Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent

Background

Since the discovery of the human CFTR gene in 1989 various mouse models for cystic fibrosis (CF) have been generated and used as a very suitable and popular tool to approach research on this life-threatening disease. Age related changes regarding the course of disease and susceptibility towards pulmonary infections have been discussed in numerous studies.

Methods

Here, we investigated Cftr^{TgH(neoim)Hgu} and Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J CF mice and their non-CF littermates during an acute lung infection with Pseudomonas aeruginosa for age dependent effects of their lung function and immune response.Mice younger than three or older than six months were intratracheally infected with P. aeruginosa TBCF10839. The infection was monitored by lung function of the animals using non-invasive head-out spirometry and the time course of physiological parameters over 192 hours. Quantitative bacteriology and lung histopathology of a subgroup of animals were used as endpoint parameters.

Results

Age-dependent changes in lung function and characteristic features for CF like a shallower, faster breathing pattern were observed in both CF mouse models in uninfected state. In contrast infected CF mice did not significantly differ from their non-CF littermates in susceptibility and severity of lung infection in both mouse models and age groups. The transgenic Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J and their non-CF littermates showed a milder course of infection than the Cftr^{TgH(neoim)Hgu} CF and their congenic C57Bl/6J non-CF mice suggesting that the genetic background was more important for outcome than Cftr dysfunction.

Conclusions

Previous investigations of the same mouse lines have shown a higher airway susceptibility of older CF mice to intranasally applied P. aeruginosa. The different outcome of intranasal and intratracheal instillation of bacteria implies that infected CF epithelium is impaired during the initial colonization of upper airways, but not in the subsequent response of host defense.     

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

Background

Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr^tm1UNC, Cftr knockout).

Methodology

Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer.

Results

In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice.

Conclusions

The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.     

Influence of language and ancestry on genetic structure of contiguous populations: A microsatellite based study on populations of Orissa

Background

Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftr ^tm1Unc ) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified.

Results

CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14.

Conclusion

Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype.     

Cholic Acid Induces a Cftr Dependent Biliary Secretion and Liver Growth Response in Mice

The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr^-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous) or chronic (three weeks via the diet). In Cftr^-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr^-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr^-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr^-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr^-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr^-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr^-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.     

Characterisation of chloride currents across the proximal colon in CftrTgH(neoim)1Hgu congenic mice

It was the aim of the present study to investigate chloride secretion across the proximal colon of Cftr ^{TgH(neoim)1Hgu} congenic mice. Stripped epithelia were incubated in Ussing chambers and the electrophysiological data were compared between cystic fibrosis (CF) animals and wild type (WT) animals. In comparison with the control animals, all Cftr ^{TgH(neoim)1Hgu} congenic mice had a distinctly reduced basal chloride secretion and a reduced chloride secretion after stimulation with carbachol and forskolin. When comparing chloride secretion across the proximal colon between WT animals, all mice showed a comparable pattern of response to carbachol and forskolin but quantitative differences, BALB/c exhibiting the highest and HsdOla:MF1 exhibiting the lowest increase in Cl current. Likewise, all CF animals showed the same reaction pattern to carbachol and forskolin, but there was no distinct difference that lasted for the whole measurement. To investigate interferences between Ca- and cyclic adenosine monophosphate-activated pathways of Cl secretion in CF animals, we studied epithelia from CF/3CF/1F1 animals with a mixed background. In these animals, the levels of the carbachol or forskolin-induced chloride currents did not depend on the prestimulation with the respective other secretagogue. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, which blocks calcium-activated chloride channels, reduced the current response to carbachol by about 23%. This result, obtained in BALB/c-Cftr ^{TgH(neoim)1Hgu} mice, indicates that alternative chloride channels might be present in the proximal colon of these mice. In contrast, there was no evidence for alternative chloride conductances in BALB/c WT animals, but we cannot exclude that in WT mice a higher chloride secretion via Cftr-channels may have masked an alternative chloride secretion.     

Cancer-Testis Antigen Expression in Serous Endometrial Cancer with Loss of X Chromosome Inactivation

Background

Cancer-testis antigens (CTAs) are potential targets for cancer immunotherapy. Many CTAs are located on the X chromosome and are epigenetically regulated. Loss of X chromosome inactivation (XCI) is observed in breast and ovarian cancers and is thought to be related to the overexpression of CTAs. We investigated the relation between expression of CTAs and loss of XCI in endometrial cancer.

