Inhibition of Receptor Signaling and of Glioblastoma-derived Tumor Growth by a Novel PDGFRβ Aptamer

Platelet-derived growth factor receptor β (PDGFRβ) is a cell-surface tyrosine kinase receptor implicated in several cellular processes including proliferation, migration, and angiogenesis. It represents a compelling therapeutic target in many human tumors, including glioma. A number of tyrosine kinase inhibitors under development as antitumor agents have been found to inhibit PDGFRβ. However, they are not selective as they present multiple tyrosine kinase targets. Here, we report a novel PDGFRβ-specific antagonist represented by a nuclease-resistant RNA-aptamer, named Gint4.T. This aptamer is able to specifically bind to the human PDGFRβ ectodomain (Kd: 9.6 nmol/l) causing a strong inhibition of ligand-dependent receptor activation and of downstream signaling in cell lines and primary cultures of human glioblastoma cells. Moreover, Gint4.T aptamer drastically inhibits cell migration and proliferation, induces differentiation, and blocks tumor growth in vivo. In addition, Gint4.T aptamer prevents PDGFRβ heterodimerization with and resultant transactivation of epidermal growth factor receptor. As a result, the combination of Gint4.T and an epidermal growth factor receptor–targeted aptamer is better at slowing tumor growth than either single aptamer alone. These findings reveal Gint4.T as a PDGFRβ-drug candidate with translational potential.     

A Nonazole CYP51 Inhibitor Cures Chagas’ Disease in a Mouse Model of Acute Infection

Chagas’ disease, the leading cause of heart failure in Latin America, is caused by the kinetoplastid protozoan Trypanosoma cruzi. The sterols of T. cruzi resemble those of fungi, both in composition and in biosynthesis. Azole inhibitors of sterol 14α-demethylase (CYP51) successfully treat fungal infections in humans, and efforts to adapt the success of antifungal azoles posaconazole and ravuconazole as second-use agents for Chagas’ disease are under way. However, to address concerns about the use of azoles for Chagas’ disease, including drug resistance and cost, the rational design of nonazole CYP51 inhibitors can provide promising alternative drug chemotypes. We report the curative effect of the nonazole CYP51 inhibitor LP10 in an acute mouse model of T. cruzi infection. Mice treated with an oral dose of 40 mg LP10/kg of body weight twice a day (BID) for 30 days, initiated 24 h postinfection, showed no signs of acute disease and had histologically normal tissues after 6 months. A very stringent test of cure showed that 4/5 mice had negative PCR results for T. cruzi, and parasites were amplified by hemoculture in only two treated mice. These results compare favorably with those reported for posaconazole. Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis of sterol composition confirmed that treatment with LP10 blocked the 14α-demethylation step and induced breakdown of parasite cell membranes, culminating in severe ultrastructural and morphological alterations and death of the clinically relevant amastigote stage of the parasite.Chagas’ disease, caused by the kinetoplastid protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The disease is transmitted naturally by hematophagous reduviid insects (6), but human infection may also occur via other routes, including blood transfusion, congenital infection, breast-feeding, organ transplant from chagasic donors, laboratory accidents, and ingestion of contaminated foods and beverages. The acute phase of infection usually occurs in children, and 5 to 10% of symptomatic patients may die. Following a subclinical “indeterminate” phase, a chronic phase involving heart failure and gastrointestinal tract lesions often ensues (37, 42). The only clinically available drugs for Chagas’ disease are nifurtimox and benznidazole, both of which have been in use for 4 decades. While they have significant efficacy in the acute phase, both drugs suffer from the twin liabilities of serious side effects and low efficacy in the chronic phase. New drugs with improved efficacy and less toxicity are needed (14, 29).The biosynthesis of membrane sterols is one of the metabolic pathways successfully targeted in the treatment of diseases caused by pathogenic fungi (48). Clinically employed antifungal azoles target sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme that catalyzes oxidative removal of the 14α-methyl group of a sterol precursor to result in Δ^14,15-desaturated intermediates in ergosterol biosynthesis (19, 20). Close similarities to fungi in sterol composition and biosynthesis, plus an absolute requirement for specific 24-methyl sterols for cell viability and proliferation, provide a basis for development of chemotherapy targeting the sterol biosynthetic pathway in T. cruzi. The successful application of antifungal drugs to anti-chagasic therapy exploits these similarities (38). In addition to compounds optimized for antifungal therapy, other CYP51 inhibitors with strong anti-T. cruzi activity have been reported (3, 8, 24, 44, 45).Inhibitors of CYP51 are in the pipeline for preclinical and clinical development for treatment of Chagas’ disease (11). Although earlier commercially available inhibitors, like ketoconazole and itraconazole, were not powerful enough to eradicate T. cruzi from infected animals or human patients (28), the recently approved inhibitor posaconazole (Noxafil; Schering-Plough) is capable of inducing parasitological cure in murine models of both acute and chronic Chagas’ disease (18). Posaconazole cured 50 to 100% of animals in the acute phase and 50 to 60% of chronically infected animals (2). Very recently, posaconazole cured an immunosuppressed patient with concomitant Chagas’ disease and systemic lupus erythematosus (34). However, the use of posaconazole as an anti-chagasic agent may be limited by the requirement for simultaneous intake of a fatty meal or a nutritional supplement to enhance absorption, the drug''s high cost, and the need for clinical monitoring during treatment (31). Another complication is the rapid appearance of laboratory-induced resistance to azoles in T. cruzi, which may predict the occurrence of drug resistance in chagasic patients (4). Although no data on the development of posaconazole resistance in patients with Chagas’ disease are available, studies of fungal infections indicate that posaconazole resistance occurs mainly by a mechanism involving mutation of the cyp51 gene (23, 33, 35). Posaconazole appears to be less susceptible to the efflux pumps that confer resistance to some other azoles (7, 25, 35). Mapping mutations in cyp51 genes in clinical posaconazole-resistant isolates on the CYP51-posaconazole structure (9) points to the mouth of the posaconazole binding tunnel as a mutation hot spot. Mutations of G54, P216, and M220 in clinical isolates of Aspergillus fumigatus (10, 12, 13, 23, 27, 32) (corresponding to G49, P210, and F214, respectively, in T. cruzi CYP51 [CYP51_Tc]) and of A61 (46) and P230 (25) in clinical isolates of Candida albicans (I45 and P210, respectively, in CYP51_Tc) map directly to the tunnel mouth, where amino acids interact with the dangling long substituent tail of posaconazole extending into the tunnel (9). Mutations of G54 in A. fumigatus to arginine or tryptophan associate with moderate and high levels of resistance, respectively, and confer cross-resistance between itraconazole and posaconazole (27). Mutations of M220 confer cross-resistance to all azole drugs tested, including itraconazole, voriconazole, ravuconazole, and posaconazole (30, 39), and therefore may interfere with the entry of the drugs. In accordance with this assumption, posaconazole is reported to induce resistance to all azole drugs in Candida parapsilosis in vitro (35). The alarming perspective emerging from antifungal therapy efforts must be taken into consideration when designing antichagasic drugs targeting CYP51_Tc. While antifungal azoles do show promise, the less than 30% sequence identity between fungal and protozoan CYP51 targets suggests that a more direct approach may be a better route toward developing novel potent therapeutic CYP51 inhibitors.Using clues from our previous work on CYP51 from Mycobacterium tuberculosis (CYP51_Mt), we focused on rationally designed nonazole inhibitors of CYP51_Tc. These inhibitors were based on an experimental hit obtained from screening a small-molecule-compound library against CYP51_Mt (36). Analysis of the X-ray structure revealed that the N-[4-pyridyl]-formamide scaffold group (Fig. ​(Fig.1A,1A, highlighted in gray) binds in the CYP51 active site via conserved residues and the heme prosthetic group. Structural characterization confirmed that these interactions in the complexes were preserved between CYP51_Mt and five different compounds (8, 36), suggesting that this scaffold could be used efficiently instead of the azole or triazole groups to target a variety of chemotypes to the CYP51 active site. Based on the similarity of the chemical structures, the expanded-spectrum compound LP10 (Fig. ​(Fig.1B)1B) was selected for its nanomolar binding affinity to CYP51_Tc and its potent efficacy against T. cruzi in mammalian cells (8). As the pyridyl group of LP10 presumably coordinates to the heme iron, the indole substituent may fill the space occupied by the 2,4-difluorophenyl ring of fluconazole or posaconazole in their structurally characterized complexes with CYP51 (9).Open in a separate windowFIG. 1.Screen hit (A) and the expanded-spectrum compound LP10 (B) containing the N-[4-pyridyl]-formamide scaffold (highlighted in gray), which unvaryingly binds in the CYP51 active site. The chiral center is labeled with an asterisk.In the present work, we evaluated the efficacy of LP10 in an animal model of acute Chagas’ disease. The curative effect of LP10 in vivo was comparable to that of the protease inhibitor K777, an antichagasic drug in preclinical development, used as a positive control (15, 16). Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis demonstrated that treatment with LP10 disrupted cell membranes in T. cruzi amastigotes and altered sterol composition via accumulation of the C-14-methylated precursors lanosterol and 24-methylene-dihydrolanosterol (eburicol). There was concomitant reduction of 14-desmethylated fecosterol and episterol. LP10-induced alterations are consistent with the inhibition of T. cruzi CYP51.     

