Munir M. Zaman

Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.

Noninvasive sizing of subcellular organelles with light scattering spectroscopy

A long-standing impediment for applications of optical techniques in cellular biology is the inability to characterize subcellular structures whose dimensions are much less than about 1 /spl mu/m. In this paper, we describe a method based on light scattering spectroscopy that can find the size distribution of subcellular organelles as small as 100 nm with an accuracy of 20 nm. We report experiments using aqueous suspensions of subcellular organelles enriched in mitochondria, zymogen granules, and microsomes. From the observed light scattering spectra, we extract size distributions that are in excellent agreement with the results of electron microscopy. Further studies are underway to extract the shapes of organelles in addition to their sizes.

Interleukin 8 secretion from monocytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered

ABSTRACT Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1β production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.

Confocal light absorption and scattering spectroscopic microscopy

We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.

Decreased expression of peroxisome proliferator activated receptor γ in CFTR-/- mice

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice.

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Resolvin D1 and Lipoxin A4 Improve Alveolarization and Normalize Septal Wall Thickness in a Neonatal Murine Model of Hyperoxia-Induced Lung Injury

Background The critical fatty acids Docosahexaenoic Acid (DHA) and Arachidonic Acid (AA) decline in preterm infants within the first postnatal week and are associated with neonatal morbidities, including bronchopulmonary dysplasia (BPD). DHA and AA are precursors to downstream metabolites that terminate the inflammatory response. We hypothesized that treatment with Resolvin D1 and/or Lipoxin A4 would prevent lung injury in a murine model of BPD. Objective To determine the effect of Resolvin D1 and/or Lipoxin A4 on hyperoxia-induced lung injury. Methods C57/BL6 pups were randomized at birth to Room Air, Hyperoxia (>90% oxygen), Hyperoxia + Resolvin D1, Hyperoxia + Lipoxin A4, or Hyperoxia + Resolvin D1/Lipoxin A4. Resolvin D1 and/or Lipoxin A4 (2 ng/g) were given IP on days 0, 3, 6, and 9. On day 10, mice were sacrificed and lungs collected for morphometric analyses including Mean Linear Intercept (MLI), Radial Alveolar Count (RAC), and Septal Thickness (ST); RT-PCR analyses of biomarkers of lung development and inflammation; and ELISA for TGFβ1 and TGFβ2. Result The increased ST observed with hyperoxia exposure was normalized by both Resolvin D1 and Lipoxin A4; while, hyperoxia-induced alveolar simplification was attenuated by Lipoxin A4. Relative to hyperoxia, Resolvin D1 reduced the gene expression of CXCL2 (2.9 fold), TIMP1 (6.7 fold), and PPARγ (4.8 fold). Treatment with Lipoxin A4 also led to a reduction of CXCL2 (2.4 fold) while selectively increasing TGFβ2 (2.1 fold) and Smad3 (1.58 fold). Conclusion The histologic and biochemical changes seen in hyperoxia-induced lung injury in this murine model can be reversed by the addition of DHA and AA fatty acid downstream metabolites that terminate the inflammatory pathways and modulate growth factors. These fatty acids or their metabolites may be novel therapies to prevent or treat lung injury in preterm infants.

GP2, a GPI-anchored protein in the apical plasma membrane of the pancreatic acinar cell, co-immunoprecipitates with src kinases and caveolin.

We previously showed that endocytosis at the apical plasma membrane (APM) of the pancreatic acinar cell is activated by the cleavage of GP2, a GPI-linked protein, from the apical cell surface. This endocytic process, as measured by horseradish peroxidase uptake into pancreatic acinar cells, is blocked by the tyrosine kinase inhibitors genistein and tyrphostin B42 as well as by disruption of actin filaments with cytochalasin. This suggests that the cleavage of GP2 from the cell membrane may activate endocytosis through a tyrosine kinase–regulated pathway. However, the mechanism by which GP2 and tyrosine kinases act together to activate endocytosis at the APM remains unknown. In this study, we demonstrate that pp60, p62yes, caveolin, and annexin, which have previously been implicated in endocytosis in other cell lines, were present in high abundance in GPI-enriched membranes by Western blot analysis. pp60, p62yes, and caveolin all co-immunoprecipitated with GP2 except annexin. An 85-kDa protein whose tyrosine-dependent phosphorylation is correlated with the activation of endocytosis in intact acinar cells also was present in these immunoprecipitates. This suggests that in pancreatic acini, GP2 may exist in a complex with src kinases, caveolin, and an 85-kDa phosphorylated substrate to regulate endocytosis at the APM.

Decreased Peroxisome Proliferator Activated Receptor α Is Associated with Bile Duct Injury in Cystic Fibrosis Transmembrane Conductance Regulator−/− Mice

Background: Primary sclerosing cholangitis (PSC) is associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. As proof of concept that CFTR dysfunction plays a role in PSC, induction of colitis in cftr−/− mice results in bile duct injury that can be prevented by pretreatment with docosahexaenoic acid (DHA). Objectives: Determine whether 1) CFTR dysfunction in cftr−/− mice through a reduction in peroxisome proliferator activated receptor (PPAR)&agr; or &ggr; leads to bile duct injury and 2) whether DHA prevents bile duct injury through an increase in PPAR. Methods: Cftr−/− and wild-type (WT) mice were treated with dextran sodium sulfate (DSS) to induce colitis with or without pretreatment with oral DHA. PPAR&agr; and &ggr; as well as tumor necrosis factor (TNF)&agr; were analyzed in liver tissue. PPAR&agr;−/− mice were also treated with DSS and histology examined. Results: PPAR&ggr; mRNA levels were low, with DSS suppressing mRNA levels equally in WT and cftr−/− mice. PPAR&agr; levels were no different between cftr−/− and WT litter mates by reverse-transcription polymerase chain reaction. After DSS, WT mice showed a 9.3-fold increase in PPAR&agr; mRNA levels and increased nuclear localization compared with no DSS (P < 0.05), with no increase seen in cftr−/− mice. This was not caused by changes in TNF&agr;. DHA treatment led to 7.0-fold increase in PPAR&agr; mRNA levels in cftr−/− mice (P < 0.01). PPAR&agr;−/− mice treated with DSS did not develop bile duct injury, indicating that PPAR&agr; alone is not sufficient to cause bile duct inflammation. Conclusion: DSS induced bile duct injury in cftr−/− mice is associated with a defect in PPAR&agr; expression, which is reversed by DHA.

Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr-/- transgenic mice.

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o(-) sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr(-/-) transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE(2), and PGF(2α) secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr(-/-) Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators.