Mycobacterium indicus pranii Supernatant Induces Apoptotic Cell Death in Mouse Peritoneal Macrophages In Vitro

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages

Designing an effective vaccine to prevent Epstein-Barr virus-associated diseases: challenges and opportunities

Introduction: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a number of clinical manifestations. Primary EBV infection in young adolescents often manifests as acute infectious mononucleosis and latent infection is associated with multiple lymphoid and epithelial cancers and autoimmune disorders, particularly multiple sclerosis. Areas covered: Over the last decade, our understanding of pathogenesis and immune regulation of EBV-associated diseases has provided an important platform for the development of novel vaccine formulations. In this review, we discuss developmental strategies for prophylactic and therapeutic EBV vaccines which have been assessed in preclinical and clinical settings. Expert commentary: Major roadblocks in EBV vaccine development include no precise understanding of the clinical correlates of protection, uncertainty about adjuvant selection and the unavailability of appropriate animal models. Recent development of new EBV vaccine formulations provides exciting opportunities for the formal clinical assessment of novel formulations

MIP supernatant induced caspase activation and PARP cleavage.

<p>(a) Measurement of cleaved (activated) caspase3 in Relative Fluorescence Units (RFU) as a direct evidence for enhanced apoptosis of macrophages on treatment with MIP supernatant (60–100 ul/ml). Macrophage monolayers were treated with different concentrations of MIP supernatant for 90 min & activated caspase3 was quantified using Sigma caspase3 FL detection kit. (b) MIP supernatant induced caspase-3 activation and PARP degradation in peritoneal macrophages. Macrophage monolayers were incubated with MIP supernatant (1 µg/ml) for 1, 2, 4 hr; the cells were harvested and examined by western blotting for procaspase-3 (32 kDa) (upper panel), cleaved caspase-3 (p17) (middle panel) and PARP (116 kDa) degradation into the main proteolytic product of PARP (85 kDa). Anti-actin Ab was used in parallel as a loading control (lower panels). Lane 1: Untreated control, 2: 1 hr, 3: 2 hr, 4: 4 hr. (c) Treatment of murine peritoneal macrophages with MIP supernatant elicited disruption of mitochondrial trans-membrane potential. Mitochondrial membrane potential was visualized with a MitoCapture Mitochondrial Apoptosis Detection kit. Pretreatment with pan-caspase inhibitor Z-VAD-fmk had no inhibitory effect on MIP supernatant induced MMP. Staurosporin (S) (0.5 uM) treated cells were taken as a positive control. Bars in the figure show % of non-apoptotic (red fluorescence) & apoptotic cells (Red & green fluorescence).</p

MIP supernatant induces apoptosis in mouse peritoneal macrophages.

<p>(a) MIP supernatant induced cell death of macrophages was not due to LPS contamination. Macrophage monolayers were treated with MIP supernatant (1 µg/ml) for 8 hr and % cytotoxicity was determined by MTT assay. Before incubation of macrophages with MIP supernatant, the supernatant was also treated either with Polymyxin B (PB). Untreated cells were taken as control with 0% cytotoxicity. (b) Murine peritoneal macrophages after treatment with MIP supernatant for 4 hr were dual stained with Sigma APOAC kit. Annexin V stained early apoptotic cells (annexin V positive, 6-CFDA positive) show red fluorescence on cell surface. Untreated viable cells (annexin V negative, 6-CFDA positive) fluoresce green with no signal for Annexin V. (c) Macrophage monolayers were treated with MIP supernatant for 6 hr, fixed & stained for DAPI. Arrows show nuclear condensation & fragmentation. Scale bar: 10 µm. (d) Macrophage monolayers were treated with MIP supernatant for 6 hr, lysed and the DNA fragmentation was detected by quantitative Nucleosome ELISA with anti-histone antibody. Untreated cells were taken as control. (e) MIP cell-free supernatant was lyophilized and re-suspended in sterile PBS & run on SDS-PAGE. The gel was stained with AgNo₃ and observed. Lane1: Low molecular weight SDS marker, lane2: MIP supernatant. The indicated molecular weights are in kDa.</p

Involvement of PKC in MIP supernatant induced cell death of macrophages.

<p>(a) Macrophage monolayers were pretreated with H7 or Rottlerin for 45 min, incubated with MIP supernatant for 8 hr and % cytotoxicity was determined by MTT assay. Untreated macrophages were taken as control with 0% cytotoxicity. (b) Macrophages monolayers were pretreated with H7, incubated with different doses of MIP supernatant & activated caspase 3 was quantified using caspase3 FL detection kit. Inhibiting PKC also resulted in decreased caspase 3 activities. RFU: Relative Fluorescence Units. (c) MIP supernatant induced nuclear translocation of PKC δ. Macrophage monolayers were treated with MIP supernatant for 1, 2 or 3 hr. Cytoplasmic and nuclear fractions were harvested and immunoblotted for PKC δ. Lanes 1–4 (Cyto): control, 1 hr, 2 hr, 3 hr; lanes 5–8 (Nuc): control, 1 hr, 2 hr, 3 hr. Cyto-cytoplasmic fraction, Nuc-nuclear fraction.</p

MIP supernatant induced release of AIF and Cytochrome c into the cytoplasm, nuclear translocation of AIF.

<p>(a) Macrophage monolayers were incubated with MIP supernatant for 3, 4 hr; the cytoplasmic and nuclear fractions were harvested and examined by western blotting for AIF and cytochrome c. Lane 1: untreated cells, lane 2: 3 hr, lane 3: 4 hr. (b) Macrophage monolayers grown overnight on cover-glasses were treated with MIP supernatant for 4 hr, washed & fixed with 4% paraformaldehyde, probed with anti-AIF-TRITC & Hoechst and visualized for nuclear accumulation of AIF under Olympus BX61 fluorescence microscope. Upper panel: untreated, lower panel: MIP supernatant treated. The areas encircled clearly demonstrate co-localization of a red signal for AIF in the nucleus in MIP supernatant treated macrophages. Scale bar: 10 µm.</p

MIP supernatant induces Bax translocation to the mitochondria.

<p>Macrophage monolayers were treated with MIP supernatant for 1–1.5 hr and fixed. Cells were probed for Bax-FITC and Hoechst, mitochondria were stained red. The upper panel (A–E) shows untreated macrophages while the lower panel (F–J) displays MIP supernatant treated macrophages. E and J show the overlay of respective panels. It is clear from the overlay that there is distinct overlapping of red & green fluorescence indicating co-localization of Bax with mitochondria in the MIP supernatant treated macrophages (J). Scale bar: 10 µm.</p

MIP Supernatant resulted into downregulation of LPS induced proinflammatory responses.

<p>Macrophage monolayers were treated with LPS without or in the presence of 40 ul of MIP supernatant for 12 hrs, supernatant was collected & ELISA was performed for IL-1β & IL-10. Supernatant led to enhanced expression of IL-10.</p

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance

Background: Application of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained. Methods: BM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells. Results: Immune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM. Conclusions: Rapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance