Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody

Pituitary adenomas sometimes show rapid growth and recurrence, and about one third invade the structures surrounding the sella turcica. In an attempt to determine aggressive behavior at an early stage, we used the MIB-1 antibody to identify the Ki-67 antigen. The present study was designed to evaluate pituitary adenomatous tissue in terms of secretion and proliferation and to correlate the Ki-67 index with hormone phenotype and invasive behavior. Material from 159 patients submitted to one or more resections of pituitary adenomas was evaluated. Forty-two non-secretory adenomas and 43 adenomas immunoreactive for growth hormone, 19 for prolactin, 18 for growth hormone and prolactin, 16 for adrenocorticotropic hormone (ACTH), and 21 cases of plurihormonal/gonadotropin adenomas were detected by immunohistochemistry. The MIB-1 antibody was positive in 139 samples and the Ki-67 index ranged from 0.16 to 15.48% (mean = 1.22 ± 2.09%), with no significant difference between genders, age groups, or secretory and non-secretory status. The Ki-67 index was higher in ACTH-secreting adenomas. Invasive pituitary adenomas had a significantly higher Ki-67 index (2.01 ± 3.15%) than macroadenomas with or without supra-sellar extension (1.12 ± 1.87%; P = 0.02). The index was not significantly different in the subgroup of adenomas with invasion of the cavernous sinus compared to groups with other types of invasion. We conclude that tumoral proliferative activity evaluated by the detection of the Ki-67 antigen is significantly higher in invasive than noninvasive adenomas, information which can be useful in therapeutic postoperative management since index cut-off values associated with aggressive behavior can be established.

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7%) and a specificity of 94.7% (95% CI, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Effect of the consumption of tomato paste on plasma prostate-specific antigen levels in patients with benign prostate hyperplasia

The consumption of tomatoes and tomato products has been associated with a reduced risk of prostate cancer. We observed a decrease of 10.77% in prostate-specific antigen (PSA) levels in patients with benign prostate hyperplasia who were submitted to daily ingestion of tomato paste. This was an experimental rather than a controlled study with a sample of 43 men ranging in age from 45 to 75 years, all with histological diagnoses of benign prostate hyperplasia and plasma PSA levels of 4-10 ng/mL. All patients received 50 g of tomato paste once a day for 10 consecutive weeks and PSA levels were analyzed before, during and after the consumption of tomato paste. ANOVA for repeated measures was used to compare PSA levels before, during and after the consumption of tomato paste. The mean ± SD PSA level was 6.51 ± 1.48 ng/mL at baseline and 5.81 ± 1.58 ng/mL (P = 0.005) after 10 weeks. Acceptance was good in 88.3, regular in 9.3, and poor in 2.3% of the patients. Dietary ingestion of 50 g of tomato paste per day for 10 weeks significantly reduced mean plasma PSA levels in patients with benign prostate hyperplasia, probably as a result of the high amount of lycopene in tomato paste. This was not a prostate cancer prevention study, but showed some action of tomato paste in prostate biology. The development of prostate cancer is typically accompanied by an increase in plasma PSA levels, thus any intervention that affects plasma PSA levels can suggest an impact in the progression of disease.

Agent-based management systems for many-core platforms : rigorous design and efficient implementation

