Abstract: The asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune dysfunction and AIDS symptoms. We have previously demonstrated that the presence of specific gp120/V3 peptides during antigen presentation can modify the activation of normal T-cells leading to altered immune function. The aim of the present study was to map the specific transcriptional profile invoked by an HIV-1/V3 epitope in uninfected T cells during antigen presentation.
Abstract: Paraoxonases and cytochromes P450 constitute two major classes of xenobiotic-metabolizing enzymes involved in the detoxification of pesticide chemicals. In this study, we examined the distribution of two common genetic polymorphisms of the paraoxonase 1 gene and one common polymorphism of the CYP1A1 gene, in relation to pathological diseases occurring in a rural population. Blood and hair samples were collected from 220 participants of an agricultural cohort in the south of Greece for genotype and pesticide analysis. Demographic information and disease status of the participants was obtained by questionnaire, medical examination and medical record. Organochlorine pesticides and metabolites (DDTs, HCHs) were extracted from hair and analyzed using gas chromatography combined with mass spectrometry techniques. Our results indicate exposure of the rural population of Amaliada to organophosphate and past exposure to organochlorine pesticides. Genotypic analysis of PON1Q192R, PON1L55M and CYP1A1*2A MspI polymorphisms was performed using PCR-RFLP. The PON1 192R and 55M alleles absence was significantly associated with hypertension (OR: 2.59; 95% CI: 1.10-6.09) and hepatitis (OR: 21.43; 95% CI: 2.53-181.50), respectively, as indicated from backward logistic regression. Although the presence of PON1 192R allele significantly affected the occurrence of prostate hyperplasia (OR: 0.35; 95% CI: 0.03-0.40), no associations were obtained between the paraoxonase serum activity or the CYP1A1 genotype and the disease status.
Abstract: Serum paraoxonase 1 (PON1) function has been associated with human cardiovascular disease. The projected mechanism postulates interaction of PON1 with lipoproteins and insulin signaling resulting in alterations in lipid homeostasis. Recently, PON2 was shown to directly regulate triglyceride accumulation in macrophages and PON1 was detected in the interstitial space of adipocytes. The aims of the present study were a) to examine the relationship of the PON1 function with serum parameters related to lipid homeostasis, and b) to examine a possible role of PON1 in the regulation of lipid composition in the human adipose tissue. Two important genetic variations with functional impact on PON1 activity in humans are the Q192R and the L55M. The present study evaluated the impact of the Q192R and the L55M polymorphisms in a cross-section of the population on the island of Crete, as regards to PON1 activity, plasma lipids/lipoproteins, parameters of the metabolic syndrome, and the fatty acid composition of the adipose tissue. We detected a significant association of the polymorphisms with blood pressure, fasting blood glucose, triglycerides, apolipoprotein B, serum iron, and homocysteine. Furthermore, a novel function is suggested for PON1 on the fatty acid composition in the adipose tissue through the positive association of the R allele with saturated fatty acid and of the Q allele with 20:5n3 fatty acid deposition.
Abstract: OBJECTIVES: Heparin acts as an extracellular stimulus capable of activating major cell signalling pathways. Thus, we examined the putative mechanisms utilized by heparin to stimulate HT29, SW1116 and HCT116 colon cancer cell growth. MATERIALS AND METHODS: Possible participation of the mitogen-activated protein kinase (MAPK) cascade on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated using specific MAPK cascade inhibitors, Western blot analysis, real-time quantitative PCR and FACS apoptosis analysis. RESULTS: Treatment with a highly specific p38 kinase inhibitor, SB203580, significantly (50-70%) inhibited heparin-induced colon cancer cell growth, demonstrating that p38 MAPK signalling is involved in their heparin-induced proliferative response. This was shown to be correlated with increased (up to 3-fold) phosphorylation of 181/182 threonine/tyrosine residues on p38 MAP kinase. Furthermore, heparin inhibited cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumour suppressor gene and protein expression up to 2-fold or 1.8-fold, respectively, and stimulated cyclin D1 expression up to 1.8-fold, in these cell lines through a p38-mediated mechanism. On the other hand, treatment with heparin did not appear to affect HT29, SW1116 and HCT116 cell levels of apoptosis. CONCLUSIONS: This study demonstrates that an extracellular glycosaminoglycan, heparin, finely modulates expression of genes crucial to cell cycle regulation through specific activation of p38 MAP kinase to stimulate colon cancer cell growth.
Abstract: Allelic variants of CYP1A1 and PON1 have been extensively studied as susceptibility factors in toxic response, although little is known about the role of these variants as risk factors for the plethora of diseases appearing in the human population. In this study we investigated the hypothesis of correlation of CYP1A1 and PON1 enzymes with the incidence of various medical examination findings in a Greek rural population professionally exposed to a variety of pesticides. The medical history of 492 individuals, randomly selected for the total population of 42,000, was acquired by interviews and their genotype determined for the CYP1A1*2A, PON1 M/L and PON1 Q/R polymorphisms. The assessment of exposure to pesticides of the population was verified by analytical methods. Analysis of the genetic data revealed that the allele frequencies of PON1 R, M and CYP1A1*2A alleles were 0.243, 0.39 and 0.107 respectively. The CYP1A1*2A polymorphism was found to have significant association with chronic obstructive pneumonopathy (p=0.045), peripheral circulatory problems (trend p=0.042), arteritis (p=0.022), allergies (trend p=0.046), hemorrhoids (trend p=0.026), allergic dermatitis (p=0.0016) and miscarriages (p=0.012). The PON1 Q/R polymorphism was found to have significant association with hypertension (p=0.046) and chronic constipation (p=0.028) whereas, the L/M polymorphism, with diabetes (p=0.036), arteritis (trend p=0.022) and hemorrhoids (trend p=0.027). Our results demonstrated an association between the CYP1A1/PON1 polymorphisms and several medical examination findings, thus indicating the possible involvement of the human detoxification system to health effects in a rural population exposed professionally to pesticides.
