristatic effect; amoxicillin/clavulanate was inactive. The combination of INH-Rif-PZA showed strong activity against 11 drug-sensitive isolates (mean, -3.8 log) but no activity against 12 multidrug-resistant (MDR) strains. The combination of levofloxacin-PZA-ethambutol had intermediate bactericidal activity against MDR isolates (mean, -1.2 log) but failed to equal that of INH-Rif-PZA against sensitive isolates (P<.001). The whole blood BACTEC method (Becton Dickinson) may be useful for the early clinical evaluation of new anti-TB drugs and in the management of individual patients.

Induction of monocyte expression of tumor necrosis factor alpha by the 30-kD alpha antigen of Mycobacterium tuberculosis and synergism with fibronectin.

Native 30-kD antigen, also known as alpha antigen, is a fibronectin-binding protein that is secreted by live Mycobacterium tuberculosis. This antigen may play an important biological role in the host-parasite interaction since it elicits delayed type hypersensitivity response and protective immunity in vivo and T lymphocyte blastogenesis and IFN-gamma production in vitro. In the present study, we show that, TNF-alpha protein is produced in monocyte culture supernatants in response to 30-kD antigen and the level is as high as that to purified protein derivative of M. tuberculosis. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan. The preincubation of monocytes with plasma fibronectin significantly enhanced the release of TNF-alpha into the culture supernatants in response to 30-kD antigen. This effect was blocked by polygonal antibody to plasma fibronectin. In contrast, the monocytic cell line U937 failed to release TNF-alpha protein in the culture supernatants in response to 30-kD antigen with or without preincubation with plasma fibronectin. To determine whether this observation was due to differential binding of the 30-kD to fibronectin on these cells, a cell based ELISA was used. Pretreatment of monocytes with fibronectin enhanced their binding of the 30-kD antigen. U937 cells bound the 30-kD antigen weakly with or without fibronectin pretreatment. These results indicate that 30-kD antigen which is a known secretary antigen of M. tuberculosis is a stimulus for human monocytes to express TNF-alpha and that stimulatory effect may be mediated through plasma fibronectin.

Induction of the Antigen 85 Complex of Mycobacterium tuberculosis in Sputum: A Determinant of Outcome in Pulmonary Tuberculosis Treatment

Sputum quantitative culture, acid-fast smear, days-to-positive by BACTEC, and Mycobacterium tuberculosis antigen 85 complex were monitored during therapy in 42 patients with pulmonary tuberculosis (TB). By BACTEC, 4 patients were persistently positive on days 90-180, and treatment ultimately failed in 2 of these. Antigen 85 expression increased in subjects in whom disease persisted (persisters) from days 0 to 14 when the difference between persisters and nonpersisters was statistically significant (P = .002). Only antigen 85 complex values at day 14 suggested TB persistence at or after day 90. All subjects with day 14 antigen 85 complex values < 60 pg/mL responded rapidly to treatment and were cured. Of those with values > 60 pg/mL, in 33% TB persisted at or after day 90 and treatment failed in 17%. Biologic factors expressed early in therapy, not related to compliance or resistance, may exert a substantial influence on outcome. The antigen 85 complex is critical in cell wall biosynthesis and is induced by isoniazid in vitro. Its induction may represent an adaptive transition to a persistent state during therapy.

Opsonizing antibodies (IgG1) up-regulate monocyte proinflammatory cytokines tumour necrosis factor-alpha (TNF-α) and IL-6 but not anti-inflammatory cytokine IL-10 in mycobacterial antigen-stimulated monocytes—implications for pathogenesis

Cachexia is one of the prominent features of advanced tuberculosis (TB) seen in association with increased expression of the monokine TNF‐α. Several mycobacterial proteins, including PPD, stimulate TNF‐α secretion from monocytes. Host factors that may play a role in cytokine expression from monocytes remain largely unknown. One such factor is the opsonizing antibodies. Monocytes have high‐affinity receptors (FcγI and FcγIII) for IgG1 and IgG3 antibodies that mediate antigen uptake. We have reported selective up‐regulation of IgG1 (which bind to Fcγ receptors) in advanced TB and have recently shown the ability of PPD‐specific IgG1 antibodies to augment TNF‐α expression in PPD‐stimulated monocytes. These observations have now been extended to other cytokines with semipurified fractions from secreted antigens of Mycobacterium tuberculosis (containing 30 kD and 58 kD) that were devoid of lipids, glycolipids and carbohydrates. In the presence of heat‐inactivated TB plasma containing known amounts of antigen‐specific IgG1 antibodies, these fractions induced significantly increased TNF‐α, IL‐6 and IL‐10 secretion. Absorption of IgG1 with Protein ‘A’ removed the augmenting activity for TNF‐α and IL‐6 secretion from the TB plasma samples. In the case of IL‐10, removal of IgG1 resulted in increased rather than decreased IL‐10 secretion. These results suggest a possible pathogenic role for antibodies in TB by enhancing proinflammatory and blocking down‐regulatory cytokines such as IL‐10 cytokines during the chronic phase of TB.

An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo. Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1–7 mm. Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.

860 Natural Killer T Cells Bearing IL-13rα2, Producing IL-13 and Having Cytotoxicity for Epithelial Cells Populate the Mucosa in Ulcerative Colitis

exposition in patients after pouch surgery (pouch patients versus IBD patients, p < 0.02). Conclusions:Increased cell shedding and transient epithelial cell destruction with subsequent bacterial translocation are newly discovered features of IBD pathophysiology. These changes can be identified and quantified in humans during ongoing colonoscopy using CLE. This might further support the notion that intrinsic micro-flora can trigger flares in patients with IBD. Thus, CLE might play a crucial role in the future for monitoring mucosal healing in patients with IBD. Epithelial changes of the terminal ileum before and after the exposition of rectal micro-flora