A narrative review on yoga: a potential intervention for augmenting immunomodulation and mental health in COVID-19.

BACKGROUND: The ongoing novel coronavirus disease 2019 (COVID-19) pandemic has a significant mortality rate of 3-5%. The principal causes of multiorgan failure and death are cytokine release syndrome and immune dysfunction. Stress, anxiety, and depression has been aggravated by the pandemic and its resultant restrictions in day-to-day life which may contribute to immune dysregulation. Thus, immunity strengthening and the prevention of cytokine release syndrome are important for preventing and minimizing mortality in COVID-19 patients. However, despite a few specific remedies that now exist for the SARS-CoV-2virus, the principal modes of prevention include vaccination, masking, and holistic healing methods, such as yoga. Currently, extensive research is being conducted to better understand the neuroendocrinoimmunological mechanisms by which yoga alleviates stress and inflammation. This review article explores the anti-inflammatory and immune-modulating potentials of yoga, along with its role in reducing risk for immune dysfunction and impaired mental health. METHODS: We conducted this narrative review from published literature in MEDLINE, EMBASE, COCHRANE databases. Screening was performed for titles and abstracts by two independent review authors; potentially eligible citations were retrieved for full-text review. References of included articles and articles of major non-indexed peer reviewed journals were searched for relevance by two independent review authors. A third review author checked the excluded records. All disagreements were resolved through discussion amongst review authors or through adjudication by a fourth review author. Abstracts, editorials, conference proceedings and clinical trial registrations were excluded. OBSERVATIONS: Yoga is a nonpharmacological, cost-effective, and safe intervention associated with several health benefits. Originating in ancient India, this vast discipline consists of postures (asanas), breathing techniques (pranayama), meditation (dhyana/dharana), and relaxation. Studies have demonstrated yoga's ability to bolster innate immunity and to inhibit cytokine release syndrome. As an intervention, yoga has been shown to improve mental health, as it alleviates anxiety, depression, and stress and enhances mindfulness, self-control, and self-regulation. Yoga has been correlated with numerous cardioprotective effects, which also may play a role in COVID-19 by preventing lung and cardiac injury. CONCLUSION AND RELEVANCE: This review paves the path for further research on yoga as a potential intervention for enhancing innate immunity and mental health and thus its role in prevention and adjunctive treatment in COVID-19.

Outcomes of uninterrupted vs interrupted Periprocedural direct oral Anticoagulants in atrial Fibrillation ablation: A meta-analysis.

BACKGROUND: Studies indicate that uninterrupted anticoagulation (UA) is superior to interrupted anticoagulation (IA) in the periprocedural period during catheter ablation of atrial fibrillation. Still IA is followed in many centers considering the bleeding risk. This meta-analysis compares interrupted and uninterrupted direct oral anticoagulation during catheter ablation of atrial fibrillation. METHODS: A systematic search into PubMed, EMBASE, and the Cochrane databases was performed and five studies were selected that directly compared IA vs UA before ablation and reported procedural outcomes, embolic, and bleeding events. The primary outcome of the study was major adverse cerebro-cardiovascular events. RESULTS: The meta-analysis included 840 patients with UA and 938 patients with IA. Median follow-up was 30 days. Activated clotting time (ACT) before first heparin bolus was significantly longer with UA (P = .006), whereas mean ACT was similar between the two groups (P = .19). Total heparin dose needed was significantly higher with IA (mean, ‒1.61; 95% CI, ‒2.67 to ‒0.55; P = .003). Mean procedure time did not vary between groups (P = .81). Overall complication rates were low, with similar major adverse cerebro-cardiovascular event (P = .40) and total bleeding (P = .55) rates between groups. Silent cerebral events (SCEs) were significantly more frequent with IA (log odds ratio, ‒0.90; 95% CI, ‒1.59 to ‒0.22; P < .01; I 2, 33%). Rates of major bleeding, minor bleeding, pericardial effusion, cardiac tamponade, and puncture complications were similar between groups. CONCLUSIONS: UA during atrial fibrillation ablation has similar bleeding event rates, procedural times, and mean ACTs as IA, with fewer SCEs.

