ANGIOTENSIN II- AND PHORBOL ESTER-INDUCED STEROIDOGENESIS BY OVINE ADRENOCORTICAL CELLS: EFFECT OF SEX AND THE GONADAL STATUS OF THE DONOR ANIMAL

1. Angiotensin II (Angll) is well recognized as a regulator of adrenal Cortisol production in the sheep in vivo, but studies to date have failed to reveal this action on cultured ovine adrenocortical cells. Similarly, phorbol myristate acetate (PMA), an activator of protein kinase C, also has little effect on Cortisol production from ovine adrenocortical cells in vitro. Previous studies have, however, only looked at the responses to single concentrations of Angll or PMA, but have led to the suggestion that important differences exist between adrenocortical cells of the sheep and other species. 2. We have extended previous studies by examining Cortisol production by ovine adrenocortical cells over 3h in response to a broad range of concentrations of either Angll (10^?10 to 10^?6mol/L) or PMA (10^?10 to 10^?5mol/L). In addition, we have investigated the possible role of gonadal factors in regulating these responses by comparing cultures derived from intact and gonadectomized male and female sheep. 3. Angiotensin II caused concentration-dependent increases (P<0.05) in Cortisol production in all cultures, with Cortisol production in response to Angll being greater (P < 0.05) in cultures from intact male sheep than in the cultures from orchid-ectomized males or intact females. Cortisol production in response to Angll was modest in all groups (two- to 2.6-fold increases). 4. Phorbol myristate acetate elicited a concentration-dependent inhibition (P<0.01) of basal Cortisol production in adrenocortical cultures from all groups. Phorbol myristate acetate produced a greater inhibition (P<0.05) of Cortisol production in cultures from intact male sheep than either the orchidectomized males or intact females. 5. These findings demonstrate that ovine adrenocortical cells are responsive in a concentration-dependent fashion to Angll and PMA. In addition, sex and gonadal factors may play an important role in regulating the responsiveness of adrenocortical cells to these factors.     

Long-term visual outcomes of optic pathway gliomas in pediatric patients without neurofibromatosis type 1

Sporadic optic pathway gliomas (OPGs) have been reported to cause more vision loss than OPGs associated with neurofibromatosis type-1, but long-term visual outcome data are limited. The purpose of this study was to report the visual outcomes of a cohort of pediatric patients with sporadic OPGs. This was a retrospective, cohort study at a tertiary care pediatric hospital and cancer institute. The study included all patients with sporadic OPGs evaluated from 1990 to 2014. The primary outcome was visual acuity at final follow-up. Secondary outcomes were risk factors for a poor visual outcome and the rate of progression. There were 59 pediatric patients included in the study. Median age at presentation was 2.5 years old and median follow-up was 5.2 years. In the worse eye at final follow-up, 16 patients (27?%) were 20/30 or better, 9 patients (15?%) were between 20/40 and 20/80, and 34 patients (58?%) were 20/100 or worse. In the better eye at final follow-up, 33 patients (56?%) were 20/30 or better, 11 patients (19?%) were between 20/40 and 20/80, and 15 patients (25?%) were 20/100 or worse. Risk factors for a poor visual outcome included younger age at presentation, optic nerve pallor, and tumor extent. Of the 54 patients (92?%) who received treatment, 40 (74?%) experienced disease progression during or after treatment. A majority of pediatric patients with sporadic OPGs had significant long-term visual impairment. In spite of treatment, tumor progression is common. Serial ophthalmic examinations with quantitative vision measurements are essential in the management of sporadic OPGs.     

Interaction of the effects of hypoxia, substrate depletion, acidosis and hyperkalaemia on the class III antiarrhythmic properties of sotalol

The effects of hypoxia on the actions of dl-sotalol 10(-4) mol . litre-1 were studied in rabbit papillary muscles at 32 degrees C. Superfusion for 30 min with a hypoxic solution (95% N2, 5% CO2) in the presence of 5 mmol . litre-1 glucose caused moderate shortening of control action potential duration from 208 +/- 3 to 138 +/- 9 ms (mean +/- SEM). In the presence of sotalol, hypoxia caused shortening of APD90 from 399 +/- 9 ms to 249 +/- ms, but the value after 30 min was still significantly greater than in controls (p less than 0.001). Superfusion with a hypoxic, glucose-free solution, however, caused profound shortening of APD90 in controls to 80 +/- 7 ms at 30 min. The highly significant lengthening of APD90 produced by sotalol in control conditions was abolished after 5 min hypoxia. The effects of hypoxia on the effective refractory period (ERP) paralleled those on APD90. Exposure to a hypoxic, acidotic, hyperkalaemic solution (80% N2, 20% CO2, pH 6.8; K+ 12 mmol . litre-1) produced moderate shortening of APD90 with convergence of the two groups. There was an increase in ERP, with the development of an equal degree of post-repolarisation refractoriness in the control and sotalol groups. The Class III effect of sotalol is preserved under mild but lost under severely hypoxic conditions. Using "simulated ischaemic" conditions, with controlled extracellular potassium concentrations, there was no difference in the relationship between APD90 and ERP in the control and sotalol groups.     

