Ibrutinib treatment inhibits breast cancer progression and metastasis by inducing conversion of myeloid-derived suppressor cells to dendritic cells.

BACKGROUND: Ibrutinib is a Bruton's tyrosine kinase (BTK) and interleukin-2-inducible kinase (ITK) inhibitor used for treating chronic lymphocytic leukaemia (CLL) and other cancers. Although ibrutinib is known to inhibit the growth of breast cancer cell growth in vitro, its impact on the treatment and metastasis of breast cancer is unclear. METHODS: Using an orthotopic mouse breast cancer model, we show that ibrutinib inhibits the progression and metastasis of breast cancer. RESULTS: Ibrutinib inhibited proliferation of cancer cells in vitro, and Ibrutinib-treated mice displayed significantly lower tumour burdens and metastasis compared to controls. Furthermore, the spleens and tumours from Ibrutinib-treated mice contained more mature DCs and lower numbers of myeloid-derived suppressor cells (MDSCs), which promote disease progression and are linked to poor prognosis. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to the induction of antitumour TH1 and CTL immune responses. CONCLUSIONS: Ibrutinib inhibits tumour development and metastasis in breast cancer by promoting the development of mature DCs from MDSCs and hence could be a novel therapeutic agent for the treatment of breast cancer.

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Use of VHH antibodies for the development of antigen detection test for visceral leishmaniasis.

We have recently developed a sensitive and specific urine-based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li-isd1), tryparedoxin1 (Li-txn1) and nuclear transport factor 2 (Li-ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li-isd1 and Li-ntf2. This new assay was specific and had analytical sensitivity of 15-45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li-txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

Unraveling Ascaris suum experimental infection in humans.

Ascaris lumbricoides and Ascaris suum are two closely related parasites that infect humans and pigs. The zoonotic potential of A. suum has been a matter of debate for decades. Here we sought to investigate the potential human infection by A. suum and its immunological alterations. We orally infected five healthy human subjects with eggs embraced by A. suum. The infection was monitored for symptoms and possible respiratory changes, by an interdisciplinary health team. Parasitological, hematological analyses, serum immunoglobulin, cytokine profiles, and gene expression were evaluated during the infection. Our results show that A. suum is able to infect and complete the cycle in humans causing A. lumbricoides similar symptoms, including, cough, headache, diarrhea, respiratory discomfort and chest x-ray alterations coinciding with larvae migration in the lungs. We also observed activation of the immune system with production of IgM and IgG and a Th2/Th17 response with downregulation of genes related to Th1 and apoptosis. PCA (Principal componts analysis) show that infection with A. suum leads to a change in the immune landscape of the human host. Our data reinforce the zoonotic capacity of A. suum and bring a new perspective on the understanding of the immune response against this parasite.

Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum / Leishmania donovani.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.

Immunohistochemical profile of cytokeratins in pseudoepitheliomatous hyperplasia of cutaneous leishmaniasis.

BACKGROUND: The purpose of this study was to evaluate the immunohistochemical profile of cytokeratins (Ck) in pseudoepitheliomatous hyperplasia of cutaneous leishmaniasis (CL). METHODS: The tissue samples, with pseudoepitheliomatous hyperplasia, were collected from 37 patients (age 6-85 years old) with indolent ulcers in skin confirmed as CL. The lesions were submitted to immunolabeling for Ck pairs 4-13, 5-14, and 6-16. RESULTS: In the 37 CL cases, Ck4 and Ck13 staining were, in the majority of cases, negative. Of them, 33 and 35 cases were negative for Ck4 and Ck13, respectively. The Ck4 immunostaining was found in basal and parabasal layers, and Ck13 was viewed in the basal compartment of epithelium. Ck5 was found in 29 cases distributed homogeneously in all layers. Ck14 and Ck6 were found in all samples in all layers, and Ck16 was positive for all cases but heterogeneously found in the basal layer. CONCLUSION: In summary, we noticed that the pattern of some Ck staining in pseudoepitheliomatous hyperplasia in CL lesions revealed intense epithelial reinforcement, protection, and proliferation.