PCSK9 expression in the ischaemic heart and its relationship to infarct size, cardiac function, and development of autophagy.

Aims: Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapy to treat hypercholesterolaemia and related cardiovascular diseases. This study determined if PCSK9 can regulate infarct size, cardiac function, and autophagy during ischaemia. Methods and results: Mice hearts were subjected to left coronary artery (LCA) occlusion. There was intense expression of PCSK9 in the zone bordering the infarct area in association with marked cardiac contractile dysfunction in the wild-type mice. This region also revealed intense autophagy. To assess the role of PCSK9 in the evolution of infarct size and function and development of autophagy, we used wild-type mice pre-treated with two different PCSK9 inhibitors (Pep2-8 and EGF-A) or mice lacking PCSK9 gene. Both strategies resulted in smaller infarcts and improved cardiac function following LCA ligation. PCSK9 inhibition also markedly reduced autophagy. Relationship between myocardial ischaemia and PCSK9 expression and autophagy was examined in cultured mouse cardiomyocytes. Exposure of cardiomyocytes to hypoxia resulted in prompt PCSK9 expression and autophagy signals; both were blocked by HIF-1α siRNA. Further, treatment of cardiomyocytes with recombinant PCSK9 during hypoxia induced, and treatment with PCSK9 siRNA reduced, autophagy suggesting a possible role of PCSK9 in the determination of autophagy. Other studies revealed activation of ROS-ATM-LKB1-AMPK axis as a possible mechanism of PCSK-induced autophagy. Hearts of humans with recent infarcts also showed expression of PCSK9 and autophagy in the border zone-similar to that in the infarcted mouse heart. Conclusion: PCSK9 is up-regulated in the ischaemic hearts and determines development of infarct size, cardiac function, and autophagy.

PCSK9 regulates expression of scavenger receptors and ox-LDL uptake in macrophages.

Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been shown to influence macrophage biology and modulate atherogenesis. We conducted this study to examine the regulation of scavenger receptors (SRs) (LOX-1, SRA, and CD36) and oxidized liporoptein cholesterol (ox-LDL) uptake in macrophages by PCSK9. Methods and results: Treatment of mouse peritoneal macrophages with tumour necrosis factor alpha (TNF-α) resulted in concentration-dependent modest, but significant, increase in PCSK9 expression. Importantly, treatment of TNF-α primed macrophages with recombinant murine PCSK9 increased the expression of LOX-1, SRA, and CD36 2-5 fold, and enhanced ox-LDL uptake by ≈five-fold. The increase in LOX-1 was much greater than in SRA or CD36. PCSK9 inhibition (by siRNA transfection or use of macrophages from PCSK9-/- mice) reduced the expression of SRs (LOX-1 ≫ SRA or CD36). Ox-LDL uptake in response to PCSK9 was also inhibited in macrophages from LOX-1-/- mice (P < 0.05 vs. macrophages from SRA-/- and CD36-/- mice). Upregulation of PCSK9 by cDNA transfection induced intense ox-LDL uptake which was inhibited by co-transfection of cells with siRNA LOX-1 (P < 0.05 vs. siRNA SRA or siRNA CD36). Further, TNF-α-mediated PCSK9 upregulation and subsequent expression of SRs and ox-LDL uptake were reduced in macrophages from gp91phox-/-, p47phox-/- and p22phox-/- mice (vs. macrophages from wild-type mice). Conclusions: This study shows that in an inflammatory milieu, elevated levels of PCSK9 potently stimulate the expression of SRs (principally LOX-1) and ox-LDL uptake in macrophages, and thus contribute to the process of atherogenesis.

Cross-Talk Between PCSK9 and Damaged mtDNA in Vascular Smooth Muscle Cells: Role in Apoptosis.