Materials and Methods

We used data generated by The Cancer Genome Atlas Genome Data Analysis Centers and data for Xist knockout mice available at the Gene Expression Omnibus.

Results

The status of XCI was estimated by methylation status, and deletion or gain of the X chromosome. The endometrial cancers were classified into the following three groups: preserved inactivated X chromosome (Xi) (n = 281), partial reactivation of Xi (n = 52), and two copies of active X group (n = 38). Loss of XCI was more common in serous adenocarcinoma. Expression of CTAs increased in endometrial cancer with loss of XCI, which was accompanied by global hypomethylation. Expression of CTAs did not increase in Xist knockout mice.

Conclusions

Loss of XCI is common in serous adenocarcinoma. Global hypomethylation, and not loss of XCI, is the main mechanism of overexpression of CTAs.     

Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr ^−/− lung when compared to wild-type (Cftr ^+/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr ^−/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr ^−/− mice compared to Cftr ^+/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr ^+/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr ^−/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr ^−/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.     

Glucocorticoid receptor gene polymorphisms associated with progression of lung disease in young patients with cystic fibrosis

Background

The variability in the inflammatory burden of the lung in cystic fibrosis (CF) patients together with the variable effect of glucocorticoid treatment led us to hypothesize that glucocorticoid receptor (GR) gene polymorphisms may affect glucocorticoid sensitivity in CF and, consequently, may contribute to variations in the inflammatory response.

Methods

We evaluated the association between four GR gene polymorphisms, TthIII, ER22/23EK, N363S and BclI, and disease progression in a cohort of 255 young patients with CF. Genotypes were tested for association with changes in lung function tests, infection with Pseudomonas aeruginosa and nutritional status by multivariable analysis.

Results

A significant non-corrected for multiple tests association was found between BclI genotypes and decline in lung function measured as the forced expiratory volume in one second (FEV₁) and the forced vital capacity (FVC). Deterioration in FEV₁ and FVC was more pronounced in patients with the BclI GG genotype compared to the group of patients with BclI CG and CC genotypes (p = 0.02 and p = 0.04 respectively for the entire cohort and p = 0.01 and p = 0.02 respectively for F508del homozygous patients).

Conclusion

The BclI polymorphism may modulate the inflammatory burden in the CF lung and in this way influence progression of lung function.     

Submucosal gland distribution in the mouse has a genetic determination localized on Chromosome 9

Submucosal glands (SMG) are important secretory glands that are present in the major airways and bronchioles of humans. In mice the structure, cellular composition, and density of SMG are similar to those seen in humans, but the glands are present only in the trachea. Characterization of SMG is important as they secrete bacteriocidal products such as lactoferrin, lysozyme, and defensins believed to be of importance in the innate defense system. Serous cells in SMG are the primary site of cystic fibrosis transmembrane conductance regulator (CFTR) gene expression and the initial site of histological abnormality in cystic fibrosis (CF) individuals. In this study, we examined four inbred strains of mice (A/J, C57BL/6N, FVB/N, and BALB/CAnN) and revealed that the extent to which glands descend in the mouse trachea varied between inbred strains. In particular, the A/J and C57BL/6N strains exhibited few SMG extending further than the first or second intercartilaginous space (mean depth of 0.4 ± 0.11 and 1.5 ± 0.32 tracheal rings respectively) in the trachea, whereas the FVB/N and BALB/CAnN strains had SMG extending beyond the fourth space (mean depths of 3.3 ± 0.46 and 5.6 ± 0.45 rings respectively). We have previously shown that in congenic C57Bl/6N Cftr mutant mice (CF mice), the SMG are distributed more distally than in wild-type C57Bl/6N but are indistinguishable from BALB/CAnN wild-type or CF mice. The implication that SMG distribution is influenced by Cftr gene expression (or a gene closely linked to Cftr) led us to investigate the genetic difference between C57Bl6/N and BALB/CAnN mice. In recombinant inbred strain (RIS) analysis (with BALB/CJ and C57BL/6J progenitors), two loci were identified as being linked to the SMG phenotype (peak likelihood statistic levels of 8.8 and 9.9 on Chrs 9 and 10 respectively, indicating suggestive linkage). A subsequent segregation analysis of an F₂ intercross between the C57BL/6N and BALB/CAnN mice indicated that there were at least two major genetic factors responsible for SMG distribution. The loci indicated in the RI analysis were included in a targeted genome scan involving 235 F₂ intercross animals (C57BL/6N and BALB/CAnN strain intercross). The genome scan confirmed the locus on Chr 9 (between genetic markers D9Mit11 and D9Mit182), designated Smgd1, as significantly linked to the SMG distribution phenotype (peak LOD score 5.8) within a 95% confidence interval of 12 cM. Received: 26 June 2000 / Accepted: 18 September 2000     

Effects of Green Tea Compound Epigallocatechin-3-Gallate against Stenotrophomonas maltophilia Infection and Biofilm

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.     