Abstract: Fibrosarcoma is an uncommon soft tissue tumor with a complex cell microenvironment, particularly rich in glycosaminoglycans/proteoglycans (GAGs/PGs). Chondroitin sulfate proteoglycans (CSPGs) participate in the modulation of various cellular functions, including adhesion and migration. The role of chondroitin sulfate (CS) chains on adhesion, chemotaxis and migration of poorly differentiated fibrosarcoma B6FS cell was studied, utilizing exogenous CS treatment and chondroitinase digestions as well as specific modulators of CS synthesis. Cleavage of cell-associated CS chains and specific inhibition of endogenous CS production severely impaired these fibrosarcoma cell functions. These results show that the reduction of endogenous CSPG expression as well as cleavage of the CS chain inhibited fibrosarcoma cell motility, migration and adhesion. Treatment with free CS chains enhanced cell chemotaxis and migration, whereas adhesion was inhibited. CS chains were found to upregulate cell motility through the MAPK pathway, specifically through JNK, whereas CS-induced migration was found to require tyrosine kinase dependent pathways. This study suggests a new role of CS on tumor cell adhesion, chemotaxis and migration.
Abstract: Platelet-derived growth factor (PDGF) is a major polypeptide mitogen for cells of mesenchymal origin such as fibroblasts. Chondroitin sulfate chains (CS), which are abundant in the extracellular matrix have been shown to physically interact with PDGF-BB modulating its biological function. The aim of the present study was to examine the involvement of CS on PDGF-BB induced proliferative responses and receptor activation in human lung fibroblasts. The addition of exogenous free CS chains caused a significant downregulation of the PDGF-BB mediated mitogenic and chemotactic responses. Similar results were obtained by the increase of endogenous CS biosynthesis after beta-D-xyloside treatment. Furthermore, removal of the membrane-bound CS chains by selective enzymatic treatment significantly increased the proliferative capacity of human fibroblasts. Analysis of PDGF-R phosphorylation in the presence of CS or beta-D-xyloside, revealed a reduction of PDGF-Rbeta phosphorylation in the tyrosine residue 1021. These results demonstrate, for the first time, that CS either soluble or surface bound downregulates the mitogenic responses of PDGF-BB in normal human lung fibroblasts through the reduction of PDGF-Rbeta phosphorylation.
Abstract: Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the pericellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by fibrosarcoma cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these cells. Fibrosarcoma cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS cells were V0 and V1. Treatment of B6FS cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF cells compared to B6FS and HT1080 cells, respectively. In HT1080 cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the pericellular matrix.
Abstract: Platelet derived growth factor (PDGF) is involved in the autocrine growth stimulation of normal and malignant cells, the stimulation of angiogenesis, and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the extracellular microenvironment. The present review discusses the effects of glycosaminoglycans on the functions mediated by the PDGF on cells of mesenchymal origin. Recent studies have demonstrated that both soluble and surface bound glycosaminoglycan chains can modulate PDGF-BB isoform signaling depending on the cell type. These data demonstrated that the microenvironment rich in GAGs/PGs is able to significantly modify the cellular response to PDGF-BB signaling in a critical way for cell growth and differentiation.
Abstract: Estrogens are related with the growth and development of target tissues and play a critical role in breast cancer progression. The effects of estrogens are mediated by the estrogen receptors ERalpha and ERbeta, which are members of the nuclear steroid receptor superfamily. To date, it is not known how these hormones elicit many of their effects on extracellular matrix molecules and how these effects can be connected with ER expression. For this purpose, the effect of estradiol on ER expression as well as on proteoglycan and metalloproteinase expression was studied. The effect of E2 on extracellular matrix molecule expression has been studied using ERalpha suppression in breast cancer cells. Our studies using ERalpha-positive MCF-7 cells show that estradiol affects the expression of syndecan-2, but not of syndecan-4, through ERalpha. Furthermore, the ability of estradiol to affect MMP-9 and TIMP-1 expression is connected with ERalpha status. Together, these data demonstrate the significant role of ERalpha on mediating the effect of estradiol on extracellular matrix molecules.
Abstract: Decorin is a multifunctional molecule of the extracellular matrix. Among the multitude of assigned functions the most intriguing is the ability to inhibit the growth and the metastasis of a wide range of cancer cells in vitro. Decorin was established to directly interact with EGFR and erb2, inducing protracted receptor internalization, which results in attenuation of the receptor-mediated intacellular signaling and induction of apoptosis. Studies by our group of osteosarcoma cells described the first exception to the established decorin-mediated growth suppression model. Osteosarcoma cells constitutively produced decorin and they were not sensitive to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells, since it was necessary for cell migration and acted as mediator, counteracting the TGFbeta2-induced cytostatic function. Importantly, decorin did not induce p21 expression whereas EGFR appeared to be overexpressed and continuously phosphorylated in our osteosarcoma model. These data provide new insight on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression.
Abstract: Decorin is an established natural oncosuppressive factor whose action is being studied in detail. Recently, decorin gene therapy formulations using adenoviral vectors have been shown in several animal models with very promising results. The present study describes the first exception to the established oncosuppression model using human osteosarcoma cells. MG-63 osteosarcoma cells were found to constitutively produce decorin, and furthermore, to be resistant to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells because it was necessary for MG-63 cell migration and acted as a mediator, counteracting the transforming growth factor-beta2-induced cytostatic function. Efforts to determine how MG-63 cells could overcome the decorin-induced cytostatic effect established that decorin in MG-63 cells does not induce p21 expression nor does it cause protracted retraction and inactivation of the epidermal growth factor receptor. Conversely, epidermal growth factor receptor seemed to be overexpressed and continuously phosphorylated. In view of the proposed design of decorin-based anticancer therapeutic strategies, our study provides new data on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression.
