Intravenous Autologous Bone Marrow-derived Mesenchymal Stromal Cells Delay Acute Respiratory Distress Syndrome in Swine.

Rationale: Early post injury mitigation strategies in ARDS are in short supply. Treatments with allogeneic stromal cells are administered after ARDS develops, require specialized expertise and equipment, and to date have shown limited benefit. Objectives: Assess the efficacy of immediate post injury intravenous administration of autologous or allogeneic bone marrow-derived mesenchymal stromal cells (MSCs) for the treatment of acute respiratory distress syndrome (ARDS) due to smoke inhalation and burns. Methods: Yorkshire swine (n = 32, 44.3 ± 0.5 kg) underwent intravenous anesthesia, placement of lines, severe smoke inhalation, and 40% total body surface area flame burns, followed by 72 hours of around-the-clock ICU care. Mechanical ventilation, fluids, pressors, bronchoscopic cast removal, daily lung computed tomography scans, and arterial blood assays were performed. After injury and 24 and 48 hours later, animals were randomized to receive autologous concentrated bone marrow aspirate (n = 10; 3 × 106 white blood cells and a mean of 56.6 × 106 platelets per dose), allogeneic MSCs (n = 10; 6.1 × 106 MSCs per dose) harvested from healthy donor swine, or no treatment in injured control animals (n = 12). Measurements and Main Results: The intravenous administration of MSCs after injury and at 24 and 48 hours delayed the onset of ARDS in swine treated with autologous MSCs (48 ± 10 h) versus control animals (14 ± 2 h) (P = 0.004), reduced ARDS severity at 24 (P < 0.001) and 48 (P = 0.003) hours, and demonstrated visibly diminished consolidation on computed tomography (not significant). Mortality at 72 hours was 1 in 10 (10%) in the autologous group, 5 in 10 (50%) in the allogeneic group, and 6 in 12 (50%) in injured control animals (not significant). Both autologous and allogeneic MSCs suppressed systemic concentrations of TNF-α (tumor necrosis factor-α). Conclusions: The intravenous administration of three doses of freshly processed autologous bone marrow-derived MSCs delays ARDS development and reduces its severity in swine. Bedside retrieval and administration of autologous MSCs in swine is feasible and may be a viable injury mitigation strategy for ARDS.

Comparison of Synthetic Computed Tomography Generation Methods, Incorporating Male and Female Anatomical Differences, for Magnetic Resonance Imaging-Only Definitive Pelvic Radiotherapy.

PURPOSE: There are several means of synthetic computed tomography (sCT) generation for magnetic resonance imaging (MRI)-only planning; however, much of the research omits large pelvic treatment regions and female anatomical specific methods. This research aimed to apply four of the most popular methods of sCT creation to facilitate MRI-only radiotherapy treatment planning for male and female anorectal and gynecological neoplasms. sCT methods were validated against conventional computed tomography (CT), with regard to Hounsfield unit (HU) estimation and plan dosimetry. METHODS AND MATERIALS: Paired MRI and CT scans of 40 patients were used for sCT generation and validation. Bulk density assignment, tissue class density assignment, hybrid atlas, and deep learning sCT generation methods were applied to all 40 patients. Dosimetric accuracy was assessed by dose difference at reference point, dose volume histogram (DVH) parameters, and 3D gamma dose comparison. HU estimation was assessed by mean error and mean absolute error in HU value between each sCT and CT. RESULTS: The median percentage dose difference between the CT and sCT was <1.0% for all sCT methods. The deep learning method resulted in the lowest median percentage dose difference to CT at -0.03% (IQR 0.13, -0.31) and bulk density assignment resulted in the greatest difference at -0.73% (IQR -0.10, -1.01). The mean 3D gamma dose agreement at 3%/2 mm among all sCT methods was 99.8%. The highest agreement at 1%/1 mm was 97.3% for the deep learning method and the lowest was 93.6% for the bulk density method. Deep learning and hybrid atlas techniques gave the lowest difference to CT in mean error and mean absolute error in HU estimation. CONCLUSIONS: All methods of sCT generation used in this study resulted in similarly high dosimetric agreement for MRI-only planning of male and female cancer pelvic regions. The choice of the sCT generation technique can be guided by department resources available and image guidance considerations, with minimal impact on dosimetric accuracy.