Variation in MSRA modifies risk of neonatal intestinal obstruction in cystic fibrosis

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10⁻⁵ to 1.08×10⁻⁶; Bonferroni P = 0.057 to 3.1×10⁻³). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10⁻⁴). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr ^−/− and Cftr ^−/− Msra ^−/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function.     

A polymorphic repetitive-sequence PR1 family evidence for meiotic instability

When EcoRI digests of mouse genomic DNA were subjected to Southern blot analysis with the polymorphic repetitive sequence PR1 as a probe, one satellite-like band of 3.5 × 10³ base-pairs, designated as PR1 family B, was detected in BALB/c-strain mice, but not in the DDD/1- or MOA-strain mice. Analysis of recombinant phage clones revealed that the repeating unit of the PR1 family B was 13.5 × 10³ base-pairs long. This family consisted of a tandem array of repeating units and occupied as much as 2% of one BALB/c chromosome. Since the BALB/c-specific PR1 family B is not present in DDD/1 or MOA mice, the unpaired portion of the BALB/c chromosome may be looped out in a synaptonemal complex during meiosis in F₁ hybrids of the BALB/c strain with DDD/1 or MOA. To determine the fate of this extra DNA, we examined the genotypes of the F₁ hybrid mice and of the segregating populations. Although the PR1 patterns of F₁ and most N₂ mice are consistent with typical Mendelian inheritance, some N₂ progeny showed an abnormal 3.5 × 10³ base-pair band of unexpectedly reduced intensity. This indicated that the extra DNA of PR1 family B occasionally underwent recombination during meiosis in F₁ mice, resulting in its apparent excision. Examination of PstI digests supported this interpretation.     

CFTR deletion affects mouse osteoblasts in a gender-specific manner

Advancements in research and care have contributed to increase life expectancy of individuals with cystic fibrosis (CF). With increasing age comes a greater likelihood of developing CF bone disease, a comorbidity characterized by a low bone mass and impaired bone quality, which displays gender differences in severity. However, pathophysiological mechanisms underlying this gender difference have never been thoroughly investigated. We used bone marrow-derived osteoblasts and osteoclasts from Cftr^+/+ and Cftr^−/− mice to examine whether the impact of CF transmembrane conductance regulator (CFTR) deletion on cellular differentiation and functions differed between genders. To determine whether in vitro findings translated into in vivo observations, we used imaging techniques and three-point bending testing. In vitro studies revealed no osteoclast-autonomous defect but impairment of osteoblast differentiation and functions and aberrant responses to various stimuli in cells isolated from Cftr^−/− females only. Compared with wild-type controls, knockout mice exhibited a trabecular osteopenic phenotype that was more pronounced in Cftr^−/− males than Cftr^−/− females. Bone strength was reduced to a similar extent in knockout mice of both genders. In conclusion, we find a trabecular bone phenotype in Cftr^−/− mice that was slightly more pronounced in males than females, which is reminiscent of the situation found in patients. However, at the osteoblast level, the pathophysiological mechanisms underlying this phenotype differ between males and females, which may underlie gender differences in the way bone marrow–derived osteoblasts behave in absence of CFTR.     

Dysregulation of X Chromosome Inactivation in High Grade Ovarian Serous Adenocarcinoma

Background

One of the two copies of the X chromosome is randomly inactivated in females as a means of dosage compensation. Loss of X chromosome inactivation (XCI) is observed in breast and ovarian cancers, and is frequent in basal-like subtype and BRCA1 mutation-associated breast cancers. We investigated the clinical implications of the loss of XCI in ovarian cancer and the association between the loss of XCI and BRCA1 dysfunction.

Materials and Methods

We used open source data generated by The Cancer Genome Atlas (TCGA) Genome Data Analysis Centers. Ward’s hierarchical clustering method was used to classify the methylation status of the X chromosome.