Abstract: Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine-rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG-63) and well-differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real-time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non-metastatic, well-differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG-63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down-regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG-63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.
Abstract: BACKGROUND: Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. METHODS: Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. RESULTS: Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. CONCLUSIONS: Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia.
Abstract: Platelet derived growth factor is involved in the autocrine growth stimulation of malignant cells, the stimulation of angiogenesis and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the fibrosarcoma cell microenvironment. Aim of the present study was to examine the effects of glycosaminoglycans on the mitogenic function of platelet derived growth factor in two human fibrosarcoma cell lines (B6FS, HT1080). For this purpose exogenously added glycosaminoglycans, regulators of endogenous glycosaminoglycan synthesis (sodium chlorate as selective inhibitor and beta-D-xyloside as a stimulator) and specific glycosidases to cleave cell-associated glycosaminoglycans, were utilized. Platelet derived growth factor demonstrated a growth stimulating effect on B6FS, whereas no effect was evident on HT1080 fibrosarcoma cells. Beta-D-xyloside had no effect on the basal level or the platelet derived growth factor-induced cell proliferation, whereas sodium chlorate severely reduced the basal level of proliferation in both cell lines. Significant co-stimulatory effects of chondroitin sulfate A in combination with platelet derived growth factor BB on the growth of HT1080 and B6FS cells were found. The co-stimulatory effect of chondroitin sulfate A was not due to transcriptional up regulation of platelet derived growth factor receptors genes, but rather to more efficient signalling of tyrosine kinase receptors. In conclusion, this study shows that chondroitin sulfate A can enhance the mitogenic activity of platelet-derived growth factor in fibrosarcoma cells utilizing a pathway which involves tyrosine kinases. This result introduces a new modulating role for chondroitin sulfate in signalling pathways critical for cancer growth.
Abstract: Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.
Abstract: OBJECTIVE: Human papillomavirus (HPV) has been identified as the principal etiologic agent for cervical cancer and its precursors. Different HPV types have been associated with different oncogenic potential. The purpose of this study was to evaluate the relationship between specific HPV type infection and expression pattern of the ras family oncogenes in different grades of HPV-associated human cervical neoplasia. METHODS: HPV typing was performed using polymerase chain reaction (PCR) in 31 HPV-positive human cervical specimens from patients with squamous intraepithelial lesions (SIL) or squamous cervical carcinoma (SCC). The mRNA expression levels of H-, K- and N-ras oncogenes were examined using the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Statistical analyses were performed using SPSS software. RESULTS: Among patients with SCC, H-, K- and N-ras expression levels were higher in HPV 16/18-associated cases compared to HPV 16/18-unassociated samples (p=0.003, p=0.004 and p=0.0001, respectively). The expression levels for H-, K- and N-ras were significantly higher in SCC patients with multiple HPV infection compared with SCC patients with single HPV infection (p=0.009, p=0.01 and p=0.021, respectively). Among patients with SIL, no statistically significant relationship was found between ras expression and HPV status. CONCLUSION: Our findings indicate the possible role of ras signaling interaction with "high-risk" HPV 16/18 and multiple HPV infection in cervical cancer development.
Abstract: AIMS: Ophthalmic pterygium is a potentially vision-threatening lesion of unknown etiology, related to an exposure to solar light. Mutations to the ras genes are frequently observed in lesions related to an exposure to solar light. The present study aims at screening pterygia for mutations at codons 12 and 13 of the ras genes. METHODS: In all, 50 pterygia were examined, together with respective blood samples and specimens of normal conjunctiva. A PCR reaction was performed to amplify sequences containing codons 12 and 13 of Ki-ras, H-ras, and N-ras. An RFLP analysis was then performed to detect point mutations at codon 12. The mutational status at codons 12 and 13 was further explored with sequencing of PCR products. RESULTS: RFLP analysis revealed Ki-ras mutations at codon 12 in five (10%) of pterygia, whereas H-ras or N-ras mutations were not observed. Sequencing confirmed Ki-ras mutations at codon 12 and revealed absence of mutations at codon 13. The presence of Ki-ras mutations was significantly correlated with postoperative recurrence (P=0.02) and young age (P=0.04). Mutations were not observed in specimens of blood or normal conjunctiva for any of the genes examined. CONCLUSIONS: The absence of N-ras mutations is in agreement with previous reports concerning mucosal lesions. The detection of Ki-ras mutations and the association with postoperative recurrence implies a possible role of Ki-ras in the clinical profile of pterygium. The mechanism of Ki-ras mutations is unclear and could be independent of the action of UV light.
Abstract: Loss of genomic rDNA has been associated with cellular and organismal ageing. The rDNA locus in humans comprises multiple copies of the 5.8S, 28S and 18S genes. Aim of the present study was to test the effect of aging on the copy number of the three rDNA genes individually in post-mitotic human tissue. We utilized real time polymerase chain reaction relative quantification to measure the copy number of 5.8S, 28S and 18S rDNA genes individually. We obtained adipose tissue from 120 male individuals aged from 9 to 94 years. The available data of each subject corresponding to the time of tissue sampling included: age, height, weight and calculated body mass index. Each rDNA gene was directly tested with Pearson correlation against age and body mass index. We found a significant negative correlation of the gene copy of 5.8S (P < 0.001) and 28S (P < 0.003) with age. Interestingly 18S gene copy displayed a different pattern with no statistically significant correlation with age. Conversely, we observed a significant negative correlation of the 18S gene copy with body mass index (P = 0.004) and a marginally non-significant negative correlation of the 5.8S (P = 0.097) gene copy with body mass index. In summary our results indicate that the rDNA recombination events in humans can be differentially targeted and regulated in response to ageing and/or fat accumulation. The proposed model generates possible implications regarding the effects of each rDNA gene loss in cell function as well as the mechanism of recombination targeting.
