Nomograms for prognosis prediction in esophageal adenocarcinoma: realities and challenges.

Prognostic assessment is of great significance for individualized treatment and care of cancer patients. Although the TNM staging system is widely used as the primary prognostic classifier for solid tumors in clinical practice, the complexity of tumor occurrence and development requires more personalized probability prediction models than an ordered staging system. By integrating clinical, pathological, and molecular factors into digital models through LASSO and Cox regression, a nomogram could provide more accurate personalized survival estimates, helping clinicians and patients develop more appropriate treatment and care plans. Esophageal adenocarcinoma (EAC) is a common pathological subtype of esophageal cancer with poor prognosis. Here, we screened and comprehensively reviewed the studies on EAC nomograms for prognostic prediction, focusing on performance evaluation and potential prognostic factors affecting survival. By analyzing the strengths and limitations of the existing nomograms, this study aims to provide assistance in constructing high-quality prognostic models for EAC patients.

A novel mutation in COL1A1 causing osteogenesis imperfecta/hearing loss

Abstract Objectives: To screen the COL1A1 and COL1A2 gene mutation sites in a family with type I osteogenesis imperfecta (OI)/hearing loss and analyze the characteristics and recovery of hearing loss in patients with osteogenesis imperfecta. Methods: The basic clinical data of Ol proband and her parents were collected, and the COL1A1 and COL1A2 genes were detected in peripheral blood by PCR amplification and generation Sanger sequencing. Literature of stapedial surgery in patients with osteogenesis imperfecta was collected. Results: The heterozygous mutation of the 26 exon c.1922_1923 ins C in the Ol progenitor COL1A1 gene led to the amino acid frameshift mutation of p.Pro 601FS, which was not detected in the phenotypic parents. The homozygous of exon 28 c.1782>G in COL1A2 was detected in the proband and her parents, resulting in changes in the protein p.Pro 549Ala. Conclusion: The clinical symptoms of the Ol proband is caused by heterozygous mutation of the 26 exon c.1922_1923 ins C in COL1A1 gene. Stapedial surgery can provide short-term and long-term hearing benefits for Ol patients with hearing loss. Level of evidence: Level 4.

A novel mutation in COL1A1 causing osteogenesis imperfecta/hearing loss.

OBJECTIVES: To screen the COL1A1 and COL1A2 gene mutation sites in a family with type I osteogenesis imperfecta (OI)/hearing loss and analyze the characteristics and recovery of hearing loss in patients with osteogenesis imperfecta. METHODS: The basic clinical data of OI proband and her parents were collected, and the COL1A1 and COL1A2 genes were detected in peripheral blood by PCR amplification and generation Sanger sequencing. Literature of stapedial surgery in patients with osteogenesis imperfecta was collected. RESULTS: The heterozygous mutation of the 26 exon c.1922_1923 ins C in the OI progenitor COL1A1 gene led to the amino acid frameshift mutation of p.Pro 601FS, which was not detected in the phenotypic parents. The homozygous of exon 28 c.1782>G in COL1A2 was detected in the proband and her parents, resulting in changes in the protein p.Pro 549Ala. CONCLUSION: The clinical symptoms of the OI proband is caused by heterozygous mutation of the 26 exon c.1922_1923 ins C in COL1A1 gene. Stapedial surgery can provide short-term and long-term hearing benefits for OI patients with hearing loss. LEVEL OF EVIDENCE: Level 4.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells in vivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

125I seed implantation enhances arsenic trioxide-induced apoptosis and anti-angiogenesis in lung cancer xenograft mice.

BACKGROUND AND PURPOSE: Arsenic trioxide (ATO) exerts anticancer effects on lung cancer. However, the clinical use of ATO is limited due to its systemic toxicity and resistance of lung cancer cells. The present study aimed to investigate the effects of ATO, alone and in combination with 125I seed implantation on tumor growth and proliferation in lung cancer xenograft mice, and investigate the possible molecular mechanisms. METHODS: The transmission electron microscope observed the tumor ultrastructure of lung cancer xenograft mice. The proliferation index of Ki-67 and the number and morphology of tumor microvessels were detected with immunohistochemical staining. The protein and mRNA expression were examined by western blot and real-time PCR assay. RESULTS: The in vivo results demonstrated that ATO combined with 125I seed significantly inhibited tumor growth and proliferation, as well as promoted apoptosis, and decreased the Ki-67 index and microvessel density in lung cancer xenograft mice. Moreover, ATO combined with 125I seed decreased the protein and mRNA expression levels of HIF-1α, VEGF, and BCL-2, and increased those of BAX and P53. CONCLUSIONS: ATO combined with 125I seed significantly inhibited tumor growth and proliferation in lung cancer, which may be accomplished by inhibiting tumor angiogenesis and inducing apoptosis.