Results

We grouped 584 high grade serous ovarian adenocarcinomas (HG-SOA) according to methylation status, loss of heterozygosity and deletion or gain of X chromosome into the following five groups: preserved inactivated X chromosome (Xi) group (n = 175), partial reactivation of Xi group (n = 100), p arm deletion of Xi group (n = 35), q arm deletion of Xi group (n = 44), and two copies of active X group (n = 230). We found four genes (XAGE3, ZNF711, MAGEA4, and ZDHHC15) that were up-regulated by loss of XCI. HG-SOA with loss of XCI showed aggressive behavior (overall survival of partial reactivation of Xi group: HR 1.7, 95% CI 1.1–2.5, two copies of active X group: HR 1.4, 95% CI 1.0–1.9). Mutation and hypermethylation of BRCA1 were not frequent in HG-SOA with loss of XCI.

Conclusions

Loss of XCI is common in HG-SOA and is associated with poor clinical outcome. The role of BRCA1 in loss of XCI might be limited. XCI induced aberrant expression of cancer-testis antigens, which may have a role in tumor aggressiveness.     

Hyperresponsiveness to inhaled but not intravenous methacholine during acute respiratory syncytial virus infection in mice

Background

To characterise the acute physiological and inflammatory changes induced by low-dose RSV infection in mice.

Methods

BALB/c mice were infected as adults (8 wk) or weanlings (3 wk) with 1 × 10⁵ pfu of RSV A2 or vehicle (intranasal, 30 μl). Inflammation, cytokines and inflammatory markers in bronchoalveolar lavage fluid (BALF) and airway and tissue responses to inhaled methacholine (MCh; 0.001 – 30 mg/ml) were measured 5, 7, 10 and 21 days post infection. Responsiveness to iv MCh (6 – 96 μg/min/kg) in vivo and to electrical field stimulation (EFS) and MCh in vitro were measured at 7 d. Epithelial permeability was measured by Evans Blue dye leakage into BALF at 7 d. Respiratory mechanics were measured using low frequency forced oscillation in tracheostomised and ventilated (450 bpm, flexiVent) mice. Low frequency impedance spectra were calculated (0.5 – 20 Hz) and a model, consisting of an airway compartment [airway resistance (Raw) and inertance (Iaw)] and a constant-phase tissue compartment [coefficients of tissue damping (G) and elastance (H)] was fitted to the data.

Results

Inflammation in adult mouse BALF peaked at 7 d (RSV 15.6 (4.7 SE) vs. control 3.7 (0.7) × 10⁴ cells/ml; p < 0.001), resolving by 21 d, with no increase in weanlings at any timepoint. RSV-infected mice were hyperresponsive to aerosolised MCh at 5 and 7 d (PC₂₀₀ Raw adults: RSV 0.02 (0.005) vs. control 1.1 (0.41) mg/ml; p = 0.003) (PC₂₀₀ Raw weanlings: RSV 0.19 (0.12) vs. control 10.2 (6.0) mg/ml MCh; p = 0.001). Increased responsiveness to aerosolised MCh was matched by elevated levels of cysLT at 5 d and elevated VEGF and PGE₂ at 7 d in BALF from both adult and weanling mice. Responsiveness was not increased in response to iv MCh in vivo or EFS or MCh challenge in vitro. Increased epithelial permeability was not detected at 7 d.

Conclusion

Infection with 1 × 10⁵ pfu RSV induced extreme hyperresponsiveness to aerosolised MCh during the acute phase of infection in adult and weanling mice. The route-specificity of hyperresponsiveness suggests that epithelial mechanisms were important in determining the physiological effects. Inflammatory changes were dissociated from physiological changes, particularly in weanling mice.     

Genetic control of resistance to Trypanosoma brucei brucei infection in mice

Background

Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.

Methods

We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC) strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem) series contains a different random subset of 12.5% genes from the parental “donor” strain STS/A and 87.5% genes from the “background” strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F₂ hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F₂ hybrid mice and their linkage with survival was tested by analysis of variance.

Results

We mapped four Tbbr (Trypanosoma brucei brucei response) loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have effects on survival independent of inter-genic interactions (main effects). Tbbr3 (chromosome 7) influences survival in interaction with Tbbr4 (chromosome 19). Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.

Conclusion

This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F₂ hybrids of inbred strains usually has a precision of 40–80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.     

Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis

Background

The mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in CF. The most common mutation, ΔF508-CFTR, is a temperature-sensitive, trafficking mutant with reduced chloride transport and exaggerated immune response. The ΔF508-CFTR is misfolded, ubiquitinated, and prematurely degraded by proteasome mediated- degradation. We recently demonstrated that selective inhibition of proteasomal pathway by the FDA approved drug PS-341 (pyrazylcarbonyl-Phe-Leuboronate, a.k.a. Velcade or bortezomib) ameliorates the inflammatory pathophysiology of CF cells. This proteasomal drug is an extremely potent, stable, reversible and selective inhibitor of chymotryptic threonine protease-activity. The apprehension in considering the proteasome as a therapeutic target is that proteasome inhibitors may affect proteostasis and consecutive processes. The affect on multiple processes can be mitigated by nanoparticle mediated PS-341 lung-delivery resulting in favorable outcome observed in this study.

Results

To overcome this challenge, we developed a nano-based approach that uses drug loaded biodegradable nanoparticle (PLGA-PEG^PS-341) to provide controlled and sustained drug delivery. The in vitro release kinetics of drug from nanoparticle was quantified by proteasomal activity assay from days 1-7 that showed slow drug release from day 2-7 with maximum inhibition at day 7. For in vivo release kinetics and biodistribution, these drug-loaded nanoparticles were fluorescently labeled, and administered to C57BL6 mice by intranasal route. Whole-body optical imaging of the treated live animals demonstrates efficient delivery of particles to murine lungs, 24 hrs post treatment, followed by biodegradation and release over time, day 1-11. The efficacy of drug release in CF mice (Cftr ^-/- ) lungs was determined by quantifying the changes in proteasomal activity (~2 fold decrease) and ability to rescue the Pseudomonas aeruginosa LPS (Pa -LPS) induced inflammation, which demonstrates the rescue of CF lung disease in murine model.

Conclusion

We have developed a novel drug delivery system to provide sustained delivery of CF "correctors" and "anti-inflammatories" to the lungs. Moreover, we demonstrate here the therapeutic efficacy of nano-based proteostasis-modulator to rescue Pa-LPS induced CF lung disease.     

Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease

Background

The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine.

Results

Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126–128 cM on SSC10. Using the IMpRH_7000rad and IMNpRH2_12000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03) and another possibly associated with age at puberty (p = 0.09).

Conclusion

AKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits. The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064–DQ474068, GenBank:DQ494488–DQ494490 and GenBank:DQ487182–DQ487184].     

Gene-specific sex effects on eosinophil infiltration in leishmaniasis

Background

Sex influences susceptibility to many infectious diseases, including some manifestations of leishmaniasis. The disease is caused by parasites that enter to the skin and can spread to the lymph nodes, spleen, liver, bone marrow, and sometimes lungs. Parasites induce host defenses including cell infiltration, leading to protective or ineffective inflammation. These responses are often influenced by host genotype and sex. We analyzed the role of sex in the impact of specific gene loci on eosinophil infiltration and its functional relevance.

Methods

We studied the genetic control of infiltration of eosinophils into the inguinal lymph nodes after 8 weeks of Leishmania major infection using mouse strains BALB/c, STS, and recombinant congenic strains CcS-1,-3,-4,-5,-7,-9,-11,-12,-15,-16,-18, and -20, each of which contains a different random set of 12.5% genes from the parental “donor” strain STS and 87.5% genes from the “background” strain BALB/c. Numbers of eosinophils were counted in hematoxylin-eosin-stained sections of the inguinal lymph nodes under a light microscope. Parasite load was determined using PCR-ELISA.

Results

The lymph nodes of resistant STS and susceptible BALB/c mice contained very low and intermediate numbers of eosinophils, respectively. Unexpectedly, eosinophil infiltration in strain CcS-9 exceeded that in BALB/c and STS and was higher in males than in females. We searched for genes controlling high eosinophil infiltration in CcS-9 mice by linkage analysis in F₂ hybrids between BALB/c and CcS-9 and detected four loci controlling eosinophil numbers. Lmr14 (chromosome 2) and Lmr25 (chromosome 5) operate independently from other genes (main effects). Lmr14 functions only in males, the effect of Lmr25 is sex independent. Lmr15 (chromosome 11) and Lmr26 (chromosome 9) operate in cooperation (non-additive interaction) with each other. This interaction was significant in males only, but sex-marker interaction was not significant. Eosinophil infiltration was positively correlated with parasite load in lymph nodes of F₂ hybrids in males, but not in females.

Conclusions

We demonstrated a strong influence of sex on numbers of eosinophils in the lymph nodes after L. major infection and present the first identification of sex-dependent autosomal loci controlling eosinophilic infiltration. The positive correlation between eosinophil infiltration and parasite load in males suggests that this sex-dependent eosinophilic infiltration reflects ineffective inflammation.