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Medial ganglionic eminence (MGE)-derived somatostatin (SST)+ and parvalbumin (PV)+ cortical interneurons (CINs), have characteristic molecular, anatomical and physiological properties. However, mechanisms regulating their diversity remain poorly understood. Here, we show that conditional loss of the Tuberous Sclerosis (TS) gene, Tsc1, which inhibits mammalian target of rapamycin (MTOR), causes a subset of SST+ CINs, to express PV and adopt fast-spiking (FS) properties, characteristic of PV+ CINs. These changes also occur when only one allele of Tsc1 is deleted, making these findings relevant to individuals with TS. Notably, treatment with rapamycin, which inhibits MTOR, reverses these changes in adult mice. These data reveal novel functions of MTOR signaling in regulating PV expression and FS properties, which may contribute to some neuropsychiatric symptoms observed in TS. Moreover, they suggest that CINs can exhibit properties intermediate between those classically associated with PV+ or SST+ CINs, which may be dynamically regulated by the MTOR signaling.

Background Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) are potential novel therapies after myocardial infarction (MI). We first established the optimal and clinically applicable dosages of these drugs in mobilizing hematopoietic stem cells (HSC), and then tested the efficacy of monotherapy and combination therapy post-MI. Methods and Results Optimal doses were established in enhanced green fluorescent protein (eGFP) + chimeric mice (n = 30). Next, mice underwent MI and randomized into 4 groups (n = 18/group): 1) GCSF; 2) EPO; 3) EPO+GCSF; and 4) control. Left ventricular (LV) function was analyzed pre-MI, at 4 hours and at 28 days post-MI. Histological assessment of infarct size, blood vessels, apoptotic cardiomyocytes, and engraftment of eGFP+ mobilized cells were analyzed at day 28. LV function in the control group continued to deteriorate, whereas all treatments showed stabilization. The treatment groups resulted in less scarring, increased numbers of mobilized cells to the infarct border zone (BZ), and a reduction in the number of apoptotic cardiomyocytes. Both EPO groups had significantly more capillaries and arterioles at the BZ. Conclusion We have established the optimal doses for EPO and GCSF in mobilizing HSC from the bone marrow and demonstrated that therapy with these agents, either as monotherapy or combination therapy, led to improvement of cardiac function post-MI. Combination therapy does not seem to have additive benefit over monotherapy in this model.

Brief Secondhand Smoke Exposure Depresses Endothelial Progenitor Cells Activity and Endothelial Function: Sustained Vascular Injury and Blunted Nitric Oxide ProductionChristian Heiss, Nicolas Amabi...

We compared therapeutic benefits of intramyocardial injection of unfractionated bone marrow cells (BMCs) versus BMC extract as treatments for myocardial infarction (MI), using closed-chest ultrasound-guided injection at a clinically relevant time post-MI. MI was induced in mice and the animals treated at day 3 with either: (i) BMCs from green fluorescent protein (GFP)-expressing mice (n = 14), (ii) BMC extract (n = 14), or (iii) saline control (n = 14). Six animals per group were used for histology at day 6 and the rest followed to day 28 for functional analysis. Ejection fraction was similarly improved in the BMC and extract groups versus control (40.6 +/- 3.4 and 39.1 +/- 2.9% versus 33.2 +/- 5.0%, P0.05) with smaller scar sizes. At day 6 but not day 28, both therapies led to significantly higher capillary area and number of arterioles versus control. At day 6, BMCs increased the number of cycling cardiomyocytes (CMs) versus control whereas extract therapy resulted in significant reduction in the number of apoptotic CMs at the border zone (BZ) versus control. Intracellular components within BMCs can enhance vascularity, reduce infarct size, improve cardiac function, and influence CM apoptosis and cycling early after therapy following MI. Intact cells are not necessary and death of implanted cells may be a major component of the benefit.

Subcutaneous injection of type 2 herpes simplex virus (HSV-2) (10(3) PFU) to newborn rabbits produced severe skin lesions and wide dissemination of the virus to various organs including the eye. Ocular lesions were characterized by retinal folds and choroiditis. HSV could be isolated from mononuclear cells (MNCs) of infected animal blood. Newborn rabbits treated with monoclonal antibody (MAb) against glycoprotein D (gD) of HSV on days 0, 2 and 4 postinfection had little or no skin lesions (0.0-2.3 mm) compared to controls (2.8-13.0 mm). In addition, the MAb treatment significantly suppressed dissemination of the virus to the eye (0% in MAb-treated vs 83% in control) and other organs and reduced the rate of chorioretinitis (0% in MAb-treated vs 50% in control). The treatment of HSV-infected MNCs with MAb resulted in 91-100% reduction in infectivity of the cells. The results suggest that anti-gD MAb protects newborn rabbits from HSV-2 eye infection by neutralizing the virus in skin and inactivating HSV-infected MNCs in blood.

Rationale: Circulating microparticles (MPs) are submicron membrane fragments shed from damaged or activated vascular cells. Endothelial MPs are a biological marker of dysfunctional endothelium. Vascular remodeling and endothelial dysfunction are involved in pulmonary hypertension (PH). Objectives: We tested the hypothesis that circulating MPs are increased in patients with PH and that identifiable subgroups of MPs predict the hemodynamic severity of this condition progression. Methods :P atients (n5 24; age, 54 6 4 yr) undergoing right heart catheterization for precapillary PH without any endothelium-active vasodilator therapy participated in the study. Age- and sex-matched healthycontrolsubjects(n 520)wereincluded.Endothelial(PECAM 1 [CD31 1 ]/CD41 2 ,VE-cadherin 1 [CD144 1 ],andE-selectin 1 [CD62e 1 ]), platelet (CD41 1 ), leukocyte-derived (CD45 1 ), and annexin V 1 MPs weremeasuredbyflowcytometryinplatelet-freeplasmafromvenous blood. Measurements and Main Results: Levels of circulating endothelial PECAM 1 , VE-cadherin 1 , E-selectin 1 , and leukocyte-derived MPs, but not platelet and annexin V1 MPs, were increased in subjects with PH compared with control subjects (P , 0.01 each). PECAM1 and VEcadherin1 MP levels significantly correlated with mean pulmonary artery pressure (r 5 0.92 and r 5 0.87, respectively), pulmonary vascular resistance (r 5 0.78 and r 5 0.73), and mean right atrial pressure(r 5 0.43,andr 5 0.46) andcorrelated inverselywithcardiac index (r 52 0.59 and r 52 0.52). These relationships were not observed for other MP subgroups, and persisted in multivariate analysis after adjustment for confounding factors. Conclusions: In subjects with precapillary PH, levels of circulating endothelialandleukocyteMPswereincreasedcomparedwithcontrol subjects. In addition, levels of PECAM1 and VE-cadherin1 ,b ut not E-selectin 1 , endothelial MPs predicted hemodynamic severity of

Cytokine therapy has been suggested to improve left ventricle (LV) function after myocardial infarction (MI). The mechanisms for this benefit remain debatable. We investigated the impact of a prolonged combined therapy with Darbepoetin alfa (DARB) and Granulocyte Colony-Stimulating Factor (G-CSF) on LV function and vascular density after MI, correlating it with circulating progenitor cells (CPC), Vascular Endothelial Growth Factor (VEGF) and Stromal-Derived Factor 1 (SDF-1) release. Methods and Results : MI was induced in swine by a 90 minutes balloon occlusion of the left anterior descending artery. Animals were divided between treatment group with DARB-GCSF combination therapy (bolus of DARB 0.9 and GCSF 10ug/kg IV at time of reperfusion, followed by 5 doses of GCSF 5ug/kg SC from day 5 to 9, and four doses of 0.45ug/kg DARB SC once per week starting at day 1, n = 8) or control group (saline injections, n = 8). White blood cells (WBC), CPC, defined by CD45, CD31, CD90 and SLA-1 expression, and circulating levels of VEGF and SDF-1 were assessed at baseline (T0), 1 (T1), 2 (T2), and 3 (T3) weeks post-MI. LV function was assessed by echocardiography at T0, T1 and T6 (6 weeks post-MI), and vascular density by histology at T6. MI size was the same in both groups by post-MI CPK peak and LV ejection fraction (EF) at T1 (41+/−1 vs. 40+/−2%). In the treatment group only, from T0 to T1, there was an increase in WBC (16 ± 2 to 42 ± 2 x 10 6 /ml, p 5 /ml, p = 0.01) and SDF-1 levels peaked from T0 to T2 (1345+/−60 to 1554 +/− 60 pg/ml; p2 , p = 0.02) and remote zone (18 +/−8 vs.11+/−5, p = 0.055) were higher compared to the control at T6. Conclusion : Our data suggest that prolonged therapy with DARB-GCSF combination after MI modulates angiogenesis and promotes stabilization of LV function by increasing CPC, and releasing SDF-1 and VEGF. This therapy provides a novel strategy to prevent post-MI LV remodeling and potentially improve outcome.

This study sought to analyze the effects of acute secondhand smoke (SHS) exposure on the number and function of endothelial progenitor cells (EPCs) over 24 h.Secondhand smoke increases the risk of vascular disease and is a major public health concern, but the mechanism(s) of action are not fully understood.Healthy nonsmokers (age SEM 30.3 +/- 1.3 years, n = 10) were exposed to 30 min of SHS yielding cotinine levels commonly observed in passive smokers and to smokefree air on 2 separate days. Measurements were taken before exposure (baseline), immediately after (0 h), and at 1 h, 2.5 h, and 24 h after. The EPCs (CD133(+)/KDR(+), CD34(+)/KDR(+)) and endothelial microparticles (EMPs: CD31(+)/CD41(-), CD144(+), CD62e(+)) were determined in blood using flow cytometry. The EPC chemotaxis toward vascular endothelial growth factor was measured. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound.Secondhand smoke exposure increased EPCs and plasma vascular endothelial growth factor and completely abolished EPC chemotaxis during 24 h after exposure. Secondhand smoke increased EMPs and decreased FMD. Although FMD returned to baseline at 2.5 h, EMPs and vascular endothelial growth factor levels remained elevated at 24 h, suggesting endothelial activation and injury with functional impairment of the vascular endothelium. Exposure to smokefree air had no effect. Incubation of EPCs from nonexposed subjects with plasma isolated from SHS-exposed subjects in vitro decreased chemotaxis by blockade of vascular endothelial growth factor-stimulated nitric oxide production.Brief exposure to real-world levels of SHS leads to sustained vascular injury characterized by mobilization of dysfunctional EPCs with blocked nitric oxide production. Our results suggest that SHS not only affects the vascular endothelium, but also the function of EPCs.

To isolate and characterize fetal trabecular meshwork (FTM) cells for study in culture. Cultured adult trabecular meshwork (TM) cells often possess a slower rate of growth and restricted number of population doublings, limiting the ability to perform expanded testing.Fetal eyes from 24-week gestation abortions were delicately dissected to isolate the developing trabecular meshwork tissue. Three primary cultures were achieved and passaged. Light microscopy was used to compare the FTM cells to two cultured adult TM cell lines. Immunocytochemistry and Western blot analysis were utilized to identify specific protein expression.The FTM cells demonstrated similar microscopic characteristics to adult TM cells, including monolayer formation, cobblestone pattern, and comparable size. FTM cells exhibited faster, more consistent doubling times when compared with adult TM cells. They grew rapidly even after passage 8, whereas their adult counterparts slowed significantly with each successive passage and failed to reach confluence at passages 4 to 5. Immunofluorescent staining was positive for actin, vimentin, fibronectin, laminin, aquaporin-1, CD-44, and myocilin in both FTM and adult TM cells. In both fetal and adult cells, Western blots showed substantial increase in myocilin after exposure to dexamethasone.Characterization by microscopy and immunocytochemistry suggest that FTM cells have properties similar to adult TM cells. Fetal tissues may be a useful source of abundant, rapidly dividing FTM cells for in vitro investigation. The ability to do expanded research in this field may contribute to a better understanding of the molecular mechanisms in glaucoma development.

We examined the ontogenic and sex specific expression of estrogen receptor-alpha and beta in mouse genital tubercles and assessed the effects of in utero estrogen exposure on these parameters.Expression of the 2 genes was detected in mouse genital tubercles from fetuses collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as the control.Estrogen receptor-alpha and beta proteins first appeared on gestational days 12 and 14, respectively. The 2 proteins were expressed in the urethral plate and mesenchyma. Staining intensity was more prominent in the mesenchyma for estrogen receptor-alpha and in the urethral plate for estrogen receptor-beta. Female genital tubercles expressed more estrogen receptor-alpha than male genital tubercles (p0.01), while estrogen receptor-alpha expression increased gradually in the 2 sexes until birth. Estrogen receptor-beta expression did not differ between males and females, and it showed no notable variation during fetal life. Ethinyl estradiol led to a 2.1 and 3.8-fold increase in estrogen receptor-alpha expression in females and in males with hypospadias (p = 0.002 and 0.04, respectively). Estrogen receptor-beta expression did not change in response to ethinyl estradiol.This study provides in vivo evidence that estrogen receptor-alpha expression in the genital tubercles of each sex increases until parturition but estrogen receptor-beta expression does not, implying genital tubercle sensitivity to estrogen increases during fetal life. Exogenous administration of estrogens results in a response of increased expression of estrogen receptor-alpha but not of estrogen receptor-beta. These differential findings for estrogen receptor-alpha and beta imply that the 2 receptors may have different roles in normal or anomalous genital tubercle development.

We studied the protective effects of passive immunization with virus specific antibody in newborn rabbits inoculated subcutaneously with type 2 herpes simplex virus (HSV-2). Newborn rabbits given anti-HSV-2 antibody intraperitoneally (IP) on days 0, 2 and 4 post infection had smaller herpetic skin lesions and reduced mortality when compared to controls. In addition, the IP treatment using this schedule reduced virus growth in the skin lesions and virus dissemination, so that it decreased the frequency of herpetic retinitis. When the IP antibody administration was started at 24 hours post virus inoculation, according to the schedule days 1, 3 and 5, there was less protection; larger skin lesions, higher mortality, and greater evidence of virus dissemination. Also HSV-infected mononuclear cells (MNCs) treated with anti-HSV serum resulted in a significant reduction in the number of infected MNCs. The results of these studies suggest that anti-HSV-2 antibody contributes to protection against HSV-2 infection of skin as well as eyes, probably by inactivation of the virus locally at the skin inoculation site, and by combating the hematogenous spread of HSV-infected MNCs as well as free virus to various organs including the eye.

When live herpes simplex virus (HSV) was injected intravitreally into the normal rabbit eye, primary uveitis developed, resolving within one month. If such a healed eye was challenged with HSV antigen, secondary uveitis developed within six hours after the challenge. We have shown previously that this secondary uveitis is mediated by an immune reaction that involves HSV antigen, sensitized T lymphocytes, and anti-HSV antibody (1,2). This paper presents our experimental data which indicate that pretreatment of the animal with a T cell suppressor, Cyclosporine (CyA), successfully prevented the induction of secondary uveitis.