Carboplatin-loaded surface modified-PLGA nanoparticles confer sustained inhibitory effect against retinoblastoma cell in vitro / Nanopartículas de PLGA com superfície modificada carregada com carboplatina conferem efeito inibitório sustentado contra células de retinoblastoma in vitro

ABSTRACT Purpose: To investigate the antiproliferative effect of carboplatin-loaded surface-modified poly(lactide-co-glycolide) on retinoblastoma cells. Methods: Carboplatin-loaded poly(lactide-co-glycolide) with or without sodium alginate surface modification was prepared using sodium alginate-poly(lactide-co-glycolide) and poly(lactide-co-glycolide). The zeta potential and carboplatin release behavior were investigated. The cellular uptake of the released drug was observed in the retinoblastoma cell line Y79. The inhibitory effect of carboplatin-loaded nanoparticles against the Y79 cell line was evaluated using methyl thiazolyl tetrazolium assay and western blot. Native carboplatin and void nanoparticles without carboplatin loading were used as controls. Results: The zeta potential was -(26.1 ± 3.1) mV for carboplatin-loaded poly(lactide-co-glycolide) and-(43.1 ± 8.1) mV for carboplatin-loaded sodium alginate-poly(lactide-co-glycolide). The burst release percentages of carboplatin-loaded poly(lactide-co-glycolide) and sodium alginate-poly(lactide-co-glycolide) were (40.0% ± 8.2%) and (18.9% ± 4.3%) at 24 hours, respectively. A significant difference was identified regarding drug release between carboplatin-loaded sodium alginate-poly(lactide-co-glycolide) and carboplatin-loaded poly(lactide-co-glycolide). Fluorescence detection revealed that intense uptake of carboplatin into the cytoplasm of the Y79 cell line that was exposed to carboplatin-loaded sodium alginate-poly(lactide-co-glycolide). Carboplatin-loaded poly(lactide-co-glycolide) or sodium alginate-poly(lactide-co-glycolide) exposure inhibited proliferating cell nuclear antigen expression in Y79 cells on day 3. Extension of exposure to day 5 revealed that the sodium alginate-poly(lactide-co-glycolide) surface modification was superior to that of poly(lactide-co-glycolide) in terms of proliferating cell nuclear antigen inhibition. The cell viability test using methyl thiazolyl tetrazolium revealed a similar inhibitory effect. Furthermore, the carboplatin-loaded nanoparticles of lower concentration inhibited cell viability more strongly than native carboplatin of higher concentration in methyl thiazolyl tetrazolium assay. Conclusions: Carboplatin-loaded sodium alginate-poly(lactide-co-glycolide) inhibited retinoblastoma cell proliferation with superior effect as compared with poly(lactide-co-glycolide) and native carboplatin. Sodium alginate surface modification offers a potential strategy for the sustained carboplatin release system.