Due to various advantages such as flexibility, scalability and updatability, software intensive systems are increasingly embedded in everyday life. The constantly growing number of functions executed by these systems requires a high level of performance from the underlying platform. The main approach to incrementing performance has been the increase of operating frequency of a chip. However, this has led to the problem of power dissipation, which has shifted the focus of research to parallel and distributed computing. Parallel many-core platforms can provide the required level of computational power along with low power consumption. On the one hand, this enables parallel execution of highly intensive applications. With their computational power, these platforms are likely to be used in various application domains: from home use electronics (e.g., video processing) to complex critical control systems. On the other hand, the utilization of the resources has to be efficient in terms of performance and power consumption. However, the high level of on-chip integration results in the increase of the probability of various faults and creation of hotspots leading to thermal problems. Additionally, radiation, which is frequent in space but becomes an issue also at the ground level, can cause transient faults. This can eventually induce a faulty execution of applications. Therefore, it is crucial to develop methods that enable efficient as well as resilient execution of applications. The main objective of the thesis is to propose an approach to design agentbased systems for many-core platforms in a rigorous manner. When designing such a system, we explore and integrate various dynamic reconfiguration mechanisms into agents functionality. The use of these mechanisms enhances resilience of the underlying platform whilst maintaining performance at an acceptable level. The design of the system proceeds according to a formal refinement approach which allows us to ensure correct behaviour of the system with respect to postulated properties. To enable analysis of the proposed system in terms of area overhead as well as performance, we explore an approach, where the developed rigorous models are transformed into a high-level implementation language. Specifically, we investigate methods for deriving fault-free implementations from these models into, e.g., a hardware description language, namely VHDL.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2%, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95%CI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Distribution of the human leukocyte antigen class II alleles in Brazilian patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a global medical problem. The current standard of treatment consists of the combination of peginterferon plus ribavirin. This regimen eradicates HCV in 55% of cases. The immune response to HCV is an important determinant of disease evolution and can be influenced by various host factors. HLA class II may play an important role in immune response against HCV. The objective of the present study was to determine the distribution of HLA class II (DRB1 and DQB1) alleles, their association with chronic HCV infection and their response to interferon therapy. One hundred and two unrelated white Brazilian patients with chronic HCV infection, 52 responders (45 males and 7 females) and 50 non-responders (43 males and 7 females) to antiviral treatment, were included in the study. Healthy Brazilian bone marrow donors of Caucasian origin from the same geographic area constituted the control group (HLA-DRB1, N = 99 and HLA-DQB1, N = 222 individuals). HLA class II genotyping was performed using a low-resolution DRB1, DQB1 sequence-specific primer amplification. There were higher frequencies of HLA-DRB1*13 (26.5 vs 14.1%) and HLA-DQB1*02 (52.9 vs 38.7%) in patients compared with controls; however, these were not significantly different after P correction (Pc = 0.39 and Pc = 0.082, respectively). There was no significant difference between the phenotypic frequencies of HLA-DRB1 (17.3 vs 14.0%) and HLA-DQB1 alleles in responder and non-responder HCV patients. The HLA-DRB1*07 allele was significantly more common in HCV patients (33.3 vs 12.1%) than in controls (Pc = 0.0039), suggesting that the HLA-DRB1*07 allele is associated with chronic HCV infection.

Effect of the human leukocyte antigen HLA-DRB1 and anti-cyclic citrullinated peptide on the outcome of rheumatoid arthritis patients

Our objective was to determine whether the presence of the human leukocyte antigen HLA-DRB1 locus is associated with production of anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) and to what extent they are associated with increased susceptibility to and severity of rheumatoid arthritis (RA) in Egyptian patients. Twenty-nine RA patients gave informed consent to participate in a case-control study that was approved by the Ain Shams University Medical Ethics Committee. RA disease activity and severity were determined using the simplified disease activity index and Larsen scores, respectively. We used a wide scale national study on the pattern of HLA typing in normal Egyptians as a control study. Anti-CCP Abs and HLA-DRB1 typing were determined for all subjects. The alleles most strongly associated with RA were HLA-DRB1 [*01 , *04 and *06] (41.4%). RA patients with serum anti-CCP Ab titers above 60 U/mL had a significantly higher frequency of HLA-DRB1*01 (58.3%) and HLA-DRB1*04 alleles (83.3%). Significant positive correlations were found between serum and synovial anti-CCP Ab titer, RA disease activity, and severity (r = 0.87, 0.66 and 0.63, respectively; P < 0.05). HLA-DRB1 SE+ alleles [*01 and *04] were highly expressed among Egyptian RA patients. The presence of these alleles was associated with higher anti-CCP Ab titer, active and severe RA disease. Early determination of HLA-DRB1 SE+ alleles and serum anti-CCP Ab could facilitate the prediction of the clinical course and prognosis of RA when first evaluated leading to better disease control.

High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Co-γ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, c-myc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Prostate-specific membrane antigen can promote in vivo osseous metastasis of prostate cancer cells in mice

Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm²) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm²). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

HLA-DR3 antigen in the resistance to idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDC) has been hypothesized as a multifactorial disorder initiated by an environment trigger in individuals with predisposing human leukocyte antigen (HLA) alleles. Published data on the association between HLA-DR3 antigen and IDC risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Studies were identified by searching the PUBMED and Embase database (starting from June 2015). A total of 19 case-control studies including 1378 cases and 10383 controls provided data on the association between HLA-DR3 antigen and genetic susceptibility to IDC. Overall, significantly decreased frequency of HLA-DR3 allele (OR=0.72; 95%CI=0.58-0.90; P=0.004) was found in patients with IDC compared with controls. When stratified by myocardial biopsy or non-biopsy cases, statistically decreased risk was found for IDC in myocardial biopsy cases (OR=0.69; 95%CI=0.57-0.84; P=0.0003). In the subgroup analysis by ethnicity, borderline statistically significantly decreased risk was found among Europeans from 12 case-control studies (OR=0.76; 95%CI=0.58-1.00; P=0.05). In conclusion, our results suggest that individuals with HLA-DR3 antigen may have a protective effect against IDC.