Transforming Growth Factor β1 Could Influence Thyroid Nodule Elasticity and Also Improve Cervical Lymph Node Metastasis in Papillary Thyroid Carcinoma

Ultrasound elastography has been a very useful tool in predicting the risk of malignant thyroid tumor for several years. The objective of this study was to determine if there is a correlation between strain ratio (SR), collagen deposition and transforming growth factor β1 (TGF-β1) expression in different types of thyroid nodules and if TGF-β1 is related to cervical lymph node metastasis. 102 nodules from 81 patients who underwent thyroid resection surgery in our hospital were retrospectively studied. All of these patients had undergone ultrasound elastography scanning before surgery. Masson staining and immunohistochemical staining were used to evaluate the ratio of expression of collagen deposition and TGF-β1. There was a significant difference between benign and malignant thyroid nodules in SR (8.913 ± 11.021 vs. 1.732 ± 0.727, p = 0.000), collagen content (0.371 ± 0.125 vs. 0.208 ± 0.057, p = 0.000) and TGF-β1 expression (0.336 ± 0.093 vs. 0.178 ± 0.071, p = 0.000). A cutoff of 2.99 for SR measurement was selected for the highest Youden index for predicting malignant thyroid nodules, which yielded 87.88% sensitivity, 100% specificity, 100% positive predictive value, 83.72% negative predictive value and 92.15% accuracy. Expression of collagen and TGF-β1 was positively correlated with SR measurements (coefficient = 0.839 for collagen and 0.855 for TGF-β1, p = 0.000). Among 61 nodules with papillary thyroid carcinoma, the average SR for the metastasis group was higher than that for the non-metastasis group (10.955 ± 13.805 and 7.852 ± 7.931, respectively), but without statistical significance (p = 0.287). Collagen deposition was significantly higher in the metastasis group than in the non-metastasis group (0.421 ± 0.091 vs. 0.353 ± 0.118, p = 0.011). TGF-β1 expression was also significantly higher in the metastasis group than in the non-metastasis group (0.378 ± 0.0.69 vs. 0.328 ± 0.091, p = 0.016). To conclude, TGF-β1 may contribute to thyroid nodule elasticity by promoting collagen deposition. In papillary thyroid carcinoma, overexpression of TGF-β1, as well as collagen deposition, may be a risk factor for cervical lymph node metastasis.     

Pharmacologic Inhibition of Porcupine,Disheveled, and β-Catenin in Wnt Signaling Pathway Ameliorates Diabetic Peripheral Neuropathy in Rats

