Salivary gland antigens of laboratory-bred Phlebotomus sergenti and their immunogenicity in human volunteers in laboratory condition

To investigate Phlebotomus (P.) sergenti Parrot, 1917 (Diptera: Psychodidae) salivary gland antigens and their immune response in human. Methods: Human volunteers were exposed to sand flies' bites in the laboratory, and following each exposure the size of induration was recorded. The mean protein concentration of salivary gland lysate and specific anti-P. sergenti saliva IgG was measured. Sand fly salivary proteins were separated by SDS-PAGE and their immunoreactivity was examined by Western blotting assays. Results: Individuals exposed to P. sergenti salivary gland lysate for 8 months showed both antibody and delayed type hypersensitivity responses, although exposure for one month did not provoke any immune responses. The trend of antibody fluctuated during the exposure time and dropped by the end of antigen loading. The mean protein content was (0.36?0.08) ug in each pair salivary glands. Salivary gland lysate showed 11 to 12 major protein bands and 3 to 6 of them were immunoreactive. Conclusions: Our study showed that the salivary gland components of P. sergenti provoked both cellular and humoral immune responses in human. Furthermore, there are some immunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Polymorphisms in the estrogen receptor beta gene and the risk of unexplained recurrent spontaneous abortion

Background: Recurrent Spontaneous Abortion [RSA] is caused by multiple genetic and non-genetic factors. Around 50% of the RSA cases have no known etiology and are considered as Unexplained RSA [URSA]. Estrogens, via binding to their receptors, play an important role in female reproduction. This study aimed to investigate whether single nucleotide polymorphisms [SNPs; +1082G/A, +1730G/A and rs1256030C/T] in the estrogen receptor beta [ESR2] gene are associated with susceptibility to URSA in a population of Iranian women

Methods: In this case-control study, the study groups consisted of 240 subjects with a history of URSA and 102 fertile women as controls. Serum levels of follicle stimulating hormone [FSH], luteinizing hormone [LH], and estradiol [E2] were measured on day 2-3 of menstrual cycle. Two functional SNPs, +1082G/A [a silent mutation in exon 5] and +1730G/A [3' untranslated region of the exon 8], and one intron, rs1256030C/T, in the ESR2 gene were genotyped, using polymerase chain reaction- restriction fragment length polymorphism [PCR-RFLP] analysis

Results: Serum levels of LH were significantly increased in URSA women. No significant differences in distribution of +1082G/A, +1730G/A and rs1256030C/T between URSA and control groups were observed

Conclusion: Our findings suggest that the studied SNPs on ESR2 gene may not be associated with URSA

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in male partners of recurrent miscarriage couples

Methylenetetrahydrofolate reductase [MTHFR] single-nucleotide polymorphisms [SNPs] C677T and A1298C have been described as strong risk factors for idiopathic recurrent miscarriage [RM]. However, very few studies have investigated the association of paternal MTHFR SNPs with RM. The aim of the present study was to evaluate the prevalence of paternal C677T and A1298C SNPs among Iranian RM couples. The study subjects comprised 225 couples with more than three consecutive pregnancy losses, and 100 control couples with no history of pregnancy complications. All females in the case group had MTHFR polymorphisms; and genotype SNPs were analyzed by PCR-RFLP. Groups were statistically compared using Mann Whitney U-test and Chi-square statistical tests. The p < 0.05 were considered significant. Statistically significant difference was detected in the frequency of MTHFR SNPs in male partners of the two groups [p=0.019]. Combined heterozygosity of MTHFR polymorphisms was a common phenomenon in the males; 52 [23.1%] and 14 [14%] of males in RM and control groups, respectively. Absence of combined homozygosity for both SNPs in all studied groups/genders was observed. The MTHFR gene composition of male partners of RM couples may contribute to increased risk of miscarriage

effect of Setarud [IMOD[TM]] on angiogenesis in transplanted human ovarian tissue to nude mice

