Sue A. Theus

Intracellular Macrophage Growth Rates and Cytokine Profiles of Mycobacterium tuberculosis Strains with Different Transmission Dynamics

Mycobacterium tuberculosis strains associated with IS6110 restriction fragment-length polymorphism (RFLP) pattern clusters and strains demonstrating unique IS6110 RFLP patterns were investigated in interferon- gamma -activated THP-1 cells by measurement of binding, intracellular growth rate, and cytokine production. Binding was the same for all strains; however, strains from clusters grew significantly more rapidly than did unique strains. Maximal concentration of tumor necrosis factor (TNF)-alpha was detected at 2 days after infection, with unique strains eliciting significantly greater amounts than did strains from clusters. Interleukin (IL)-10 levels peaked at 1 day after infection with strains from clusters, whereas they peaked at 5 days after infection with unique strains. Rapid growth demonstrated by strains from clusters was highly correlated with rapid production of IL-10 and suppression of TNF-alpha in THP-1 cells during the early stages of infection. Characterization of this phenotype will further advance the investigation of virulence factors in M. tuberculosis.

Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging.

To assess the specificity of metabolite trafficking among gap junction‐coupled astrocytes, we developed novel, real‐time, single‐cell enzymatic fluorescence assays to assay cell‐to‐cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose‐6‐phosphate (P) and two analogs, deoxyglucose (DG)‐6‐P, and 2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐DG‐6‐P, compared with DG and 2‐ and 6‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐DG, (ii) extensive junctional diffusion of glyceraldehyde‐3‐P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na+ that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux‐regulating compounds (e.g. glucose‐6‐P) may be necessary for local metabolic control, whereas ‘syncytial sharing’ may dissipate the work load on peri‐synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG‐6‐P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron–astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.

Comparison of the immunotoxicity of propanil and its metabolite, 3,4-dichloroaniline, in C57Bl/6 mice

Propanil (3,4-dichloropropionaniline), used extensively as a postemergence herbicide in rice and wheat, has as its major metabolite, 3,4-dichloroaniline (DCA). Propanil has previously been shown to affect the T cell-dependent antibody response. To determine the immunotoxicity of DCA, as well as extend the previous immunotoxicity studies, several T cell-dependent and -independent immune responses were determined after DCA or propanil exposure. Unlike propanil, DCA caused a significant reduction in T-dependent antibody production (anti-SRBC response) only at a high dose (150 mg/kg). DCA or propanil at 150 or 200 mg/kg, respectively, caused a significant reduction in the number of anti-DNP antibody producing cells. However, doses of 37 or 50 mg/kg of DCA or propanil, respectively, caused an increase in the number of anti-DNP antibody producing cells. These data indicate that both propanil and DCA have a differential effect on the T-independent antibody response depending on the dose. Similar to propanil, DCA (at 150 mg/kg) caused a significant increase in spleen weight and cellularity. The effect of DCA or propanil on selected cellular immune functions was also determined. DCA caused a significant decrease in the natural killer (NK) cell activity at doses of 75 or 150 mg/kg, and propanil caused a significant decrease at 100 or 200 mg/kg. Cytotoxic T lymphocyte activity, however, was unaffected even at 150 or 200 mg/kg DCA or propanil, respectively. Thus, it appears that T cells are relatively resistant to the effects of propanil and DCA, whereas, other immune cell types, e.g., NK cells are sensitive to its effects.

Differences in the Growth of Paired Ugandan Isolates of Mycobacterium tuberculosis within Human Mononuclear Phagocytes Correlate with Epidemiological Evidence of Strain Virulence

ABSTRACT Previous studies have suggested that isolates of Mycobacterium tuberculosis responsible for tuberculosis outbreaks grow more rapidly within human mononuclear phagocytes than do other isolates. Clinical scenarios suggesting virulence of specific M. tuberculosis isolates are readily identified. Determination of appropriate “control” isolates for these studies is more problematic, but equally important for validating these assays and, ultimately, for identifying biologic differences between M. tuberculosis strains that contribute to virulence. We utilized the database from a study of Ugandan tuberculosis patients and their household (HH) contacts to identify M. tuberculosis isolates transmitted within HH and nontransmitted control isolates. Isolate pairs were evaluated from matched HH in each of three clinical scenarios: (i) coprevalent disease and no disease, (ii) incident disease and no disease, and (iii) M. tuberculosis infection (purified protein derivative [PPD] positive) and no infection (PPD negative). Intracellular growth of paired organisms was determined in a blinded fashion using two models of intracellular infection in which we have previously demonstrated correlation between intracellular growth and strain virulence, primary human monocytes (MN) and THP-1 human macrophage-like cells. In both models, transmitted isolates from coprevalent disease HH displayed more rapid growth than nontransmitted control isolates. In the THP-1 model, this was also true of transmitted isolates from HH with incident disease and their controls. Differences in production of tumor necrosis factor alpha and interleukin-10 by matched isolates showed correlation with growth patterns in the THP-1 cells but not in MN. Paired isolates characterized in this manner may be of particular interest for further investigations of the virulence of M. tuberculosis.

