Rajaa Al-Attiyah

Restoration of mycobacterial antigen‐induced proliferation and interferon‐γ responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.

Human Th1 Cell Lines Recognize the Mycobacterium tuberculosis ESAT-6 Antigen and its Peptides in Association with Frequently Expressed HLA Class II Molecules

We have used a synthetic‐peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT‐6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT‐6‐specific CD4+ T‐cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA‐DR‐typed tuberculosis patients with complete antigen in vitro. The established T‐cell lines were then screened for proliferation and interferon‐γ (IFN‐γ) secretion in response to eight overlapping 20‐mer peptides covering the ESAT‐6 sequence. The response of the T‐cell lines to ESAT‐6 and peptides from a human leucocyte antigen (HLA)‐heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti‐HLA class I and class II antibodies suggested that the T‐cell lines recognized ESAT‐6 in association with HLA‐DR and ‐DQ molecules. Furthermore, testing of selected T‐cell lines with ESAT‐6 and the peptides in the presence of autologous and allogeneic HLA‐DR‐ and ‐DQ‐typed antigen‐presenting cells identified HLA‐DR2, ‐DR52 and ‐DQ2 amongst the HLA molecules involved in the presentation of ESAT‐6 and its peptides to human Th1 cells. In addition, the T‐cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT‐6 and peptides. In conclusion, the recognition of ESAT‐6 by IFN‐γ‐secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.

Mammalian Cell-Entry Proteins Encoded by the mce3 Operon of Mycobacterium tuberculosis are Expressed During Natural Infection in Humans

The mammalian cell‐entry (mce)3 operon is one of four homologous mce operons on Mycobacterium tuberculosis genome that encodes six putative invasin/ adhesin‐like proteins (Mce3A–F) possibly involved in the entry and survival of this bacterium inside macrophages. To study the in vivo expression of the mce3 operon‐encoded proteins during natural human infection, the genes encoding Mce3A–F were cloned and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST) at the N‐terminal and a ×6 histidine (His) tag at the C‐terminal end. The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels and reacted with anti‐GST and antipenta‐His antibodies at the expected molecular mass of 72, 61, 78, 80, 66 and 78 kDa for GST‐Mce3A, GST‐Mce3B, GST‐Mce3C, GST‐Mce3D, GST‐Mce3E and GST‐Mce3F, respectively. In Western immunoblots, all the six fusion proteins, particularly GST‐Mce3A, GST‐Mce3C, GST‐Mce3D and GST‐Mce3E, reacted with antibodies in combined human serum from 11 tuberculosis (TB) patients. Pure Mce3A, Mce3D and Mce3E could be isolated by specific proteolytic cleavage by thrombin protease of the respective purified fusion protein followed by preparative SDS‐PAGE. The pure Mce3A, Mce3D and Mce3E reacted to various extents with antibodies in serum samples from TB patients. The Mce3E reacted with 51 of 55 (93%) and all the three proteins reacted with 34 of 55 (62%) serum samples. The Mce3A, Mce3D and Mce3E proteins also reacted, albeit at lower frequency, with one of 23 (4%) serum sample obtained from M. bovis bacillus Calmette–Guérin‐vaccinated healthy subjects and four of 18 (22%) serum samples from long‐term contacts of TB patients showing reactivity with all the three Mce3 proteins. The data show that Mce3A, Mce3D and Mce3E encoded by mce3 operon of M. tuberculosis are expressed and elicit antibody responses in humans during natural infection with this pathogen.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell‐mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette–Guérin (BCG)‐vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen‐induced proliferation and interferon (IFN)‐γ secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8·4, Mtb9·8, Mtb9·9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen‐induced proliferation and IFN‐γ secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9·8 and Mtb39A in proliferation assays (median SI = 6·2 and 6·4, respectively) and of Mtb9·8, Mtb39A, Mtb40 and Ag85B in IFN‐γ assays (median delta IFN‐γ= 15·5, 10·8, 7·8 and 8·1 U/ml, respectively). BCG‐vaccinated healthy donors showed weak (<30% responders) to moderate (31–50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐12] and the immunosuppressive cytokine IL‐10, the complex CF and CW antigens as well as the recombinant Mtb9·8, Mtb9·9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL‐10, while Mtb8·4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL‐10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Mycobacterial antigen-induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non-diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)-vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.

Computer‐Assisted Prediction of HLA‐DR Binding and Experimental Analysis for Human Promiscuous Th1‐Cell Peptides in the 24 kDa Secreted Lipoprotein (LppX) of Mycobacterium tuberculosis

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)‐cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA‐DR molecules, by using a virtual matrix‐based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to ≥10 alleles from more than or equal to three serologically defined HLA‐DR molecules. The Th1‐cell reactivity of all the peptides was assessed in antigen‐induced proliferation and interferon‐γ (IFN‐γ)‐secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette–Guérin (BCG)‐vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA‐DR‐heterogeneous group, responded to one or more peptides of Rv2945c in the Th1‐cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196–220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin‐like exported proteins (Mce3A–F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A–F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A–F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A–F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S‐transferase (GST) as the fusion partner (GST‐Mce3A–F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro‐grown M. tuberculosis cells. The presence of mRNA for mce3A–F genes was also shown by using mce3A–F gene‐specific primers, and total RNA isolated from in vitro‐grown M. tuberculosis cells by reverse transcription‐polymerase chain reaction (RT‐PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT‐PCR confirming that mce3A–F mRNA rather than genomic DNA was being amplified. The data show that Mce3A–F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.

Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes.

Objective: To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference (RD)1, deleted in Mycobacterium bovis Bacille Calmette-Guérin(BCG), by using synthetic peptides and whole blood from tuberculosis (TB) patients. Materials and Methods: Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients (n = 16) attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon-γ (IFN-γ) secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames (ORFs) of RD1 (RD1mix), peptide pools of RD1 ORF5 (ORF5mix), ORF6 (ORF6mix) and ORF7 (ORF7mix), and individual peptides of ORF6 (P6.1–P6.6) and ORF7 (P7.1–P7.6). M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. Results: The complex mycobacterial antigens (culture filtrate, cell walls and M.bovis BCG) and RD1mix induced comparable (p > 0.05) positive antigen-induced proliferation and IFN-γ responses with whole blood from TB patients. However, the positive IFN-γ responses induced by ORF6mix and ORF7mix were higher than ORF5mix. Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN-γ responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. Conclusion: The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions.

c-myc Antisense Oligonucleotides Sensitize Human Colorectal Cancer Cells to Chemotherapeutic Drugs

Background/Aims: Overexpression of the c-myc oncogene frequently occurs in both colon tumors and colon carcinoma cell lines. We examined the sensitization of human colorectal cancer cells to chemotherapeutic drugs using c-myc antisense (AS) phosphorothioate oligonucleotides ([S]ODNs). Methods: Cancer cells were treated with c-myc [S]ODNs, taxol, 5-fluorouracil (5-FU), doxorubicin and vinblastine individually and in combination. The antiproliferative effects, type of interaction between c-myc [S]ODNs and cytotoxic drugs, cell cycle, apoptosis and expression of cell-cycle- and apoptosis-regulatory genes were evaluated. Results: After treatment withc-myc AS[S]ODNs, the growth of cancer cells was markedly inhibited in a dose- and time-dependent manner and the levels of c-myc mRNA and protein were greatly decreased (p < 0.0001). The combinations of c-myc AS[S]ODNs and cytotoxic drugs produced greater growth inhibition of human colorectal cancer cells compared to single treatment with either c-myc AS[S]ODNs (p < 0.006) or cytotoxic drugs (p < 0.0001). These combinations exhibited time- and dose-dependent additive and/or synergistic antiproliferative effects. Cancer cells treated with cytotoxic drugs were growth arrested in the S phase. In contrast, cells treated with either c-myc AS[S]ODNs or by the combination of c-myc AS[S]ODNs and cytotoxic drugs were growth arrested in the G2/M and S phases. The combination treatments also exhibited a marked apoptotic effect compared to single treatments. c-myc AS[S]ODN treatment reduced the mRNA levels of Bcl2, BclxL, cdk2, cyclin E1, cdk1 and cyclin B1, while increasing the mRNA levels of p21, p27, bax and caspase-3. Conclusion: This two-hit approach may be important in the quest to overcome drug resistance in cancer patients whose tumors carry an overexpressed c-myc gene.

Comparative Analysis of Spontaneous and Mycobacterial Antigen‐Induced Secretion of Th1, Th2 and Pro‐Inflammatory Cytokines by Peripheral Blood Mononuclear Cells of Tuberculosis Patients

The cytokines produced by T helper (Th)1 cells (IFN‐γ, IL‐2 and TNF‐β) correlate with protection, whereas the cytokines released by Th2 cells (IL‐4, IL‐5) and the anti‐inflammatory cytokine IL‐10 correlate with pathogenesis of tuberculosis (TB). However, the pro‐inflammatory cytokines (IL‐1β, IL‐6, IL‐8, TNF‐α and IL‐12p70) are responsible for both protection and pathogenesis of TB. The aim of this work was to carry out a comparative analysis of cytokines present in early (day 2) and late (day 6) cultures of peripheral blood mononuclear cells (PBMCs) obtained from pulmonary tuberculosis patients. PBMCs were cultured in vitro in the absence and presence of exogenously added complex mycobacterial antigens and RD1 peptide pool. The supernatants were collected on day 2 and day 6 of culture and assayed for secreted cytokines using the flow cytomix assay. All of the cytokines, except for IL‐12p70, were spontaneously secreted by PBMCs of 27–100% TB patients, but only TNF‐α concentration was significantly higher on day 2 than day 6 (P < 0.05). Two days following antigenic stimulation, only IL‐1β, IL‐6, TNF‐α and IL‐10 were secreted in response to some mycobacterial antigens. However, 6 days later, all of the cytokines, except for IL‐2, IL‐4, IL‐5 and IL‐8, were secreted significantly in response to all complex antigens and RD1 peptides, compared with the non‐stimulated cultures (P < 0.05). In conclusion, the study shows that the longer in vitro stimulation time (6 days) was necessary for the optimal induction of IFN‐γ and TNF‐β, and practically convenient for the detection of IL‐10, IL‐1 β, TNF‐α and IL‐6.