Wnt signaling pathway has been investigated extensively for its diverse metabolic and pain-modulating mechanisms; recently its involvement has been postulated in the development of neuropathic pain. However, there are no reports as yet on the involvement of Wnt signaling pathway in one of the most debilitating neurovascular complication of diabetes, namely, diabetic peripheral neuropathy (DPN). Thus, in the present study, involvement of Wnt signaling was investigated in DPN using Wnt signaling inhibitors namely LGK974 (porcupine inhibitor), NSC668036 (disheveled inhibitor), and PNU74654 (β-catenin inhibitor). Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg) to male Sprague–Dawley rats. Diabetic rats after 6 weeks of diabetes induction showed increased expression of Wnt signaling proteins in the spinal cord (L4–L6 lumbar segment), dorsal root ganglions and sciatic nerves. Subsequent increase in inflammation, endoplasmic reticulum stress and loss of intraepidermal nerve fiber density was also observed, leading to neurobehavioral and nerve functional deficits in diabetic rats. Intrathecal administration of Wnt signaling inhibitors (each at doses of 10 and 30 µmol/L) in diabetic rats showed improvement in pain-associated behaviors (heat, cold, and mechanical hyperalgesia) and nerve functions (motor, sensory nerve conduction velocities, and nerve blood flow) by decreasing the expression of Wnt pathway proteins, inflammatory marker, matrix metalloproteinase 2, endoplasmic reticulum stress marker, glucose-regulated protein 78, and improving intraepidermal nerve fiber density. All these results signify the neuroprotective potential of Wnt signaling inhibitors in DPN.PerspectiveThis study emphasizes the involvement of Wnt signaling pathway in DPN. Blockade of this pathway using Wnt inhibitors provided neuroprotection in experimental DPN in rats. This study may provide a basis for exploring the therapeutic potential of Wnt inhibitors in DPN patients.     

Systemic Administration of Ribbon-type Decoy Oligodeoxynucleotide Against Nuclear Factor κB and Ets Prevents Abdominal Aortic Aneurysm in Rat Model

Currently, there is no effective clinical treatment to prevent abdominal aortic aneurysm (AAA). To develop a novel therapeutic approach, we modified decoy oligodeoxynucleotide (ODN) against nuclear factor κB (NFκB) and ets, to a ribbon-shaped circular structure without chemical modification, to increase its resistance to endonuclease for systemic administration. Intraperitoneal administration of ribbon-type decoy ODNs (R-ODNs) was performed in an elastase-induced rat AAA model. Fluorescent isothiocyanate (FITC)-labeled R-ODNs could be detected in macrophages migrating into the aneurysm wall, and NFκB and ets activity were simultaneously inhibited by chimeric R-ODN. Treatment with chimeric R-ODN significantly inhibited aortic dilatation, whereas conventional phosphorothioate decoy ODN failed to prevent aneurysm formation. Significant preservation of elastic fibers was observed with chimeric R-ODN, accompanied by a reduction of secretion of several proteases from macrophages. Activation of matrix metalloproteinase (MMP)-9 and MMP-12, but not MMP-2, was suppressed in the aneurysm wall by chimeric R-ODN, whereas recruitment of macrophages was not inhibited. Treatment with chimeric R-ODN also inhibited the secretion of cathepsin B and K from macrophages. Overall, the present study demonstrated that systemic administration of chimeric R-ODNs prevented aneurysm formation in a rat model. Further modification of the decoy strategy would provide a means of less invasive molecular therapy for human AAA.     

Transforming Growth Factor β (TGF-β)-dependent Inhibition of T Helper Cell 2 (Th2)-induced Autoimmunity by Self–Major Histocompatibility Complex (MHC) Class II–specific,Regulatory CD4+ T Cell Lines