RESUMO Objetivo: Investigar o efeito antiproliferativo de poli (lactídeo-coglicolídeo) com superfície modificada carregada com carboplatina contra células de retinoblastoma. Métodos: Preparou-se poli (lactídeo-co-glicolídeo) carregado com carboplatina com ou sem alginato de sódio para modifição da superfície, poli com alginato de sódio (lactídeo-co-glicolídeo) e poli (lactídeo-co-glicolídeo). O potencial zeta e o comportamento de liberação de carboplatina foram investigados. A captação celular do fármaco liberado foi observada na linha celular de retinoblastoma Y79. O efeito inibitório das nanopartículas carregadas com carboplatina contra a linha celular Y79 foi avaliado através do ensaio de metiltiazol tetrazólio e Western-blot. Carboplatina nativa e nanopartículas vazias sem carga de carboplatina serviram como controles. Resultados: O potencial zeta de poli carregado com carboplatina (lactídeo-co-glicolídeo) foi - (26,1 ± 3,1) mV versus - (43,1 ± 8,1) mV em poli com alginato de sódio carregado com carboplatina (lactídeo-co-glicolídeo). A percentagem de libertação de explosão de poli carregado com carboplatina (lactídeo-co-glicolídeo) e poli com alginato de sódio (lactídeo-co-glicolídeo) foram (40,0 ± 8,2)% e (18,9 ± 4,3)% às 24 horas, respectivamente. Uma diferença significativa foi identificada em relação à liberação de fármaco entre poli com alginato de sódio carregado com carboplatina (lactídeo-co-glicolídeo) e poli carregado com carboplatina (lactídeo-co-glicolídeo). A detecção de fluorescência revelou que a carboplatina foi assimilada intensamente no citoplasma da linha celular Y79 que foi exposta ao poli com alginato de sódio carregado com carboplatina (lactídeo-co-glicolídeo). A exposição de poli carregada com carboplatina (lactídeo-co-glicolídeo) ou poli com alginato de sódio (lactídeo-co-glicolídeo) inibiu a expressão de antígeno nuclear de proliferação celular em células Y79 no 3º dia. A extensão da exposição no 5º dia revelou que poli com alginato de sódio (lactídeo-co-glicolídeo) para modificação da superfície foi superior a poli (lactídeo-co-glicolídeo) em termos de inibição do antígeno nuclear de proliferação celular. O teste de viabilidade celular via metiltiazol tetrazólio mostrou um efeito inibitório semelhante. Além disso, as nanopartículas carregadas com carboplatina de concentração mais baixa inibiram a viabilidade celular mais fortemente em comparação com a carboplatina nativa de concentração mais alta no ensaio de metiltiazol tetrazólio. Conclusões: Poli com alginato de sódio carregado com carboplatina (lactídeo-co-glicolídeo) inibiu a proliferação de células de retinoblastoma com efeito superior em contraste com poli (lactídeo-co-glicolídeo) e carboplatina nativa. O alginato de sódio para modificação da superfície oferece uma estratégia potencial para o sistema de liberação de carboplatina sustentada.

Carboplatin-loaded surface modified-PLGA nanoparticles confer sustained inhibitory effect against retinoblastoma cell in vitro.

PURPOSE: To investigate the antiproliferative effect of carboplatin-loaded surface-modified poly(lactide-co-glycolide) on retinoblastoma cells. METHODS: Carboplatin-loaded poly(lactide-co-glycolide) with or without sodium alginate surface modification was prepared using sodium alginate-poly(lactide-co-glycolide) and poly(lactide-co-glycolide). The zeta potential and carboplatin release behavior were investigated. The cellular uptake of the released drug was observed in the retinoblastoma cell line Y79. The inhibitory effect of carboplatin-loaded nanoparticles against the Y79 cell line was evaluated using methyl thiazolyl tetrazolium assay and western blot. Native carboplatin and void nanoparticles without carboplatin loading were used as controls. RESULTS: The zeta potential was -(26.1 ± 3.1) mV for carboplatin-loaded poly(lactide-co-glycolide) and-(43.1 ± 8.1) mV for carboplatin-loaded sodium alginate-poly(lactide-co-glycolide). The burst release percentages of carboplatin-loaded poly(lactide-co-glycolide) and sodium alginate-poly(lactide-co-glycolide) were (40.0% ± 8.2%) and (18.9% ± 4.3%) at 24 hours, respectively. A significant difference was identified regarding drug release between carboplatin-loaded sodium alginate-poly(lactide-co-glycolide) and carboplatin-loaded poly(lactide-co-glycolide). Fluorescence detection revealed that intense uptake of carboplatin into the cytoplasm of the Y79 cell line that was exposed to carboplatin-loaded sodium alginate-poly(lactide-co-glycolide). Carboplatin-loaded poly(lactide-co-glycolide) or sodium alginate-poly(lactide-co-glycolide) exposure inhibited proliferating cell nuclear antigen expression in Y79 cells on day 3. Extension of exposure to day 5 revealed that the sodium alginate-poly(lactide-co-glycolide) surface modification was superior to that of poly(lactide-co-glycolide) in terms of proliferating cell nuclear antigen inhibition. The cell viability test using methyl thiazolyl tetrazolium revealed a similar inhibitory effect. Furthermore, the carboplatin-loaded nanoparticles of lower concentration inhibited cell viability more strongly than native carboplatin of higher concentration in methyl thiazolyl tetrazolium assay. CONCLUSIONS: Carboplatin-loaded sodium alginate-poly(lactide-co-glycolide) inhibited retinoblastoma cell proliferation with superior effect as compared with poly(lactide-co-glycolide) and native carboplatin. Sodium alginate surface modification offers a potential strategy for the sustained carboplatin release system.

