Alia Albawardi

Cellular bioenergetics, caspase activity and glutathione in murine lungs infected with influenza A virus

Inhibition of cellular respiration, oxidation of glutathione and induction of apoptosis have been reported in epithelial cells infected in vitro with influenza A virus (IAV). Here, the same biomarkers were investigated in vivo by assessing the lungs of BALB/c mice infected with IAV. Cellular respiration declined on day 3 and recovered on day 7 post-infection. For days 3-5, the rate (mean±SD) of respiration (µMO2min(-1)mg(-1)) in uninfected lungs was 0.103±0.021 (n=4) and in infected lungs was 0.076±0.025 (n=4, p=0.026). Relative cellular ATP (infected/uninfected) was 4.7 on day 2 and 1.07 on day 7. Intracellular caspase activity peaked on day 7. Cellular glutathione decreased by ≥10% on days 3-7. Lung pathology was prominent on day 3 and caspase-3 labeling was prominent on day 5. IAV infection was associated with suppression of cellular respiration, diminished glutathione, and induction of apoptosis. These functional biomarkers were associated with structural changes noted in infected mice.

Epithelioid trophoblastic tumor: report of two cases in postmenopausal women with literature review and emphasis on cytological findings.

Background: Epithelioid trophoblastic tumor (ETT) is a rare gestational trophoblastic neoplasm of intermediate trophoblasts. It was first described by Shih and Kurman [Am J Surg Pathol 1998;22:1393-1403] who outlined its clinicopathologic characteristics in 14 cases, establishing it as a distinct entity of gestational trophoblastic tumors. It represents 1.39% of all gestational trophoblastic diseases. Most cases are reported in reproductive-age women following a prior gestation with a time interval between 2 weeks and 30 years. ETT is extremely rare in postmenopausal women. It is commonly misdiagnosed as a squamous cell carcinoma (SCC), poorly differentiated carcinoma or another gestational trophoblastic tumor. Limited data is available regarding its cytological features on Pap smears. Cases: We report 2 cases of uterine ETT occurring in postmenopausal women. In both cases, an initial diagnosis of an SCC and a poorly differentiated carcinoma was rendered. We highlight the features of ETT helpful in differentiating it from other mimickers with emphasis on rarely reported cytological features of this neoplasm. Conclusion: ETT is a rare tumor with characteristic cytological features, but is commonly confused with SCC. A high index of suspicion is needed to make the correct diagnosis or to raise the consideration of ETT, especially in cases with an increased β-human chorionic gonadotropin.

Lung toxicities of core-shell nanoparticles composed of carbon, cobalt, and silica.

We present here comparative assessments of murine lung toxicity (biocompatibility) after in vitro and in vivo exposures to carbon (C–SiO2-etched), carbon–silica (C–SiO2), carbon–cobalt–silica (C–Co–SiO2), and carbon–cobalt oxide–silica (C–Co3O4–SiO2) nanoparticles. These nanoparticles have potential applications in clinical medicine and bioimaging, and thus their possible adverse events require thorough investigation. The primary aim of this work was to explore whether the nanoparticles are biocompatible with pneumatocyte bioenergetics (cellular respiration and adenosine triphosphate content). Other objectives included assessments of caspase activity, lung structure, and cellular organelles. Pneumatocyte bioenergetics of murine lung remained preserved after treatment with C–SiO2-etched or C–SiO2 nanoparticles. C–SiO2-etched nanoparticles, however, increased caspase activity and altered lung structure more than C–SiO2 did. Consistent with the known mitochondrial toxicity of cobalt, both C–Co–SiO2 and C–Co3O4–SiO2 impaired lung tissue bioenergetics. C–Co–SiO2, however, increased caspase activity and altered lung structure more than C–Co3O4–SiO2. The results indicate that silica shell is essential for biocompatibility. Furthermore, cobalt oxide is the preferred phase over the zerovalent Co(0) phase to impart biocompatibility to cobalt-based nanoparticles.

