Raja’a Al-Attiyah

Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the Secreted Mycobacterium tuberculosis Antigen 85B Recognized by Antigen-Specific Human CD4+ T-Cell Lines

ABSTRACT Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+ T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1,DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells

ABSTRACT Comparative genomics has identified several regions of difference (RDs) of Mycobacterium tuberculosis that are deleted or absent in Mycobacterium bovis BCG vaccines. To determine their relevance for diagnostic and vaccine applications, it is imperative that efficient methods are developed to test the encoded proteins for immunological reactivity. In this study, we have used 220 synthetic peptides covering sequences of 12 open reading frames (ORFs) of RD1 and tested them as a single pool (RD1pool) with peripheral blood mononuclear cells obtained from pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays that measure antigen-induced proliferation and IFN-γ secretion. The results showed that RD1pool induced strong responses in both TB patients and BCG-vaccinated healthy subjects. The subsequent testing of peptide pools of individual ORFs revealed that all ORFs induced positive responses in a portion of donors, but PPE68, CFP10, and ESAT6 induced strong responses in TB patients and PPE68 induced strong responses in BCG-vaccinated healthy subjects. In addition, HLA-DR and -DQ typing of donors and HLA-DR binding prediction analysis of proteins suggested HLA-promiscuous presentation of PPE68, CFP10, and ESAT6. Further testing of individual peptides showed that a single peptide of PPE68 (121-VLTATNFFGINTIPIALTEMDYFIR-145) was immunodominant. The search for sequence homology revealed that a part of this peptide, 124-ATNFFGINTIPIAL-137, was present in several PPE family proteins of M. tuberculosis and M. bovis BCG vaccines. Further experiments limited the promiscuous and immunodominant epitope region to the 10-amino-acid cross-reactive sequence 127-FFGINTIPIA-136.

Synthetic Peptides Identify Promiscuous Human Th1 Cell Epitopes of the Secreted Mycobacterial Antigen MPB70

ABSTRACT MPB70 is a secreted protein of Mycobacterium bovis and Mycobacterium tuberculosis which stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cell M. tuberculosis and M. tuberculosis culture filtrates), with MPB70 purified from the culture filtrate of M. bovis BCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-γ) assays. Testing of antigen-specific CD4+ T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-γ assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-γ-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.

Clinical, immunologic and genetic profiles of DOCK8-deficient patients in Kuwait

Deficiency of dedicator of cytokinesis 8 (DOCK8) is a newly described combined primary immunodeficiency disease. It was found to account for 15% of combined immune deficiency cases in the National Primary Immunodeficiency Disorders Registry in Kuwait, a country with high prevalence of consanguinity. We present the clinical, immunologic and molecular characteristics of 9 Kuwaiti patients with DOCK8 deficiency and discuss differences that distinguish DOCK8 deficiency from atopic dermatitis. Clinical immunologists in areas with high incidence of consanguinity should have a high index of suspicion of DOCK8 deficiency in children with recalcitrant eczema, recurrent non-cutaneous infections and lymphopenia.

Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer

BackgroundNatural products with diverse bioactivities are becoming an important source of novel agents with medicinal potential. Cancer is a devastating disease that causes the death of millions of people each year. Thus, intense research has been conducted on several natural products to develop novel anticancer drugs.MethodsChromatographic and spectral techniques were used for the isolation and identification of naringenin (Nar). MTT, flow cytometry, western blotting, Real Time PCR were used to test anticancer and chemosensitizing effects of Nar, cell cycle, apoptosis, and expression of cell cycle, apoptosis, pro-survival and anti-survival-related genes.ResultsIn the present study, Thymus vulgaris ethanol extract was purified repeatedly to produce several compounds including the known flavanone, Nar which was identified using different spectral techniques. Nar was shown to inhibit both human colorectal and breast cancer cell growth in a dose- and time-dependent manner through cell cycle arrest at S- and G2/M-phases accompanied by an increase in apoptotic cell death. Additionally, Nar altered the expression of apoptosis and cell-cycle regulatory genes by down-regulating Cdk4, Cdk6, Cdk7, Bcl2, x-IAP and c-IAP-2 and up-regulating p18, p19, p21, caspases 3, 7, 8 and 9, Bak, AIF and Bax in both colorectal and breast cancer cells. Conversely, it diminished the expression levels of the cell survival factors PI3K, pAkt, pIκBα and NFκBp65. Moreover, Nar enhanced the sensitivity of colorectal and breast cancer cells to DNA-acting drugs.DiscussionThese findings provide evidence that Nar’s pro-apoptotic and chemo-sensitizing effects are mediated by perturbation of cell cycle, upregulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes and inhibition of pro-survival signaling pathways.ConclusionIn conclusion, Nar might be a promising candidate for chemoprevention and/or chemotherapy of human cancers. However, further studies exploring this therapeutic strategy are necessary.