One of the promising methods in fertility preservation among women with cancer is cryopreservation of ovarian cortex but there are many drawbacks such as apoptosis and considerable reduction of follicular density in the transplanted ovary. One solution to reduce ischemic damage is enhancing angiogenesis after transplantation of ovarian cortex tissue. The aim of this study was to investigate the effect of Setarud, on angiogenesis in transplanted human ovarian tissue. In this case control study, twenty four nude mice were implanted subcutaneously, with human ovarian tissues, from four women. The mice were randomly divided into two groups [n=12]: the experimental group was treated with Setarud, while control group received only vehicle. Each group was divided into three subgroups [n=4] based on the graft recovery days post transplantation [PT]. The transplanted fragments were removed on days 2, 7, and 30 PT and the expression of Angiopoietin-1, Angiopoietin-2, and Vascular endothelial growth factor at both gene and protein levels and vascular density were studied in the grafted ovarian tissues. On the 2[nd] and 7[th] day PT, the level of Angiopoietin-1 gene expression in case group was significantly lower than that in control group, while the opposite results were obtained for Angiopoietin-2 and Vascular endothelial growth factor. These results were also confirmed at the protein level. The density of vessels in Setarud group elevated significantly on day 7 PT compared to pre-treatment state. Our results showed that administration of Setarud may stimulates angiogenesis in transplanted human ovarian tissues, although further researches are needed before a clear judgment is made

Production and characterization of monoclonal antibodies against human prostate specific antigen

Prostate Specific Antigen [PSA] is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies [mAbs] against PSA have been presented. Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer [PC alpha], Benign Prostatic Hyperplasia [BPH] and brain cancer tissues by Immunohistochemistry [IHC]. Five anti-PSA mAbs [clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/K] and clones [2C8-E9, 2G3-E2, IgG2 alpha/K] were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kD alpha in human seminal plasma in western blot. These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids

Lipopolysaccharide- and lipoteichoic acid-mediated pro-inflammatory cytokine production and modulation of tlr2, tlr4 and myd88 expression in human endometrial cells

Toll-like receptor [TLR]-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells [ESCs] and whole endometrial cells [WECs] to lipopolysaccharide [LPS] and lipoteichoic acid [LTA] Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr [p<0.05]. At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs [p<0.05]. LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner [p<0.05]. Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-alpha in response to LPS activation [p<0.05]. Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus

Effect of administration of single dose GnRH agonist in luteal phase on outcome of ICSI-ET cycles in women with previous history of IVF/ICSI failure: a randomized controlled trial

GnRH agonist administration in the luteal phase has been suggested to beneficially affect the outcome of intracytoplasmic sperm injection [ICSI] and embryo transfer [ET] cycles. This blind randomized controlled study evaluates the effect of GnRH [Gonadotropine Releasing Hormone] agonist administration on ICSI outcome in GnRH antagonist ovarian stimulation protocol in women with 2 or more previous IVF/ICSI-ET failures. One hundred IVF failure women who underwent ICSI cycles and stimulated with GnRH antagonist ovarian stimulation protocol, were included in the study. Women were randomly assigned to intervention [received a single dose injection of GnRH agonist [0.1 mg of Decapeptil] subcutaneously 6 days after oocyte retrieval] and control [did not receive GnRH agonist] groups. Implantation and clinical pregnancy rates were the primary outcome measures. Although the age of women, the number of embryos transferred in the current cycle and the quality of the transferred embryos were similar in the two groups, there was a significantly higher rate of implantation [Mann Whitney test, p=0.041] and pregnancy [32.6% vs. 12.5%, p=0.030, OR=3.3, 95%CI, 1.08 to 10.4] in the in-tervention group. Our results suggested that, in addition to routine luteal phase support using progesterone, administration of 0.1 mg of Decapeptil 6 days after oocyte re-trieval in women with previous history of 2 or more IVF/ICSI failures led to a signif-icant improvement in implantation and pregnancy rates after ICSI following ovarian stimulation with GnRH antagonist protocol

Investigation on estrogen receptor alpha gene polymorphisms in Iranian women with recurrent pregnancy loss

Recurrent pregnancy loss [RPL] is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes. Conflicting data suggest an association between estrogen receptor alpha [ESR1] gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL. In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment length polymorphism [PCR-RFLP], we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects. The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups [p=0.20 and p=0.09, respectively]. A significantly negative correlation was observed between -397C/T and -351A/G [r=-0.852, p<0.001] in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found [D': 0.959; r-square= 0.758, p<0.001]. This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population

effect of sortilin silencing on ovarian carcinoma cells

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin [NTR3], the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis [27.5 +/- 0.48%] in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation [40.1%] in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart [p<0.05]. As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma

McL[-1] is ip regulated by prenylated coumarin, umbelliprenin in jurkat cells

Chronic lymphocytic leukaemia [CLL] is the most common B-cell malignancy in the western world and exists as subtypes with very different clinical courses. Myeloid cell leukemia 1 [McL[-1]] is one member of Bcl-2 family proteins that has been shown to be expressed in various tissues and malignant cells, including CLL, where its expression is significantly associated with a failure to achieve complete remission following cytotoxic therapy. Induction of apoptosis by prenylated coumarin, umbelliprenin, in Jurkat cells was previously shown. We examined whether umbelliprenin can down-regulate McL[-1] gene and protein in Jurkat cells. In this regard cells were incubated by umbelliprenin, and then down- regulation of McL[-1] gene was studied by Real Time PCR method. Moreover, down-regulation of McL[-1] protein was studied by western blot analysis. We showed that, expression of McL[-1] mRNA was increased from 1 hour to 3 hours incubation, but this increase has a scale down pattern. Moreover umbelliprenin could inhibit McL[-1] protein. In conclusion umbelliprenin treatment modulates McL[-1] expression at both the transcriptional and posttranslational levels

Sero-prevalence of antibodies against varicella zoster virus in children under seven-years old in 2012 in Tehran, Iran

Varicella zoster virus [VZV] is a member of herpes family viruses, which causes varicella [chickenpox] after primary infection and herpes zoster [shingles] because of latent virus reactivation from dorsal root ganglia. Generally, prevalence of varicella antibodies increases with age. We aimed to compare the prevalence of anti-VZV antibody in children under seven years old, in order to obtain a preliminarily picture of general presence of these antibodies to design an immunization plan. In this cross-sectional study, performed from September 2011 to September 2012 in Tehran, Iran, 267 serum samples including sera from 7 month old infants, n= 87; 18 month old children, n= 86; and 6 year old children, n= 94 were assessed for the presence of specific IgG antibodies against VZV, using ELISA technique. 4.6% of 7 month, 12.8% of 18 month and 21.3% of 6-year-old children were seropositive. No relation was found between demographic variables [e.g. age and birth weight] and seropositivity in these age groups. VZV antibodies increased with age. Serum levels of varicella antibodies were elevated in 18 months old compared to 7 months old children, significantly [P < 0.001]. In view of the significant elevation of VZV antibodies in children from 7 months to 18 months of age and rate of seronegative children, our results support the necessity of varicella immunization between 7 and 18 months of age in order to prevent viral infection

novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells

Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem cells [HSCs] and the small- vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a marker for diagnosis and classification of leukemia. Anti CD34 antibodies are used for isolation and purification of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody [Clone 2C10-D3] was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 [Human Erythroblast] cell line. ELISA experiment revealed that the antibody recognized CD34 peptide. Western blot analysis on TF1 cell lysate confirmed the reactivity of the antibody with a 42 Kda protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody. Our data suggest that the anti CD34 monoclonal antibody [Clone 2C10-D3] is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

Production and characterization of a marine monoclonal antibody against human ferritin

Ferritin is an iron storage protein, which plays a hey role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody [mAb] against human ferritin was reported. Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma [clone: 2F9-C9] was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity [2.34 10[9] A/[1]] and the isotype was determined to be lgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic bit if other requirements of the hit are met

Propagation of human germ stem cells in long-term culture

Spermatogonial stem cells [SSCs], a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. The aim of this study was in vitro propagation of human spermatogonial stem cells [SSCs] and improvement of presence of human Germ Stem Cells [hGSCs] were assessed by specific markers POU domain, class 5, transcription factor 1 [POU5F1], also known as Octamer-binding transcription factor 4 [Oct-4] and PLZF [Promyelocytic leukaemia zinc finger protein]. Human testicular cells were isolated by enzymatic digestion [Collagenase IV and Trypsin]. Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions

Cloning, expression and characterization of recombinant human Fc receptor like 1, 2 and 4 molecules