Abstract: It is well documented that inflammation plays a major role in the establishment and progression of atherosclerosis. Endothelial cells, vascular smooth muscle cells and monocytes/macrophages are involved in this process by expressing inflammatory factors. The aim of the present study was to evaluate potential association and risk of VEGF-A and TGF-beta1 in human coronary atherosclerotic lesions. Twenty-six fresh human coronary artery segments were collected at autopsy. Conventional histology was performed and samples were classified into: no lesion group (NL), fatty streak group (FS), plaque group (P) and complicated lesion group (CL) based on the atherosclerotic lesion type. RNA extraction-analysis with RT-PCR and immunohistochemistry was also performed. We observed that VEGF-A protein and mRNA expression increased during atherogenesis. The expression levels (protein and mRNA levels) of TGF-beta1 were decreased from NL to the FS group while, strong protein-staining and signal of mRNA expression in P and CL groups were observed. Our findings suggest a crucial role of VEGF-A in the development of coronary artery disease. The high protein and mRNA expression levels of TGF-beta1 in P and CL suggest that this factor may be implicated in the deposition of excessive extracellular matrix in the intima of the vessel wall, contributing to the expansion of the atheromatic plaque.
Abstract: Osteoblastic cells produce a complex extracellular matrix (ECM) composed of a mixture of proteoglycans (PGs), collagens and non-collagenous proteins. The interaction of proteoglycans with matrix effector macromolecules via either their glycosaminoglycan (GAG) chains or their protein core is critical in regulating a variety of cellular events. Alterations in the structural composition of the GAG/PG component of the ECM may have important consequences on cell proliferation and/or differentiation. Human osteoblasts and two osteosarcoma cell lines, able to produce galactosaminoglycan (GalAGs) and heparan sulphate (HS)-containing proteoglycans, were treated with their main GAG chain types, and the effects on cell growth were examined. Chondroitin sulphate (CSA) and dermatan sulphate (DS) inhibited cell proliferation of all osteoblastic cell lines at high concentration (100 microg/ml). DS showed the stronger inhibitory effect, probably due to the presence of flexible IdoA residues that provide a greater variety in conformation to these macromolecules. Heparin strongly inhibited the proliferation rates of both normal osteoblasts and transformed osteoblastic cells at concentrations > or = 1 microg/ml. The presence of large amounts of IdoA-derived trisulphated disaccharides, responsible for the overall negative charge of heparin, should be considered as a critical factor for the inhibition of cell proliferation. The obtained results suggest that matrix GAGs are factors which affect cell growth of both malignant and normal cells of the osteoblastic lineage in a concentration-dependent manner. This effect is closely related to the fine chemical structure of GAGs, i.e. the presence of L-iduronic acid and the degree of sulphation.
Abstract: Deregulation of the apoptotic machinery plays a major role in cell death, cellular transformation and cancer. p53, Bcl-2, Bcl-XL, Bax and Mdm2 mRNA expression patterns were evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer compared to those of normal cervical tissues, and correlated with the underlying cervical lesions. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. p53, Bcl-2, Bax and Mdm2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (p=0.003, p=0.009, p=0.040 and p=0.001, respectively). Specifically, p53, Bax and Bcl-2 exhibited substantially lower transcript levels in CIN lesions compared to controls, whereas Bax mRNA levels showed a significant decrease in cancer compared to normal specimens. Mdm2 mRNA expression was considerably lower in cancer than in CIN lesions or normal cervix. High-grade squamous intraepithelial lesions exhibited lower p53 and Bcl-2 mRNA levels than controls (p=0.002, p=0.016). Coexpression analysis revealed more correlations between the above apoptosis-related molecules in normal tissues compared to CIN or cancer specimens. p53 showed significant coexpression with Bax, Bcl-2 and Mdm2 (p=0.040, p=0.013 and p=0.015, respectively) in normal cervical specimens. Bax and Bcl-XL mRNA expression was negatively correlated. Mdm2 transcriptional levels correlated significantly with those of Bax, Bcl-XL and Bcl-2. Our findings show that p53, Bax, Bcl-2 and Mdm2 mRNA expression levels correlate with the malignant transformation of the uterine cervix. mRNA coexpression patterns of the members of the pro- and anti-apoptotic family examined in cervical carcinogenesis were found to be disrupted in CIN and cancer, as already demonstrated at the protein level.
Abstract: Human carcinogenesis is a multistep process involving complicated genetic events in which several onco-genes and oncosuppressor genes are implicated. The role of ras oncogenes in cellular transformation and apoptosis has been extensively examined and the dual role of ras as oncogene and oncosuppressor gene has been supported. Activation of ras occurs either by genomic alterations such as point mutations or by modulation of Ras protein expression. Many molecular and immunohistochemical studies have focused on ras activation in a wide range of human tumours. In this review, we summarize available information regarding the genomic and expression alterations of ras oncogenes in cervical, endometrial and ovarian cancer. Gynecological malignancies represent some of the most well- studied types of human cancer concerning ras activation and its possible use in clinical practice.
