Smanla Tundup

Ex Vivo Programming of Dendritic Cells by Mitochondria-Targeted Nanoparticles to Produce Interferon-Gamma for Cancer Immunotherapy

One of the limitations for clinical applications of dendritic cell (DC)-based cancer immunotherapy is the low potency in generating tumor antigen specific T cell responses. We examined the immunotherapeutic potential of a mitochondria-targeted nanoparticle (NP) based on a biodegradable polymer and zinc phthalocyanine (ZnPc) photosensitizer (T-ZnPc-NPs). Here, we report that tumor antigens generated from treatment of breast cancer cells with T-ZnPc-NPs upon light stimulation activate DCs to produce high levels of interferon-gamma, an important cytokine considered as a product of T and natural killer cells. The remarkable ex vivo DC stimulation ability of this tumor cell supernatant is a result of an interleukin (IL)-12/IL-18 autocrine effect. These findings contribute to the understanding of how in situ light activation amplifies the host immune responses when NPs deliver the photosensitizer to the mitochondria and open up the possibility of using mitochondria-targeted-NP-treated, light-activated cancer cell supernatants as possible vaccines.

Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: Evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other

About 10% of the coding capacity of the Mycobacterium tuberculosis (M. tb) genome is devoted to the PE/PPE family of genes scattered throughout the genome. We have identified 28 PE/PPE operons which are organized within the M. tb genome in such a way that most PE members are upstream to PPE members. One example of such a gene arrangement is the PPE gene Rv2430c, earlier shown by us to code for a highly antigenic protein eliciting strong B‐cell responses in TB patients [Choudhary, R.K., Mukhopadhyay, S., Chakhaiyar, P., Sharma, N., Murthy, K.J.R., Katoch V.M. and Hasnain, S.E. (2003) PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B cell response. Infect. Immun. 71, 6338–6343], situated downstream to PE gene Rv2431c. Rv2431c and Rv2430c are transcribed as an operon. Expression of either rRv2431c or rRv2430c alone in E. coli limited their localization to the inclusion bodies. However, when they were co‐expressed, both the proteins appeared in the soluble fraction. These two proteins interact with each other and form oligomers when alone, however, when present together they exist as heteromer.

The Co-Operonic PE25/PPE41 Protein Complex of Mycobacterium tuberculosis Elicits Increased Humoral and Cell Mediated Immune Response

Background Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. Methodology/Principal Finding We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15–22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8–10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-γ, TNF-α and IL-2 cytokines. Conclusion While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis.

Polarization of host immune responses by helminth‐expressed glycans

Helminth parasites bias host CD4+ T helper (Th) cells toward Th2 responses, drive alternative activation of macrophages, and expand T regulatory cells. Helminth‐expressed carbohydrates play critical roles in driving much of this immune cell biasing. Studies on helminth glycans have focused on Lewis X, LDN, LDN‐DF, other fucosylated structures, chitin, tyvelose, and trehalose, which interact with host antigen presenting cells (APCs) minimally via C‐type lectins and/or Toll‐like receptors (TLR). Here, we review recent findings on helminth glycan activation of APCs via C‐type lectin/TLRs and introduce the concept that glycosylated helminth molecules require endocytosis to function as immune modulators. Second, we describe unpublished data showing that in vivo glycoconjugates comprising multiple copies of glycans on carriers are directly immune modulatory. Lastly, we discuss the observation that CD14 negatively regulates alternative activation of APCs during helminth infection. We close with a discussion on the use of immune modulatory glycans as vaccine adjuvants and as antiinflammatory therapeutics.

Transcriptional Regulation of Mycobacterium tuberculosis PE/PPE Genes: A Molecular Switch to Virulence

The PE/PPE family of proteins, which constitute 10% of the coding capacity of the mycobacterial genome, comprises a unique set of genes which have no known homologs and have expanded throughout their evolution. Their association with virulence has been implicated by several researchers in tuberculosis, but the molecular basis of their virulence is yet to be completely explored. PE/PPE genes are mostly associated with the pathogenic strains of mycobacteria as many of them are known to be deleted in non-pathogenic ones. The non-essentiality of these genes for their in vitrogrowth but essentiality during infection highlights their active role in the host-pathogen interaction and consequently virulence. Even within the different strains of pathogenic mycobacteria and clinical isolates, many of the PE/PPE genes show sequence variation, pointing to their importance in providing antigenic variations, and have also been speculated to perform varied roles by differential expression during host-pathogen interaction. The transcriptional regulators of these genes could therefore act as a molecular switch for the pathogenesis of Mycobacterium tuberculosis. This review focuses on the expression and regulation of PE/PPE genes in the context of infection and pathogenicity and discusses the potential of these proteins as drug targets.