Lateral femoral bypass for prosthetic arterial graft infections in the groin.

OBJECTIVE: Prosthetic arterial graft infections (PAGIs) in the groin pose significant challenges in terms of revascularization options and risk of limb loss as well as associated morbidities. Although obturator canal bypass (OCB) has been suggested for revascularization of the extremity in these cases, moderate success rates and technical challenges have limited widespread use. Our study analyzed lateral femoral bypass (LFB) as an alternative approach for the treatment of groin PAGIs. METHODS: This is a retrospective review of a prospectively maintained database of patients who underwent LFB for groin PAGIs at a single center from 2000 to 2017. Patients' data including demographics, comorbidities, perioperative complications, graft patency, and need for reintervention were used. Patients were observed after LFB with duplex ultrasound surveillance in an accredited noninvasive vascular laboratory every 3 months during the first year, followed by every 6 months for the second year and yearly thereafter. After isolation of the infected wound with sterile dressings, remote proximal and distal arterial exposure incisions were made. LFBs were tunneled under the inguinal ligament and lateral to the infected wound from an uninvolved inflow artery or bypass graft to an uninvolved outflow vessel. RESULTS: A total of 19 LFBs were performed in 16 patients (mean age, 69 ± 12.6 years). Three LFBs were performed urgently for acute bleeding. Choice of conduit included 6 (31.6%) autogenous vein grafts, 10 (52.6%) cadaveric grafts, 2 (10.5%) rifampin-soaked Dacron grafts, and 1 (5.3%) polytetrafluoroethylene graft. Average follow-up was 33 months (range, 0-103 months). Major adverse events occurring within 30 days of the operation included one (5.3%) death and one (5.3%) graft excision for pseudoaneurysm. Primary patency and primary assisted patency at 12 and 24 months were 73% and 83%, respectively. One patient required an amputation 17 months after surgery after failure of repeated revascularization attempts. Overall limb salvage was 93.8% during this follow-up period. CONCLUSIONS: In this series, LFB for management of groin PAGIs demonstrated higher patency and limb salvage rates compared with previous reports of OCB. Diligent postoperative duplex ultrasound surveillance is critical to the achievement of limb salvage and maintenance of graft patency. These results suggest that LFB, which is technically less complex than OCB, should be considered the first choice for revascularization in select cases of PAGIs involving the groin.

Low carotid stump pressure as a predictor for ischemic symptoms and as a marker for compromised cerebral reserve in octogenarians undergoing carotid endarterectomy.

BACKGROUND: Carotid artery occlusive disease can cause stroke by embolization, thrombosis, and hypoperfusion. The majority of strokes secondary to cervical carotid atherosclerosis are believed to be of embolic etiology. However, cerebral hypoperfusion could be an important factor in perioperative stroke. We retrospectively reviewed the stump pressure (SP) of carotid endarterectomy (CEA) of patients at Pennsylvania Hospital to identify whether physiologic perfusion differences account for differences in perioperative stroke rates, particularly in octogenarians. METHODS: We conducted a retrospective review of our prospectively maintained database for CEA performed between 1992 and 2015. SP was measured and recorded for 1190 patients. A low SP was defined as systolic pressure <50 mm Hg. Shunts were used only for patients under general anesthesia with SP <50 mm Hg, for awake patients with neurologic changes with carotid clamping, and in some patients with recent stroke. RESULTS: Symptomatic patients were more likely to have SP <50 mm Hg compared with asymptomatic patients (35.6% vs 26.2%; P = .0015). Patients having SP <50 mm Hg had a higher postoperative stroke rate compared with patients with SP >50 mm Hg (2.9% vs 0.9%; P = .0174). Octogenarians were more likely to have a lower SP compared with patients younger than 80 years (35.7% vs 27.7%; P = .0328). Symptomatic patients with low SP were at highest risk for perioperative stroke (6.4% vs 1.2%; P = .001) compared with patients without these factors. CONCLUSIONS: SP is a marker for decreased cerebrovascular reserve and along with symptomatic status identifies those at highest risk for periprocedural stroke with CEA. Whereas patients older than 80 years may benefit from carotid intervention, they are likely to be at somewhat elevated stroke risk because of higher prevalence of low SP, and shunting does not eliminate this risk.

Liposome-entrapped GABA modulates the expression of nNOS in NG108-15 cells.

BACKGROUND: Liposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA (γ-aminobutyric acid) is the most prevalent inhibitory neurotransmitter in the central nervous system. NEW METHOD: Using GABA-containing liposomes (GL) prepared by the freeze-thawing method, we determined the effect of sustained release of GABA on expression of neuronal nitric oxide synthase (nNOS) and GABAA receptor (GABAAR) in an in vitro neuronal model. RESULTS: Neuronal cell line NG108-15 treated with different doses of GL during 24h showed an increase in expression of GABAAR (54 and 50% with 10 and 20ng doses, respectively) and nNOS (138, 157 and 165% with 20, 50 and 100ng doses, respectively) compared with cells treated with empty liposomes (EL). Additionally, cells treated with 50ng of GL showed an increase in GABAAR (23%) after 1h followed by an increase in nNOS (55, 46 and 55%) at 8, 12 and 24h time points, respectively. Immunofluorescence experiments confirmed an increase in nNOS (134%) and basal intracellular levels of nitric oxide (84%) after GL treatment. Further, treatment of cells with GL showed a decrease in expression of a protein inhibitor of nNOS (PIN) (26, 66 and 57% with 20, 50 and 100ng doses respectively) compared with control. COMPARISON WITH EXISTING METHODS: This is first demonstration for the development of GL that allows sustained slow release of this neurotransmitter. CONCLUSION: These results suggest that a slow release of GABA can change the expression of nNOS possibly via alteration in PIN levels in neuronal cells.

Repair of aortoenteric fistula secondary to graft placement for middle aortic syndrome.

When an aortoenteric fistula (AEF) arises secondary to suprarenal or more proximal aortic repair, mortality and the complexity of the surgery increases. We present the first reported case to our knowledge of a secondary AEF arising 13 years after surgical repair of middle aortic syndrome. We performed the original surgery on a 22-year-old male who presented with hypertension and claudication by placing a Dacron prosthetic patch on the juxtarenal and infrarenal aorta, bilateral vein bypasses to the left and right renal artery, and a Dacron bypass to the proximal superior mesenteric artery. Thirteen years later, he presented with massive gastrointestinal bleeding and syncope. We performed a distal descending thoracic aortic rifampin-soaked bifurcated Dacron graft to the left renal artery and to a large meandering mesenteric artery followed by excision of all previous prosthetic graft and insertion of a rifampin-soaked tube graft from the distal descending thoracic aorta to the distal abdominal aorta with omental flap coverage. After a complicated postoperative course, he was discharged 2 months later and remains on dialysis at his 6-month postoperative follow-up without evidence of recurrent infection.

Methylprednisolone in patients undergoing cardiopulmonary bypass (SIRS): a randomised, double-blind, placebo-controlled trial

BACKGROUND:Cardiopulmonary bypass initiates a systemic inflammatory response syndrome that is associated with postoperative morbidity and mortality. Steroids suppress inflammatory responses and might improve outcomes in patients at high risk of morbidity and mortality undergoing cardiopulmonary bypass. We aimed to assess the effects of steroids in patients at high risk of morbidity and mortality undergoing cardiopulmonary bypass.METHODS:The Steroids In caRdiac Surgery (SIRS) study is a double-blind, randomised, controlled trial. We used a central computerised phone or interactive web system to randomly assign (1:1) patients at high risk of morbidity and mortality from 80 hospital or cardiac surgery centres in 18 countries undergoing cardiac surgery with the use of cardiopulmonary bypass to receive either methylprednisolone (250 mg at anaesthetic induction and 250 mg at initiation of cardiopulmonary bypass) or placebo. Patients were assigned with block randomisation with random block sizes of 2, 4, or 6 and stratified by centre. Patients aged 18 years or older were eligible if they had a European System for Cardiac Operative Risk Evaluation of at least 6. Patients were excluded if they were taking or expected to receive systemic steroids in the immediate postoperative period or had a history of bacterial or fungal infection in the preceding 30 days. Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcomes were 30-day mortality and a composite of death and major morbidity (ie, myocardial injury, stroke, renal failure, or respiratory failure) within 30 days, both analysed by intention to treat. Safety outcomes were also analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00427388...