The Fc receptor like [FCRL] molecules belong to the immunoglobulin [Ig] superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identified for the human FCRL1, 2 and 4 molecules. Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b [+] and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies. Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b [+] vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37[degree]C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% [FCRL1] to 25% [FCRL2 and 4] of the total bacterial lysate proteins. These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.

vitro culture of human testicular stem cells on feeder-free condition

Spermatogonial stem cells are subpopulation of spermatogonial cells in testis tissue that support beginning and maintenance of spermatogenesis. Ubiquitin carboxy-terminal hydrolase L1 [UCHL1] could be a specific marker for identification of spermatogonial stem cells including spermatogonial sperm cells [SSCs] in testis tissue and during the culture; therefore we undertook this study to culture these human testicular stem cells [hTSCs] in vitro and approved the presence of human testicular stem cells [hTSCs] by UCHL1, also known as PGP9.5. Enzymatic digestion of human testicular biopsies was done by collagenase IV [4 mg/ml] and trypsin [0.25%]. Differential plating of testicular cells in DMEM/F12 and 10% FBS was applied for 16 hr. Floating cells were collected and transferred onto laminin-coated plates with Stem-Pro 34 media supplemented with growth factors of GDNF, bFGF, EGF and LIF to support self-renewal divisions; testicular stem cell clusters were passaged every 14 days for two months. Spermatogonial cells propagation was studied through Expression of UCHL1 in testis tissue and the entire testicular stem cell culture. Testicular stem cell clusters from 10 patients with obstructive azoospermia were cultured on laminin-coated plates and subsequently propagated for two months. The average of harvested viable cells was approximately 89.6%. UCHL1 was expressed as specific marker in testicular stem cells entire the culture. Human testicular stem cells could be obtained from human testicular tissue by a simple digestion, culturing and propagation method for long-term in vitro conditions. Propagation of these cells approved by specific marker UCHL1, during the culture period

Effects of Chlamydia trachomatis infection on fertility; a case-control study

Nowadays, Chlamydia trachomatis is known as a causative agent of infertility. Because of, asymptomatic nature of infection, many may suffer from its lasting complications such as infertility. This study was performed in Tehran during April 2007 to April 2008 to compare the prevalence of Chlamydia trachomatis infection in fertile and infertile women using ELISA and PCR methods. Overall, 234 infertile and 223 pregnant women, as the fertile group, participated in this hospital-based case-control study. After completing an informed consent form and the questionnaire, first catch urine and blood sample were obtained for PCR and ELISA [IgG, IgM] tests, respectively. Logistic regression analysis was used to control possible confounding factors, and determine adjusted odds ratio of infertility due to the infection. PCR results revealed that 29 [12.4%] of the infertile and 19 [8.5%] of the fertile women were positive for C. trachomatis infection [p=0.440]. IgG was positive in 21 [9.0%] of the infertile and 11 [5.0%] in the fertile group [p=0.093]. IgM assays identified that 2 [0.9%] of the infertile and 4 [1.8%] of the fertile women were positive for the micro-organism [p=0.375]. We found no significant differences among fertile and infertile women for Chlamydia trachomatis infection. Nevertheless, molecular techniques which are more sensitive, more specific and non-invasive can be used to detect C. trachomatis infection

Immune reactivity of Brucella melitensis-vaccinated rabbit serum with recombinant Omp31 and DnaK proteins

Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East. In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein [DnaK] and an outer membrane protein [Omp31] of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis. It was proved that immunized serum contains antibodies against recombinant Omp31 [rOmp31] and DnaK [rDnaK] by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy

Expression, purification and characterization of three overlapping immunodominant recombinant fragments from Bordetella pertussis filamentous hemagglutinin

Filamentous hemagglutinin [FHA] is one of the most important immunoprotective antigens of Bordetella pertussis [B. pertussis] and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli [E. coli] BL21[DE3] strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells [PBMC] proliferation and IFN- gamma production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21[DE3]. SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN- gamma production. Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immunoprotective

Umbelliprenin from Ferula szowitsiana activates both intrinsic and extrinsic pathways of apoptosis in jurkat T-CLL cell line

Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest [Golestan Province, north of Iran]. Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases [caspase-8 and -9] and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway