Clearance In Mice Research Articles

Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

S4‐03‐02: Discovery of novel [18F]‐PET agents for imaging neurofibrillary tangles (NFTs)

There are two main neuropathologic hallmarks in AD, senile neuritic plaques (SP) around an amyloid beta core and neurofibrillar protein aggregates containing tau protein. In AD patients tau is abnormally phosphorylated and loses its normal function. Unlike ß-amyloid plaque deposition, human post-mortem studies indicate that neurofibrillary tangle density correlates with neurodegeneration and cognitive impairment. Furthermore, abundant neurofibrillary tangles are not observed in cognitively unimpaired individuals, in contrast to ß-amyloid plaques that can be present also in cognitively normal people. Therefore, a tau PET tracer might be a good diagnostic tool for Alzheimer's disease that can be correlated with the progression of clinical symptoms. In order to screen many compounds as potential tau binders, we developed a competitive autoradiography screening method utilizing human AD brain sections. Brain sections from over 40 human AD brains were immunostained for tau and Aß and quantified by area measurements. Selectivity of tau binding compounds was determined by competition experiments in tau rich or Aß rich brain sections. Grey and white matter binding was measured for each candidate. Double staining of fluorescent analogs confirms binding of these compounds to tau tangles in AD brains. Brain uptake of [F18]-labeled tracers was measured in rodents and monkeys. Metabolism and pharmacokinetic studies were performed in mice. We have discovered several novel, small molecule tau binding compounds that show high selectivity for native tau aggregates in human AD brain sections over ß-amyloid. Several of these candidates were further optimized to show a high brain uptake/fast clearance in mice, rats, and monkeys. White matter distribution in monkeys is very low. Metabolism studies of our lead candidates show very good stability in plasma and no metabolite presence in mouse brains. We have identified several novel small heterocyclic tau binders. Our in vitro and in vivo studies confirm that our lead candidate [F18]-T808 binds to tau selectively over ß-amyloid, shows good PK and metabolic properties, and exhibits excellent brain uptake/washout kinetics in rodent and monkey brains.

T Cells from Programmed Death-1 Deficient Mice Respond Poorly to Mycobacterium tuberculosis Infection

BackgroundProgrammed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.Methodology/Principal FindingsHere, we report that PD-1 deficient (PD-1−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.Conclusions/SignificanceOur findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.

Contributions of Antinucleoprotein IgG to Heterosubtypic Immunity against Influenza Virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Toll-like Receptor 3 Is Required for Development of Retinopathy Caused by Impaired All-trans-retinal Clearance in Mice

Chronic inflammation is an important component that contributes to many age-related neurodegenerative diseases, including macular degeneration. Here, we report a role for toll-like receptor 3 (TLR3) in cone-rod dystrophy (CORD) of mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), proteins critical for all-trans-retinal clearance in the retina. Increased expression of toll-like receptor-signaling elements and inflammatory changes were observed in Rdh8(-/-)Abca4(-/-) eyes by RNA expression analysis. Unlike 3-month-old Rdh8(-/-)Abca4(-/-) mice that developed CORD, 6-month-old Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice did not evidence an abnormal retinal phenotype. Light-induced retinal degeneration in Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice was milder than that in Rdh8(-/-)Abca4(-/-) mice, and a 2-fold increased TLR3 expression was detected in light-illuminated retinas of Rdh8(-/-)Abca4(-/-) mice compared with nonilluminated retinas. Poly(I-C), a TLR3 ligand, caused caspase-8-independent cellular apoptosis. Whereas poly(I-C) induced retinal cell death in Rdh8(-/-)Abca4(-/-) and WT mice both in vivo and ex vivo, this was not seen in mice lacking Tlr3. Far fewer invasive macrophage/microglial cells in the subretinal space and weaker activation of Muller glial cells were exhibited by Tlr3(-/-)Rdh8(-/-) Abca4(-/-) mice compared with Rdh8(-/-)Abca4(-/-) mice at 3 and 6 months of age, indicating that loss of TLR3 inhibits local inflammation in the retina. Both poly(I-C) and endogenous products emanating from dying/dead retinal cells induced NF-κB and IRF3 activation. These findings demonstrate that endogenous products from degenerating retina stimulate TLR3 that causes cellular apoptosis and retinal inflammation and that loss of TLR3 protects mice from CORD.