Autoreactive anti–MHC class II T cells are found in Brown Norway (BN) and Lewis (LEW) rats that receive either HgCl₂ or gold salts. These T cells have a T helper cell 2 (Th2) phenotype in the former strain and are responsible for Th2-mediated autoimmunity. In contrast, T cells that expand in LEW rats produce IL-2 and prevent experimental autoimmune encephalomyelitis, a cell-mediated autoimmune disease. The aim of this work was to investigate, using T cell lines derived from HgCl₂-injected LEW rats (LEWHg), the effect of these autoreactive T cells on the development of Th2-mediated autoimmunity. The five LEWHg T cell lines obtained protect against Th2-mediated autoimmunity induced by HgCl₂ in (LEW × BN)F1 hybrids. The lines produce, in addition to IL-2, IFN-γ and TGF-β, and the protective effect is TGF-β dependent since protection is abrogated by anti-TGF-β treatment. These results identify regulatory, TGF-β–producing, autoreactive T cells that are distinct from classical Th1 or Th2 and inhibit both Th1- and Th2-mediated autoimmune diseases.Mercuric chloride or gold salts induce in Brown Norway (BN)¹, in (Lewis [LEW] × BN)F1 hybrids, and in susceptible mice, a transient Th2-dependent B cell polyclonal activation (1–4) responsible for an increase in serum IgE concentration and for the production of various autoantibodies including anti-DNA and antilaminin antibodies (for review see reference 1). These latter antibodies are associated with the occurrence of an autoimmune glomerulonephritis as observed in some patients treated with gold salts (5). T cells that recognize either self–MHC class II molecules or an ubiquitous self-peptide presented by MHC class II molecules play an important role in the induction of B cell polyclonal activation in this model (6, 7). In contrast, HgCl₂ provokes in LEW rats, a nonantigen-specific suppression and protects from autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) to which this strain is otherwise highly susceptible. Autoreactive anti-self– MHC class II T cells have also been detected in HgCl₂- injected LEW rats (8). Autoreactive T cell lines have been derived from gold-injected BN rats and HgCl₂-injected LEW rats (LEWHg T cell lines) by repeated stimulations with syngeneic APCs. In both strains, these anti–MHC class II T cell lines are RT1.B- (mouse IA equivalent), but not RT1.D- (mouse IE equivalent) restricted (7, 8). Whereas T cell lines derived from BN rats produce IL-4 and are able to passively transfer autoimmunity into CD8-depleted naive BN rats (7), the LEWHgA T cell line, derived from HgCl₂-injected LEW rats, produces IL-2 and IFN-γ and protects LEW rats against EAE, a Th1-mediated autoimmune disease, by inducing regulatory CD8⁺ T cells (8).The aim of this study was to assess the effect of adoptive transfer of LEWHg T cell lines on the course of Th2mediated autoimmunity induced by HgCl₂ in (LEW × BN)F1 hybrids. We show that (a) the lines produce TGF-β in addition to IL-2 and IFN-γ, (b) adoptive transfer of the lines prevents HgCl₂-induced autoimmunity, and (c) this protection is abrogated after anti-TGF-β mAb administration.     

Heart Rate and Heart Rate Variability Changes Are Not Related to Future Cardiovascular Disease and Death in People With and Without Dysglycemia: A Downfall of Risk Markers? The Whitehall II Cohort Study

OBJECTIVEHigher resting heart rate (rHR) and lower heart rate variability (HRV) are associated with increased risk of cardiovascular disease (CVD) and all-cause mortality in people with and without diabetes. It is unknown whether temporal changes in rHR and HRV may contribute to this risk. We investigated associations between 5-year changes in rHR and HRV and risk of future CVD and death, taking into account participants’ baseline glycemic state.RESEARCH DESIGN AND METHODSIn this prospective, population-based cohort study we investigated 4,611 CVD-free civil servants (mean [SD] age, 60 [5.9] years; 70% men). We measured rHR and/or six indices of HRV. Associations of 5-year change in 5-min rHR and HRV with fatal and nonfatal CVD and all-cause mortality or the composite of the two were assessed, with adjustments made for relevant confounders. Effect modification by glycemic state was tested.RESULTSAt baseline, 63% of participants were normoglycemic, 29% had prediabetes, and 8% had diabetes. During a median (interquartile range) follow-up of 11.9 (11.4; 12.3) years, 298 participants (6.5%) experienced a CVD event and 279 (6.1%) died of non–CVD-related causes. We found no association between 5-year changes in rHR and HRV and future events. Only baseline rHR was associated with all-cause mortality. A 10 bpm–higher baseline HR level was associated with an 11.4% higher rate of all-cause mortality (95% CI 1.0–22.9%; P = 0.032). Glycemic state did not modify associations.CONCLUSIONSChanges in rHR and HRV and possibly also baseline values of these measures are not associated with future CVD or death in people with or without dysglycemia.     

Selective Imaging of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Atherosclerotic Lesions in Diabetic and Non-diabetic ApoE<Superscript>−/−</Superscript> Mice

Purpose

Plaque vulnerability is associated with inflammation and angiogenesis, processes that rely on vascular endothelial growth factor (VEGF) signaling via two receptors, VEGFR-1 and VEGFR-2. We have recently reported that enhanced uptake of scVEGF-PEG-DOTA/Tc-99m (scV/Tc) single photon emission computed tomography (SPECT) tracer that targets both VEGFR-1 and VEGFR-2, identifies accelerated atherosclerosis in diabetic relative to non-diabetic ApoE^?/? mice. Since VEGFR-1 and VEGFR-2 may play different roles in atherosclerotic plaques, we reasoned that selective imaging of each receptor can provide more detailed information on plaque biology.

