A Kidney Biopsy Simulation Training Program for Renal Fellows: Two Years of Results

Renal interventions could re-foster interest in Nephrology and attract more medical graduates. Percutaneous kidney biopsy (PKB) is an important diagnostic tool and should be taught through simulation. We initiated a PKB simulation training program and designed a 2-year study to examine its effect on the confidence level, the procedural competence and the satisfaction with this training of Nephrology fellows compared to historical controls. All fellows were consented and trained at UNM’s simulation center (BATCAVE) with a simulation training model (CAE Healthcare Blue PhantomTM). Trainees’ demographics and previous PKB experience were collected. We utilized pre-assigned readings, online videos and hands-on simulation practice. Performance of each trainee during each session was graded with a procedural competence evaluation form. Drs. JO and MER were present in all sessions and completed these forms. Each session lasted 1 to 1-1 1/2 h. Pre-and post-simulation surveys evaluated the participants’ confidence level quantitatively on a 5-point Likert scale. All participants completed the satisfaction with PKB simulation experience scale (PKB-SSE). All three 1st and 2nd year renal fellows completed the simulation training in 2018 and two first year fellows completed the training in 2019. Independent of their previous experience on PKBs all renal fellows expressed a high level of satisfaction from their participation (4 to 5) and increased their confidence level. This year’s trainees increased their performance level from 2 to 5 and from 1 to 5, respectively. PKB simulation may improve trainees’ confidence level and their satisfaction with the training. The procedural competence of the trainees on PKBs will be evaluated during the 2nd year of their fellowship and will be compared to the procedural competence of historical controls. We expect that the simulation training will reduce the discomfort and minimize the adverse PKB outcomes in patients undergoing PKB in UNMH

Exosomes Released from Mycoplasma Infected Tumor Cells Activate Inhibitory B Cells

Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity

A Kidney Biopsy Simulation Program for Nephrology Fellows: The Phantom Project

A proposed curriculum development plan providing an educational and instruction tool for Nephrology fellows. During the two-year fellowship program Nephrology fellows will perform real-time ultrasound-guided kidney biopsies using a simulation tool

Myco+ exosomes induce B cell activation and expansion.

<p>Splenocytes were treated with 1 µg/ml of B16 myco+ exosomes or B16 myco− exosomes, or cultured untreated for 72 hr. Cells were harvested and analyzed by FACS. (A) Expression of CD25, CD40, CD86, CD80, CD23, IgD, IgM, CD1d, CD5 and CD43 in the B cell gate (CD19+B220+). (B) Percentage of B cells in total splenocytes within the live cell gate after exosome treatment. Data represents the mean ± SD of four independent experiments. Significance at: *, P<0.05. (C) Expression of CD25, CD69, CD44, CD62L, CD80 and CD86 in the CD4+ T cell gate and the CD8+ T cell gate. Black line: B16 myco+ exosome treatment; grey line: B16 myco− exosome treatment; grey solid: untreated.</p

The induction of IFN-γ-producing T cells by myco+ exosomes increases in the absence of B cells.

<p>WT or μMT spleen cells were cultured with or without 1 µg/ml of B16 myco+ exosome for 48 hr and stained for intracellular IFN-γ. (A) Induction of IFN-γ+CD8+ T cells in WT and μMT splenocyte cultures. Data shows one representative experiment of three with similar results. Numbers in each plot represent % cells in CD8+ cell gate. (B) Fold increase of % IFN-γ+ cells in the CD8+ cell gate in WT and μMT splenocytes upon B16 myco+ exosome treatment. Data shows the mean ± SD of three independent experiments. Significance at: *, P<0.05. (C) Induction of IFN-γ+CD4+ T cells in WT and μMT splenocyte cultures. Data shows one representative experiment of three with similar results. Numbers in each plot represent % cells in the CD4+ cell gate. (D) Fold increase of % IFN-γ+ cells in the CD4+ cell gate in WT and μMT splenocytes upon B16 myco+ exosome treatment. Data shows the mean ± SD of three independent experiments. Significance at: *, P<0.05.</p

Cytokine induction by myco+ exosomes after exosome membrane disruption or mycoplasma removal reagent treatment of parental cells.

<p>(A) Mycoplasma-infected cells were treated with Plasmocin for 2 wk and tested to be mycoplasma-free. (B) B16 and EL4 myco+ exosomes were subjected to 5 cycles of freeze/thaw (F/T) or sonication (sonic). Splenocytes were treated with 1 µg/ml of myco+ exosomes, F/T exosomes, sonic exosomes or exosomes derived from Plasmocin-treated cells (plasmo) for 72 hr. IL-10 production was measured by ELISA. (C) IFN-γ production measured by ELISA. Induction of IL-10 and IFN-γ by plasmo exosomes was significantly reduced compared with intact, F/T and sonic exosomes. Significance at: *, P<0.05.</p

Intracellular cytokine staining of myco+ exosome-treated splenocytes.

