PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

Introduction

Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis.

Methods

PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination.

Results

PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion.

Conclusions

PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease.     

Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4

Background

Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simpliCity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation.

Results

SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe.

Conclusions

Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.     

The challenge for genetic epidemiologists: how to analyze large numbers of SNPs in relation to complex diseases

Background

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.

Results

Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.

Conclusion

The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.     

Successful establishment of primary small airway cell cultures in human lung transplantation

Background

Although both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.

Methods

Pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.

Results

After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.

Conclusion

In this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.     

Potential of (GATA)n microsatellites from rice for inter- and intra-specific variability studies

Background

The microsatellite, (GATA)_n has been frequently used for DNA fingerprinting. However, very few attempts have been made to analyze (GATA)_n-containing loci in rice.

Results

Three polymorphic (GATA)_n-harboring loci viz. OS1A6, OS1H10 and OS2E7, containing 7–13 repeat motifs were identified from a genomic library of a cultivated rice, Oryza sativa var. Basmati-370 using oligonucleotide probe (GATA)₄. When (GATA)_n flanking primers were used to screen 26 wilds (representing different genomes of rice), 16 cultivars, 47 Indian elite rice varieties and 37 lines resistant/susceptible to bacterial blight, up to 22 alleles were obtained at an individual locus. Also, interestingly the bacterial blight resistant lines clustered into a separate group from the remaining rice genotypes, when a dendrogram was constructed based on the polymorphism obtained at the three loci. This may be due to the partial homology of the clones OS1H10 and OS2E7 to regions encoding O. longistaminata receptor kinase-like protein and pathogenesis-related protein. The ability of these O. sativa flanking primers to amplify DNA of maize, wheat, barley and oat indicates that these (GATA)_n-containing loci are conserved across different cereal genera.

Conclusions

The large allele number obtained reveals the potential of (GATA)_n-containing loci as powerful tools to detect simple sequence length polymorphism (SSLP). The (GATA)_n-flanking primers were not only useful in distinguishing between closely related genotypes, but could also be used for cross-species amplification and are also conserved across different cereal genera. These loci could also cluster the bacterial blight resistant/susceptible lines into different groups based on the resistance genes present in them.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

LoxP-FRT Trap (LOFT): a simple and flexible system for conventional and reversible gene targeting

Background

Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.

Results

We describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.

Conclusions

LOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.

     

Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice

Background

Mice lacking the preproenkephalin (ppENK) gene are hyperalgesic and show more anxiety and aggression than wild-type (WT) mice. The marked behavioral changes in ppENK knock-out (KO) mice appeared to occur in supraspinal response to painful stimuli. However the functional role of enkephalins in the supraspinal nociceptive processing and their underlying mechanism is not clear. The aim of present study was to compare supraspinal nociceptive and morphine antinociceptive responses between WT and ppENK KO mice.

Results

The genotypes of bred KO mice were confirmed by PCR. Met-enkephalin immunoreactive neurons were labeled in the caudate-putamen, intermediated part of lateral septum, lateral globus pallidus, intermediated part of lateral septum, hypothalamus, and amygdala of WT mice. Met-enkephalin immunoreactive neurons were not found in the same brain areas in KO mice. Tail withdrawal and von Frey test results did not differ between WT and KO mice. KO mice had shorter latency to start paw licking than WT mice in the hot plate test. The maximal percent effect of morphine treatments (5 mg/kg and 10 mg/kg, i.p.) differed between WT and KO mice in hot plate test. The current source density (CSD) profiles evoked by peripheral noxious stimuli in the primary somatosenstory cortex (S1) and anterior cingulate cortex (ACC) were similar in WT and KO mice. After morphine injection, the amplitude of the laser-evoked sink currents was decreased in S1 while the amplitude of electrical-evoked sink currents was increased in the ACC. These differential morphine effects in S1 and ACC were enhanced in KO mice. Facilitation of synaptic currents in the ACC is mediated by GABA inhibitory interneurons in the local circuitry. Percent increases in opioid receptor binding in S1 and ACC were 5.1% and 5.8%, respectively.

Conclusion

The present results indicate that the endogenous enkephalin system is not involved in acute nociceptive transmission in the spinal cord, S1, and ACC. However, morphine preferentially suppressed supraspinal related nociceptive behavior in KO mice. This effect was reflected in the potentiated differential effects of morphine in the S1 and ACC in KO mice. This potentiation may be due to an up-regulation of opioid receptors. Thus these findings strongly suggest an antagonistic interaction between the endogenous enkephalinergic system and exogenous opioid analgesic actions in the supraspinal brain structures.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Background

Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved.

