Drops of Lactiplantibacillus plantarum CRL 759 culture supernatant attenuates eyes inflammation induced by lipopolysaccharide.

Anti-inflammatory effect of soluble secreted compounds of probiotic bacteria was widely demonstrated as therapy for different inflammatory diseases, but was not investigated in inflammatory eye disorders. The aim of this study was to determine whether Lactiplantibacillus plantarum CRL759 cell-free supernatant reduced inflammatory parameters and clinical signs in ocular inflammations. First, we evaluated the effect of L. plantarum CRL759 supernatant in vitro on human retinal cell line, ARPE-19 cells, stimulated with lipopolysaccharide (LPS). Then, we investigated in vivo its capacity to decrease inflammation by local administration on the eyes of mice with endotoxin induced inflammation. In vitro assays demonstrated that L. plantarum CRL759 supernatant reduced the production of interleukin (IL)-6, IL-8, nitric oxide and thiobarbituric acid reactive substances in LPS-stimulated ARPE-19 cells. Our in vivo data proved that L. plantarum supernatant significantly reduced the clinical score of endotoxin treated mice and diminished levels of tumour necrosis factor alpha, interferon gamma and protein concentration in aqueous humour. Histological examination showed reduction of infiltrating inflammatory cells in the posterior segment of the eyes. As far as we know, this is the first report showing that Lactobacillus spp. supernatant administered as drops reduces some parameters of ocular inflammation. This promising strategy is safe and could alleviate symptoms and signs of ocular inflammation in people that are refractories to the conventional therapies.

Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs.

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

Trypanocidal Activity of Smallanthus sonchifolius: Identification of Active Sesquiterpene Lactones by Bioassay-Guided Fractionation.

In order to find novel plant-derived biologically active compounds against Trypanosoma cruzi, we isolated, from the organic extract of Smallanthus sonchifolius, the sesquiterpene lactones enhydrin (1), uvedalin (2), and polymatin B (3) by bioassay-guided fractionation technique. These compounds showed a significant trypanocidal activity against the epimastigote forms of the parasite with IC50 values of 0.84 µ M (1), 1.09 µ M (2), and 4.90 µ M (3). After a 24 h treatment with 10 µ g/mL of enhydrin or uvedalin, parasites were not able to recover their replication rate. Compounds 1 and 2 showed IC50 values of 33.4 µ M and 25.0 µ M against T. cruzi trypomastigotes, while polymatin B was not active. When the three compounds were tested against the intracellular forms of T. cruzi, they were able to inhibit the amastigote replication with IC50 of 5.17 µ M, 3.34 µ M, and 9.02 µ M for 1, 2, and 3, respectively. The cytotoxicity of the compounds was evaluated in Vero cells obtaining CC50 values of 46.5 µ M (1), 46.8 µ M (2), and 147.3 µ M (3) and the selectivity index calculated. According to these results, enhydrin and uvedalin might have potentials as agents against Chagas disease and could serve as lead molecules to develop new drugs.

An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

Elicitation of specific, Th1-biased immune response precludes skeletal muscle damage in cruzipain-vaccinated mice.

Cruzipain (Cz), the major cystein proteinase of Trypanosoma cruzi, is able to induce protective immunity against parasite challenge. However, some concern has arisen regarding its potential to elicit pathogenic autoimmune reactivity. To determine whether the adverse myopathic effects of Cz-based immunization could be prevented, we evaluated the co-administration of Cz with different adjuvants. Mice were immunized with Cz adjuvantized by alum (Cz+alum), oligodeoxynucleotides containing CpG motifs (Cz+ODN-CpG) or Freund's preparation (Cz+CFA). Cz triggered a vigorous specific humoral response, irrespective of the adjuvant used. Alum mainly drove response towards Th2 phenotype, characterized by specific IgG1 antibodies and IL-10 induction, whereas Cz+ODN-CpG mice exhibited Th1-dominant immunity, with antibodies of the IgG2a isotype and enhanced IFN-gamma production. Histological examination of cardiac tissue demonstrated lesions in Cz+CFA but not in Cz+alum nor Cz+ODN-CpG immunized animals, suggesting that CFA is critical for Cz-mediated injury. Analysis of skeletal muscle revealed that mice receiving Cz+CFA exhibited disrupted and hyalinized myofibers, whereas [Cz+alum]-immunized animals showed hyalinization, architecture modifications and small inflammatory foci. Conversely, no abnormalities were observed in the striated muscle from the Cz+ODN-CpG group. Hence, generation of specific immune response skewed towards Th1, as that recorded for the ODN-CpG adjuvant, may preclude triggering of Cz-mediated muscle tissue damage.