<p>WT splenocytes were cultured with or without 1 µg/ml of B16 myco+ exosome for 48 hr in 24-well-plate at 5×10⁶ cells/1.5 ml media/well with 30 U/ml of rmIL-2. Brefeldin A was added to the culture for the last 6 hr before cells were harvested. Cells were first surface stained for CD19, B220, CD4 and CD8, and then stained for intracellular IL-10 and IFN-γ. (A) Percentage of IL-10+ cells in the B cell, CD4+ T cell and CD8+ T cell gates. Numbers in each plot represent % cells in each cell gate. Figures show the data of one representative experiment of three with similar results. (B) Fold increase of % IL-10+ cell in the B cell, CD4+ cell and CD8+ cell gate. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05. (C) Percentage of IL-10+ B cells, IL-10+ CD4+ cells and IL-10+ CD8+ cells in total splenocytes in untreated or B16 myco+ exosome-treated splenocytes. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05. (D) Percentage of IFN-γ+ cells in the B cell, CD4+ T cell and CD8+ T cell gates. Numbers in each plot represent % cells in each cell gate. Figures show the data of one representative experiment of three with similar results. (E) Fold increase of % IFN-γ+ cell in the B cell, CD4+ cell and CD8+ cell gate. Data represents the mean ± SD of three independent experiments. (F) Percentage of IFN-γ+ B cells, IFN-γ+ CD4+ cells and IFN-γ+ CD8+ cells in total splenocytes in untreated or B16 myco+ exosome-treated splenocytes. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05.</p

Cytokine induction by myco+ exosomes in WT and μMT splenocytes.

<p>Splenocytes from either WT mice or μMT mice were cultured in 24-well-plate at 5×10⁶ cells/1.5 ml media/well with 30 U/ml of rmIL-2 and treated with 1 µg/ml of B16 myco+ exosomes or left untreated for 72 hr. IL-10 and IFN-γ levels in the culture supernatants were measured by ELISA. Treatments were conducted in triplicates in each experiment. Data represents the averaged cytokine levels ± SD of three independent experiments. Significance at: *, P<0.05.</p

T cell proliferation is inhibited when co-cultured with myco+ exosome-treated splenocytes or purified B cells.

<p>Splenocytes (T cell-depleted) or purified splenic B cells were cultured in 24-well-plate at 2.5×10⁶ cells/well with or without 1 µg/ml of B16 myco+ exosomes for 24 hr, then 0.5×10⁶ of CFSE-labeled T cells (CD45.1+) were added to the culture and stimulated with 10 µg/ml of anti-CD3e. Cells were co-cultured for another 3 days and T cell proliferation was analyzed by CFSE dilution. (A) Gating of CD45.1+CD8+ T cells and CD45.1+CD4+ T cells. Expression of CD44 and CD62L were shown within each T cell gate in non-treated and B16 myco+ exosome treated co-cultures. Non-treated cells without anti-CD3e were included as an unstimulated control. T cells that are CD44^highCD62L^low represent the activated T cell subset. (B) Proliferation of CD8+ T cells and CD4+ T cells in myco+ exosome-treated splenocytes shown by CFSE dilution. Total T cells: total CD8+ or CD4+ T cells. CD44^hiCD62L^lo T cells: T cell subsets that are CD44^highCD62L^low. Unstimulated: Non-treated T cells without anti-CD3 stimulation. (C) Proliferation of CD8+ T cells and CD4+ T cells when co-cultured with myco+ exosome-treated B cells, shown by CFSE dilution.</p

Cytokine induction in splenocytes by myco+ exosome treatment.

<p>(A) Splenocytes from C57BL/6 mice were cultured in a 24-well-plate at the density of 5×10⁶ cells/1.5 ml media/well in the presence of 30 U/ml rmIL-2 and were treated with either myco+ exosomes or myco− exosomes (1 µg/ml), or left untreated for 72 hr. The IL-10 and IFN-γ levels (pg/ml) in the culture supernatants were measured by ELISA. Treatments were conducted in duplicates or triplicates in each experiment. Data represent the averaged cytokine levels ± SD of three independent experiments. (B) Dose-dependent cytokine induction by myco+ exosomes. Splenocytes were treated with an increasing dose of myco+ exsosomes (0.1, 1 and 10 µg/ml) for 72 hr, and the cytokine levels were measured by ELISA. Treatments were conducted in duplicates. Data represent the averaged cytokine levels ± SD of three independent experiments. Significance at: *, P<0.05.</p