Optimisation and validation of an integrated magnetic resonance imaging-only radiotherapy planning solution.

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI)-only treatment planning is gaining in popularity in radiation oncology, with various methods available to generate a synthetic computed tomography (sCT) for this purpose. The aim of this study was to validate a sCT generation software for MRI-only radiotherapy planning of male and female pelvic cancers. The secondary aim of this study was to improve dose agreement by applying a derived relative electron and mass density (RED) curve to the sCT. METHOD AND MATERIALS: Computed tomography (CT) and MRI scans of forty patients with pelvic neoplasms were used in the study. Treatment plans were copied from the CT scan to the sCT scan for dose comparison. Dose difference at reference point, 3D gamma comparison and dose volume histogram analysis was used to validate the dose impact of the sCT. The RED values were optimised to improve dose agreement by using a linear plot. RESULTS: The average percentage dose difference at isocentre was 1.2% and the mean 3D gamma comparison with a criteria of 1%/1 mm was 84.0% ± 9.7%. The results indicate an inherent systematic difference in the dosimetry of the sCT plans, deriving from the tissue densities. With the adapted REDmod table, the average percentage dose difference was reduced to -0.1% and the mean 3D gamma analysis improved to 92.9% ± 5.7% at 1%/1 mm. CONCLUSIONS: CT generation software is a viable solution for MRI-only radiotherapy planning. The option makes it relatively easy for departments to implement a MRI-only planning workflow for cancers of male and female pelvic anatomy.

A System for Continuous Pre- to Post-reperfusion Intra-carotid Cold Infusion for Selective Brain Hypothermia in Rodent StrokeModels.

Intra-carotid cold infusion (ICCI) appears as a promising method for hypothermia-mediated brain protection from ischemic stroke. Recent clinical pilot studies indicate easy implementation of ICCI into endovascular acute ischemic stroke treatment. Current rodent ICCI-in-stroke models limit ICCI to the post-reperfusion phase. To establish a method for continuous ICCI over the duration of intra-ischemia to post-reperfusion in rodent stroke models, a novel system was developed. Eighteen male Sprague-Dawley rats were included. Intraluminal filament method was used for transient middle cerebral artery occlusion (MCAO). Normal saline (~ 0 °C) was delivered (≤ 2.0 mL/min) into the internal carotid artery via a customized infusion system without interruption during MCAO (intra-ischemia) to after filament withdrawal (post-reperfusion). Bilateral cortical and striatal temperatures were monitored. Hypothermia goals were a temperature reduction in the ischemic hemisphere by 2 °C prior to reperfusion and thereafter maintenance of regional brain hypothermia at ~ 32 °C limiting the administered ICCI volume to ½ of each rat's total blood volume. During ischemia, maximum brain cooling rate was achieved with ICCI at 0.5 mL/min. It took 2 min to reduce ischemic striatal temperature by 2.3 ± 0.3 °C. After reperfusion, brain cooling was continued at 2 mL/min ICCI first (over 42 s) and maintained at 32.1 ± 0.3 °C at 0.7 mL/min ICCI over a duration of 15 ± 0.8 min. ICCI (total 12.6 ± 0.6 mL) was uninterrupted over the duration of the studied phases. First system that allows continuous ICCI during the phases of intra-ischemia to post-reperfusion in small animals for selective brain cooling and for investigations of other neuroprotective infusions.

Selective brain hypothermia.

Selective brain hypothermia is a powerful concept for neuroprotection that has been successfully investigated in a variety of animal models of global and focal ischemia. Its major advantages over systemic hypothermia include rapid induction of cooling, ability to achieve profound target brain temperatures, organ-selective cooling, and temperature control. Clinical systems and devices are available or are currently under development that utilize conductive (surface-cooling pads, closed-loop catheters), convective (transnasal coolant delivery), or mass and energy transport (cold intra-arterial infusion) methods to achieve and maintain selective brain hypothermia. The "ideal" brain-cooling system that is characterized by rapid cooling to profound hypothermia, its ability to maintain selective cooling over several days, and is noninvasive in nature, remains unrealistic. Instead, systems may be identified by their distinct advantages to meet a specific need in the care of a patient. This involves the consideration of the timing of ischemic injury (preischemic, intraischemic, postischemic), extent of ischemic damage (excitotoxicity, inflammation, necrosis, edema), and type and setting of therapeutic intervention (intensive care, interventional therapy, surgery). The successful translation of these systems into clinical practice will depend on smart engineering, safety and efficacy, and usability in current clinical work flow.

Impact of Concomitant Mitral Valve Surgery With LVAD Placement: Systematic Review and Meta-Analysis.

The aim of this systematic review and meta-analysis was to evaluate the outcomes of concomitant mitral valve surgery for significant preexisting mitral regurgitation (MR) in patients undergoing continuous-flow left ventricular assist device (CF-LVAD) implantation. Electronic search was performed to identify all studies in the English literature examining concurrent mitral valve surgery in patients with CF-LVAD implantation. Identified articles were systematically assessed for inclusion and exclusion criteria. Of 2319 studies identified, 8 studies were included. Among 445 patients with moderate to severe or severe MR, 113 (25.4%) patients received concurrent mitral valvular intervention during CF-LVAD implantation. There were no significant differences in cardiopulmonary bypass time (MR Surgery 154 min vs. no MR Surgery 119 min, P = 0.64) or hospital length of stay (MR Surgery 21 days vs. no MR Surgery 18 days, P = 0.93). On follow-up, there were no significant differences in freedom from greater than moderate MR (MR Surgery 100% vs. no MR Surgery 74%, P = 0.12) or left ventricular end-diastolic diameter (MR Surgery: 60 mm vs. no MR Surgery 65 mm, P = 0.51). Survival was comparable at 6-months (MR Surgery 77% vs. no MR Surgery 81%, P = 0.75), 1-year (MR Surgery 72% vs. no MR Surgery 80%, P = 0.36), and 2-years of follow-up (MR Surgery 65% vs. no MR Surgery 70%, P = 0.56). The results of our systematic review and meta-analysis of 8 studies consisting of 445 patients demonstrates that the addition of mitral valve intervention to CF-LVAD implantation appears to be safe with comparable survival to those undergoing CF-LVAD implantation alone. Large prospective randomized clinical trials are needed to elucidate whether concomitant mitral valve intervention during CF-LVAD implantation in patients with severe MR is necessary.

Identification of a functional nuclear translocation sequence in hPPIP5K2.

BACKGROUND: Cells contain several inositol pyrophosphates (PP-InsPs; also known as diphosphoinositol polyphosphates), which play pivotal roles in cellular and organismic homeostasis. It has been proposed that determining mechanisms of compartmentation of the synthesis of a particular PP-InsP is key to understanding how each of them may exert a specific function. Human PPIP5K2 (hPPIP5K2), one of the key enzymes that synthesizes PP-InsPs, contains a putative consensus sequence for a nuclear localization signal (NLS). However, such in silico analysis has limited predictive power, and may be complicated by phosphorylation events that can dynamically modulate NLS function. We investigated if this candidate NLS is functional and regulated, using the techniques of cell biology, mutagenesis and mass spectrometry. RESULTS: Multiple sequence alignments revealed that the metazoan PPIP5K2 family contains a candidate NLS within a strikingly well-conserved 63 amino-acid domain. By analyzing the distribution of hPPIP5K2-GFP in HEK293T cells with the techniques of confocal microscopy and imaging flow cytometry, we found that a distinct pool of hPPIP5K2 is present in the nucleus. Imaging flow cytometry yielded particular insight into the characteristics of the nuclear hPPIP5K2 sub-pool, through a high-throughput, statistically-robust analysis of many hundreds of cells. Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization. Proximal to the NLS is a Ser residue (S1006) that mass spectrometry data indicate is phosphorylated inside cells. The degree of nuclear localization of hPPIP5K2 was increased when S1006 was rendered non-phosphorylatable by its mutation to Ala. Conversely, a S1006D phosphomimetic mutant of hPPIP5K2 exhibited a lower degree of nuclear localization. CONCLUSIONS: The current study describes for the first time the functional significance of an NLS in the conserved PPIP5K2 family. We have further demonstrated that there is phosphorylation of a Ser residue that is proximal to the NLS of hPPIP5K2. These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event. Our study also increases general insight into the consensus sequences of other NLSs, the functions of which might be similarly regulated.

Trends in the management of intracranial vascular malformations in the USA from 2000 to 2007.

Objective. To assess prevalence, clinical characteristics, trends in treatment pattern, and outcome in patients with intracranial vascular malformations (IVMs). Methods. Nationwide inpatient sample. Patients with the diagnosis of an IVM admitted to US hospitals from 2000 to 2007. Results. In 58,051 IVM-related admissions (detection rate 2.4/100,000 person-years; mean age 49 ± 17 years; 52% women) major diagnoses were intracranial hemorrhage (ICrH) in 15%, seizure 32%, ischemia 5%, and headache 9%. Procedures included surgery (13%), embolization (13%), radiation therapy (2%), aneurysm clipping (1%), and mechanical ventilation (6%). Ventilation and ICrH were associated with death (2%), whereas ventilation, ICrH, surgery, seizure, and ischemia were associated with unfavorable outcome (20%). IVM detection rate and hospital outcome remained stable over time, whereas mean age and comorbid diagnosis of cerebral ischemia increased (ICrH and seizure decreased). Conclusion. IVMs are infrequent and present in 1/6 patients with some form of ICrH. Overall, seizure is the dominant comorbid diagnosis (1/3 patients). IVMs are equally prevalent among race-ethnic groups and are increasingly detected later in life. The inpatient care of IVM patients results in death or discharge into specialized care in 1/5 patients.

Unified immune modulation by 4-1BB triggering leads to diverse effects on disease progression in vivo.

4-1BB (CD137) is a powerful T-cell costimulatory molecule in the treatment of virus infections and tumors, but recent studies have also uncovered regulatory functions of 4-1BB signaling. Since 4-1BB triggering suppresses autoimmunity by accumulating indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) in an interferon (IFN)-γ-dependent manner, we asked whether similar molecular and cellular changes were induced by 4-1BB triggering in virus-infected mice. 4-1BB triggering increased IFN-γ and IDO, and suppressed CD4(+) T cells, in C57BL/6 mice infected with the type 1 KOS strain of Herpes simplex virus (HSV-1), as it does in an autoimmune disease model. Detailed analysis of the CD4(+) T suppression showed that freshly activated CD62L(high) T cells underwent apoptosis in the early phase of suppression, and CD62L(low) effector/memory T cells in the later phase. Although 4-1BB triggering resulted in similar cellular changes - increased CD8(+) T and decreased CD4(+) T cells, it had different effects on mortality in mice infected with HSV-1 RE, influenza, and Japanese encephalitis virus (JEV); it increased mortality in influenza-infected mice but decreased it in JEV-infected mice. Since the dominant type of immune cell generated to protect the host was different for each virus - CD4(+) T cells and neutrophils in HSV-1 RE infection, both CD4(+) T and CD8(+) T cells in influenza infection, and a crucial role for B cells in JEV infection, 4-1BB triggering resulted in different therapeutic outcomes. We conclude that the therapeutic outcome of 4-1BB triggering is determined by whether the protective immunity generated against the virus was beneficially altered by the 4-1BB triggering.

Cellular energetic status supervises the synthesis of bis-diphosphoinositol tetrakisphosphate independently of AMP-activated protein kinase.