Preparation and in vivo biological investigations on a novel radioligand for bone scanning: technetium-99m-labeled zoledronic acid derivative

To enable imaging at an earlier time after injection, a radiopharmaceutical with higher affinity for bone, larger ratio of bone-to-soft tissue uptake and more rapid clearance from blood is required. The nature of diphosphonic acid is a key factor to determine the advantages of the radiopharmaceuticals. The purpose of this study is to optimize the linker chain between the imidazolyl and geminal diphosphonate group in the zoledronic acid (ZL) to develop novel single photon emission computed tomography (SPECT) bone imaging agent. A novel ZL derivative, 1-hydroxy-3-(1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (IPrDP), was successfully prepared and labeled with (99m)Tc in a high labeling yield. Biodistribution of (99m)Tc-IPrDP and (99m)Tc-ZL in normal mice were studied and compared. SPECT bone scanning was performed on the rabbit and a series of dynamic and static images were recorded by Philips SKY Light emission computed tomography. In the biodistribution studies, (99m)Tc-IPrDP exhibits significant advantages on the bone resorption and the clearance from soft tissues compared with (99m)Tc-ZL. Kinetics of blood clearance in mice showed that T(1/2α) and T(1/2β) of (99m)Tc-IPrDP were 1.47 min and 46.47 min, while those of (99m)Tc-ZL were 2.28 and 52.63 min respectively. Excellent images of the rabbit skeleton can be quickly obtained for (99m)Tc-IPrDP, which was faster than (99m)Tc-ZL and the clinically widely used bone imaging agent (99m)Tc-MDP (technetium-99m labeled with methylenediphosphonate). (99m)Tc-IPrDP possesses excellent characteristics for the potential application as a novel bone scanning agent.

Efficient Lung Recruitment of Respiratory Syncytial Virus-Specific Th1 Cells Induced by Recombinant Bacillus Calmette-Guérin Promotes Virus Clearance and Protects from Infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.

Indocyanine green clearance varies as a function of N-acetylcysteine treatment in a murine model of acetaminophen toxicity

Standard assays to assess acetaminophen (APAP) toxicity in animal models include determination of ALT (alanine aminotransferase) levels and examination of histopathology of liver sections. However, these assays do not reflect the functional capacity of the injured liver. To examine a functional marker of liver injury, the pharmacokinetics of indocyanine green (ICG) were examined in mice treated with APAP, saline, or APAP followed by N-acetylcysteine (NAC) treatment.Male B6C3F1 mice were administered APAP (200 mg/kg IP) or saline. Two additional groups of mice received APAP followed by NAC at 1 or 4 h after APAP. At 24 h, mice were injected with ICG (10 mg/kg IV) and serial blood samples (0, 2, 10, 30, 50 and 75 min) were obtained for determination of serum ICG concentrations and ALT. Mouse livers were removed for measurement of APAP protein adducts and examination of histopathology. Toxicity (ALT values and histology) was significantly increased above saline treated mice in the APAP and APAP/NAC 4 h mice. Mice treated with APAP/NAC 1 h had complete protection from toxicity. APAP protein adducts were increased in all APAP treated groups and were highest in the APAP/NAC 1 h group. Pharmacokinetic analysis of ICG demonstrated that the total body clearance (Cl T) of ICG was significantly decreased and the mean residence time (MRT) was significantly increased in the APAP mice compared to the saline mice. Mice treated with NAC at 1 h had Cl T and MRT values similar to those of saline treated mice. Conversely, mice that received NAC at 4 h had a similar ICG pharmacokinetic profile to that of the APAP only mice. Prompt treatment with NAC prevented loss of functional activity while late treatment with NAC offered no improvement in ICG clearance at 24 h. ICG clearance in mice with APAP toxicity can be utilized in future studies testing the effects of novel treatments for APAP toxicity.