AIMS: The present study was designed to investigate a possible interaction between vascular smooth muscle cell (SMC)-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) and mitochondrial DNA (mtDNA) damage. RESULTS: Treatment of cultured SMCs with the proinflammatory stimulus lipopolysaccharide (LPS) stimulated PCSK9 release and induced mtDNA damage. PCSK9 inhibition by its siRNA reduced, and its enhancement increased, mtDNA damage. Induction of mitochondria-derived reactive oxygen species (mtROS) (by rotenone, thenoyltrifluoroacetone, or antimycin A) enhanced mtDNA damage as well as PCSK9 release, suggesting a role of mtROS in PCSK9-mtDNA damage interplay. Induction of mtDNA damage (with the autophagy inhibitor, 3-methyladenine, or DNase II inhibition) enhanced PCSK9 expression, and inhibition of mtDNA damage (with the autophagy inducer, rapamycin) reduced PCSK9 expression, indicating bidirectional interplay between PCSK9 and mtDNA damage. Other studies showed that p38 MAPK is involved in PCSK9-induced mtDNA damage, and mammalian target of rapamycin activation plays a role in mtDNA damage-induced PCSK9 release. Functional impact of PCSK9-mtDNA damage cross-talk was evident in the form of SMC apoptosis, which was enhanced in cells treated with recombinant human PCSK9, but inhibited in cells treated with PCSK9 siRNA. Last, LPS administration in wild-type mice resulted in simultaneous PCSK9 release and mtDNA damage, but mtDNA damage was minimal in PCSK9-null mice given LPS. INNOVATION: Vascular SMC-derived PCSK9 induces mtDNA damage, and damaged mtDNA fragments stimulate PCSK9 release mediated, at least in part, by mtROS. CONCLUSIONS: These observations suggest positive feedback interplay between SMC-derived PCSK9 and mtDNA damage in the proinflammatory milieu involving mtROS. This interaction results in cellular injury, characterized by apoptosis-a hallmark of atherosclerosis. Antioxid. Redox Signal. 25, 997-1008.

Modulation of myocardial injury and collagen deposition following ischaemia-reperfusion by linagliptin and liraglutide, and both together.

Studies have indicated that dipeptidyl peptidase-4 (DPP-4) inhibitors and glucagon-like peptide-1 (GLP-1) agonists reduce infarct size after myocardial ischaemia. Whether these agents modify cardiac remodelling after ischaemia is unclear. Furthermore, it is not known if combination of the two types of drugs is superior to either agent alone. We investigated the modulatory effect of the DPP-4 inhibitor linagliptin alone, the GLP-1 activator liraglutide alone, or the two agents together on myocardial infarct size, left ventricular contractile function and cardiac remodelling signals after a brief period of left coronary artery (LCA) occlusion. C57BL/6 mice were treated with vehicle, the DPP-4 inhibitor linagliptin, the GLP-1 activator liraglutide, or both agents together for 5 days, and then subjected to LCA occlusion (1 h) and reperfusion (3 h). Ischaemia-reperfusion increased reactive oxygen species (ROS) generation and expression of NADPH oxidase (p47(phox), p22(phox) and gp91(phox) subtypes), collagens, fibronectin and proinflammatory cytokines (interleukin 6, tumour necrosis factor α and monocyte chemoattractant protein-1) in the LCA-supplied regions. Pre-treatment with linagliptin or liraglutide reduced infarct size, protected cardiomyocytes from injury and preserved cardiac contractile function in a similar fashion. It is interesting that profibrotic (collagen deposition) signals were expressed soon after ischaemia-reperfusion. Both linagliptin and liraglutide suppressed ROS generation, NADPH oxidase and proinflammatory signals, and reduced collagen deposition. Addition of linagliptin or liraglutide had no significant additive effect above and beyond that of liraglutide and linagliptin given alone. In conclusion, linagliptin and liraglutide can improve cardiac contractile function and indices of cardiac remodelling, which may be related to their role in inhibition of ROS production and proinflammatory cytokines after ischaemia.

