Mahmood Jeddi-Tehrani

Durable Carcinoembryonic Antigen (CEA)-Specific Humoral and Cellular Immune Responses in Colorectal Carcinoma Patients Vaccinated with Recombinant CEA and Granulocyte/Macrophage Colony-Stimulating Factor

Purpose: Previous studies have indicated that carcinoembryonic antigen (CEA) might be a suitable immunotherapeutic target in colorectal carcinoma (CRC). The aim of the present study was to analyze the immunological and clinical effects of vaccination with CEA together with the adjuvant granulocyte/macrophage colony-stimulating factor (GM-CSF). Experimental Design: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with recombinant CEA at four different dose levels over a 12-month period. Half of the patients received GM-CSF (80 μg/day for 4 consecutive days) at each immunization. Patients were monitored immunologically for 36 months and clinically for 76 months. T-cell response was evaluated by a [3H]thymidine incorporation assay, and IgG response was determined by ELISA. Results: Minor local side effects were common. All 12 patients (100%) in the GM-CSF group developed a CEA-specific T-cell as well as an IgG response. The corresponding figures in the CEA alone group were 9 of 12 (75%) and 8 of 12 (66%), respectively. GM-CSF significantly augmented the amplitude of the T-cell response and the IgG titers. No dose–response relationship was noted. The immune responses at 12 months persisted 24 months after the last vaccination. Anti-CEA IgG titers were associated with increased survival (P < 0.05), whereas standard prognostic factors had no relationship, with the exception of serum CEA value. Conclusions: Vaccination with recombinant CEA and GM-CSF appears to be a nontoxic regimen inducing potent and durable antigen-specific IgG and T-cell response. The results of this study justify more extensive trials with recombinant CEA protein for immunotherapy of CRC.

Peripheral blood T cell expansions in patients with Behçet's disease

Behc¸ets disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) Vα/Vβ gene product expression as well as Jβ gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V‐specific MoAbs was used for TCRV analyses. Jβ gene segment usage by T cell populations expressing certain Vβs was determined by polymerase chain reaction (PCR) technique with Vβ‐ and Cβ‐specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jβ gene segment‐specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V‐gene products, only expansions among the CD4+ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jβ gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen‐specific T lymphocytes in the pathogenesis of BD.

A persistent T cell expansion in the peripheral blood of a normal adult male: a new clinical entity?

A dramatic and persistent T cell expansion in a healthy adult male was initially identified, using anti‐T cell receptor for antigen (TCR)‐specific MoAbs. The expanded T cells were found to be expressing TCR containing Vα 12.1 and V β 5.2, and they composed approximately one third of all the CD8+ T cells. The cells were shown to be not only non‐activated (HLA‐DR−, IL‐2R−) but also of ‘virgin’ cell type (CD45RA+/CD45RO−) and they persisted over the observation period of more than one and a half years. Various T and B cell markers, and all other laboratory and physical parameters analysed, were normal. The expanded CD8+ T cells were further characterized by polymerase chain reaction (PCR) amplification, using Vβ‐ and Cβ‐specific primers, followed by hybridization with Jβ‐specific probes. Close to 90% of the Vα 12.1+ Vβ 5.2+ T cells were found to utilize the Jβ 2.5 gene segment, thus strongly suggesting the expanded T cells to be monoclonal. The condition may constitute a T cell counterpart to ‘monoclonal gammopathy of undetermined significance’ (MGUS), and by analogy we suggest it should be designated ‘monoclonal T cell expansion of undetermined significance’ (MTUS).

Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real‐time reverse transcription polymerase chain reaction (RT‐PCR) revealed that granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were γ‐interferon (IFN), interleukin (IL)‐2 and tumour necrosis factor (TNF)‐α, but not IL‐4. This profile suggests a type 1 anti‐B‐CLL T‐cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T‐cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T‐cell DNA synthesis proliferation. The proliferative T‐cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T‐cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T‐cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti‐CLL T‐cell immunity is warranted that may facilitate the development of effective anti‐tumour vaccines in CLL.

The citrus flavonoid hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in NALM-6 cells: involvement of PPARγ-dependent mechanism

BackgroundHesperidin, a flavanone present in citrus fruits, has been identified as a potent anticancer agent because of its proapoptotic and antiproliferative characteristics in some tumor cells. However, the precise mechanisms of action are not entirely understood.AimThe main purpose of this study is to investigate the involvement of peroxisome proliferator-activated receptor-gamma (PPARγ) in hesperidin’s anticancer actions in human pre-B NALM-6 cells, which expresses wild-type p53.MethodsThe effects of hesperidin on cell-cycle distribution, proliferation, and caspase-mediated apoptosis were examined in NALM-6 cells in the presence or absence of GW9662. The expression of peroxisome proliferator-activated receptor-gamma (PPARγ), p53, phospho-IκB, Bcl-2, Bax, and XIAP proteins were focused on using the immunoblotting assay. The transcriptional activities of PPARγ and nuclear factor-kappaB (NF-κB) were analyzed by the transcription factor assay kits. The expression of PPARγ and p53 was analyzed using the RT-PCR method.ResultsHesperidin induced the expression and transcriptional activity of PPARγ and promoted p53 accumulation and downregulated constitutive NF-κB activity in a PPARγ-dependent and PPARγ-independent manner. The growth-inhibitory effect of hesperidin was partially reduced when the cells preincubated with PPARγ antagonist prior to the exposure to hesperidin.ConclusionsThe findings of this study clearly demonstrate that hesperidin-mediated proapoptotic and antiproliferative actions are regulated via both PPARγ-dependent and PPARγ-independent pathways in NALM-6 cells. These data provide the first evidence that hesperidin could be developed as an agent against hematopoietic malignancies.