Responses against a Subdominant CD8+ T Cell Epitope Protect against Immunopathology Caused by a Dominant Epitope

CD8(+) T cell responses are critical for the control of virus infections. Following infection, epitope-specific responses establish an unpredictable but reproducible pattern of dominance that is dictated by a large number of both positive and negative factors. Immunodomination, or diminution of subdominant epitope-specific responses by dominant epitopes, can play a substantial role in the establishment of epitope hierarchy. To determine the role of a dominant (K(d)M2(82-90)) and a subdominant (D(b)M(187-195)) epitope of respiratory syncytial virus in viral control and immunodomination, MHC-binding anchor residues in the two epitopes were mutated individually in recombinant infectious viruses, greatly reducing or deleting the epitope-specific CD8(+) T cell responses. Neither mutation negatively affected viral clearance in mice, and compensation by the unmutated epitope was seen in both cases, whereas compensation by five other subdominant epitopes was minimal. Mutation of the dominant K(d)M2(82-90) response resulted in effective viral clearance by the subdominant epitope with less illness, whereas mutation of the subdominant D(b)M(187-195) response resulted in overcompensation of the already dominant K(d)M2(82-90) epitope, and increased severity of illness. Increased illness was associated with poor functionality of the abundant population of CD8(+) T cells specific to the dominant K(d)M2(82-90) epitope, as measured by the percentage and magnitude of IFN-γ production. These data demonstrate efficient viral clearance, and a protective effect of subdominant CD8(+) T cell responses.

Mast cells protect against airway Mycoplasma pneumoniae under allergic conditions

Infection with Mycoplasma pneumoniae (Mp) in asthma can occur both acutely and chronically with an associated Th2 inflammatory response and/or increased numbers of bronchial mast cells. Mast cells have previously been shown to promote mycoplasma clearance in mice; however, it is unknown whether mast cells would aid Mp clearance under allergic conditions. Our aim was to determine the impact of allergic inflammation on mast cell-mediated lung Mp clearance. Furthermore, as we have previously demonstrated an essential role for IL-6 in lung Mp clearance we also investigated the role of mast cell-derived IL-6. Mast cell-deficient (WBB6F1/J-Kit(W)/Kit(W-v)) mice were challenged with ovalbumin to induce airway inflammation before Mp infection. The role of mast cell-derived IL-6 in bacterial clearance was further investigated by reconstitution of mast cell-deficient mice with IL-6(-/-) mast cells. Allergic mast cell-deficient mice exhibited increased lung Mp burden compared with control littermates. Intravenous adoptive transfer of wild-type and IL-6(-/-) mast cells significantly improved Mp clearance in mast cell-deficient mice. Acutely after Mp infection, allergen-challenged mast cell-deficient mice had increased levels of the pro-inflammatory cytokines IL-6 and TNF-alpha in the bronchoalveolar lavage (BAL) fluid. The total number of neutrophils was also increased in mast cell-deficient mice. Our results establish that mast cells aid host defense against Mp in an allergic setting and that while IL-6 is necessary for lung Mp clearance, mast cell-derived IL-6 is not required.