Effects of Selected Inhibitors of Protein Kinases and Phosphatases on Cellular Respiration: An In Vitro Study

Inhibitors of protein kinases/phosphatases are known to alter cellular metabolism. Effects of these rapidly identified small molecules on cellular respiration (mitochondrial O2 consumption) have not been adequately investigated, especially in healthy organs. This in vitro study measured cellular respiration in tissues from C57BL/6 mice with and without GSK2126458 (PI3K/mTOR inhibitor), BEZ235 (PI3K/mTOR inhibitor), GDC0980 (PI3K/mTOR inhibitor), GSK1120212 (trametinib, MEK inhibitor), sorafenib, regorafenib (multikinase inhibitors), and cyclosporine (calcineurin inhibitor). Cellular respiration was measured by the phosphorescence oxygen analyzer, aided by the O2 probe Pd(II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the respiratory chain. Renal cellular respiration decreased 26-34% in the presence of 10 μM GSK2126458 (p<0.001), 10 μM BEZ235 (p<0.001), or 1.0 μM GDC0980 (p<0.001). Liver cellular respiration decreased 20-32% with 10 μM GSK2126458 (p=0.048), 0.1 μM BEZ235 (p=0.028), or 0.1 μM GDC0980 (p=0.016). Heart cellular respiration decreased 19-27% with 10 μM GSK2126458 (p=0.078), 10 μM BEZ235 (p=0.040), or 10 μM GDC0980 (p=0.036). GSK1120212, sorafenib, regorafenib, and cyclosporine had no effects on cellular respiration. Thus, cellular bioenergetics (the biochemical processes involved in energy conversion) is interconnected with PI3K/PTEN/Akt/mTOR; and inhibitors of this cascade impair cellular respiration. This biomarker (cellular respiration) senses the activity/toxicity of this class of molecularly targeted agents.

Bioenergetics of murine lungs infected with respiratory syncytial virus

BackgroundCellular bioenergetics (cellular respiration and accompanying ATP synthesis) is a highly sensitive biomarker of tissue injury and may be altered following infection. The status of cellular mitochondrial O2 consumption of the lung in pulmonary RSV infection is unknown.MethodsIn this study, lung fragments from RSV-infected BALB/c mice were evaluated for cellular O2 consumption, ATP content and caspase activity. The disease was induced by intranasal inoculation with the RSV strain A2 and lung specimens were analyzed on days 2–15 after inoculation. A phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor respiration. The caspase-3 substrate analogue N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspases.ResultsO2 concentration declined linearly with time when measured in a sealed vial containing lung fragment and glucose as a respiratory substrate, revealing its zero-order kinetics. O2 consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Cellular respiration increased by 1.6-fold (p<0.010) and ATP content increased by 3-fold in the first week of RSV infection. Both parameters returned to levels found in uninfected lungs in the second week of RSV infection. Intracellular caspase activity in infected lungs was similar to uninfected lungs throughout the course of disease.ConclusionsLung tissue bioenergetics is transiently enhanced in RSV infection. This energy burst, triggered by the virus or virus-induced inflammation, is an early biomarker of the disease and may be targeted for therapy.

Lung tissue bioenergetics and caspase activity in rodents

BackgroundThis study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed.ResultsFor Wistar rats, the values of kc and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.ConclusionsLung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.

Respiratory syncytial virus increases lung cellular bioenergetics in neonatal C57BL/6 mice.

We have previously reported that lung cellular bioenergetics (cellular respiration and ATP) increased in 4-10 week-old BALB/c mice infected with respiratory syncytial virus (RSV). This study examined the kinetics and changes in cellular bioenergetics in ≤ 2-week-old C57BL/6 mice following RSV infection. Mice (5-14 days old) were inoculated intranasally with RSV and the lungs were examined on days 1-10 post-infection. Histopathology and electron microscopy revealed preserved pneumocyte architectures and organelles. Increased lung cellular bioenergetics was noted from days 1-10 post-infection. Cellular GSH remained unchanged. These results indicate that the increased lung cellular respiration (measured by mitochondrial O2 consumption) and ATP following RSV infection is independent of either age or genetic background of the host.

A 4-month-old baby presenting with dermal necrotizing granulomatous giant cell reaction at the injection site of 13-valent pneumococcal conjugate vaccine: a case report