Rationale and design of the steroids in cardiac surgery trial

Background Steroids may improve outcomes in high-risk patients undergoing cardiac surgery with the use of cardiopulmonarybypass (CBP). There is a need for a large randomized controlled trial to clarify the effect of steroids in such patients.Methods We plan to randomize 7,500 patients with elevated European System for Cardiac Operative Risk Evaluation whoare undergoing cardiac surgery with the use of CBP to methylprednisolone or placebo. The first coprimary outcome is 30-day allcausemortality, and the most second coprimary outcome is a composite of death, MI, stroke, renal failure, or respiratory failurewithin 30 days. Other outcomes include a composite of MI or mortality at 30 days, new onset atrial fibrillation, bleeding andtransfusion requirements, length of intensive care unit stay and hospital stay, infection, stroke, wound complications,gastrointestinal complications, delirium, postoperative insulin use and peak blood glucose, and all-cause mortality at 6 months.Results As of October 22, 2013, 7,034 patients have been recruited into SIRS in 82 centers from 18 countries. Patient’smean age is 67.3 years, and 60.4% are male. The average European System for Cardiac Operative Risk Evaluation is 7.0with 22.1% having an isolated coronary artery bypass graft procedure, and 66.1% having a valve procedure.Conclusions SIRS will lead to a better understanding of the safety and efficacy of prophylactic steroids for cardiacsurgery requiring CBP. (Am Heart J 2014;167:660-5.)BackgroundWorldwide, N2 million patients undergo cardiacsurgery annually. Most cardiac surgeries use cardiopulmonarybypass (CPB). Although CPB serves an importantrole, it.

Construction and validation of lentiviral vector carrying rat neuronal nitric oxide synthase in vitro and in vivo.

In the present study, we developed a lentiviral vector with human cytomegalovirus promoter permitting high-level of nNOS expression. Neuronal cell line NG108 was used as an in vitro model to check the validity of gene transfer. The cells were infected with lenti-EGFP or lenti-nNOS particles for 24h. Lenti-nNOS infection in the NG108 cells induced dose dependent increase in mRNA and protein for nNOS; with a dose of 2.5 × 104 pfu/ml, nNOS mRNA expression increased by 40-fold while protein expression was increased by 2.5-fold compared to lenti-EGFP. Moreover, lenti-nNOS infection caused a greater increase in nNOS immunoreactivity in NG108 cells compared to lenti-EGFP as shown by immonocytochemistry. nNOS expression showed time dependent increases with lenti-nNOS infection with maximum up-regulation observed after two weeks of infection. Moreover, in vivo, unilateral injection of lenti-nNOS into the paraventricular nucleus (PVN) of rats induced a 27-fold increase of nNOS protein level in the injected side compared to non-injected side and this escalation was sustained up to three weeks. Overall, lenti-EGFP injection in the PVN did not show any significant change in nNOS expression. Furthermore, NADPH-diaphorase staining of nNOS in the PVN infected with lenti-nNOS induced a visible increase in nNOS expression compared with contralateral non-injected side up to three weeks. These results indicate that this approach of lentiviral mediated gene transfer of nNOS may provide a new means to up-regulate the nNOS expression for longer periods of time compared to adenoviral transfection and can be used as a research tool and potentially a therapy for chronic diseases involving impaired nNOS expression.

Contribution of the paraventricular nucleus in autonomic adjustments to heat stress.

We assessed the contribution of the paraventricular nucleus (PVN) in the heat stress-mediated changes in sympathetic nerve activity and blood flow redistribution from the core to the skin surface. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR), and body and tail temperatures were recorded in anesthetized rats after bilateral microinjection of cerebrospinal fluid (CSF), lidocaine or NG-monomethyl-L-arginine (L-NMMA) into the PVN during heat stress. Heat stress was induced by a graded increase in the temperature of a heating pad for 30 min. Heat stimulus after blockade of the PVN with lidocaine resulted in a blunted RSNA response (ΔRSNA: 117.6 ± 17.0% versus 11.3 ± 7.3%), as well as blunted MAP and HR (ΔMAP: 22 ± 2 versus -0.04 ± 7.2 mmHg; ΔHR: 93.4 ± 9.3 versus 43.4 ± 18.8 bpm). Body temperature threshold for tail vasodilation was unaffected by lidocaine treatment. The increase in RSNA, MAP and HR due to heat stress in L-NMMA-treated rats reached similar levels as CSF-treated control rats. However, a higher body temperature threshold for tail vasodilation was observed after L-NMMA injection (37.3 ± 0.1 versus 37.8 ± 0.2 °C). In conclusion, an intact PVN contributes to an increase in renal sympathetic activity provoked by heat stress, resulting in cardiovascular adjustments that influence core blood redistribution to the periphery. Furthermore, during heat stress, the effect of the PVN on cutaneous vasodilation is dependent on a nitric oxide mechanism.