Species differences in methanol and formic acid pharmacokinetics in mice, rabbits and primates

Methanol (MeOH) is metabolized primarily by alcohol dehydrogenase in humans, but by catalase in rodents, with species variations in the pharmacokinetics of its formic acid (FA) metabolite. The teratogenic potential of MeOH in humans is unknown, and its teratogenicity in rodents may not accurately reflect human developmental risk due to differential species metabolism, as for some other teratogens. To determine if human MeOH metabolism might be better reflected in rabbits than rodents, the plasma pharmacokinetics of MeOH and FA were compared in male CD-1 mice, New Zealand white rabbits and cynomolgus monkeys over time (24, 48 and 6 h, respectively) following a single intraperitoneal injection of 0.5 or 2 g/kg MeOH or its saline vehicle. Following the high dose, MeOH exhibited saturated elimination kinetics in all 3 species, with similar peak concentrations and a 2.5-fold higher clearance in mice than rabbits. FA accumulation within 6 h in primates was 5-fold and 43-fold higher than in rabbits and mice respectively, with accumulation being 10-fold higher in rabbits than mice. Over 48 h, FA accumulation was nearly 5-fold higher in rabbits than mice. Low-dose MeOH in mice and rabbits resulted in similarly saturated MeOH elimination in both species, but with approximately 2-fold higher clearance rates in mice. FA accumulation was 3.8-fold higher in rabbits than mice. Rabbits more closely than mice reflected primates for in vivo MeOH metabolism, and particularly FA accumulation, suggesting that developmental studies in rabbits may be useful for assessing potential human teratological risk.

Major contribution of tubular secretion to creatinine clearance in mice

This study was performed to quantify the fraction of excreted creatinine not attributable to creatinine filtration for accurately determining the glomerular filtration rate in mice. To measure this we compared creatinine filtration with the simultaneous measurement of inulin clearance using both single-bolus fluorescein isothiocyanate (FITC)-inulin elimination kinetics and standard FITC-inulin infusion. During anesthesia, creatinine filtration was found to be systematically higher than inulin clearance in both male and female C57BL/6J mice. The secretion fraction was significantly less in female mice. Administration of either cimetidine or para-aminohippuric acid, competitors of organic cation and anion transport respectively, significantly reduced the secretion fraction in male and female mice and both significantly increased the plasma creatinine level. Creatinine secretion in both genders was not mediated by the organic cation transporters OCT1 or OCT 2 since secretion fraction levels were identical in FVB wild-type and OCT1/2 knockout mice. Thus, secretion accounts for about 50 and 35% of excreted creatinine in male and female mice, respectively. Increasing plasma creatinine threefold by infusion further increased the secretion fraction. Renal organic anion transporter 1 mRNA expression was higher in male than in female mice, reflecting the gender difference in creatinine secretion. Hence we show that there is a major secretory contribution to creatinine excretion mediated through the organic anion transport system. This feature adds to problems associated with measuring endogenous creatinine filtration in mice.

Method for evaluating pulmonary clearance in mice and its pharmacological applications

The purpose of this study was to develop a method for evaluating pulmonary clearance in mice that inhaled aerosolized 99mTc-labeled human serum albumin (33mTc-HSA). Mice were ventilated with an aerosol of 99mTc-HSA for 10 min with an ultrasonic nebulizer in an acrylic chamber. Pulmonary clearance was estimated from lung retention ratio at various times after inhalation of 99mTc-HSA. Lung retention ratios in mice were 72.4 +/- 3.3% at 1 hr, 60.1 +/- 2.3% at 3 hr and 34.8 +/- 2.0% at 24 hr after inhalation of 99mTc-HSA, similar to the previous results obtained in humans by cinescintigraphy. The data were analyzed on the basis of two-compartment model. Mabuterol, a beta 2-adrenoceptor agonist, stimulated the pulmonary clearance. In contrast, metyrapone, an adrenal 11-beta steroid hydroxylase inhibitor, suppressed the clearance with a decreased corticosterone content in plasma. These findings suggest that the developed method is useful for evaluating the pharmacological effects on pulmonary clearance.