IntroductionAdjuvants (for example, aluminum salts) are frequently incorporated in licensed vaccines to enhance the host immune response. Such vaccines include the pneumococcal conjugate, combinations of diphtheria–tetanus/acellular pertussis, tetanus– diphtheria/acellular pertussis, hepatitis B, some Haemophilus influenzae type b, hepatitis A, and human papillomavirus. These preparations have been associated with complicated local adverse events, especially if administered subcutaneously or intradermally in comparison to deep intramuscular injection. We describe a severe inflammatory reaction at the site of an injection of 13-valent pneumococcal conjugate vaccine.Case presentationA 4-month-old Arab baby boy developed dermal necrotizing granulomatous giant cell reaction at the injection site (right anterior thigh) of the second dose of 13-valent pneumococcal conjugate vaccine. Ziehl–Neelsen and periodic-acid Schiff were negative. This reaction probably resulted from improper intramuscular administration because the first (at 2 months of age) and third (at 10 months of age) doses were uneventful.ConclusionsDermal necrotizing granulomatous reactions are a serious complication of the 13-valent pneumococcal conjugate vaccine. Health care providers need to administer this preparation deeply into a muscle mass. Completing the vaccine series is an acceptable option. Physicians are encouraged to report their experience with completing vaccine series following adverse events.

Modulation of Cardiomyocyte and Hepatocyte Bioenergetics by Biguanides

Biguanides (metformin, buformin and phenformin) have been developed for oral treatment of non-insulindependent diabetes mellitus. Metformin, the drug of choice in this class, controls blood glucose primarily by lowering hepatic gluconeogenesis (e.g., decreasing glucagon-mediated hepatic glucose output). Its mode-of-action, however, is more complex and may involve “refining” cellular bioenergetics (improving energy efficiency) in various cell types including myocytes. Buformin and phenformin presumably have similar mechanisms of action. The main purpose of this in vitro study was to assess the effects of these drugs on bioenergetics - cellular respiration (mitochondrial O2 consumption) and ATP content - in tissue fragments from the heart muscle (cardiomyocytes) and liver (hepatocytes) of C57BL/6 mice. Cardiomyocyte respiration decreased by 10-26% in the presence of 100 μM metformin (p=0.093), buformin (p=0.028) or phenformin (p=0.015). Similar effects on cardiomyocyte respiration were noted with 1.0 mM drugs. Cardiomyocyte ATP, on the other hand, increased by 17-31% in the presence of 100 μM metformin (p=0.093), buformin (p=0.445) or phenformin (p=0.093). Hepatocyte respiration and ATP decreased by 11-26% and 8-25%, respectively in the presence of 1.0 mM drugs. Decreased respiration and ATP were also noted in hepatocytes exposed to 100 μM metformin for 1 ≤ t ≤ 6 hours (13% and 5%, respectively). Thus, the effects of biguanides on cardiomyocyte bioenergetics differed from that on hepatocyte bioenergetics. These findings suggest that biguanides regulate cardiomyocyte energy conversion, favoring better fuel efficiency (↓respiration/↑ATP). The drug effects in hepatocyte are ↓respiration/↓ATP, favoring less fuel production (↓hepatic gluconeogenesis). Biguanide activities in various tissues may be coupled.

In vitro assessment of antitumor activities of the PI3K/mTOR inhibitor GSK2126458

BackgroundUp-regulation of the PI3K/mTOR (phosphatidylinositol-3′ kinase/mammalian target of rapamycin) signaling is common in carcinoma. Consistently, targeting these molecules has been shown to halt the growth of many tumors. The main purpose of this study was to develop surrogate biomarkers of the antitumor activity of PI3K/mTOR inhibitors.MethodsFragments from eight tumors were collected immediately after resection in ice-cold RPMI gassed with 95% O2 :5% CO2. Viability was determined by measuring tumor cellular respiration (mitochondrial O2 consumption). The specimens were incubated at 37°C with and without 50 nM GSK2126458 (a highly potent and selective inhibitor of PI3K/mTOR) for 90 min. The tissue was then processed for histology, measurement of intracellular caspase-3 activity (using the caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin), and immunohistochemical detection of the apoptotic biomarkers caspase-3, cytochrome C, and annexin A2.ResultsGSK2126458 induced morphologic changes in four tumors (two invasive ductal carcinomas, one invasive lobular carcinoma, and one ovarian dysgerminoma), intracellular caspase-3 activity in three tumors (two invasive ductal carcinomas and one poorly differentiated signet ring adenocarcinoma of gastric origin), and immunohistochemical evidence of apoptosis in at least four tumors (three invasive ductal carcinomas and one adenocarcinoma of gastric origin). Two tumors (ovarian serous carcinoma and moderately differentiated adenocarcinoma of colorectal origin) demonstrated no treatment effect.ConclusionThese preliminary results demonstrate the feasibility of using in vitro biomarkers for detecting antitumor activities of the rapidly emerging PI3K/mTOR inhibitors.