Abstract: Angiogenesis is a complex procedure induced by the secretion of numerous growth factors from endothelial cells. Vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (FGF2), transforming growth factor-beta1, 2, 3 (TGFB1, 2, 3), and transforming growth factor-beta receptors (TGFBR1, 2, 3) mRNA expression pattern was evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer, compared to that of normal cervical tissues, and correlated to the clinical stage of the disease. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. VEGF, TGFB1, TGFBR1, and FGF2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (P=0.015, 0.001, 0.008, and 0.029, respectively). Higher TGFBR1 mRNA levels were observed in parallel with increased severity of the lesion, whereas FGF2 exhibited lower transcript levels. A highly significant increase of VEGF mRNA expression was found upon cervical neoplastic transformation (P<0.0001). High-grade squamous intraepithelial lesions exhibited higher VEGF mRNA levels than low-grade lesions (P=0.039). TGFBR1 and TGFBR3 receptors demonstrated significant co-expressions with TGFB2 (P<0.0001), and TGFB1 (P=0.005 and 0.002, respectively) in normal cervical specimens. However, a disruption of co-expression patterns was observed in the groups of CIN and cancer cases, compared to normal tissues. Our findings show that VEGF, FGF2, TGFB1 and TGFBR1 mRNA expression levels correlate with the malignant transformation of the uterine cervix. The involvement of the examined markers in cervical carcinogenesis is furthermore supported by the observed disruption of their mRNA co-expression patterns.
Abstract: BACKGROUND: The chemokine receptor polymorphisms CCR5Delta32, CXCL12 3'A, CCR2-64I and CCR5-59029 G/A have been demonstrated to affect HIV-1 infection and progression. OBJECTIVE: We studied the impact of the above polymorphisms on the effectiveness of a 30-month treatment with highly active antiretroviral therapy (HAART) in 149 HIV-1 patients. STUDY DESIGN: We stratified the patients according to CD4 CDC criteria and applied Kaplan-Meier analysis using the following end-point criteria: (a) the time from HAART initiation to undetectable viral load (VL) counts (VL<50 copies/ml), (b) the duration of undetectable VL status and (c) the time required for CD4+ T-cell counts to pass over the 500 cells/ml threshold. RESULTS: Our results in the second group (CD4 201-500) revealed that patients with the CCR2-64I allele achieved undetectable VL counts at 3.5+/-0.48 months as compared to 10.26+/-1.42 months in the control group (p=0.018). The VL remained undetectable for 28+/-2 months, in contrast to 20+/-2 months in the control group (p=0.048). Patients carrying CXCL12 3'A restored the CD4 population faster than the control group (9+/-2 and 14+/-2 months, respectively, p=0.023). The CCR5Delta32 and CCR5-59029 G/A alleles did not appear to affect the parameters studied. CONCLUSIONS: Our results suggest that patients carrying either CCR2-64I or CXCL12 3'A have a more favorable prognosis during HAART treatment.
Abstract: The main cell population affected by the human immunodeficiency virus-1 (HIV-1) infection belongs to the CD4+ T-lymphocyte family. Recent convincing evidence indicates that the majority of the cells that die due to HIV-1 are not actually infected by the virus. Instead, these cells are being led to programmed cell death after the activation of apoptotic mechanisms by the virus or its components. We propose here from accumulated evidence that the virus appears to deregulate the physiological function of these cells during the process of antigen presentation. Ionic interactions between the variable V3 domain of the HIV-1 coat glycoprotein gp120 and the amino terminal of the chemokine receptor CCR5 play a prominent role in this process, and we speculate that nature has evolved simple electrostatic interaction mechanisms which, coupled to specific recognition systems on the cell surface, can initiate and modulate certain cellular events without the need for specific molecular structures. HIV-1 utilizes such a mechanism to ensure activation of the target host cell.
Abstract: ND10 structures are disrupted during herpes simplex virus type 1 (HSV-1) infection by viral regulatory protein ICP0. The significance of this effect remains controversial, partly because of a report that high-level expression of the major ND10 promyelocytic leukemia (PML) protein precludes ND10 disruption yet does not inhibit HSV-1 infection. Here we demonstrate dramatic reorganization of ND10 during HSV-1 infection by live-cell microscopy, even in the presence of overexpressed PML.
Abstract: Schizophrenia is a severe and common psychiatric disorder afflicting 1% of the world population. A role of many neurotransmitter receptors in schizophrenia was suggested by an association with several polymorphisms located in their coding regions. In this study we examined the contribution of the T-102C and A-206G transitions in the 5-HTR2a and DRD3 receptor genes respectively to genetic susceptibility and phenotypic expression of schizophrenia disorder within the Greek population. We determined by PCR and RFLP analysis the genotype for the above polymorphisms in 114 schizophrenic hospitalized individuals and 192 control samples. In contrast to previous reports from large European multicentre studies, which indicate significant correlation between schizophrenia and C-102 allele of the T-102C polymorphism, in this study we observed a statistically significant overall association between the disorder and allele T-102 (P<0.0001, odds ratio (OR)=2.11, 95% CI=1.48-3.02). We also found a highly significant excess of the T-102/C-102 and C-102/C-102 genotypes in the normal group (P<0.001). Comparison of the patients with the controls for the DRD3 polymorphism (A-206G transition) showed marginally nonsignificant differences in the genotypic (P=0.054) and no significance in the allelic (P=0.163) frequencies. However, the A-206/A-206 genotype seems to positively contribute to the disorder appearance, when compared to A-206/G-206 as genotype base line risk (P=0.016, OR=1.88, 95% CI=1.09-3.26). In conclusion, from genetic association analysis of this schizophrenic population, a significant association is clearly determined between the HTR2 genetic polymorphism and the presence of schizophrenic disorder, manifested as increased risk of schizophrenia for carriers of the T-102 allele.