Procedures

Recently described VEGFR-1 and VEGFR-2 selective mutants of scVEGF, named scVR1 and scVR2, were site-specifically derivatized with Tc-99m chelator DOTA via 3.4 kDa PEG linker, and their selectivity to the cognate receptors was confirmed in vitro. scVR1 and scVR2 conjugates were radiolabeled with Tc-99m to specific activity of 110 ± 11 MBq/nmol, yielding tracers named scVR1/Tc and scVR2/Tc. 34–40 week old diabetic and age-matched non-diabetic ApoE^?/? mice were injected with tracers, 2–3 h later injected with x-ray computed tomography (CT) contrast agent and underwent hybrid SPECT/CT imaging. Tracer uptake, localized to proximal aorta and brachiocephalic vessels, was quantified as %ID from. Tracer uptake was also quantified as %ID/g from gamma counting of harvested plaques. Harvested atherosclerotic arterial tissue was used for immunofluorescent analyses of VEGFR-1 and VEGFR-2 and various lineage-specific markers.

Results

Focal, receptor-mediated uptake in proximal aorta and brachiocephalic vessels was detected for both scVR1/Tc and scVR2/Tc tracers. Uptake of scVR1/Tc and scVR2/Tc was efficiently inhibited only by “cold” proteins of the same receptor selectivity. Tracer uptake in this area, expressed as %ID, was higher in diabetic vs. non- diabetic mice for scVR1/Tc (p = 0.01) but not for scVR2/Tc. Immunofluorescent analysis revealed enhanced VEGFR-1 prevalence in and around plaque area in diabetic mice.

Conclusions

Selective VEGFR-1 and VEGFR-2 imaging of atherosclerotic lesions may be useful to explore plaque biology and identify vulnerability.

     

Effects of Medicated Diet to Eradicate Helicobacter spp. on Growth,Pathology, and Infection Status in Rag1–/– and Nude Mice

