Investigation of cellular and molecular responses to pulsed focused ultrasound in a mouse model

Continuous focused ultrasound (cFUS) has been widely used for thermal ablation of tissues, relying on continuous exposures to generate temperatures necessary to induce coagulative necrosis. Pulsed FUS (pFUS) employs non-continuous exposures that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing effects created by non-thermal mechanisms of FUS (i.e., acoustic radiation forces and acoustic cavitation). pFUS has shown promise for a variety of applications including drug and nanoparticle delivery; however, little is understood about the effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, and cell adhesion molecules). We examined changes in murine hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g., IL-1α, IL-1β, TNFα, INFγ, MIP-1α, MCP-1, and GMCSF) creating a local cytokine gradient on days 0 and 1 post-pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced upregulation of other signaling molecules (e.g., VEGF, FGF, PlGF, HGF, and SDF-1α) and cell adhesion molecules (e.g., ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies by increasing homing to areas of pathology.     

Dexamethasone induces different cellular protein synthetic responses in PNA+ and PNA- mouse thymocyte subpopulations

Thymocytes from adrenalectomized BALB/c male mice were separated by peanut agglutination (PNA) into cortical, corticosensitive, PNA+ cells and larger, medullary, corticoresistant, PNA- cells; the extent of cross-contamination of PNA+ and PNA- cells, and vice versa, was checked by flow microfluorometry. Glucocorticoid receptor profiles were established with 3H-dexamethasone as probe; no differences in receptor affinity or cellular concentration, or in cytoplasmic and nuclear compartmentalization were seen between PNA+ and PNA- cells. On two-dimensional gel electrophoresis, PNA+ and PNA- thymocytes from oil-injected (control) adrenalectomized mice showed patterns of incorporation of 35S-methionine into protein that differed in at least 12 spots, as revealed by autoradiography. PNA+ and PNA- cells from mice treated with submaximal (6 micrograms/day) or near-maximal thymolytic doses of dexamethasone (20 micrograms/day) were also examined by two-dimensional gel electrophoresis. Both PNA+ and PNA- cells showed substantial, overlapping dexamethasone-induced changes in protein synthetic profiles.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.

The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2.     

Uteroglobin induces the development and cellular proliferation of the mouse early embryo

Two-cell mouse embryos cultured in vitro in the presence of either purified rabbit uteroglobin (UG) or recombinant human UG developed and proliferated faster than controls cultured in the absence of this protein. Both the percentage of embryos developing to the blastocyst stage and the number of cells per embryo were increased. Treatment with UG for 3 hr was enough to trigger this response. The effect of UG was blocked by genistein, an inhibitor of tyrosine protein kinases, suggesting the involvement of these kinases in the stimulation of the embryo by UG. To further support this suggestion, embryos were metabolically labeled in vitro with [32P] and the phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine. Analysis of the immunoprecipitates by SDS-PAGE showed that UG induced the phosphorylation of several proteins of M(r) between 200 and 37 kDa. This induction was observed after 1 hr of stimulation with UG and further increased after 3 hr of treatment. Since UG is synthesized and secreted in the uterus and the oviduct, these results suggest a physiological role of this protein in the correct development of the embryo in vivo.     

DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants

Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes many minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA. In this study, we focused on the comparison of DNA immunization protocols using different routes of administrations of DNA (intradermal tattoo versus intramuscular injection) and molecular adjuvants (cardiotoxin pre-treatment or GM-CSF DNA co-delivery). For this comparison we used the major capsid protein L1 of human papillomavirus type 16 as a model antigen. L1-specific immune responses were detected after three and four immunizations with 50 μg plasmid DNA. Cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered intramuscularly by needle injection but had virtually no effect on the intradermal tattoo vaccination. The promoting effect of both adjuvants was more pronounced after three rather than four immunizations. However, three DNA tattoo immunizations without any adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than intramuscularly delivered DNA in combination with adjuvants. In addition, the lymphocytes of mice treated with the tattoo device proliferated more strongly after mitogen stimulation suggesting the presence of inflammatory responses after tattooing. The tattoo delivery of DNA is a cost-effective method that may be used in laboratory conditions when more rapid and more robust immune responses are required.     

Selected contribution: acute cellular and molecular responses to resistance exercise.

Training protocols apply sequential bouts of resistance exercise (RE) to induce the cellular and molecular responses necessary to produce compensatory hypertrophy. This study was designed to 1) define the time course of selected cellular and molecular responses to a single bout of RE and 2) examine the effects of interbout rest intervals on the summation of these responses. Rat muscles were exposed to RE via stimulation of the sciatic nerve in vivo. Stimulated and control muscles were obtained at various time points post-RE and analyzed via Western blot and RT-PCR. A single bout of RE increased intracellular signaling (i.e., phosphorylations) and expression of mRNAs for insulin-like growth factor-I system components and myogenic markers (e.g., cyclin D1, myogenin). A rest interval of 48 h between RE bouts resulted in much greater summation of myogenic responses than 24- or 8-h rest intervals. This experimental approach should be useful for studying the regulatory mechanisms that control the hypertrophy response. These methods could also be used to compare and contrast different exercise parameters (e.g., concentric vs. eccentric, etc.).     

The cellular and molecular basis for divergent allergic responses to chemicals.

Chemicals vary with respect to the nature of allergic reactions which they will elicit preferentially. A wide variety of environmental and industrial chemicals are known to cause allergic contact dermatitis (contact sensitivity). Some of these are able also to induce respiratory allergy. This article reviews the characteristics of immune responses to different classes of chemical allergens and the role which functional subpopulations of T helper (TH) cells and their soluble cytokine products play in the induction of allergic sensitization. In addition, new opportunities to identify and classify chemical allergens based upon characterization of divergent allergic responses is discussed.     

内质网应激的细胞效应分子机制

内质网应激（endoplasmic reticulum stress,ERs）是内质网腔内错误折叠蛋白聚积的一种适应性反应,适度ERs通过激活未折叠蛋白反应起适应性的细胞保护作用,而过高和持久的ERs则通过诱导转录因子CHOP表达、激活caspase-12和c—Jun氨基末端激酶（JNK）等导致细胞凋亡。近年来,越来越多的研究提示内质网应激是神经退行性病变、2型糖尿病以及肥胖等疾病发生过程中的重要环节。对内质网应激的细胞效应分子机制进行综述。随着对ERs机制理解的深入,有可能会发现新的分子标志物或新的诊疗策略。     

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48 h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24 h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.     

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A·C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.     

IL-13 induces airways hyperreactivity independently of the IL-4R alpha chain in the allergic lung

The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Ralpha1-IL-4Ralpha complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4(+) T cells that were wild type (IL-13(+/+) T cells) or deficient in IL-13 (IL-13(-/-) T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13(+/+) T cells induce these features (except mucus production) of allergic disease independently of the IL-4Ralpha chain. By contrast, IL-13(+/+) T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Ralpha chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.