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP](2)-InsP(4)), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP](2)-InsP(4) synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP](2)-InsP(4) synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP](2)-InsP(4) levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP](2)-InsP(4) synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2'-amino-3'-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP](2)-InsP(4) is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.

Role of endogenous 4-1BB in the development of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies directed against nuclear antigens including nucleosomes and DNA. To determine the role of T-cell costimulatory molecule 4-1BB in the regulation of SLE, MRL-Fas(lpr) (lpr) mice deficient in 4-1BB (lpr/4-1BB(-/-)) were generated and their disease phenotype was compared to that of control lpr mice. The main finding of this study is that the lpr/4-1BB(-/-) mice had more pronounced skin lesions which appeared earlier, increased lymphadenopathy, increased renal damage, and higher mortality than 4-1BB-intact control lpr mice. The increased severity of lesions in lpr/4-1BB(-/-) mice was closely associated with increases in CD4(+) T, CD3(+) B220(+) double-negative T cells, serum immunoglobulin, anti-dsDNA autoantibodies, and tissue immunoglobulin deposits. These data suggest that the 4-1BB-4-1BB ligand signalling pathway plays an important role in SLE and that deletion of 4-1BB confers susceptibility to lpr mice, leading to accelerated induction of disease and early mortality.

scyllo-inositol pentakisphosphate as an analogue of myo-inositol 1,3,4,5,6-pentakisphosphate: chemical synthesis, physicochemistry and biological applications.

myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.

Invasive treatment of unruptured brain arteriovenous malformations is experimental therapy.

PURPOSE OF REVIEW: Brain arteriovenous malformations (AVMs) are currently being treated in a variety of ways, including medical management, endovascular procedures, neurosurgery and radiotherapy. The widespread diffusion of these various treatment approaches is partially driven by the existence of variations in the perception about the risks of rupture, and how devastating such events would be. RECENT FINDINGS: Data from the most recent studies suggest the majority of AVM patients are diagnosed without signs of hemorrhage, further, that the natural history risk for the unruptured cohort is far more benign than for those presenting with rupture. In cases where hemorrhage occurs, the clinical syndrome is significantly less disabling than in patients with non-AVM related hemorrhage. For unruptured AVMs, current morbidity data suggest a higher risk for invasive management than for the natural history of untreated patients. SUMMARY: No randomized clinical trial data exist on the benefit of invasive AVM treatment, and the most contentious issue at present is whether intervention should be considered for AVMs that have not bled. In a scientific sense, invasive treatment for unruptured brain AVMs may be considered experimental therapy awaiting the results from 'A Randomized Trial of Unruptured Brain AVMs' (ARUBA), which is currently underway.

Endovascular recanalization therapy in acute ischemic stroke.

BACKGROUND AND PURPOSE: To assess the outcome in acute ischemic stroke patients not eligible for systemic thrombolysis (outside the 3-hour time window, after surgery, or on anticoagulant) undergoing endovascular recanalization therapy (ERT) at the Columbia University Medical Center (CUMC) and to determine US nationwide usage and outcome of ERT in acute ischemic stroke. METHODS: Patients treated at CUMC from 2001 to 2004 and the Nationwide Inpatient Sample (NIS) comprising 20% of all admissions in the United States from 1999 to 2002 were analyzed retrospectively. RESULTS: Thirty-one patients underwent ERT. Mean age was 68+/-14 years, 68% were female, and 45% nonwhite (occlusion sites: internal carotid artery 29%; middle cerebral artery 39%; posterior circulation 32%). Pharmacological or mechanical ERT was initiated beyond 3 hours after symptom onset (median time 4.4 hours) in 61%, 29% had surgery, and 39% were on anticoagulant medication. At discharge, 32% had modified Rankin Scale scores of 0 to 2 (52% discharged home or to rehabilitation facilities); overall mortality was 29%, of which 19% were fatal intracerebral hemorrhages. From the NIS cohort, 477 patients (0.17% of all strokes and 14% of all thrombolysis cases) underwent ERT. Fifteen percent died, and approximately 50% were discharged home or to rehabilitation facilities. Intracerebral hemorrhage occurred in 6%. Fewer good outcomes of the CUMC cohort may be explained by more unfavorable premorbid patient characteristics compared with the NIS cohort. CONCLUSIONS: Despite significant variability in patient characteristics and treatment methods among 2 sources of data analyzed, ERT in stroke patients not eligible for intravenous thrombolysis appears to be a relatively safe and effective treatment alternative that is being used increasingly nationwide.

