An intranasal administration of Lactococcus lactis strains expressing recombinant interleukin-10 modulates acute allergic airway inflammation in a murine model.

BACKGROUND: Around 300 million people world-wide suffer from asthma, and the prevalence of allergic diseases has increased. Much effort has been used in the study of mechanisms involved in the immune response observed in asthma to intervene for the treatment of this condition. During inflammation in asthma, Th2 cytokines and eosinophils are essential components of the host immune system. Furthermore, for therapeutic interventions against this disease, IL-10 is an important cytokine because it has a central role in the regulation of inflammatory cascades. OBJECTIVE: To evaluate the immunomodulatory effect of Lactococcus lactis strains expressing recombinant IL-10 in a mouse model of ovalbumin (OVA)-induced acute airway inflammation. METHODS: L. lactis expressing recombinant IL-10 in a cytoplasmic (LL-CYT) or secreted form (LL-SEC) and wild-type (LL-WT) were used. IL-10 production by the recombinant strains was evaluated by ELISA. After an intranasal administration of L. lactis producing recombinant IL-10 and the induction of acute allergic airway inflammation in mice, blood samples were collected to detect IgE anti-OVA, and bronchoalveolar lavage (BAL) was harvested for eosinophil count. Additionally, the lungs were collected for the detection of the eosinophil peroxidase (EPO) activity, measurement of cytokines and chemokines and evaluation of pathology. RESULTS: Mice that received LL-CYT and LL-SEC strains showed a significant decrease in eosinophils numbers, EPO activity, anti-OVA IgE and IgG1 levels, IL-4 and CCL3 production and pulmonary inflammation and mucus hypersecretion, compared with the asthmatic group. Only the LL-CYT/OVA group showed reduced levels of IL-5, CCL2, CCL5 and CCL11. CONCLUSION: Treatment with L. lactis producing recombinant IL-10 used in this study (LL-CYT and LL-SEC) modulated experimental airway inflammation in the mouse model independently of Treg cells. Additionally, the LL-CYT strain was more efficient in the suppression of lung inflammation.

Effects of sample re-sequencing and trimming on the quality and size of assembled consensus sequences.

The production of nucleic acid sequences by automatic DNA sequencer machines is always associated with some base-calling errors. In order to produce a high-quality DNA sequence from a molecule of interest, researchers normally sequence the same sample many times. Considering base-calling errors as rare events, re-sequencing the same molecule and assembling the reads produced are frequently thought to be a good way to generate reliable sequences. However, a relevant question on this issue is: how many times the sample needs to be re-sequenced to minimize costs and achieve a high-fidelity sequence? We examined how both the number of re-sequenced reads and PHRED trimming parameters affect the accuracy and size of final consensus sequences. Hundreds of single-pool reaction pUC18 reads were generated and assembled into consensus sequences with CAP3 software. Using local alignment against the published pUC18 cloning vector sequence, the position and number of errors in the consensus were identified and stored in MySQL databases. Stringent PHRED trimming parameters proved to be efficient for the reduction of errors; however, this procedure also decreased consensus size. Moreover, re-sequencing did not have a clear effect on the removal of consensus errors, although it was able to slightly increase consensus.

Effects of sample re-sequencing and trimming on the quality and size of assembled consensus sequences

The production of nucleic acid sequences by automatic DNA sequencer machines is always associated with some base-calling errors. In order to produce a high-quality DNA sequence from a molecule of interest, researchers normally sequence the same sample many times. Considering base-calling errors as rare events, re-sequencing the same molecule and assembling the reads produced are frequently thought to be a good way to generate reliable sequences. However, a relevant question on this issue is: how many times the sample needs to be re-sequenced to minimize costs and achieve a high-fidelity sequence? We examined how both the number of re-sequenced reads and PHRED trimming parameters affect the accuracy and size of final consensus sequences. Hundreds of single-pool reaction pUC18 reads were generated and assembled into consensus sequences with CAP3 software. Using local alignment against the published pUC18 cloning vector sequence, the position and number of errors in the consensus were identified and stored in MySQL databases. Stringent PHRED trimming parameters proved to be efficient for the reduction of errors; however, this procedure also decreased consensus size. Moreover, re-sequencing did not have a clear effect on the removal of consensus errors, although it was able to slightly increase consensus.