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.

LOX-1 - dependent mitochondrial DNA damage and NLRP3 activation during systemic inflammation in mice.

BACKGROUND: Lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) is known to be involved in many pathophysiological events, such as inflammation. METHODS: To clarify the role of LOX-1 in mtDNA damage and NLRP3 inflammasome activation, we studied wild-type (WT) and LOX-1 knockout (KO) mice given thioglycollate, an inflammatory stimulus. RESULTS: We observed intense inflammatory response (CD45 and CD68 expression) and mtDNA damage in spleen and kidneys of WT mice given thioglycollate. The abrogation of LOX-1 (use of LOX-1 knockout mice) reduced the inflammatory response as well as mtDNA damage (P<0.05 vs. WT mice). We also observed that mice with LOX-1 deletion had markedly reduced expression of caspase-1 (P10 and P20 subunits) as well as cleaved IL-1ß and IL-18. These mice also had much less mtDNA damage and only limited NLRP3 inflammasome expression. CONCLUSIONS: These in vivo observations indicate that LOX-1 plays a key role in mtDNA damage which then leads to NLRP3 inflammasome activation during inflammation.

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 Iкßα 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.

Proximal tubule PPARα attenuates renal fibrosis and inflammation caused by unilateral ureteral obstruction.

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-ß1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-ß, demonstrating PPARα signaling reduces epithelial TGF-ß production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1ß, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

Preliminary findings of the relationship of lower heart rate variability with military sexual trauma and presumed posttraumatic stress disorder.

Decreased heart rate variability (HRV) occurs with physical and psychological disorders and is a predictor of cardiac and all-cause mortality. This study was the first of which we are aware to examine and report the relationship between military sexual trauma (MST) and HRV measures. In a historical cohort study of female veterans with (n = 27) and without (n = 99) MST who received Holter and electrocardiogram evaluation at a Veteran Affairs medical center during 2007-2010, we examined the relationship between MST and the standard deviation of all R-R intervals (SDNN) and the square root of the mean of the sum of the squares of differences between adjacent R-R intervals (RMSSD). Female veterans with MST were younger, p = .002, frequently had a probable posttraumatic stress disorder diagnosis, 80% versus 15%, p = < .0001, and had lower SDNN, p = .0001, and RMSSD, p = .001, than those without MST. The SDNN and RMSSD of a 25-year-old female veteran with MST were comparable to that of female veterans aged 69 to 81 years without MST. Further research is needed to evaluate relationships between MST and HRV measures.

Lower heart rate variability associated with military sexual trauma rape and posttraumatic stress disorder.

Low heart rate variability (HRV) can occur with psychological disorders such as posttraumatic stress disorder (PTSD). The purpose of this study was to examine the association between PTSD by trauma type and decreased HRV measures in female veterans with cardiac symptoms. This secondary analysis utilized data from a previous study of female veterans (n = 125) examined for cardiac symptoms by Holter and electrocardiogram recordings at a Veterans Affairs medical center. The mean HRV measure from three 10-s data segments with spontaneous respirations was obtained for each subject. PTSD diagnosis and type of trauma exposure were collected from mental health consult notes. Chi-square was used for frequency of subject characteristics; independent t tests and one-way analysis of variance (ANOVA) compared means of HRV measures between trauma types. Statistical significance was set at p < .05 a priori. By ANOVA, significantly lower log-transformed standard deviation of all normal sinus rhythm R-R intervals (SDNN) and log-transformed square root of the mean of the sum of the squares of differences between adjacent normal sinus rhythm R-R intervals (RMSSD) were found in the PTSD group with documented rape military sexual trauma (MST) compared to other groups including no PTSD, PTSD following MST with rape not specified, combat exposure, and nonmilitary-related trauma; lower HRV measures were not found with other PTSD types of trauma. This study suggests rape MST with concomitant PTSD may be a risk factor for decreased HRV in female veterans examined for cardiac symptoms.