The use of a commercial 4-drug diet has been shown to eradicate Helicobacter spp. from immunocompetent mice and those with innate immunodeficiencies. However the efficacy of this diet has not been confirmed in mice with altered adaptive immunity. We hypothesized that an 8-wk treatment with medicated diet would eradicate H. hepaticus and H. typhlonius from young naturally infected nude and Rag1 mice lacking functional T cells (Foxn1^nu) or T and B cells (B6.129S7-Rag1^tm1Mom/J), respectively. We evaluated helicobacter status, body weight, and gross and histologic changes between medicated and control diet in groups of infected and uninfected mice throughout treatment and at 8 wk after treatment completion. Initial infection status was confirmed by fecal PCR at weaning and 3 wk later, with study initiation in 7-wk-old mice. PCR testing demonstrated that independent of strain and sex, all treated mice tested negative for Helicobacter spp. after 4 wk of treatment and remained negative for the duration of the study. Irrespective of infection status, nude and Rag1 mice fed 8 wk of medicated diet gained less weight than did their untreated controls. Both strains normalized body weight while on control diet for the 8 wk after treatment. Mice fed medicated diet developed severe gastroesophageal hyperkeratosis, suggestive of reduced feed consumption, and enlarged ceca. These conditions improved or resolved after the return to control diet. This report is the first to demonstrate the efficacy and physical effects of providing medicated diet for the eradication of Helicobacter spp. from mice with adaptive immune deficiencies.Abbreviation: IBD, inflammatory bowel disease; NU/J, Foxn1^nu1; Rag1^tm1Mom, B6.129S7- Rag1^tm1Mom/JDespite 2 decades of reports documenting Helicobacter-associated gastrointestinal disease in mice, infections continue to persist widely not only at academic institutions in the United States but also at commercial vendors in other regions of the world.^1,3,31,44 Within the academic setting, health monitoring and exclusion policies vary markedly between universities and even within different animal facilities at a single institution. Often these policies are based on financial factors (the costs of screening by PCR and the resources required to rederive infected animals) in addition to the potential for Helicobacter infections to confound research.Attempting to predict the overall effect of infection on research can also be problematic. The development and severity of gastrointestinal pathology can vary considerably by mouse strain, species of Helicobacter, and disease model. Some strains of mice, including A/JCr, BALB/cAnNCr, C3H/HeNCr, and SJL/NCr, are particularly susceptible and develop chronic enterohepatic disease of considerable severity.^{19,21,30,49,50} In addition, several immunodeficient strains of mice develop severe disease after chronic infection. C.B-17/Icr-Prkdc^scid (SCID/NCr) mice, which lack functional T and B cells, develop progressive hepatitis and proliferative typhlocolitis after natural infection with H. hepaticus.³⁰ IL10^−/− mice such as B6.129P2-IL-10^tm1Cgn/J develop severe typhlocolitis after infection with several Helicobacter spp.^51,52 In contrast to immunocompetent strains, immunodeficient mice may manifest clinical signs such as diarrhea, perianal bleeding and rectal prolapse of variable severity.^18,29,51The species of Helicobacter affects the severity of disease. Although H. hepaticus remains the most well-studied enterohepatic mouse species, other closely related Helicobacter spp. also result in gastrointestinal disease.^15,31 Natural and experimental monoinfection with H. typhlonius led to typhlocolitis in C57BL/6J IL-10^−/− and SCID/NCr mice.^18,22,23 H. mastomyrinus infection led to granulomatous typhlocolitis (inflammatory bowel disease, IBD) in telomerase-deficient C57BL/6J mice during crucial early-senescence studies.¹⁵ Interestingly, gastrointestinal disease was significantly more severe in mice infected with H. mastomyrinus than in those infected with H. hepaticus.¹⁵In addition, the research impact of Helicobacter infections varies with disease model in a complex dynamic resulting from interactions between host gastrointestinal immunity, microflora, diet, and environmental conditions. Mouse models of IBD highlight these complexities. Helicobacter spp. infection rather than genetic modification was found to be responsible for the susceptibility and pattern of IBD development in T cell receptor αβ mutant mice.^6,10 Intentional inoculation has been used to study Helicobacter spp.- associated alterations in resident intestinal microflora and induction and severity of IBD in immunodeficient mice.⁵¹Given the difficulties of predicting the research impact of Helicobacter spp. infection in mice, perhaps tolerance for enzootic infections should be reconsidered. Benefits to eradication include not only elimination of the agent as an experimental confounder but also as a means to improve welfare through reduced clinical disease.¹ Complete exclusion of infected animals may serve as the least labor intensive and most cost effective strategy. A 10-y institution-wide exclusion policy that required all imported mice to be either rederived by embryo transfer or purchased from an approved Helicobacter-free vendor resulted in either complete elimination or significant reduction in 4 facilities tested in 1999 and again in 2009.³¹ Although effective, this method has the potential to interfere with the ability of individual investigators to receive mice from collaborating institutions that maintain facilities of unknown or positive infection status. In addition, this strategy would be ineffective for inhouse breeding colonies of genetically modified mice. In such cases, rederivation by in vitro fertilization, embryo transfer, and postpartum cross-fostering have all been proven successful. Although often effective, embryo transfer can be impeded by factors such as insufficient response to superovulation, unavailability of stud males, inadequate yield of fertilized eggs, unsuccessful embryo transfer, and the need for genotyping—all of which delay the initiation of research.