Long-term glucose control and risk of perioperative complications

BACKGROUND The impact of long-term preoperative glucose control on short-term surgical complications is unclear. We investigated whether preoperative hemoglobin A1c (HA(1c)) levels correlated with the risk of postoperative complications. METHODS A database of 38,989 patients undergoing major surgical procedures from October 1996 to May 2007 was reviewed. Of these patients, 2,960 were diagnosed diabetic with a HA(1c) level within 30 days before their operation. National Surgical Quality Improvement Program (NSQIP) definitions were used in determining postoperative complications. RESULTS Of 36,039 nondiabetic patients, 5,095 experienced 1 or more complications (14.1%). In 2,960 diabetic patients, 780 diabetic patients had 1 or more complications (26.4%) (P <or=.001). Mortality was higher in diabetics than nondiabetics (4.2% vs 1%, respectively; P <.0001). There was no correlation between HA(1c) levels and risk of complication, type of complication, or death. CONCLUSIONS Although our data show an increased risk of surgical complications in diabetics, there was no detectable correlation between risk of complication and HA(1c) level. This suggests that poor long-term glucose control may not play a major role in determining short-term surgical morbidity.

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase–3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase–3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

Cytokine regulation of bone marrow natural suppressor cell activity in the suppression of lymphocyte function

Natural suppressor (NS) cells, which nonspecifically suppress immune responses, are present in the spleen following exposure to radiation, chronic graft-versus-host disease, or cancer and in normal bone marrow. A model system is described which allows the study of cytokines activating and inhibiting NS cells, cytokines mediating NS activity, and NS effects on cytokine synthesis. Recombinant interleukin-3 (rIL-3) and granulocyte-macrophage colony-stimulating factor (rGM-CSF) efficiently activated NS cells present in normal bone marrow and were effective at concentrations as low as 5 U/ml. At high concentrations, GM-CSF, but not IL-3, did not activate NS cells. Recombinant interferon-gamma (rIFN-gamma) blocked the activation of bone marrow NS cells by rIL-3, but did not down-regulate NS cells once activated. The NS cells secreted one or more soluble suppressor factors, which blocked IL-2 synthesis and also inhibited IL-2-dependent T cell proliferation in the presence of excess IL-2.

Macrophage tumoricidal mechanisms are selectively altered by prenatal chlordane exposure

Macrophages (mø) derived from mice treatedin utero with chlordane show a significant delay of tumoricidal induction activity. In this study, mø from chlordane-treated animals required a 48 hin vitro period of induction with interferon-ψ and lipopolysaccharide (IFN/LPS) before they could kill P815 targets. Similarly, mø from chlordane-treated animals also failed to produce an immediate H2O2 burst upon perturbation. Conversely, their stimulated control mø counterparts were tumoricidal by 2 h and exhibited a respiratory burst without any delay. Moreover, levels of the second messenger, inositol triphosphate (IP3), were significantly delayed in chlordane-treated animals following interaction with IFN/LPS. When nitrate/nitrite production was analyzed as an alternate mechanism for killing tumors, stimulated mø from both normal and chlordane-treated animals responded equally. The data show that chlordane differentially introduces defects in mø biochemical mechanisms associated with tumor killing.

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

Background It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models. Methodology/Principal Findings By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates. Conclusions/Significance These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.

Is the lack of adiponectin associated with increased ER/SR stress and inflammation in the heart?

Objective To study whether there is an association between adiponectin and endoplasmic reticulum/sarcoplasmic reticulum (ERSR) stress. Research design Eleven-month-old male wild-type (WT) and adiponectin knockout (ADKO) mice were placed on chow or high fat diet for 12 weeks. The changes in ER stress and inflammatory genes were determined in the epididymal adipose, as well as heart tissue of adult WT and ADKO mice. To understand the role of ER/SR stress in the regulation of adiponectin, we studied the effect of tunicamycin or palmitate on H9C2 cardiomyoblasts in culture. To demonstrate the protective role of adiponectin, we studied the effect of purified adiponectin on the regulation of ERSR stress genes and inflammation in H9C2 cardiomyoblasts. Results (1) High fat diet increased TNFα in adipose tissue of ADKO mice. (2) ERSR stress genes, HSPa5, ERN1, and GADD34, and inflammation response genes, TNFα and CD68, were increased in heart of ADKO mice. High fat diet did not further increase the effect. (3) Induction of ERSR stress by tunicamycin in H9C2 resulted in the upregulation of ERSR stress response genes along with downregulation of adiponectin, adiponectin receptors 1 and 2, and Serca2A. ER stress was accompanied by down regulation of Iкβα and an increase in HSPa5 proteins. (4) Adiponectin decreased ERSR stress and inflammation response genes and increased Serca2A in to H9C2 cardiomyoblasts. Conclusion The lack of adiponectin is associated with increased ER/SR stress and inflammation in the heart. Adiponectin provides a protective effect by lowering inflammation and ER/SR stress along with increasing Serca2A in H9C2 cells.