Dissociated effects of glucose-dependent insulinotropic polypeptide vs glucagon-like peptide–1 on β-cell secretion and insulin clearance in mice

Glucagon-like peptide–1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) potently augment insulin response to glucose. It is less known what their effects are insulin clearance, which also contributes to peripheral hyperinsulinemia observed after administration of incretins together with glucose. The aims of this study were the quantification of C-peptide secretion and the evaluation of insulin clearance after administration of GIP with glucose. This allows the assessment of GIP's effects on hyperinsulinemia. In addition, GIP's effects were compared with those of GLP-1. Anesthetized female NMRI mice were injected intravenously with glucose alone (1 g/kg, n = 35) or glucose together with GIP (50 μg/kg, n = 12). Samples were taken through the following 50 minutes, and C-peptide and insulin concentrations were used to reconstruct C-peptide secretion rate and insulin clearance. In a previous study, GLP-1 (10 μg/kg) was used in 12 mice; and we used those GLP-1 results to compare GIP effects with those of GLP-1. C-peptide secretion rate peaked at 1 minute after glucose injection, and the immediate part of the insulin-releasing process was markedly augmented by both incretin hormones (1-minute suprabasal increment secretory rate was 20 ± 2 pmol/min for GIP and 28 ± 2 pmol/min for GLP-1, vs only 9 ± 1 pmol/min for glucose alone; P < .001). Until 10 minutes after administration, C-peptide secretion remained higher with incretins ( P < .0001), whereas starting from 20 minutes, the 3 patterns were undistinguishable ( P > .2). Insulin clearance, previously shown to be abridged by 46% with GLP-1, was reduced only by a nonsignificant ( P = .27) 21% with GIP. This study thus shows that the 2 incretins markedly augment glucose-stimulated insulin secretion in mice by a preferential action on the immediate response to glucose of insulin secretion. However, the action of GIP is less effective than that of GLP-1. Insulin clearance with GIP is not significantly reduced. We conclude that GIP is less potent than GLP-1 in inducing glucose-stimulated hyperinsulinemia in the mouse.

Abstract C220: Genz-644282, a non-camptothecin topoisomerase one inhibitor, demonstrates a wide spectrum of in vitro and in vivo antitumor activity