The clinician's role in promoting smoking cessation among clinic patients.

Like other chronic conditions, nicotine dependence offers both challenges and rewards to clinicians. The treatment of this condition frequently requires experience in pharmacology, behavioral science, and social aspects of medicine. Physicians are uniquely qualified to assist patients in their efforts to overcome the multifaceted condition of tobacco addiction. In providing this treatment, the clinical challenges are far outweighed by the benefit to patients who stop smoking. For many patients, smoking cessation is, by far, the most important step they can take to improve their health and increase their life span. Physicians who help patients accomplish this difficult goal provide a life-saving service. There is sufficient scientific evidence to guide physicians in their approach to smoking patients. Brief, systematic interventions have been shown to increase patient smoking cessation rates. The intervention can be described in four steps: ask about smoking, advise smokers to stop, assist those who want to stop, and arrange adequate follow-up. These interventions are used consistently when a smoking cessation program is adopted by an entire office practice. The components of this office-based program include defining staff roles, maintaining a smoke-free office, stocking appropriate materials, making use of the medical record to identify smokers and to remind staff to intervene, and monitoring patient progress. The potential public health impact of physician intervention with smoking patients is enormous. Even with very modest expectations of cessation rates, 100,000 physicians using effective intervention can produce over 3 million new ex-smokers in the United States each year. In conjunction with other community-based tobacco control efforts, this physician-lead effort will result in a marked reduction in the morbidity and mortality caused by smoking and, thus, control of "the most important public health issue of our time."     

Fetal parathyroids are not required to maintain placental calcium transport

We used Hoxa3 knockout mice and other mouse models to study the role of the fetal parathyroids in fetal calcium homeostasis. Hoxa3-null fetuses lack parathyroid glands, and absence of parathyroid hormone (PTH) was confirmed with a rodent PTH immunoradiometric assay. The ionized calcium level of Hoxa3-null fetuses was significantly lower than that of wild-type or heterozygous littermates or of the mother. Both the rate of placental calcium transfer and the plasma PTHrP level were normal in Hoxa3 mutants and their heterozygous siblings. Because we had previously observed an increase in placental calcium transfer in PTH/PTHrP receptor 1-null (Pthr1-null) fetuses, we assayed plasma PTHrP in those mice. Pthr1-null fetuses had plasma PTHrP levels 11-fold higher than those of their littermates. Northern analysis, immunohistochemical, and in situ hybridization studies of Pthr1-null fetuses indicated that liver and placenta had increased expression of PTHRP: In summary, loss of fetal parathyroids in Hoxa3-null fetuses caused marked hypocalcemia but did not alter placental calcium transfer or the circulating PTHrP level. The findings in the Pthr1-null fetuses indicate that several tissues may contribute to the circulating PTHrP level in fetal mice.     

Generation of HIV-1-specific CD8+ cell responses following allogeneic hematopoietic cell transplantation

This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active antiretroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infusion of allogeneic donor peripheral blood cells and posttransplantation cyclosporine and mycophenolate mofetil. Viral load, lymphocyte counts, and HIV-1-specific CD8(+) cell immune responses were compared before and after hematopoietic cell transplantation (HCT). Uninterrupted administration of HAART was feasible during nonmyeloablative conditioning and after HCT. The HIV RNA remained undetectable and no HIV-associated infections were observed. CD8(+) T-cell responses targeting multiple epitopes were detected before HCT. After HCT a different pattern of donor-derived HIV-specific CTL responses emerged by day +80, presumably primed in vivo. We conclude that allogeneic HCT offers the unique ability to characterize de novo HIV-1-specific immune responses. This clinical trial was registered at ClinicalTrials.gov (identifier: NCT00112593).     

Pro-Regenerative Signaling after Acetaminophen-Induced Acute Liver Injury in Mice Identified Using a Novel Incremental Dose Model

Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/β-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of β-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.Acetaminophen (APAP) is one of the most widely used over-the-counter analgesic and antipyretic drugs in the world. APAP is safe at therapeutic doses, but overdose can cause acute liver failure (ALF). In fact, APAP overdose is associated with 56,000 emergency department visits and 26,000 hospitalizations every year in the United States.¹ The only pharmacological intervention, at present, is N-acetyl cysteine (precursor of glutathione), which is successful only if given within a few hours after APAP overdose.² An ultimate option is liver transplantation, which is complicated by issues such as donor availability, long-term immunosuppression, and exorbitant costs.³Previous studies suggest that liver regeneration after APAP overdose plays a critical role in determination of outcome of injury.^4–7 α-Feto protein, a marker of liver regeneration, was found to be associated with better survival rate in patients with APAP-induced ALF.⁴ Several other studies in animal models suggest that timely stimulation of liver regeneration, such as with stem cell factor⁶ and vascular endothelial growth factor,⁷ improves survival after APAP overdose in mice. These studies highlight stimulating liver regeneration in APAP-induced patients with ALF as a plausible therapeutic option. However, the mechanisms of liver regeneration after APAP-induced ALF are not well known. Although liver regeneration has been extensively studied in the past,⁸ most of the studies are on the basis of a partial hepatectomy (PHX) model, a mechanistically different model from APAP-induced ALF.Data on hepatotoxicants, in general, suggest that liver regeneration follows the principles of dose-response.⁹ Studies indicate that liver regeneration after toxic injury to liver increases proportionately to injury but only up to a threshold dose. Doses higher than the threshold dose actually inhibit liver regeneration, resulting in progression of injury to ALF and death.^9–11 These studies have suggested that at higher doses, regeneration is inhibited because of blockade in critical proregenerative signaling pathways.^9,11,12 On the basis of this principle, we developed a novel incremental dose model to delineate the mechanisms of liver regeneration after APAP-induced acute liver injury (ALI). We used two doses of APAP, a lower dose (300 mg/kg), after which liver regeneration is intact, and a higher dose (600 mg/kg), after which liver regeneration is inhibited. We performed a comprehensive analysis of several signaling pathways known to be involved in liver regeneration and identified pathways that are potentially important for liver regeneration after APAP-induced ALI; these pathways can be targeted therapeutically.     

A phase II trial of a multi‐agent oral antiangiogenic (metronomic) regimen in children with recurrent or progressive cancer

Background

Preclinical models show that an antiangiogenic regimen at low‐dose daily (metronomic) dosing may be effective against chemotherapy‐resistant tumors. We undertook a prospective, open‐label, single‐arm, multi‐institutional phase II study to evaluate the efficacy of a “5‐drug” oral regimen in children with recurrent or progressive cancer.

Procedure

Patients ≤21 years old with recurrent or progressive tumors were eligible. Treatment consisted of continuous oral celecoxib, thalidomide, and fenofibrate, with alternating 21‐day cycles of low‐dose cyclophosphamide and etoposide. Primary endpoint was to assess, within eight disease strata, activity of the 5‐drug regimen over 27 weeks. Blood and urine angiogenesis markers were assessed.

Results

One hundred one patients were enrolled; 97 began treatment. Median age was 10 years (range: 191 days–21 years); 47 (49%) were female. Disease strata included high‐grade glioma (HGG, 21 patients), ependymoma (19), low‐grade glioma (LGG, 12), bone tumors (12), medulloblastoma/primitive neuroectodermal tumor (PNET, 8), leukemia (4), neuroblastoma (3), and miscellaneous tumors (18). Treatment was generally well tolerated; most common toxicities were hematologic. Twenty‐four (25%) patients completed 27 weeks therapy without progression, including HGG: 1 (5%), ependymoma: 7 (37%), LGG: 7 (58%), medulloblastoma/PNET: 1, neuroblastoma: 1, and miscellaneous tumors: 7 (39%). Best response was complete response (one patient with medulloblastoma), partial response (12), stable disease (36), progressive disease (47), and inevaluable (1). Baseline serum thrombospondin levels were significantly higher in patients successfully completing therapy than in those who progressed (P = 0.009).

Conclusion

The 5‐drug regimen was well tolerated. Clinical activity was demonstrated in some but not all tumor strata. Pediatric Blood Cancer 2014;61:636–642. © 2013 The Authors Pediatric Blood & Cancer Published by Wiley Periodicals, Inc.     

Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection

Although cytomegalovirus (CMV) expresses proteins that interfere with antigen presentation by class I major histocompatibility complex (MHC) molecules, CD8+ cytotoxic T cells (CTLs) are indispensable for controlling infection and maintaining latency. Here, a cytokine flow cytometry assay that employs fibroblasts infected with a mutant strain of CMV (RV798), which is deleted of the 4 viral genes that are responsible for interfering with class I MHC presentation, was used to examine the frequency and specificity of the CD8+ CTLs to CMV in immunocompetent CMV-seropositive individuals. A large fraction of the CD8+ CTL response was found to be specific for viral antigens expressed during the immediate early and early phases of virus replication and presented by fibroblasts infected with RV798 but not wild-type CMV. These results demonstrate that the inhibition of class I antigen presentation observed in CMV-infected cells in vitro is not sufficient to prevent the induction of a broad repertoire of CD8+ CTLs after natural infection in vivo. Thus, reconstitution of T-cell immunity in immunodeficient patients by cell therapy or by vaccination may need to target multiple viral antigens to completely restore immunologic control of CMV.