Hossein Navabi

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Induction of heat shock proteins in B-cell exosomes

Exosomes are nanometer-sized vesicles secreted by a diverse range of live cells that probably have physiological roles in modulating cellular immunity. The extracellular factors that regulate the quantity and phenotype of exosomes produced are poorly understood, and the properties of exosomes that dictate their immune functions are not yet clear. We investigated the effect of cellular stress on the exosomes produced by B-lymphoblastoid cell lines. Under steady-state conditions, the exosomes were positive for hsp27, hsc70, hsp70 and hsp90, and other recognised exosome markers such as MHC class I, CD81, and LAMP-2. Exposing cells to heat stress (42°C for up to 3 hours), resulted in a marked increase in these heat shock proteins (hsps), while the expression of other stress proteins such as hsp60 and gp96 remained negative, and other exosome markers remained unchanged. Stress also triggered a small increase in the quantity of exosomes produced [with a ratio of 1.245±0.07 to 1 (mean±s.e.m., n=20) of 3-hour-stress-exosomes to control-exosomes]. Flow-cytometric analysis of exosome-coated beads and immuno-precipitation of intact exosomes demonstrated that hsps were located within the exosome lumen, and not present at the exosome-surface, suggesting that such exosomes may not interact with target cells through cell-surface hsp-receptors. Functional studies further supported this finding, in that exosomes from control or heat-stressed B cells did not trigger dendritic cell maturation, assessed by analysis of dendritic-cell-surface phenotype, and cytokine secretion profile. Our findings demonstrate that specific alterations in exosome phenotype are a hitherto unknown component of the cellular response to environmental stress and their extracellular function does not involve the direct activation of dendritic cells.

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I] :poly [C12U] (Ampligen®)

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

A clinical grade poly I:C-analogue (Ampligen®) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro

Maturation of dendritic cells (DC) can be triggered in vitro by inflammatory cytokines or Toll-like receptor (TLR) ligands such as CpG or polyI:C. Corresponding, well-characterized agents which can be applied in clinical settings are sparse. We have evaluated a clinical grade, non-toxic analogue of polyI:C, poly(I:C12U) (Ampligen), as a potential adjuvant for cancer immunotherapy, for its ability to drive maturation of human myeloid DC. Our results provide evidence that poly(I:C12U) is effective in inducing optimal phenotypic (elevated levels of MHC-Class I/Class II, CD83, CCR7, CD86 and CD40 molecules) and functional maturation of human DC in vitro, capable of promoting the production of the inflammatory (Th1-type) cytokine IL-12, with significantly lower levels of IL-10 production, compared to that induced by the parent compound polyI:C. Importantly, poly(I:C12U) has a comparable effect on the maturation and function of DC derived either from healthy donors or cancer patients indicating that it is able to overcome any immune suppressive factors associated with the tumour bearing state. These characteristics make poly(I:C12U) a suitable agent for use as an adjuvant in cancer directed immunotherapeutic regimes.

ROLE OF CALCIUM CHELATION IN HIGH-TEMPERATURE ANTIGEN RETRIEVAL AT DIFFERENT pH VALUES

Recent work by Shi et al.1 on the mechanism of high‐temperature antigen retrieval has claimed that the antigen retrieval process is pH‐dependent, with different antigens benefitting at high or low pH values of antigen retrieval solutions. It has previously been claimed2 that chelation of Ca2+ at high temperature is an essential feature of the antigen retrieval process. In order to resolve this apparent dichotomy, the relative antigen retrieval effects were analysed using the buffers employed by Shi et al. in both a facilitating and an inhibitory mode. The results show that calcium‐related effects are optimal at high pH values and do not operate at very low pH. The relative antigen retrieval effectiveness of hydrochloric acid and its metal halide solutions were also investigated in relation to pH. The results of these experiments showed that whilst HCl alone produced antigen retrieval (AR), it also produced severe tissue damage, which was reduced by the inclusion of inorganic salts. These results suggest that antigen retrieval at low pH may be achieved through the dissociation of Ca2+ complexes by high concentrations of H+ ions and/or the breaking up of cross‐links from formalin fixation. Results are also presented to show that chaotropic denaturants such as urea and guanidine hydrochloride also function principally through calcium chelation, whilst detergents have no role to play in high‐temperature retrieval.

Clonal overexpression of metallothionein is induced by somatic mutation in morphologically normal colonic mucosa

Metallothionein (MT) overexpression occurs frequently in human tumours but the underlying mechanism is unknown. Morphologically normal‐appearing mucosa from human colorectal carcinoma resection specimens and of the colons of ageing laboratory mice contains scattered single crypts whose cells show uniformly increased MT immunostaining, suggesting that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny. This hypothesis has now been tested by quantifying the frequency of immunocytochemically detectable monocryptal colorectal MT overexpression, 5 and 30 days after injection of 8‐week‐old mice with a single dose of the mutagen dimethylhydrazine (DMH, 30 mg/kg subcutaneous). Otherwise normal‐appearing MT‐positive crypts were recorded as either wholly or partially involved by the overexpressing phenotype. Five days after DMH injection, the median frequency of partially involved MT‐positive crypts was 11·7×10declining significantly to 1·8×10−4 at 30 days (Mann–Whitney U, P<0·01). In contrast, the median frequency of wholly involved crypts was 0·2×10−4 at 5 days, increasing significantly (P<0·005) to 12·9×10−4 at 30 days. The frequency of MT‐positive crypts and the time course of evolution of partially involved to wholly involved forms were similar to those described for mutation‐induced crypt‐restricted loss of glucose‐6‐phosphate dehydrogenase activity in mice treated with an identical DMH regimen. The findings indicate that cellular MT overexpression can occur as a direct consequence of somatic mutation, either cis‐activating mutation(s) of the MT gene itself, or trans‐activating mutation(s) of other genes involved in controlling MT expression. This is likely to be an important mechanism underlying MT overexpression in neoplasia. Such mutation‐induced aberrant MT expression may be involved in the acquisition of selective cellular growth or survival advantage during tumour progression.

Immunoreactive p53 and metallothionein expression in duct carcinoma in situ of the breast

Abstract Immunocytochemically detectable MT and p53 have been found more commonly in comedo DCIS of the breast with high-grade cytology. The aim of this study is to confirm these findings and to investigate the relationship between MT and p53 in a single large series of cases of DCIS of the breast. To this end, 127 cases of DCIS were classified histologically according to architecture, cytonuclear differentiation (grade), presence and extent of intraduct necrosis, and using the Van Nuys system. Sections were immunostained for p53 and MT (E9) using established techniques, and the extent and intensity of staining were assessed semi-quantitively. The results confirmed that there was generally more MT and p53 positivity in poorly differentiated (grade 3) DCIS with extensive necrosis and that MT expression was greater in grade 2 lesions than p53 expression. However, overall there was no statistically significant correlation between p53 and MT staining. The results indicate that MT and p53 overexpression may arise from independent mechanisms in early breast neoplasia.

Clinical studies of human papilloma vaccines in cervical cancer

Cervical cancer is the second most common cause of cancer death world wide’ and is a particular problem in developing countries. Even with optimum treatment approximately 40% continue to die from this disease.2There is therefore a need for new strategies to reduce global mortality, including immunotherapeutic intervention.

Generation of in Vitro Autologous Human Cytotoxic T-Cell Response to E7 and HER-2/Neu Oncogene Products Using Ex-Vivo Peptide Loaded Dendritic Cells

Cytotoxic T-cells (CTL) have been shown to be capable of causing tumour-specific cell lysis when primed with tumour-specific antigenic peptides presented in conjunction with haplotype matched cell surface MHC class I molecules. For this approach to be successful for immunotherapy of tumours in vivo, it is essential that it is shown to be capable of generating adequate numbers of tumour-specific CTL either in vivo for active immunisation, or ex vivo for adoptive transfer therapy. A powerful means for ex vivo expansion of CTL has been recently shown to be antigen loaded autologous dendritic cells (DC)1. It has also become possible to generate large numbers of these cells from bone marrow or blood derived precursor cells cultured in the presence of GM-CSF and 1L42. Furthermore, murine studies have confirmed the efficacy of these cells to evoke significant ex vivo and in vivo responses against tumour specific antigenic peptides, in particular, HPV 16 E7 and HER-2/neu oncogene products3, thereby creating the possibility of using these antigenic targets4 for effective immunotherapy of gynaecological cancers such as cervical and ovarian carcinomas in which these tumour associated antigens are expressed with 93%5 and 30%6 frequency, respectively.

Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure: A 10 year review of its principle reagents and applications

Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labelled primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or anti-DNP linked marker enzyme. In this article the development of these reagents and various modifications of the basic technique are reviewed in conjunction with the special applications accruing from their use.