Abstract: OBJECTIVE: Overexpression of p21 protein has been detected in human cervical cancer. However, to date, there are no data on the differential activation of the three ras genes at the transcriptional level in cervical lesions. The purpose of this study was to evaluate the quantitative and qualitative changes of expression of the ras family genes in the development of human cervical cancer. METHODS: The expression of ras mRNA levels in 35 human cervical specimens [11 normal cervix, 15 cervical intraepithelial neoplasia (CIN), 9 cervical cancer] was examined using the RT-PCR technique. In addition, we studied the incidence of point mutations in codon 12 of each ras gene using RFLP analysis and human papilloma virus (HPV) status. RESULTS: The transcript levels for H-ras and N-ras were significantly higher in cancer cases compared to normal cervical tissues (P=0.0002 and P=0.001, respectively) and CIN lesions (P<0.0001 and P=0.002, respectively). The transcript levels for K-ras were similar in normal cervical tissue, CIN and cervical cancer. A strong positive correlation was found between H- and N-ras expression (P=0.001) and no correlation between H- and K- or N- and K-ras expression. Point mutations were detected only in three samples, located in codon 12 of K-ras gene. No relationship was found between expression levels of each ras gene and the presence of HPV. CONCLUSION: Our findings indicate the expression pattern of the three ras genes in cervical tissue and the involvement of H- and N-ras up-regulation in the pathogenesis of cervical cancer independent of HPV infection.
Abstract: BACKGROUND: Macrophages can produce vascular endothelial growth factor (VEGF) in response to hypoxia, transforming growth factor beta1 (TGF-beta1), angiotensin II, basic fibroblast growth factor (bFGF), and interleukin-1. These factors have been found in the serum of coronary artery disease (CAD) patients as well as in atherosclerotic lesions. The aim of the present study was to test the hypothesis that the expression of VEGF, TGF-beta1 and bFGF in peripheral monocytes and lymphocytes is related to CAD. METHODS: Human Mononuclear cells and lymphocytes from peripheral blood were isolated from 53 donors undergoing angiography. Seventeen were found to be healthy and 36 were diagnosed with CAD. The respective mRNAs were extracted and quantified. RESULTS: The statistical analysis revealed a significant increase of the basal level expression for macrophage VEGF and bFGF in the CAD SA (stable angina) patient group compared to the noCAD (control) (p = 0.041 and p = 0.022 respectively) and CAD UA (unstable angina) (p = 0.024 and p = 0.005 respectively) groups, which was highly dependent on the diabetic status of the population. Furthermore, we demonstrated with an in vitro cell culture model that the levels of VEGF and bFGF in monocytes of healthy donors are not affected by short term exposure to increased glucose levels (usually observed in the diabetic patients) and/or statin. CONCLUSION: Our findings display a statistically significant association of the increased VEGF and bFGF levels in peripheral monocytes, with stable angina and diabetes in coronary artery disease. The results give new insight to CAD and the impaired collateral vessel formation in diabetics.
Abstract: Using PCR-based microsatellite DNA analysis with 48 markers we examined sputum and bronchial washing for genetic alterations compared with lymphocyte extracted DNA from 124 lung cancer patients and 36 healthy donors as normal control. Microsatellite alterations (MA) in at least one locus were detected in all cancer patient-derived specimens but only in 22.2% of the healthy donors. Loss of heterozygosity (LOH) was detected in bronchial washings from 101 non-small cell lung cancer (NSCLC) predominantly on 17p13.1-p13.3 (69.7%), 9p13.3-p24.1 (63.3%), 1p34.2-p36.22 (48.5%), 13q12.1-q13.1 (47.7%) and 3p22.3-p23 (42.7%). In bronchial washings from 23 small cell lung cancer (SCLC) LOH was detected mostly on 3p22.3-p23 (88.6%), 17p13.1-p13.2 (82.3%), 5q32-q33.1 (66.6%), 13q12.2-q13.1 (65.6%) and 9q22.33-q31.3 (52.9%). The different LOH patterns indicate that different genetic background may be responsible for the different physiology of NSCLC and SCLC. The fractional allele loss (FAL) mean value of all cancer specimens was 0.243+/-0.021 compared with 0.007+/-0.008 of healthy donors with a confidence interval (CI) 99.5%. Only seven out of 124 lung cancer specimens (5.2%) exhibited FAL value less than 0.083, the highest was observed in the healthy donors group. FAL appears to be a likely indicator for lung cancer detection. Microsatellite instability (MIN) was detected in 8.7% of SCLC and 4.0% of NSCLC bronchial washings in at least three loci tested. LOH and MIN detection in sputum and bronchial washing from the same patient was 77.6%. Calculation of these indexes per marker exhibits significant variations that could be attributed to diffuse lung disorders or non-cancer specific genetic alterations.
Abstract: BACKGROUND: Genetic polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and transcobalamin (TC) genes influence homocysteine metabolism which in turn may influence the risk of spontaneous abortion. It was hypothesized that there may be a significant interaction between MTHFR and TC genotypes which affects the pathogenesis of spontaneous abortion. METHODS AND RESULTS: A total of 76 fetal tissue samples from spontaneous abortions between weeks 6 and 20 of pregnancy, and 114 control samples from healthy blood donors were genotyped for the MTHFR 677C>T and 776C>G polymorphisms. Subjects with combined MTHFR 677TT/TC 776GG and combined MTHFR 677TT/TC 776CG genotypes gave an odds ratio for spontaneous abortion of 3.8 (95% confidence interval 1.4-9.9, P = 0.005). CONCLUSIONS: Embryos that have combined MTHFR 677TT and TC 776CG or 776GG genotypes; genotypes that individually are associated with impaired homocysteine metabolism in adults, are at increased risk for spontaneous abortion compared with embryos that have only one of these genotypes.