Smaller cardiac cell size and reduced extra-cellular collagen might be beneficial for hearts of Ames dwarf mice.

PURPOSE: To test the hypothesis that cardiac morphologic differences between Ames dwarf and wild-type littermates might correlate with the increased longevity observed in the Ames dwarf mice. METHODS: Hearts removed from young adult (5-7 mo) and old (24-28 mo) Ames dwarf and wild-type littermates underwent histological and morphometric analysis. Measurements of cell size, nuclear size, and collagen content were made using computerized color deconvolution and particle analysis methodology. RESULTS: In the young mice at six months of age, mean cardiomyocyte area was 46% less in Ames dwarf than in wild-type mice (p<0.0001). Cardiomyocyte size increased with age by about 52% in the wild-type mice and 44% in the Ames dwarf mice (p<0.001). There was no difference in nuclear size of the cardiomyocytes between the young adult wild-type and Ames dwarf mice. There was an age-associated increase in the cardiomyocyte nuclear size by approximately 50% in both the Ames and wild-type mice (p<0.001). The older Ames dwarf mice had slightly larger cardiomyocyte nuclei compared to wild-type (2%, p<0.05). The collagen content of the hearts in young adult Ames dwarf mice was estimated to be 57% less compared to wild-type littermates (p<0.05). Although collagen content of both Ames dwarf and wild-type mouse hearts increased with age, there was no significant difference at 24 months. CONCLUSIONS: In wild-type and Ames dwarf mice, nuclear size, cardiomyocyte size, and collagen content increased with advancing age. While cardiomyocyte size was much reduced in young and old Ames dwarf mice compared with wild-type, collagen content was reduced only in the young adult mice. Taken together, these findings suggest that Ames dwarf mice may receive some longevity benefit from the reduced cardiomyocyte cell size and a period of reduced collagen content in the heart during adulthood.

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.

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

A correlation between growth rate, apoptosis, and tumor necrosis factor-α in umbilical cord blood cells infected with two strains of Mycobacterium tuberculosis.

BACKGROUND: Using umbilical cord blood (UCB) cells, it was demonstrated that three virulent isolates, two highly transmissible clinical isolates and the virulent laboratory strain, demonstrated rapid growth in the UCB cells, which was significantly faster than the growth rate observed for a unique isolate. There was also a significant increase in the amount of tumor necrosis factor (TNF)-a elicited from the UCB cells after infection with the unique isolate compared to the hypervirulent isolates. This study investigated whether neutralization or addition of TNF-a within this system would alter growth rates and apoptosis. STUDY DESIGN AND METHODS: Ten UCB samples were obtained for these experiments and adherent cells were isolated. Two clinical isolates, one virulent and one unique, were used. Colony-forming units were assessed at 3 hours postinfection (Day 0) and on Day 7 to generate growth ratios. TNF-a antibody or exogenous TNF-a was added after the 3-hour incubation period. Viability of the UCB cells was assessed. Apoptosis was measured using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining. RESULTS: There was no significant difference in the growth ratio for the virulent strain regardless of the presence of TNF-a antibody. There was a significant increase for the nonvirulent strain after the addition of antibody. There was an increase in viability for the UCB cells in the presence of antibody, suggesting a decrease in TNF-a­dependent apoptosis. Addition of exogenous TNF-a to the UCB cells after infection with the virulent strain decreased the growth ratio with a significant increase in apoptosis. CONCLUSIONS: The TNF-a response of the UCB cells is related to the infecting strain and the intracellular growth of the strain of Mycobacterium tuberculosis is not directly controlled by the level of TNF-a. This cytokine is at the start of a powerful cascade of transcription factors with numerous pleiotropic effects; consequently abrogating/enhancing a single direct outcome was difficult, with only slight alterations in growth.

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.