Safety profile of a nicotine lozenge compared with that of nicotine gum in adult smokers with underlying medical conditions: a 12-week, randomized, open-label study.

BACKGROUND: Nicotine polacrilex lozenges deliver 25% to 27% more nicotine compared with equivalent doses of nicotine polacrilex gum. The increased nicotine exposure from the lozenge has raised questions about the relative safety of the lozenge and gum. OBJECTIVE: The objective of this study was to compare the safety profiles of the 4-mg nicotine lozenge and 4-mg nicotine gum in smokers with selected label-restricted diseases. METHODS: This was a multicenter, randomized, open-label study in adult smokers with heart disease, hypertension not controlled by medication, and/or diabetes mellitus. Patients were randomized in a 1:1 ratio to receive the 4-mg nicotine lozenge or 4-mg nicotine gum. Safety assessments were made at baseline and at 2, 4, 6, and 12 weeks after the start of product use. RESULTS: Nine hundred one patients were randomized to treatment, 447 who received the lozenge and 454 who received the gum (safety population). The majority were women (52.7%). Patients' mean age was 53.9 years, their mean weight was 193.9 pounds, and they smoked a mean of 25.2 cigarettes per day at baseline. Five hundred fifty-three patients, 264 taking the lozenge and 289 taking the gum, used the study product for > or =4 days per week during the first 2 weeks (evaluable population). The nicotine lozenge and nicotine gum were equally well tolerated, despite increased nicotine exposure from the lozenge. The incidence of adverse events in the 2 groups was similar during the first 2 weeks of product use (evaluation population: 55.3% lozenge, 54.7% gum), as well as during the entire study (safety population: 63.8% and 58.6%, respectively). Stratification of patients by sex, age, extent of concurrent smoking, extent of product use, and severity of adverse events revealed no clinically significant differences between the lozenge and gum. The most common adverse events were nausea (17.2% and 16.1%; 95% CI, -3.7 to 6.0), hiccups (10.7% and 6.6%; 95% CI, 0.5 to 7.8), and headache (8.7% and 9.9%; 95% Cl, -5.0 to 2.6). Serious adverse events were reported in 11 and 13 patients in the respective groups. Fewer than 6% of patients in either group were considered by the investigator to have a worsening of their overall disease condition during the study. The majority of patients (>60%) experienced no change in their disease status from baseline. CONCLUSION: The 4-mg nicotine lozenge and 4-mg nicotine gum had comparable safety profiles in these patients with label-restricted medical conditions.

Interaction of Pik1p and Sjl proteins in membrane trafficking.

Phosphatidylinositol (PtdIns) phosphates are involved in signal transduction, cytoskeletal organization, and membrane traffic. PtdIns 4-phosphate [PtdIns(4)P], produced in yeast by PtdIns 4-kinase (Pik1p), appears to regulate Golgi secretory function. PtdIns(4)P is also produced by dephosphorylation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], catalyzed by one of the three yeast Sjl proteins, homologs of the mammalian synaptic vesicle-associated PtdIns(4,5)P2 5-phosphatase, synaptojanin. To determine whether Pik1p and Sjl proteins operate in the same pathway or regulate the same process, we used a genetic approach. Mutation in the PIK1 gene displays synthetic genetic interactions with deletions of individual SJL genes. Deletion of SJL3 gene is synthetically lethal with pik1ts, and deletions of SJL1 or SJL2 genes in pik1ts cells exacerbate the temperature sensitivity, neomycin sensitivity, and defect in invertase secretion. A diminished level of PtdIns(4)P and increased level of PtdIns(4,5)P2 in pik1(ts)sjl1delta and pik1(ts)sjl2delta cells, compared with pik1ts cells, indicate that PtdIns(4)P is specifically required for secretion. Collectively, our results suggest that Pik1p and the Sjl proteins coordinately function to regulate the dynamic phosphorylation-dephosphorylation of the polar heads of phosphoinositides, and this process appears to be important for membrane trafficking pathways.

4-1BB-mediated immunotherapy of rheumatoid arthritis.

Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.

Pharmacokinetics of a nicotine polacrilex lozenge.

To evaluate the pharmacokinetic characteristics of the 2-mg and 4-mg nicotine polacrilex lozenges, the following four separate studies were conducted in healthy adult smokers: (a) A single-dose, four-way crossover (replicate design) study to compare the 4-mg lozenge and the 4-mg nicotine polacrilex gum, (b) a single-dose, two-way crossover study to compare the 2-mg lozenge and the 2-mg gum, (c) a multiple-dose, four-way crossover study to compare the lozenges administered every 90 min and the gums administered every 60 min at 2- and 4-mg dose levels, and (d) a single-dose, three-way crossover study to compare the pharmacokinetic profiles of the 4-mg lozenge when administered in three different ways: (i) Used as directed, (ii) chewed and immediately swallowed, and (iii) chewed, retained in the mouth for 5 min, and then swallowed. The single-dose studies consistently demonstrated 8%-10% higher maximal plasma concentrations and 25%-27% higher AUC values (area under the concentration-time curve) from the lozenges compared with the gums at the 2- and 4-mg dose levels, probably owing to the residual nicotine retained in the gum. The multiple-dose study applying different dosing intervals (i.e., every 90 min for the lozenges and every 60 min for the gums) resulted in approximately 30% lower AUC(0-t) values for the lozenges compared with those for the gums. Administration of the lozenge contrary to the label-specified instructions for use did not lead to a faster or higher absorption of nicotine. These pharmacokinetic characteristics should allow the lozenge to become an effective and safe therapeutic alternative for smoking cessation.

Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is a chronic inflammatory process in corneal stroma that results from recurrent HSV type 1 infection. We used the murine model of HSK to demonstrate the importance of the interaction between an inducible T cell costimulatory receptor, 4-1BB, and its ligand, 4-1BB ligand (4-1BBL), in the development of this disease. In BALB/c mice, HSK ordinarily induced by infection with the RE strain of herpes was prevented by blocking 4-1BB/4-1BBL interaction, either by deleting 4-1BB (in mutant 4-1BB(-/-) mice) or by introducing mAbs against 4-1BBL. The majority of T cells infiltrating the infected corneas were 4-1BB(+) activated effector cells that expressed cell surface markers CD44, CD25, and/or CD62L, as well as chemokine receptors CCR1, CCR2, and CCR5, and a limited number of TCR Vbeta chains (Vbeta8.1/8.2, Vbeta8.3, Vbeta10b, and Vbeta5.1/5.2, in order of abundance). Analysis of cell surface phenotypes showed that the failure to develop HSK in the 4-1BB(-/-) mice was associated with a reduced expression of CD62L at the time of T cell migration into the corneal stroma.

The FKBP12-rapamycin-associated protein (FRAP) is a CLIP-170 kinase.

CLIP-170/Restin belongs to a family of conserved microtubule (MT)-associated proteins, which are important for MT organization and functions. CLIP-170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP-170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12-rapamycin-associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP-170. CLIP-170 is phosphorylated in vivo at multiple sites, including rapamycin-sensitive and -insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin-sensitive sites. In addition, rapamycin inhibited the ability of CLIP-170 to bind to MTs. Our observations suggest that multiple CLIP-170 kinases are involved in positive and negative control of CLIP-170, and FRAP is a CLIP-170 kinase positively regulating the MT-binding behavior of CLIP-170.