Infective Endocarditis Associated with Streptococcal Toxic Shock Syndrome due to Streptococcus dysgalactiae subsp. equisimilis Infection in a Hemodialysis Patient

The risk of infective endocarditis in chronic hemodialysis patients is markedly higher than that in the general population. We report the first case of a hemodialysis patient with infective endocarditis caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE) who presented with streptococcal toxic shock syndrome. In the last decade, there has been an increase in the incidence of SDSE infections. Therefore, it is important to recognize SDSE as a possible causative agent of infective endocarditis in an immunocompromised population, such as hemodialysis patients

In small cell lung cancer patients treated with RRx-001, a downregulator of CD47, decreased expression of PD-L1 on circulating tumor cells significantly correlates with clinical benefit.

BackgroundSmall cell lung cancer (SCLC) is the most aggressive lung tumor, characterized by a rapid doubling time and the development of widespread metastases, for which immune checkpoint inhibitors have been approved to overcome T cell anergy. In light of its dismal prognosis, and lack of curative options, new therapies for extensive-disease SCLC are desperately needed.MethodsRRx-001 is a small molecule Myc inhibitor and down-regulates CD47 expression on tumor cells. We evaluated the programmed death-ligand 1 (PD-L1) status of circulating tumor cells (CTCs) pre and post RRx-001 treatment in a phase 2 clinical trial, called QUADRUPLE THREAT, where patients with previously treated SCLC received RRx-001 in combination with a platinum doublet. The trial was registered with ClinicalTrials.gov, number NCT02489903. Fourteen patients with SCLC were analyzed to investigate the association between clinical outcome and PD-L1 expression on CTCs pre and post RRx-001. The correlation between the binary clinical outcome (clinical benefit vs. progressive disease) and the change of PD-L1 expression on CTCs after RRx-001 was analyzed using a logistic regression adjusting for baseline PD-L1 expression.ResultsThe logistic model McFadden goodness of fit score was 0.477. The logistic model analyzing the association between decreased PD-L1 expression on CTCs after RRx-001 and response to reintroduced platinum doublet had an approximate 92.8% accuracy in its prediction of clinical benefit. The estimated receiver operating characteristic (ROC) displayed a ROC area under the curve (AUC) of 0.93 (95% confidence interval, 0.78-0.99).ConclusionsThese results suggest that PD-L1 expression on CTCs decreased after RRx-001 was significantly correlated with response to reintroduced platinum-based doublet therapy. Monitoring PD-L1 expression on CTCs during RRx-001 treatment may serve as a biomarker to predict response to RRx-001-based cancer therapy

Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology

<div><p>We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. <i>In vitro</i> stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4⁺ and CD8⁺ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8⁺ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.</p></div

Induction of CD8⁺ T cell lines that are reactive to antigens by CD14-ML-DC that express antigenic proteins.

<p>(A) The lentivirus constructs for CMVpp65 (EF-CMV-IP) and MART1 (EF-MART1-IP) are shown. CD14-ML were transduced with the lentivirus vector and cultured in the presence of puromycin (5 μg/ml) to select and expand the cell population carrying the transgene, resulting in the generation of CD14-ML/CMV and CD14-ML/MART1. (B) Expression of CMVpp65 by CD14-ML/CMV was analyzed by flow cytometric analysis. The staining profiles of the specific mAb (black lines) and isotype-matched control mAb (gray area) are shown. (C) CD14-ML-DC/CMV derived from a HLA-A*24:02-positive healthy donor were cultured with autologous CD8⁺ T cells. On day 9, the number of T cells reactive to the CMVpp65_341-349 peptide was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are shown (Day 0). (D) On day 9, the T cells were recovered and stained with an anti-CD8 mAb and a tetramer of HLA-A*24:02/CMVpp65_341-349 complex. The numbers in the figure indicate the percentage of the CD8⁺ T cells positively stained with the tetramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (E) CD8⁺ T cells obtained from an HLA-A*24:02-negative healthy donor were co-cultured with autologous CD14-ML-DC/CMV. On day 9, the number of IFN-γ producing CD8⁺ T cells was analyzed by ELISPOT assay, using CD14-ML and CD14-ML/CMV as stimulators. The results of the T cells before stimulation culture are also shown (Day 0). (F) Expression of MART1 by CD14-ML/MART1 was analyzed by flow cytometric analysis. The staining profiles of the specific mAbs (black lines) and isotype-matched control mAbs (gray area) are shown. (G) CD8⁺ T cells obtained from an HLA-A*02:01-positive healthy donor were co-cultured with autologous CD14-ML-DC/MART1 cells. On day 21, the frequency of CD8⁺ T cells reactive to MART1_26-35 was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are also shown (Day 0). (H) On day 21, the T cells were recovered and stained with an anti-CD8 mAb and HLA-A*02:01/MART1_26-35 dextramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the dextramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (I) CD8⁺ T cells obtained from an HLA-A*02:01-negative healthy donor were co-cultured with autologous CD14-ML-DC/MART1. On day 21, the frequency of CD8⁺ T cells reactive to MART1 was analyzed by ELISPOT assay, using CD14-ML-DC and CD14-ML-DC/MART1 as stimulators. The results of the T cells before stimulation culture are also shown (Day 0).</p

Morphology of CD14-ML and CD14-ML-DC generated by the current procedure.

<p>(A) Phase-contrast images of live cells (left) and cytospin samples stained with May–Grünwald Giemsa (right) of the human monocytes and monocyte-derived myeloid cell lines (CD14-ML) are shown. (B) Morphology of OK432-stimulated mo-DC and CD14-ML-DC are shown. mo-DC and CD14-ML-DC were stimulated with OK432 for 2 days and subjected to microscopic analysis. The data are representative of 2 experiments.</p

Induction of CD4⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC.

<p>(A) Protocol for the induction of cancer antigen-specific CD4⁺ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with a mixture of 6 peptides (CDCA1_39-64, CDCA1_55-78, KIF20A_60-84, KIF20A_809-833, LY6K_119-142 and LY6K_172-191) for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD4⁺ T cells in AIM-V with 5% human decomplemented plasma. On day 7, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rIL-7 (5 ng/ml). After two days, these cultures were supplemented with rIL-2 (10 IU/ml). CD14-ML-DC were added with only IL-4 (did not add OK432). On day 14, the stimulated CD4⁺ T cells in each well were analyzed for specificity in IFN-γ ELISPOT assays. The T cells showing a specific response to the cognate peptide were transferred to 24-well plates and restimulated with the autologous peptide-pulsed CD14-ML-DC, and subsequently supplemented with rhIL-7 (5 ng/ml) and rhIL-2 (20 IU/ml). On day 21, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rhIL-7 and rhIL-2. IFN-γ ELISPOT assays were performed after 6 or 7 days from the fourth round of peptide stimulation. (B, C) After the stimulation (more than three times), the number of CD4⁺ T cells reacting to each peptide was analyzed with an IFN-γ ELISPOT assay (Day 28). The results of the T cells before stimulation culture are shown (Day 0). Dimethyl sulfoxide was used as a control. The results for the healthy donor 3 (B) and donor 2 (C) are shown.</p

Induction of CD4⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC obtained from HNC patients.

<p>CD4⁺ T cells isolated from PBMCs of HNC patients were stimulated with CD14-ML-DC that were pulsed with a mixture of 6 kinds of peptides (CDCA1_39-64, CDCA1_55-78, KIF20A_60-84, KIF20A_809-833, LY6K_119-142 and LY6K_172-191). After more than three rounds of stimulation, the number of CD4⁺ T cells that reacted to each peptide was analyzed by ELISPOT assay. CD14-ML-DC were used as stimulators in the assay, because only a few amount of blood samples could be obtained from cancer patients. The results for cancer patient 1 (A) and cancer patient 2 (B) are shown.</p

Induction of CD8⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC obtained from HNC patients.

<p>Peripheral blood CD8⁺ T cells obtained from HLA-A*24:02-positive HNC patients were co-cultured with autologous CD14-ML-DC pre-loaded with a peptide mixture (CDCA1_56-64, KIF20A_66-75, LY6K_177-186 and IMP-3_508–516) to induce T cell lines that were reactive to the peptides under a schedule similar to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152384#pone.0152384.g004" target="_blank">Fig 4A</a>. On days 7 and 14, the CD8⁺ T cells were re-stimulated with peptide-loaded CD14-ML-DC. (A, C) On day 21, the number of CD8⁺ T cells responding to the peptides were analyzed by an IFN-γ ELISPOT assay. An HIV-peptide was used as a control peptide. (B, D) On day 21, the T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*24:02/CDCA1_56-64, HLA-A*24:02/KIF20A_66-75 or HLA-A*24:02/LY6K_177-186 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the tetramer from the HLA-peptide complex. The results for the cancer patient 1 (A, B) and the cancer patient 2 (C, D) are shown.</p

Induction of CD8⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC.

<p>(A) Protocol for the induction of cancer antigen-specific CD8⁺ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with peptides for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD8⁺ T cells. Cells were cultured with rIL-7 (10 ng/ml) in AIM-V with 5% human decomplemented plasma. On days 7 and 14, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and on days 9 and 16, and were supplemented with rIL-2 (20 IU/ml). CD14-ML-DC were prepared each time, and we only added IL-4 (did not add OK432). IFN-γ ELISPOT assay and flow cytometry were performed after 6 or 7 days from the third round of peptide stimulation. (B, C) Peripheral blood CD8⁺ T cells were obtained from a HLA-A*24:02-positive healthy donor (healthy donor 1) and were co-cultured with 4 peptides (CDCA1_56-64, KIF20A_66-75, LY6K_177-186 and IMP-3_508–516)-loaded autologous CD14-ML-DC. (B) On day 21, the number of IFN-γ producing CD8⁺ T cells were analyzed by ELISPOT assay (Day 21). The results of the T cells before stimulation culture are also shown (Day 0). The HIV-peptide was used as a control peptide. (C) On day 21, the T cells were recovered and stained with anti-CD8 mAb and the HLA-A*24:02/CDCA1_56-64 or HLA-A*24:02/LY6K_177-186 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the tetramer of the HLA-peptide complex (Day 21). The results of the T cells before stimulation culture are also shown (day 0). (D, E) A similar experiment as in (B, C) was done with the cells obtained from a HLA-A*02:01-positive donor (healthy donor 2). We used 4 peptides (CDCA1_351-359, KIF20A_809-817, MART1_26-35 and IMP3_515-523) for the stimulation of the T cells. (D) The number of IFN-γ producing CD8⁺ T cells was analyzed by ELISPOT assay. (E) The T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*02:01/MART1_26-35 dextramer, HLA-A*02:01/CDCA1_351-359 tetramer or HLA-A*02:01/IMP3_515-523 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the dextramer or tetramer of HLA-peptide complex.</p

Summary of blood donors.

<p>Summary of blood donors.</p