Abstract Genz-644282 (GZ) is a novel non-camptothecin topoisomerase I (Top1) inhibitor. The in vitro and in vivo activity of GZ and its M1 and M2 metabolites were explored and compared with the activity of camptothecin Top1 inhibitors. In vitro in mouse, rat, dog, and human GZ exhibited high metabolic stability, plasma binding of 88–93% and exhibits concentration dependent partitioning into red blood cells. In vivo, GZ has a large volume of distribution and low-to-moderate clearance in mouse, rat and dog. In nude mice, the t1/2 for GZ is 3.6 h (po), 10.4 h (ip) and 5.1h (iv) and longer in tumor-bearing mice. In human HCT-116 colon ca, HT-29 colon ca and NCI-H460 NSCLC cells the concentration response for Genz-6244282, M1 and M2 are the same. Upon 72h exposure of the cells to GZ, M1 or M2 the IC50 concentrations were 0.5-0.65 nM and the IC90 concentrations were 1.8–2 nM. In order to evaluate the antitumor activity of GZ as compared to several approved anticancer agents, the compound was tested in seven xenograft models: LOX-IMVI melanoma, DLD-1 and HCT-15 colon, MDA-MB-231 breast, NCI-H292 and NCI-H1299 lung ca. GZ was compared against two of its metabolites, Genz-649974 (GZ-74) and Genz-649975 in the HCT-116 colon ca resulting in comparable activity with GZ-74. GZ was administered intravenously on a QODx3 schedule for 2 cycles. The tumor growth delay, TGD, (T-C) and increase in lifespan, ILS, (T/C) for each study are listed in the table below. All of the GZ dosages were well tolerated resulting in a maximum body weight loss of ≤20%, except for the high dosages in the HCT-15 and NCI-H292 in which there was a maximum body weight loss of 25.7 and 20.9%, respectively. Based on these findings and other data, GZ was selected to be a development candidate. Treatments Dose (mg/kg/day) Route/Schedule TGD (T-C) ILS (T/C) Tumor Line GZ 1 IV/QODx3 2 days 1.1x for 2 cycles GZ 4 IV/QODx3 25 days 2.8x LOX-IMVI Dacarbazine 90 IP/QDx5 14 days 2.0x GZ 4 IV/QODx3 8 days 1.2x for 2 cycles CPT-11 60 IV/Q4Dx3 5 days 1.1x DLD-1 for 2 cycles GZ 1 IV/QODx3 14 days 1.3x for 2 cycles GZ 2 IV/QODx3 35 days 1.8x HCT-15 for 2 cycles CPT-11 60 IV/Q4Dx3 28 days 1.7x GZ 1 IV/QODx3 21 days 1.7x for 2 cycles GZ 1.36 IV/QODx3 &amp;gt;47 days &amp;gt;2.3x MDA-MB-231 for 2 cycles GZ 1.7 IV/QODx3 35 days 2.0x for 2 cycles Docetaxel 20 IV/QODx3 &amp;gt;47 days &amp;gt;2.3x GZ 1 IV/QODx3 18 days 1.5x for 2 cycles GZ 1.36 IV/QODx3 21 days 1.6x for 2 cycles NCI-H292 GZ 1.7 IV/QODx3 21 days 1.6x for 2 cycles Docetaxel 20 IV/QODx3 39 days 2.1x GZ 1 IV/QODx3 20 days 1.7x for 2 cycles GZ 1.36 IV/QODx3 24 days 1.8x for 2 cycles NCI-H1299 GZ 1.7 IV/QODx3 34 days 2.1x for 2 cycles Docetaxel 20 IV/QODx3 17 days 1.6x GZ-74 4 IV/QODx3 25 days 1.8x for 2 cycles GZ-74 6 IV/QODx3 28 days 1.9x for 2 cycles HCT-116 GZ-74 8 IV/QODx3 32 days 2.0x for 2 cycles GZ 1.7 IV/QODx3 28 days 1.9x for 2 cycles Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C220.

Noninvasive real-time measurement of nasal mucociliary clearance in mice by pinhole gamma scintigraphy.

Mucociliary clearance (MCC) is the key defense mechanism in the upper airways, as the removal of debris-laden mucus in the sinuses completely depends on MCC. So far, how the nasal MCC is regulated remains unknown. Recently, mice deficient in genes encoding the components of MCC apparatus have been generated, which will allow investigators to conduct more in-depth nasal MCC studies. However, the methodology necessary to comprehensively evaluate the nasal MCC in this species is not well established. We therefore developed a novel method to measure nasal MCC in live mice using pinhole gamma camera. Insoluble radiolabeled particles were delivered into the noses of lightly anesthetized mice. The nasal clearance of these particles was measured continuously in a real-time manner. The effect of three different anesthetics-avertin, pentobarbital, and isoflurane-on nasal MCC was also determined. In mice anesthetized by 1.1% isoflurane, radiolabeled particles were immediately moved into the oropharynx, which was significantly accelerated by the treatment of hypertonic but not isotonic saline. According to the clearance rate, the mouse nasal MCC presented two distinct phases: a rapid phase and a slow phase. In addition, we found that isoflurane had a very small inhibitory effect on nasal MCC vs. both avertin and pentobarbital. This was further supported by its dose response. Collectively, we have developed a noninvasive method to monitor the real-time nasal MCC in live mice under physiological conditions. It provides more comprehensive evaluation on nasal MCC rather than assessing a single component of the MCC apparatus in isolation.

Serotonin Decreases Alveolar Epithelial Fluid Transport via a Direct Inhibition of the Epithelial Sodium Channel

Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.

Mouse Eosinophils Possess Potent Antibacterial Properties In Vivo

Eosinophils are best known as the predominant cellular infiltrate associated with asthma and parasitic infections. Recently, numerous studies have documented the presence of Toll-like receptors (TLRs) on the surfaces of eosinophils, suggesting that these leukocytes may participate in the recognition and killing of viruses and bacteria. However, the significance of this role in the innate immune response to bacterial infection is largely unknown. Here we report a novel role for eosinophils as antibacterial defenders in the host response. Isolated mouse eosinophils possessed antipseudomonal properties in vitro. In vivo, interleukin-5 transgenic mice, which have profound eosinophilia, demonstrated improved clearance of Pseudomonas aeruginosa introduced into the peritoneal cavity. The findings of improved bacterial clearance following adoptive transfer of eosinophils, and impaired bacterial clearance in mice with a congenital eosinophil deficiency, established that this effect was eosinophil specific. The data presented also demonstrate that eosinophils mediate this antibacterial effect in part through the release of cationic secondary granule proteins. Specifically, isolated eosinophil granules had antibacterial properties in vitro, and administration of eosinophil granule extracts significantly improved bacterial clearance in vivo. These data suggest a potent yet underappreciated antibacterial role for eosinophils in vivo, specifically for eosinophil granules. Moreover, the data suggest that the administration of eosinophil-derived products may represent a viable adjuvant therapy for septic or bacteremic patients in the intensive care unit.

Identification of conserved antigens for early serodiagnosis of relapsing fever Borrelia

Borrelia hermsii is a blood-borne pathogen transmitted by the argasid tick Ornithodoros hermsi. Since spirochaete clearance in mice is associated with an IgM-mediated response, an immunoproteomic analysis was used to identify proteins reactive with IgM. We report that IgM from both mice and human patients infected with B. hermsii not only reacted with the previously identified variable membrane proteins but also identified candidate antigens including heat-shock proteins, an adhesin protein, ABC transporter proteins, flagellar proteins, housekeeping proteins, an immune evasion protein, and proteins with unknown function. Furthermore, IgM reactivity to recombinant glycerophosphodiester phosphodiesterase was detected during early spirochaete infection and prior to a detectable IgG response. Lastly, a conserved hypothetical protein was produced in Escherichia coli and tested with immune serum against B. hermsii and Borrelia recurrentis. These results identify a much larger set of immunoreactive proteins, and could help in the early serodiagnosis of this tick-borne infection.

Pseudomonas aeruginosa Microevolution during Cystic Fibrosis Lung Infection Establishes Clones with Adapted Virulence

During long-term lung infection in patients with cystic fibrosis (CF), Pseudomonas aeruginosa strains develop mutations leading to clonal expansion. This microevolution is believed to be correlated with a reduced virulence. We tested this hypothesis in models of lung infection, using mice with different genetic backgrounds. From infected airways of six patients with CF, 25 P. aeruginosa clones were isolated during a period of up to 16.3 years and genotypically and phenotypically characterized. Virulence of the 8 early, 6 intermediate, and 11 late CF isolates and 5 environmental strains was assessed by monitoring acute mortality versus survival and P. aeruginosa chronic persistence versus lung clearance in mice of different genetic backgrounds, including CF mice. Different patients harbored clonally unrelated strains, but early, intermediate, and late P. aeruginosa isolates from single patients were clonally related, allowing comparative in vivo analysis. Although late isolates were attenuated in causing acute mortality in the mouse models, compared with early and intermediate clonal isolates and environmental strains, they did not differ from early and intermediate clonal isolates in their capacity to establish chronic infection and cause extensive inflammation in the murine respiratory tract. Our findings indicate that clonal expansion of P. aeruginosa strains during microevolution within CF lungs leads to populations with altered but not reduced virulence. These P. aeruginosa clones with adapted virulence play a critical role in the pathogenesis of chronic infections and may serve to define virulence determinants as targets for novel therapies.