Immunomodulatory Glycan Lacto-N-Fucopentaose III Requires Clathrin-Mediated Endocytosis To Induce Alternative Activation of Antigen-Presenting Cells

ABSTRACT The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewisx trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewisx trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewisx-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses.

New Approaches with Different Types of Circulating Cathodic Antigen for the Diagnosis of Patients with Low Schistosoma mansoni Load

Background Schistosomiasis mansoni is a debilitating and sometimes fatal disease. Accurate diagnosis plays a key role in patient management and infection control. However, currently available parasitological methods are laborious and lack sensitivity. The selection of target antigen candidates has turned out to be a promising tool for the development of more sensitive diagnostic methods. In our previous investigations, the use of crude antigens led to false-positive results. Recently, focus has been given to highly purified Schistosoma mansoni antigens, especially to circulating antigens. Method Thus, our main goal was to test different types of circulating cathodic antigen glycoprotein (CCA), as “crude antigen,” the protein chain of recombinant CCA and two individual peptides. These schistosome proteins/peptides were tested in a new diagnostic method employing immunomagnetic separation based on the improvement of antigen–antibody binding. Principal Findings Use of recombinant CCA as a diagnostic antigen allowed us to develop a diagnostic assay with high sensitivity and specificity with no false-negative results. Interestingly, the “crude antigen” worked as a good marker for control of cure after praziquantel treatment. Conclusions/Significance Our new diagnostic method was superior to enzyme-linked immunosorbent assay in diagnosing low endemicity patients.

CD14 Influences Host Immune Responses and Alternative Activation of Macrophages during Schistosoma mansoni Infection

ABSTRACT Antigen-presenting cell (APC) plasticity is critical for controlling inflammation in metabolic diseases and infections. The roles that pattern recognition receptors (PRRs) play in regulating APC phenotypes are just now being defined. We evaluated the expression of PRRs on APCs in mice infected with the helminth parasite Schistosoma mansoni and observed an upregulation of CD14 expression on macrophages. Schistosome-infected Cd14 −/− mice showed significantly increased alternative activation of (M2) macrophages in the livers compared to infected wild-type (wt) mice. In addition, splenocytes from infected Cd14 −/− mice exhibited increased production of CD4+-specific interleukin-4 (IL-4), IL-5, and IL-13 and CD4+Foxp3+IL-10+ regulatory T cells compared to cells from infected wt mice. S. mansoni-infected Cd14 −/− mice also presented with smaller liver egg granulomas associated with increased collagen deposition compared to granulomas in infected wt mice. The highest expression of CD14 was found on liver macrophages in infected mice. To determine if the Cd14 −/− phenotype was in part due to increased M2 macrophages, we adoptively transferred wt macrophages into Cd14 −/− mice and normalized the M2 and CD4+ Th cell balance close to that observed in infected wt mice. Finally, we demonstrated that CD14 regulates STAT6 activation, as Cd14 −/− mice had increased STAT6 activation in vivo, suggesting that lack of CD14 impacts the IL-4Rα-STAT6 pathway, altering macrophage polarization during parasite infection. Collectively, these data identify a previously unrecognized role for CD14 in regulating macrophage plasticity and CD4+ T cell biasing during helminth infection.

Ex Vivo Generation of Functional Immune Cells by Mitochondria-Targeted Photosensitization of Cancer Cells

Stimulating the immune system for potent immune therapy against cancer is potentially a revolutionary method to eradicate cancer. Tumors stimulated with photosensitizers (PSs) not only kill cancer cells but also help to boost the immune system. We recently reported that tumor-associated antigens (TAAs) generated by delivery of a mitochondria-acting PS zinc phthalocyanine (ZnPc) to MCF-7 breast cancer cells followed by laser irradiation can lead to ex vivo stimulation of mouse bone marrow-derived dendritic cells (BMDCs). The antigens generated from the breast cancer cells were also found to cause significant DC maturation and the activated DCs were able to stimulate T cells to cytotoxic CD8(+) T cells. In this protocol, we describe methods to engineer a mitochondria-targeted biodegradable nanoparticle (NP) formulation, T-ZnPc-NPs for delivery of ZnPc to the mitochondria of MCF-7 cells, subsequent photodynamic therapy (PDT) using a long wavelength laser irradiation to produce TAAs, DC stimulation by the TAAs to secrete interferon-gamma (IFN-γ), and matured DC-driven T-cell activation.