Syringic acid from Tamarix aucheriana possesses antimitogenic and chemo-sensitizing activities in human colorectal cancer cells

Abstract Context: For its variety of biological activities, Tamarix aucheriana (Decne.) Baum. (Tamaricaceae) has an extensive history as a traditional Arab medicine. Objectives: Antimitogenic and chemo-sensitizing activities of syringic acid (SA) were studied against human colorectal cancer. Materials and methods: Chromatographic and spectral data were used for the isolation and identification of SA. MTT, flow cytometry, in vitro invasion and angiogenesis assays, fluoremetry, ELISA and Real Time qPCR were used to test antimitogenic and chemo-sensitizing activities of SA, cell cycle, apoptosis, proteasome and NFκB-DNA-binding activities, cancer cell invasion and angiogenesis, and expression of cell cycle/apoptosis-related genes. Results: SA showed a time- and dose-dependent (IC50 = 0.95–1.2 mg mL−1) antimitogenic effect against cancer cells with little cytotoxicity on normal fibroblasts (≤20%). SA-altered cell cycle (S/G2-M or G1/G2-M phases) in a time-dependent manner, induced apoptosis, inhibited DNA-binding activity of NFκB (p ≤ 0.0001), chymotrypsin-like/PGPH (peptidyl-glutamyl peptide-hydrolyzing) (p ≤ 0.0001) and the trypsin-like (p ≤ 0.002) activities of 26S proteasome and angiogenesis. SA also differentially sensitized cancer cells to standard chemotherapies with a marked increase in their sensitivity to camptothecin (500-fold), 5FU (20 000-fold), doxorubicin (210-fold), taxol (3134-fold), vinblastine (1000-fold), vincristine (130-fold) and amsacrine (107-fold) compared to standard drugs alone. Discussion: SA exerted its chemotherapeutic and chemo-sensitizing effects through an array of mechanisms including cell-cycle arrest, apoptosis induction, inhibition of cell proliferation, cell migration, angiogenesis, NFκB DNA-binding and proteasome activities. Conclusion: These results demonstrate the potential of SA as an antimitogenic and chemo-sensitizing agent for human colorectal cancer.

Valproic acid, an anti-epileptic drug and a histone deacetylase inhibitor, in combination with proteasome inhibitors exerts antiproliferative, pro-apoptotic and chemosensitizing effects in human colorectal cancer cells: Underlying molecular mechanisms

Although the therapeutic efficacy of valproic acid (VPA) has been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical utility. The present study focused on the development of combined molecular targeted therapies using VPA and proteasome inhibitors (PIs: MG132, PI-1 and PR-39) to determine whether this combination of treatments has synergistic anticancer and chemosensitizing effects against colorectal cancer. Furthermore, the potential molecular mechanisms of action of the VPA/PI combinations were evaluated. The effects of VPA in combination with PIs on the growth of colorectal cancer cells were assessed with regard to proliferation, cell cycle, apoptosis, reactive oxygen species (ROS) generation and the expression of genes that control the cell cycle, apoptosis and pro-survival/stress-related pathways. Treatment with combinations of VPA and PIs resulted in an additive/synergistic decrease in colorectal cancer cell proliferation compared to treatment with VPA or PIs alone. The combination treatment was associated with a synergistic increase in apoptosis and in the number of cells arrested in the S phase of the cell cycle. These events were associated with increased ROS generation, pro-apoptotic gene expression and stress-related gene expression. These events were also associated with the decreased expression of anti-apoptotic genes and pro-survival genes. The combination of VPA with MG132 or PI-1 enhanced the chemosensitivity of the SW1116 (29-185‑fold) and SW837 (50-620-fold) colorectal cancer cells. By contrast, the combination of VPA/PR-39 induced a pronounced increase in the chemosensitivity of the SW837 (16-54-fold) colorectal cancer cells. These data provide a rational basis for the clinical use of this combination therapy for the treatment of colorectal cancer.

Superior antimitogenic and chemosensitization activities of the combination treatment of the histone deacetylase inhibitor apicidin and proteasome inhibitors on human colorectal cancer cells

Despite the effectiveness of histone deacetylase inhibitors, proteasome inhibitors and cytotoxic drugs on human cancers, none of these types of treatments by themselves has been sufficient to eradicate the disease. The combination of different modalities may hold enormous potential for eliciting therapeutic results. In the current study, we examined the effects of treatment with the histone deacetylase inhibitor (HDACI) apicidin (APC) in combination with proteasome inhibitors on human colorectal cancer cells. The molecular mechanisms of the combined treatments and their potential to sensitize colorectal cancer cells to chemotherapies were also investigated. Cancer cells were exposed to the agents alone and in combination, and cell growth inhibition was determined by MTT and colony formation assays. HDAC, proteasome and NF-κB activities as well as reactive oxygen species (ROS) were monitored. Cell cycle perturbation and induction of apoptosis were assessed by flow cytometry. The expression of cell cycle/apoptosis- and cytoprotective/stress-related genes was determined by quantitative PCR and EIA, respectively. The potentiation of cancer cell sensitivity to chemotherapies upon APC/PI combination treatment was also studied. The combination of APC and MG132, PI-1 or epoxomicin potently inhibited cancer cell growth, disrupted the cell cycle, induced apoptosis, decreased NF-κB activity and increased ROS production. These events were accompanied by the altered expression of genes associated with the cell cycle, apoptosis and cytoprotection/stress regulation. The combination treatment markedly enhanced the chemosensitivity of colorectal cancer cells (50-3.7 x 10(4)-fold) in a drug-, APC/PI combination- and colorectal cancer subtype-dependent manner. The results of this study have implications for the development of com-binatorial treatments that include HDACIs, PIs and conventional chemotherapeutic drugs, suggesting a potential therapeutic synergy with general applicability to various types of cancers.