The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Hemodynamic and morphological vasculature response to a burn monitored using a combined dual-wavelength laser speckle and optical microangiography imaging system

A multi-functional imaging system capable of determining relative changes in blood flow, hemoglobin concentration, and morphological features of the blood vasculature is demonstrated. The system combines two non-invasive imaging techniques, a dual-wavelength laser speckle contrast imaging (2-LSI) and an optical microangiography (OMAG) system. 2-LSI is used to monitor the changes in the dynamic blood flow and the changes in the concentration of oxygenated (HbO), deoxygenated (Hb) and total hemoglobin (HbT). The OMAG system is used to acquire high resolution images of the functional blood vessel network. The vessel area density (VAD) is used to quantify the blood vessel network morphology, specifically the capillary recruitment. The proposed multi-functional system is employed to assess the blood perfusion status from a mouse pinna before and immediately after a burn injury. To our knowledge, this is the first non-invasive, non-contact and multifunctional imaging modality that can simultaneously measure variations of several blood perfusion parameters.     

Age and diet act through distinct isoforms of the class II transactivator gene in mouse intestinal epithelium

BACKGROUND & AIMS: Normal weaning induces class II major histocompatibility complex (Ia) and invariant chain (Ii) expression in the mouse intestinal epithelium. Because the class II transactivator protein (CIITA) induces Ia and Ii in most cell types, we hypothesized that diet-induced expression of these genes was through CIITA. METHODS: Mouse litters were split and weaned onto an elemental diet or a normal (complex) chow diet. On days 24, 31, and 45, epithelial cells were isolated from small intestine with EDTA, and the RNA was extracted from both wild-type and interferon (IFN)-gamma receptor knockout mice. Messenger RNA (mRNA) was measured by Northern blotting, RNase protection assay, and real-time polymerase chain reaction and Ia localized by immunohistochemistry. RESULTS: By day 31, CIITA mRNA was induced in the intestinal epithelium of normally weaned wild-type mice, and this mirrored the expression of Ii chain mRNA. Mice weaned onto an elemental diet did not exhibit Ii mRNA or increased CIITA mRNA in the intestinal epithelium by day 31, but low levels of Ii mRNA were detectable by day 45. Of the 3 isoforms of CIITA, weaning onto a complex diet induced only CIITA IV by day 31. Mice deficient in the IFN-gamma receptor expressed Ia in the epithelium and they also accumulated Ii mRNA (at low levels) by day 45, irrespective of diet. CIITA III mRNA accumulation mirrored the dietary-independent changes of Ii mRNA. CONCLUSIONS: Two mechanisms regulate Ii in the mouse intestinal epithelium: a rapid one, which is diet-induced acting through CIITA IV; and a slower, dietary-independent pathway, acting through CIITA III.     

Optical microangiography of retina and choroid and measurement of total retinal blood flow in mice

We present a novel application of optical microangiography (OMAG) imaging technique for visualization of depth-resolved vascular network within retina and choroid as well as measurement of total retinal blood flow in mice. A fast speed spectral domain OCT imaging system at 820nm with a line scan rate of 140 kHz was developed to image the posterior segment of eyes in mice. By applying an OMAG algorithm to extract the moving blood flow signals out of the background tissue, we are able to provide true capillary level imaging of the retinal and choroidal vasculature. The microvascular patterns within different retinal layers are presented. An en face Doppler OCT approach [Srinivasan et al., Opt Express 18, 2477 (2010)] was adopted for retinal blood flow measurement. The flow is calculated by integrating the axial blood flow velocity over the vessel area measured in an en face plane without knowing the blood vessel angle. Total retinal blood flow can be measured from both retinal arteries and veins. The results indicate that OMAG has the potential for qualitative and quantitative evaluation of the microcirculation in posterior eye compartments in mouse models of retinopathy and neovascularization.OCIS codes: (170.4500) Optical coherence tomography, (170.3880) Medical and biological imaging     

Ovarian steroid action on tryptophan hydroxylase protein and serotonin compared to localization of ovarian steroid receptors in midbrain of guinea pigs

The effect of estrogen (E) and progesterone (P) on the protein expression of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase (TPH), and the level of serotonin, in the hypothalamic terminal field was examined in guinea pigs. In addition, we questioned whether serotonin neurons of guinea pigs contain ovarian steroid receptors (estrogen receptor-α[ERα], estrogen receptor β[ERβ], progestin, receptors [PRs]) that could directly mediate, the actions of E or P. Western blot and densitometric analysis for TPH were used on raphe extracts from untreated-ovariectomized (OVX), OVX-E-treated (28d), and OVX-E+P-treated (14 d E+ 14 d E+P), guinea pigs. The medial basal hypothalami from the same animals were extracted and subjected to high-performance liquid chromatography analysis for serotonin, dopamine, 5-hydroxyindole acetic acid, and homovanillic acid. The brains from other aminals treated in an identical manner were perfusion fixed and examined for the colocalization of ERα plus serotonin and PR plus serotonin with double immunohistochemistry or for expression of ERβ mRNA with in situ hybridization. E and E+P treatment significantly increased TPH protein levels compared to the untreated control group (p<0.05), but TPH levels were similar in the E and E+P-treated groups. By contrast, serotonin (nanogram/milligram of protein) in the hypothalamus was significantly increased by E+P treatment, but not by E alone. Neither ERα nor PR proteins were detected within serotonin neurons of the guinea pig raphe nucleus. However, ERβ mRNA was expressed in the dorsal raphe. In summary, E alone increased TPH protein expression and the addition of P had no further effect, whereas E+P increased, hypothalamic serotonin and E alone had no effect. The localization of ERβ, but not ERα or PR, in the dorsal raphe nucleus suggests that E acting via ERβ within serotonin neurons increases expression of TPH, but that P acting via other neurons and transsynaptic stimulation may effect changes in TPH enzymatic activity, which in turn, would lead to an increase in serotonin synthesis.     

Therapy with recombinant T-cell receptor ligand reduces infarct size and infiltrating inflammatory cells in brain after middle cerebral artery occlusion in mice

Stroke induces a biphasic effect on the peripheral immune response that involves early activation of peripheral leukocytes followed by severe immunosuppression and atrophy of the spleen. Peripheral immune cells, including T lymphocytes, migrate to the brain and exacerbate the developing infarct. Recombinant T-cell receptor (TCR) Ligand (RTL)551 is designed as a partial TCR agonist for myelin oligodendrocyte glycoprotein (MOG)-reactive T cells and has demonstrated the capacity to limit infarct volume and inflammation in brain when administered to mice undergoing middle cerebral artery occlusion (MCAO). The goal of this study was to determine if RTL551 could retain protection when given within the therapeutically relevant 4 h time window currently in clinical practice for stroke patients. RTL551 was administered subcutaneously 4 h after MCAO, with repeated doses every 24 h until the time of euthanasia. Cell numbers were assessed in the brain, blood, spleen and lymph nodes and infarct size was measured after 24 and 96 h reperfusion. RTL551 reduced infarct size in both cortex and striatum at 24 h and in cortex at 96 h after MCAO and inhibited the accumulation of inflammatory cells in brain at both time points. At 24 h post-MCAO, RTL551 reduced the frequency of the activation marker, CD44, on T-cells in blood and in the ischemic hemisphere. Moreover, RTL551 reduced expression of the chemokine receptors, CCR5 in lymph nodes and spleen, and CCR7 in the blood and lymph nodes. These data demonstrate effective treatment of experimental stroke with RTL551 within a therapeutically relevant 4 h time window through immune regulation of myelin-reactive inflammatory T-cells.