Abstract: We examined the possible involvement of human herpes viruses in sporadic non-melanoma skin cancer of Greek patients. Polymerase chain reaction (PCR) based detection assays were utilized for the detection of viral cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) genomes in 24 squamous cell carcinomas (SCC), five Bowen's disease, 72 basal cell carcinomas (BCC) specimens and eight premalignant lesions. Forty-two of 109 (38.5%) skin lesions were found positive for CMV DNA. The highest incidence was 6/8 (75%) observed in specimens with premalignant lesions. The incidence was 37.5% (27/72) in BCC, 33% (8/24) in SCC and 20% (1/5) in extragenital Bowen's disease. All samples were negative for HSV-1/2 and EBV DNA as assessed by our PCR based assay. The CMV infection showed no statistically significant correlation with the histological type, age, site of lesion or sex. Our results give a strong indication of the possible involvement of CMV in non-melanoma skin cancer development.
Abstract: BACKGROUND: Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population. OBJECTIVES: The purpose of our study was to determine the involvement of CDKN2A genes in the development of sporadic nonmelanoma skin cancer in Greek patients. PATIENTS AND METHODS: Allelic imbalance analysis was performed in 22 SCC and five Bowen's disease specimens. Mutational analysis was performed on exons 1alpha, 1beta and 2 of the CDKN2A locus in 22 SCC, five Bowen's disease and 39 BCC specimens. Exon 1alpha was additionally screened in 28 BCC specimens to complete the mutational analysis of a previous study. RESULTS: Overall, 52% (14 of 27) of the SCC and Bowen's disease specimens exhibited loss of heterozygosity (LOH) in at least one microsatellite marker, whereas, only two of 27 (7%) exhibited microsatellite instability. LOH in 9p appears to be equally involved in both BCC and SCC tumours. Exons 1alpha, 1beta and 2 of the CDKN2A locus were screened for mutations. A Val28Gly substitution in exon 1alpha and a CCC-->TTT (Ala57Val and Arg58Ter) substitution in exon 2, resulting in a change in the amino acid sequence, are reported for the first time in two SCCs, the latter being indicative of a combination of an ultraviolet (UV) radiation-induced mutation and a point mutation. A previously described polymorphism of CDKN2A, the gene for p16INK4a, Ala148Thr, was also detected in an allelic frequency of 3.72%. No mutation was found in any of the five Bowen's disease specimens, or in exon 1beta of CDKN2A, also the gene for p14ARF. CONCLUSIONS: Mutations and the high incidence of 9p LOH detected in our SCC samples imply that inactivation of CDKN2A genes, via allelic loss and/or mutation (probably UV-induced) may play a significant role in nonmelanoma skin cancer development, particularly in the more aggressive SCC type.
Abstract: Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.
Abstract: Two poly (vinyl pyrrolidone) (PVP) families with amino-acid residues (glycine, beta-alanine, gamma-aminobutiric acid and epsilon-aminocaproic acid) on the base of the co-polymer N-vinyl pyrrolidone and allyl-glycidyl ether (VP-AGE) and on the base of epoxidized PVP (EPVP) were synthesized. Static and dynamic light scattering measurements of these PVP derivatives in water showed that their structure/ behavior were similar to that of PVP. The bioreactivity was also similar to that of PVP. Further investigation of the immunoreactive properties of the derivatives in in vitro proliferation assays with fresh normal human peripheral blood lymphocytes and monocytes led to the determination of a costimulatory profile for each derivative in terms of polyclonal stimulation, specific antigen presentation, and immunoglobulin secretion. This profile allows the selection of an appropriate derivative as a carrier that would suit the immunoreactivity needs of the immobilized ligand.
Abstract: BACKGROUND: The remethylation cycle of methionine is folate and vitamin B(12) (cobalamin) dependent and appears to be crucial for embryonic development, probably through effects on synthesis of DNA, proteins and polyamines. Transcobalamin (TC) transports vitamin B(12) to the tissues. The objective of the present investigation was to explore the putative association between the major TC genetic polymorphism (Pro259Arg) and human spontaneous abortion. METHODS: The prevalence of the TC Pro259Arg polymorphism was determined in DNA samples from embryos that had been spontaneously aborted between the 6th and 20th week after conception, and adult controls using solid-phase minisequencing technique. RESULTS: The 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57.0% respectively; P = 0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2% respectively; P < 0.001). CONCLUSIONS: The 259-Pro allele seems to have beneficial influences during embryogenesis, conceivably through its positive effect on vitamin B(12) intracellular bioavailability. Our results warrant additional investigations addressing the question if vitamin B(12) supplementation in addition to folic acid supplementation may prevent spontaneous abortion in women planning a pregnancy.
Abstract: We have reported that the principal neutralizing domain of V3 of the HIV-1 gp120 induces an antigen-specific activation apoptosis of responding effector CD4+ T lymphocytes, a phenomenon inhibited by RANTES, an agonist of CCR5. Here, addressing the question of how a hypervariable region could induce such a selective reaction, we demonstrated that the magnitude of the activation phase was dependent on the number of basic amino acids present in the V3 peptide, an observation confirmed by using V3 peptides with appropriate basic amino acid substitutions. The relative position of the amino acids in the V3 peptide did not affect the biological phenomenon. Using surface plasmon resonance biosensor analysis, we also provided direct evidence of the influence of basic amino acids in the interaction between V3 and the amino terminal domain of CCR5. Sulphation of tyrosines in the CCR5 peptide was essential. Our results confirm gp120 modelling predictions and demonstrate simple molecular ionic interactions as capable of affecting key cell events, the wider biological implications of which need to be further explored.
Abstract: The ras family of oncogenes has been extensively studied for its implication in several types of human malignancies. Activation of ras genes involves mutations that alter the catalytic activity of the protein enhancing the downstream signals mostly towards cell proliferation and malignant transformation. Ras genes are also involved in induction of senescence or apoptosis, suggesting activation of alternative pathways that may be anti-oncogenic. Early experiments showed that transfection of wild-type ras in transformed cells reversed the oncogenic phenotype suggesting that wild-type ras has onco-suppressive properties. Indeed, expression of wild-type ras genes in several human malignancies is associated with good prognosis. In tumors carrying mutant ras genes the levels of expression of the wild-type allele never exceeded the mutant counterpart, indicating that the wild-type protein suppresses the effect of the mutant one. Recent development of the Kras2 deficient mice provided the tool to study the role of wild-type ras genes in tumorigenesis.
