Generation of self‐renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells

Pal, Rubina; Marwaha, Sarandeep; Pepponi, Ilaria; Mann, J. Adin; Paul, Mathew; Reljić, Rajko

doi:10.1111/j.1600-0463.2010.02650.x

Cited by 6 publications

(4 citation statements)

References 25 publications

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“…Unexpectedly, our results also showed an intracellular distribution of M. tuberculosis in CD3 ϩ T lymphocytes. Previous studies have shown that dendritic cells, a derivative from the lymphocyte precursor, can phagocytize M. tuberculosis and exert their antigenpresenting functions (11,23). These findings together prompt us to propose that lymphocytes play a similar role in TBM, compensating for the decreased antigen-presenting function of macrophages.…”

Section: Discussionmentioning

confidence: 74%

A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

et al. 2012

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Tuberculous meningitis leads to a devastating outcome, and early diagnosis and rapid chemotherapy are vital to reduce morbidity and mortality. Since Mycobacterium tuberculosis is a kind of cytozoic pathogen and its numbers are very few in cerebrospinal fluid, detecting M. tuberculosis in cerebrospinal fluid from tuberculous meningitis patients is still a challenge for clinicians. Ziehl-Neelsen stain, the current feasible microbiological method for the diagnosis of tuberculosis, often needs a large amount of cerebrospinal fluid specimen but shows a low detection rate of M. tuberculosis. Here, we developed a modified Ziehl-Neelsen stain, involving cytospin slides with Triton processing, in which only 0.5 ml of cerebrospinal fluid specimens was required. This method not only improved the detection rate of extracellular M. tuberculosis significantly but also identified intracellular M. tuberculosis in the neutrophils, monocytes, and lymphocytes clearly. Thus, our modified method is more effective and sensitive than the conventional Ziehl-Neelsen stain, providing clinicians a convenient yet powerful tool for rapidly diagnosing tuberculous meningitis.T uberculous meningitis (TBM) is the most severe form of tuberculosis and causes substantial morbidity and mortality (18). The early diagnosis of and prompt initiation of chemotherapy for TBM are crucial to a successful outcome. However, the early and accurate detection of Mycobacterium tuberculosis in the cerebrospinal fluid (CSF) of TBM patients still remains a challenge for clinicians, mainly due to the lack of rapid, efficient, and practical detection methods (30).Currently, mycobacterial culture is the gold standard for detecting M. tuberculosis, but it is time-consuming and requires specialized safety procedures in laboratories (19,26). Serological methods are convenient but lack sensitivity and specificity (4, 7). Although the PCR technique is rapid, it is costly for routine use in developing countries where most tuberculosis cases occur (5,17,21,24). Conventional smear microscopy with the Ziehl-Neelsen (ZN) stain is a rapid and practical method for detecting acid-fast bacilli (AFB), especially in low-income countries, due to its rapidity, low cost, and high positive predictive value for tuberculosis (14). However, the Ziehl-Neelsen method is severely handicapped by its low detection rate, ranging from 0 to 20% for CSF specimens (31-33). One of the main reasons behind this is that M. tuberculosis can hardly be stained by acid-fast dyes once it enters the cells. Another important reason is that the Ziehl-Neelsen method requires a large volume of CSF for TBM diagnosis, as it is incapable of detecting bacilli that are fewer than 10,000 in number per slide or per ml of specimen (32, 33). Therefore, it is important to develop an alternative, cost-effective method for detecting intracellular M. tuberculosis. Additionally, knowing which cell type is infected by M. tuberculosis in the CSF of TBM patients could help us to unravel new antituberculotic candidates (10).To reve...

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Section: Discussionmentioning

confidence: 74%

A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

et al. 2012

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“…3A). For testing the ability of ICM to bind to APC, we generated mouse spleen derived APC as described previously [25]. Their phenotype was analysed by surface molecule staining and they were found to be positive for CD16/32 (93%), CD11b (98%), CD11c (65%) and MHC II (51%) (not shown), consistent with myeloid dendritic cells and macrophages.…”

Section: Resultsmentioning

confidence: 99%

“…1×10 6 spleen-derived APC, prepared as described by Pal et al [25], were re-suspended in 100 µL of PBS containing 5% BSA. ICMs (10 µg/mL) were added and cells incubated on ice for 2 h. Unbound protein was removed by repeated (3x) addition of the binding buffer and recovery of cells by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

et al. 2013

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Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role.

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“…On the other hand, immature dendritic cells established from the mouse spleen grow in suspension and can take up mycobacteria (21). We therefore thought that cells of a lymphocyte cell line could be a good host for reconstituting apoptotic cell engulfment, when coupled with the pHrodo-labeled apoptotic cell engulfment assay.…”

Section: Discussionmentioning

confidence: 99%

Two-Step Engulfment of Apoptotic Cells

Toda

Hanayama

Nagata

2012

Molecular and Cellular Biology

113

116

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Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin-and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin ␣ v ␤ 3 complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin ␣ v ␤ 3 . These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin ␣ v ␤ 3 complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages. Every day, billions of cells that are toxic, useless, and senescent die by apoptosis and are engulfed by macrophages, presumably to prevent the release of noxious materials from the dead cells (18). The system that efficiently removes apoptotic cells from the body appears to be quite elaborate, and some details of this process are unclear (25). Apoptotic cells present an "eat me" signal to macrophages, triggering their own engulfment. Among the various molecules proposed to be involved in this process, phosphatidylserine (PS) is a strong candidate for the "eat me" signal (11). PS is transferred caspase dependently from the inner leaflet to the outer leaflet of the plasma membrane (13), and masking PS inhibits the engulfment of apoptotic cells by macrophages (2, 6, 11).By identifying monoclonal antibodies that have positive or negative effects on the engulfment of apoptotic cells, we previously identified two molecules, milk fat globule epidermal growth factor (EGF) factor VIII (MFG-E8) and T cell immunoglobulinand mucin-domain-containing molecule 4 (Tim4), that enhance engulfment (6, 15). MFG-E8 is a secreted protein of 75 kDa that binds to PS via its factor VIII-homologous domain. It also binds to the integrin ␣ v ␤ 3 complex in macrophages via an RGD motif in MFG-E8's EGF domain, thus bridging apoptotic cells and macrophages. Tim4 is a type I membrane protein of 70 kDa that binds to PS via the immunoglobulin-like domain in its extracellular region. Its cytoplasmic region is 43 amino acids long and is dispensable for...

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Generation of self‐renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells

Cited by 6 publications

References 25 publications

A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Two-Step Engulfment of Apoptotic Cells

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