Thrombophilic mutations in Iranian patients with infertility and recurrent spontaneous abortion.

Factor V Leiden (FVL) G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T, and factor II (FII) G20210A mutations are three important causes of thrombophilia, the condition that might be related to infertility and recurrent spontaneous abortion (RSA). In this study we evaluated the presence of these three mutations in 36 female patients with unexplained infertility, 65 female patients with unexplained RSA, and 62 healthy fertile women as control group. DNA was extracted from peripheral blood samples and PCR-RFLP was performed for the molecular diagnosis of each mutation. In addition, activated protein C resistance (APC-R) was also evaluated. The frequencies of FVL, MTHFR, and FII mutations (heterozygous and homozygous) in the control group were 0.0%, 38.7%, and 3.2%, respectively. The frequency of FVL mutation in patients with infertility (30.6%) or RSA (20.0%) was significantly higher than that of the control group. A significantly higher MTHFR mutation rate was also observed in patients with RSA (63.1%) as compared to controls. However, the mutation rate of MTHFR in patients with infertility (50.0%) was not statistically different from that in controls. No significant difference was observed in the frequencies of FII mutations between the patients and controls. Decreased levels of APC-R were observed in 25.0% of infertile patients and 18.9% of patients with RSA. In conclusion, our results show a skew towards higher mutation frequencies of FVL and MTHFR in patients that may necessitate detection of such mutations in these Iranian patients.

Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B‐CLL)

Abstract: Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B‐CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL‐2, IL‐4 and GM‐CSF as compared to healthy donors and patients with non‐progressive CLL, which was not the case for TNF‐α and IFN‐γ producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non‐progressive disease to control donors. Polyclonal activation of B‐CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM‐CSF, TNF‐α and IL‐4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine‐producing T cells was noted in patients with progressive disease as compared to those with non‐progressive disease. The augmented number of cytokine‐producing T cells in CLL may indicate an up‐regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM‐CSF, TNF‐α, IL‐4 and IL‐2 is interesting, as these cytokines have previously been shown to support growth of B‐CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo. The findings may suggest a role for T cells in the pathogenesis of B‐CLL.

The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments.

The rapidly growing applications of antibody-based therapeutics requires novel approaches to develop efficient drug delivery systems in which biodegradable polymeric nanoparticles are amongst the best candidates. In the present study bevacizumab loaded PLGA nanoparticles were formulated by water-in-oil-in-water emulsion method. Protein inactivation and aggregation are the major drawbacks of this technique. Therefore protective ability of various stabilizers was studied during entrapment process. Probable changes in VEGF₁₆₅ binding capability of bevacizumab was assayed by ELISA which portrays the antibodys bio-efficiency. Probable breakage of bevacizumab and its secondary and tertiary structural integrity upon entrapment were analyzed by SDS-PAGE and circular dichroism spectroscopy, respectively. In vitro and ex vivo released bevacizumab from the prepared nanoparticles was also investigated. Results revealed that the protein interfacial adsorption is the foremost destabilizing factor in the double emulsion method and incorporation of appropriate concentrations of albumin could protect bevacizumab against entrapment stress. Ex vivo release results, in rabbit vitreous, indicated the ability of prepared nanoparticles in prolonged release of the active antibody. Consequently this approach was an attempt to achieve sustained release PLGA nanoparticle formulation with the aim of protecting integrity and performance of entrapped bevacizumab.

Kinetics of Cytokine Gene Expression in Human CD4+ and CD8+ T-Lymphocyte Subsets Using Quantitative Real-Time PCR

The time kinetics of five cytokines [interleukin‐2 (IL‐2), IL‐5, interferon‐γ (IFN‐γ), granulocyte macrophage‐colony stimulating factor (GM‐CSF) and tumour necrosis factor‐α (TNF‐α)] and one cytotoxic effector protein (granzyme B) was analysed by real‐time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T‐cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell‐shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD‐activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4–8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48–72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen‐specific T lymphocytes. It might be recommended that cytokine gene expression (IL‐2, IL‐5 and IFN‐γ) should be measured after 4–8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48–72 h of activation.

HIV infection leads to differential expression of T-cell receptor V-beta genes in CD4+ and CD8+ T cells

ObjectiveTo analyse variation in T-cell receptor (TCR) Vβ gene expression in T cells in HIV-infected individuals. DesignBecause there are very few monoclonal antibodies available for studying TCR VP gene expression, we used polymerase chain reaction (PCR) to analyse the TCR Vβ repertoire in HIV-infected individuals using specific primers for 20 distinct families of TCR Vβ. MethodsEvaluation of TCR Vβ gene expression in peripheral blood from HIV-1-infected individuals [two in Centers for Disease Control (CDC) stage II, five in CDC stage III and four in CDC stage IV]. Complementary DNA was produced from CD4+ and CD8 + T cells, amplified by PCR and analysed after Southern blotting and hybridization with a Cp-specific oligonucleotide probe. ResultsVβ gene expression was dramatically modified, especially in AIDS patients. The CD4+ T-cell subset showed both overexpression (Vβ2) and deletions or underexpression (Vβ9-Vβ20), whereas these gene segments were expressed normally in the CD8+ subset. Only Vβ 3 was deleted or underexpression in both CD4+ and CD8+ populations in AIDS patients. ConlusionsHIV-1 infection induces CD4+ T-cell deficiency, both in total numbers and by causing a paucity of select Vβ gene expression in this subset. In addition, the Vβ3 gene family was deleted or underexpressed was observed in both CD4+ and CD8 + T-cell subsets from patients in CDC stage IV. These results are compatible with changes in Vβ gene expression known to occur under the action of endogenous or exogenous superantigens.