Results

Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished.

Conclusion

In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.     

Calcium sparks in the intact gerbil spiral modiolar artery

Background

We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity.

Results

Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1^st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice.

Conclusions

These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1^st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge.     

Mesoporous silicon nanotechnology for cancer applications

Abstract

The commonest genetic defects leading to α-thalassemia are genomic deletions encompassing one or both α-globin loci. The aim of this work was to evaluate the interest of melting curves characteristics obtained in gap real-time polymerase chain reaction for the diagnosis of α-thalassemia deletions. Five patients with known deletional α-thalassemia, and five control subjects were tested. The melting temperature and melting curve shapes of the PCR products allowed us to discriminate controls from α-thalassemic patients. This promising strategy should be further tested with samples carrying other α-thalassemic deletions.

Background

The commonest genetic defects leading to α-thalassemia are genomic deletions encompassing one or both α-globin loci. The aim of this work was to evaluate the interest of melting curves characteristics obtained in gap real-time polymerase chain reaction for the diagnosis of α-thalassemia deletions.

Methods

Five patients with known deletional α thalassemia, —(MED)(2), —(SEA)(2), —(FIL)(1), and five control subjects were tested. Specific primers were used for each deletion. Real-time-gap PCRs, using SYBER-Green mix, were performed on a « light-cycler 480 from Roche® ». At the melting temperature, the released of the SYBER-Green dye bonded to double strained DNA is associated with a dramatic decrease in fluorescence intensity. The rate of this variation was determined by plotting the negative derivative of fluorescence vs temperature (lightcycler 480 software, Roche®).

Results

The melting temperature (Tm) and melting curve shapes of the PCR products derived from α-thalassemic patients or control subjects, allowed us to discriminate controls from α-thalassemic patients (eg —Phil Tm = 91°C whereas control Tm = 81 and 85,6°C). Some non specific amplified products are still present, probably because of the high GC content and high homology of sequences in the α-globin cluster.

Conclusion

This promising strategy should be further tested with samples carrying other α-thalassemic deletions. Nevertheless it appears reliable, cost-effective and safe offering additional benefits including minimal labor, rapid turnaround time and decreased risk of PCR carryover contamination. It may be one alternative technology available for routine diagnosis of all types of deletional α-thalassemia.     

Aberrant Sporogonic Development of Dmc1 (a Meiotic Recombinase) Deficient Plasmodium berghei Parasites

Background

In Plasmodium, meiosis occurs in diploid zygotes as they develop into haploid motile ookinetes inside the mosquito. Further sporogonic development involves transformation of ookinetes into oocysts and formation of infective sporozoites.

Methodology/Principal Findings

Reverse genetics was employed to examine the role of the meiotic specific recombinase Dmc1, a bacterial RecA homolog during sporogony in Plasmodium berghei. PbDmc1 knockout (KO) parasites showed normal asexual growth kinetics compared to WT parasites; however oocyst formation in mosquitoes was reduced by 50 to 80%. Moreover, the majority of oocysts were retarded in their growth and were smaller in size compared to WT parasites. Only a few Dmc1 KO parasites completed maturation resulting in formation of fewer sporozoites which were incapable of infecting naive mice or hepatocytes in vitro. PbDmc1 KO parasites were shown to be approximately 18 times more sensitive to Bizelesin, a DNA alkylating drug compared to WT parasites as reflected by impairment of oocyst formation and sporogonic development in the mosquito vector.

Conclusions/Significance

Our findings suggest that PbDmc1 plays a critical role in malaria transmission biology.     

Including endophenotypes as covariates in variance component heritability and linkage analysis

Background

Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous.

Results

A novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus ) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified.

Conclusion

PCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.     

Reduced susceptibility to pyrethroid insecticide treated nets by the malaria vector Anopheles gambiae s.l. in western Uganda

Background

Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.

Methods

Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.

Results

The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.

Conclusion

The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.     

A role for B cells in organic dust induced lung inflammation

Background

Agriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity.

Methods

Using an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected.

Results

ODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c⁺CD11b⁺ exudative macrophages and compensatory increase in CD8⁺ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19⁺CD11b⁺CD5^+/?)-, and memory (CD19⁺CD273^+/-CD73^+/?) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals.

Conclusions

Lung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.

     

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification

Background

Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing.

Results

To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software.

Conclusions

In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.