⁴⁷ Furthermore, the detection of H. typhlonius from sex organs of both female and male Hsd:Athymic Nude-Foxn1^nu mice has demonstrated the potential for transmission from vasectomized male and recipient female mice.³⁸Rederivation by cross fostering has been used to eliminate enzootic Helicobacter spp. infections.^1,46 This technique is less costly and labor intensive and requires less expertise than embryo transfer.^1,42,46 One study comparing 2 cross fostering paradigms to eliminate Helicobacter spp., murine norovirus, mouse hepatitis virus and Syphacia obvelata found success was dependent on both pup age at the time of transfer and bedding changing patterns.¹ Pups transferred within 24 h of birth from cages that underwent bedding changes every 24 h tested negative more frequently than did pups transferred within 48 h of birth from cages containing up to 7-d-old dirty bedding.¹ However neither paradigm was completely successful in Helicobacter elimination, as evidenced by follow-up PCR testing of cross-fostered offspring.¹ Furthermore, isolation of H. hepaticus from the viscera of several late-stage C.B-17/Icr-Prkdc^scid (SCID/NCr) embryos belonging to an infected dam suggests that transplacental transmission is possible in immunodeficient mice.³⁰The administration of antibiotics to eliminate Helicobacter spp. in rodents has evolved considerably since first used to prevent chronic active hepatitis and colitis in young male SCID/NCr mice naturally infected with H. hepaticus.³⁶ In that initial report, various combinations of amoxicillin, bismuth, metronidazole, neomycin, and tetracycline were either added to the drinking water or administered orally 3 times daily for 14 d.³⁶ Although clearly labor-intensive, the cited study³⁶ determined that H. hepaticus could be eliminated by using all treatment regimens that included amoxicillin. A similar dosing regimen using either amoxicillin or tetracycline-based triple therapy was used to eliminate H. hepaticus from infected A/JCr mice.¹⁶ The first medicated diet formulation intended to eliminate infection in mice was a triple therapy of amoxicillin, metronidazole, and bismuth.¹⁷ When fed continuously for 2 wk to 6- to 10-mo-old DBA/J mice, the diet successfully eliminated chronic H. hepaticus infection as confirmed by posttreatment culture and PCR testing 1 mo later.¹⁷ Despite this success, the same amoxicillin-based triple combination diet fed for an unspecified duration failed to eliminate H. hepaticus from an immunodeficient breeding colony of Rag1 ^−/− mice.^16,51 When administered to a SCID (Prkc^scid/Tpr53^tm1tyi on a B6.129/Sv × C.B17 background) breeding colony severely affected by diarrhea associated with H. rodentium and H. bilis coinfection, the triple therapy reduced clinical illness during treatment period but did not eliminate infection or persistent diarrhea.⁴¹Although a newer medicated diet containing amoxicillin, clarithromycin, metronidazole, and omeprazole has been available for a decade, reports of efficacy are still quite limited. One study demonstrated successful eradication of H. hepaticus and H. bilis from a colony of 129 × 1/SvJ desmin-null and heterozygotic mice after 8 wk of continual treatment and 19 mo of PCR follow-up testing.²⁸ In addition, the 4-drug therapy was successful in eliminating Helicobacter spp. from several genetically modified rat strains whereby infected male rats were medicated for 3-two week cycles and pregnant rat dams and offspring were fed continuously from day 7 of gestation through weaning.²⁶ Posttreatment follow-up testing for 8 mo by fecal PCR confirmed that all treated rats remained negative.²⁶ More recently, we have reported the successful eradication of H. hepaticus, H. bilis, and H. rodentium from 2 strains of mice with innate immune deficiencies.⁹Questions remain, however, regarding the broad applicability of medicated diet for the elimination of Helicobacter spp., particularly with regard to strains with modifications of the immune system. The 4-drug medicated diet was unable to eradicate Helicobacter spp. from in B6.129P2-IL10^tm1Cgn/J mice,⁴⁰ and there is no information regarding efficacy in mice with deficiencies in adaptive immunity. Given the widespread use of these mice in gastrointestinal cancer and IBD research, evaluation of medicated diet to eradicate Helicobacter spp. from such strains is warranted. Although many immunodeficient inbred strains are available Helicobacter-free from commercial vendors, these strains often are genetically modified further, maintained, and imported from collaborating institutions with endemic Helicobacter spp. infections that need to be eliminated in a timely manner. We therefore conducted a prospective controlled study to evaluate the potential of the 4-drug medicated diet to eradicate H. hepaticus and H. typhlonius from young, naturally infected nude and Rag1 mice lacking functional T cells (Foxn1^nu) or T and B cells (B6.129S7-Rag1^tm1Mom/J), respectively. We also evaluated the physical effect of both Helicobacter infection and medicated diet in growing mice by weekly recordings of body weight and assessment of gross and histologic changes between medicated and control diet in groups of infected and uninfected mice after an 8-wk treatment course and again 8 wk after treatment completion.     

Therapeutic Effects of Focused Ultrasound on Expression of Notch1, C-Fos and Transforming Growth Factor-β3 in Vulvar Skin of SD Rats with Vulvar Lichen Simplex Chronicus

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-β3 (TGF-β3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-β3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-β3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC.     

Acid-Induced Acute Lung Injury in Mice is Associated With p44/42 and c-Jun N-Terminal Kinase Activation and Requires the Function of Tumor Necrosis Factor α Receptor I

ABSTRACT: Aspiration of hydrochloric acid (HCl)-containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor α (TNF-α) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-α receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-α receptor I, but not by deletion of the TNF-α gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-α receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-α receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-α receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.