Abstract: It was recently demonstrated that the semiconserved domain of the V3 region of the HIV-1 surface glycoprotein gp120 can induce an activation-apoptosis phenomenon to memory CD4+ cells from healthy individuals. Studying the effects of V3 on the interaction of antigen presentation between monocyte-derived macrophages and resting memory CD4+ T cells, we observed that V3 affects both cell populations. Macrophages exposed to composite liposomes containing V3 on the surface and tetanus toxoid (TT) as the recall antigen entrapped in the aqueous phase (lipoV3/TT liposomes) were able to activate CD4+ T cells during primary stimulation, but not after restimulation nine days later. Unstimulated macrophages or macrophages exposed to soluble TT responded to second stimuli, lipoV3/TT liposomes, and soluble TT in activating CD4+ T cells. Soluble TT-activated CD4+ T cells could be restimulated by soluble TT but not by lipoV3/TT liposomes, whereas lipoV3/TT liposome-activated CD4+ T cells became unresponsive to a second stimulus. These results show that resting memory CD4+ cells activated by macrophages presenting the recall antigen together with V3 become unresponsive to restimulation.
Abstract: It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy.
Abstract: The proteolytic activity of Escherichia coli periplasmic proteases can affect the expression efficiency of many heterologous proteins such as antibody fragments that are transported to the host periplasm for folding. We investigated whether four E. coli strains that were deficient in the periplasmic proteases tsp, protease III, degP and ompT, in different combinations, affect the expression levels of an anti-MUC1 scFv fragment. The ompT protease appeared to be involved in partial degradation of the scFv since degradation products were observed in all ompT unmutated strains in Western blotting, whereas such products were absent in the ompT mutated strains. The HM120 strain that contained most mutations, expressed the scFv protein efficiently but the level of functional antibody activity was low. This was probably due to an accumulation of incorrectly folded antibody molecules in the periplasm as it was characterised by low enzyme immunoassay reactions in contrast to the intense staining of the tag in Western blots. Improved understanding of the periplasmic protease involvement in the process of the antibody expression in bacteria may allow us to design host E. coli strains that are more efficient in producing functional antibodies.
Abstract: The semi-conserved domain of V3 of HIV-1 was synthesised in a lipopeptide form to be presented on the surface of liposome particles. Composite liposomes were constructed with entrapped tetanus toxoid as a recall antigen (lipo-V3/TT liposomes) to study the influence of V3 on effector T cells of human normal peripheral lymphocyte populations. We demonstrated that lipo-V3/TT liposomes induce a V3-specific response characterised by an early, enhanced proliferation of effector CD4+ T cells, followed by a sharp apoptosis. The phenomenon required the presence of monocyte-derived macrophages and CD4+ T cells, but it was qualitatively and quantitatively distinct from the normal soluble antigen-mediated antigen presenting cell: T cell interaction. Presence of the beta-chemokine RANTES in the culture medium inhibited the phenomenon, suggesting that V3 plays a costimulatory role that involves the chemokine receptor CCR5 pathway during the process of antigen presentation to T cells. This observation may be very important if it occurs also in HIV-1 infection, as it may explain the selective and progressive depletion of non-infected effector CD4+ T cells.
Abstract: The major cellular and molecular factors that lead to the induction of somatic mutations in human B cells remain unclear. This study describes an approach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism analysis on the descendant cultures we were able to demonstrate that the Ig gene of B cells was induced to mutate. Three clones were isolated from a single cell culture of naive B cells immunized against the semi-conserved region of V3 of HIV-1, which contained two point mutations at the CDR2 region leading to amino acid codon change. Two clones were also obtained from a single cell culture of memory B cells immunized against the recall antigen tetanus toxoid. These clones showed one point mutation at the CDR1-FR2 border region leading to amino acid codon change. Fab constructs from the mutated culture of naive B cells immunized against V3 were cloned to a procaryotic expression vector. The expression products of two clones showed anti-V3 specificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through specific interaction with antigen and antigen-activated T cells.
Abstract: The use of in vitro immunization technology for the generation of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in vivo primary immune response. We now describe a detailed reproducible protocol for a two-step in vitro immunization, which yields isotype switched, antigen-specific human antibodies. The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-1) and a T helper cell epitope (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetanus toxoid; (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. None of the donors which were examined after primary immunization showed at any time an IgG anti-V3 specific antibody response, while all the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/8).
Abstract: We describe a method for retrieving sequences with one or two point mutations of a given target sequence, which are present in a DNA population at a frequency of 1 in 466 x 10(3) and 1 in 28 x 10(3) molecules, respectively. By stringent hybridization to a stable, chemically immobilized probe, a large excess of unrelated fragments is removed, and the bound sequences are dissociated and amplified. By repeating the hybridization-amplification cycles twice, we achieved an estimated enrichment of 404,000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedure can be applied to retrieve mutated sequences that exist at an extremely low frequency in a DNA population.
Abstract: beta 2-glycoprotein I (beta 2I) is a 50kDa serum glycoprotein of ill defined function. Based upon its capacity to bind negatively charged phospholipids a number of possible inhibitory roles for beta 2I have been proposed. We have cloned and sequenced a full length mouse beta 2I cDNA clone and demonstrated that mouse beta 2I does not behave as an acute phase reactant following an experimentally induced inflammation. Phylogenetic analysis of the known mammalian beta 2I homologues has provided evidence that mouse beta 2I is the most divergent and is evolving at a faster rate than beta 2I in other species.
