T regulatory cells-derived extracellular vesicles and their contribution to the generation of immune tolerance.

T regulatory (Treg) cells have a major role in the maintenance of immune tolerance against self and foreign antigens through the control of harmful inflammation. Treg cells exert immunosuppressive function by several mechanisms, which can be distinguished as contact dependent or independent. Recently, the secretion of extracellular vesicles (EVs) by Treg cells has been reported as a novel suppressive mechanism capable of modulating immunity in a cell-contact independent and targeted manner, which has been identified in different pathologic scenarios. EVs are cell-derived membranous structures involved in physiologic and pathologic processes through protein, lipid, and genetic material exchange, which allow intercellular communication. In this review, we revise and discuss current knowledge on Treg cells-mediated immune tolerance giving special attention to the production and release of EVs. Multiple studies support that Treg cells-derived EVs represent a refined intercellular exchange device with the capacity of modulating immune responses, thus creating a tolerogenic microenvironment in a cell-free manner. The mechanisms proposed encompass miRNAs-induced gene silencing, the action of surface proteins and the transmission of enzymes. These observations gain relevance by the fact that Treg cells are susceptible to converting into effector T cells after exposition to inflammatory environments. Yet, in contrast to their cells of origin, EVs are unlikely to be modified under inflammatory conditions, highlighting the advantage of their use. Moreover, we speculate in the possibility that Treg cells may contribute to infectious tolerance via vesicle secretion, intervening with CD4+ T cells differentiation and/or stability.

Stevioside inhibits experimental fibrosis by down-regulating profibrotic Smad pathways and blocking hepatic stellate cell activation.

Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co-cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non-canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up-regulated the protein expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases-9,-2 and -13 and overstimulate the canonical and non-canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up-regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down-regulating canonical and non-canonical profibrotic Smad pathways.

Gene expression profiles of primary retinal pigment epithelial cells from apolipoprotein E knockout and human apolipoprotein E2 transgenic mice.

Age-related macular degeneration (AMD) causes visual impairment in the elderly. In non-neovascular AMD, studies involving human subjects have suggested potential involvement of aberrant lipid metabolism. However, there have been no reports on gene expression patterns in animal models of non-neovascular AMD with abnormal lipid metabolism such as apolipoprotein E knockout and human apolipoprotein E2 transgenic mice. Transcriptome analysis was performed using retinal pigment epithelium cells of apoE knockout and apolipoprotein E2 mice using microarray analysis. C57BL/6, Rxrb, Pparbp, Vldlr, and Edf1, which are primarily related to lipid metabolism, were upregulated, while Tgfbr1 and Pdgfb, which are related to pathologic angiogenesis in AMD, were downregulated in both types of mice. Apolipoprotein E knockout and apolipoprotein E2 mice showed characteristic gene expression patterns in the transcriptome analysis of primary retinal pigment epithelium cells. These results suggest that specific genes associated with lipid metabolism and angiogenesis are involved in the pathogenesis and progression of AMD.

Practices in primary health care oriented toward the harmful consumption of drugs / Las prácticas en la atención primaria de salud relacionados con el consumo nocivo de las drogas / Práticas na Atenção Básica voltadas para o consumo prejudicial de drogas

Objective To analyze the practices of primary care focused on the harmful consumption of drugs. Method This is a qualitative study, developed with a dialectical-critical approach. Data collection was carried out through semi-structured interviews with 10 employees of a basic health unit (UBS). Results The demands are not accepted, and if they go beyond the barriers shaped by the historical absence of health care practices for drug users and moralistic and preconceived ideologies, they are not reinterpreted as health needs; practices that meet these demands and go beyond the barriers are poor; the functionalist approach, which explains drug use as a disease and considers drug users as deviants, supports the few existing practices. Conclusion primary health care is mistakenly focused on addiction; it lacks structural elements of the production process in health and internal dynamics of the working processes that would foster the development of collective practices. .

Objetivo El estudio tiene como objetivo analizar las prácticas de atención primaria dirigidos a lo consumo prejudicial de drogas. Método Se trata de un estudio cualitativo, desarrollado en la perspectiva dialéctica crítica. La recolección de datos se realizó a través de entrevistas semiestructuradas con 10 empleados de una Unidad Básica de Salud. Resultados Muestran que: las demandas no son aceptadas, y si van más allá de las barreras - formadas por la ausencia histórica de la práctica de la atención de salud para los consumidores de drogas y las ideologías morales y preconcebidas -, no son reinterpretados como necesidades de salud; las prácticas que satisfagan esas demandas son pobres; detrás de estas escasas prácticas, está la perspectiva funcionalista, que considera el uso de drogas como una enfermedad y los usuarios de drogas como desviados; los trabajadores valoran la formación clínica y culpan a los usuarios por los problemas que enfrentan. Conclusión Se pode concluir que la atención primaria: es equívoca hacia el objeto de la dependencia; carece de los elementos estructurales del proceso de producción en la salud y las dinámicas internas de los procesos de trabajo que fomenten el desarrollo de las prácticas colectivas.

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Objetivo Analisar as práticas de atenção básica voltadas ao consumo prejudicial de drogas. Método Estudo qualitativo, desenvolvido na perspectiva dialético-crítica. A coleta de dados foi realizada através de entrevistas semiestruturadas com 10 trabalhadores de uma Unidade Básica de Saúde (UBS). Resultados As demandas não são acolhidas e, quando ultrapassam as barreiras - conformadas pela ausência histórica de práticas de atenção à saúde ao usuário de drogas e por ideologias moralistas e preconceituosas -, não são reinterpretadas como necessidades de saúde; as práticas que atendem essas demandas são precárias; a perspectiva funcionalista, que compreende o consumo de drogas como doença e considera usuários de drogas como desviantes, embasa as escassas práticas existentes. Conclusão A atenção básica encontra-se equivocamente voltada para a dependência; carece de elementos estruturais do processo de produção em saúde e da dinamicidade interna aos processos de trabalho, que favoreceriam o desenvolvimento de práticas coletivas. .

Emancipatory practices of nurses in primary health care: the home visit as an instrument of health needs assessment / Prácticas emancipadoras del enfermero en la atención primaria de salud: la visita domiciliaria como instrumento de reconocimiento de necesidades de la salud / Práticas emancipatórias de enfermeiros na Atenção Básica à Saúde: a visita domiciliar como instrumento de reconhecimento de necessidades de saúde

Objective Identify nurses’ emancipatory practices in primary care, to contribute to the improvement of health care. Method A case study type social research of qualitative nature, in which nurses of a primary health care service unit in São Paulo were interviewed. Results The home visit was identified as a nursing practice possible to be expanded in order to identify social determinants of health, triggering emancipatory practices in the service. This expansion occurred because the design of health care labour intended by the service team changed its focus from the traditional object of health services, the disease. Conclusion First, it is advocated that social policies lead projects with the purpose of improving health needs. On the other hand, the daily labour needs to provide opportunities for reflection and discussion of healthcare projects, leading workers to propose labour-processes targeted to both the social determinants of health and people’s illness. .

Objetivo Identificar las prácticas emancipadoras de enfermeras en Unidad de Salud Familiar fueron el objeto de este estudio. Método La investigación social cualitativa tipo estúdio de caso. Fueron entrevistados enfermeros de una Unidad de Salud Familiar en Sao Paulo. Resultados Se identificó que la Visita Domiciliaria ha ampliado su alcance y identificado determinantes del proceso salud-enfermedad, lo que provocó en la Unidad de Salud Familiar prácticas emancipadoras. Esta expansión se produjo debido a que el diseño de la atención en propósito por la USF amplió el tradicional objeto de los servicios de salud. Conclusión Se aboga que las directrices de las políticas sociales basen proyectos que tengan como fin el mejoramiento de las necesidades de salud y que el trabajo diario proporcione la reflexión y discusión de los proyectos de atención, para proponer prácticas que enfoquen en los determinantes del proceso salud-enfermedad, tanto cuanto en sus resultados - la enfermedad en el cuerpo individual. .

Objetivo Identificar as práticas emancipatórias de enfermeiros da Atenção Primária, com a finalidade de contribuir para o aprimoramento do cuidado em saúde. Método Pesquisa social de natureza qualitativa, do tipo estudo de caso. Foram entrevistados os enfermeiros de uma Unidade de Saúde da Família em São Paulo. Resultados Identificou-se que a visita domiciliária, prática protocolar, ampliou seu escopo e identificou determinantes do processo saúde-doença, desencadeando na Unidade de Saúde da Família práticas emancipatórias. Essa ampliação ocorreu porque o projeto de cuidado intencionalizado ampliou o objeto tradicional dos serviços de saúde. Conclusão Advoga-se que as diretrizes das políticas sociais ancorem projetos que tomem como finalidade o aprimoramento das necessidades de saúde e que o cotidiano do trabalho proporcione reflexão e discussão dos projetos de cuidado, para intencionalizar práticas que incidam nos determinantes do processo saúde-doença, tanto quanto nos resultados - a doença expressa no corpo individual. .

Angiogenesis and expression of PDGF-C, VEGF, CD105 and HIF-1α in human glioblastoma.

Glioblastoma (GBM), the most frequent and aggressive brain tumor, is characterized by marked angiogenesis directly related to invasiveness and poor prognosis. Hypoxia is considered to be an important stimulus for angiogenesis by inducing hypoxia-inducible factor 1-alpha (HIF-1α) overexpression that activates platelet-derived growth factor (PDGF) and VEGF. The aim of this study is to analyze the expression of PDGF-C, VEGF in endothelial and tumor cells of GBM and their relation to HIF-1α expression. Two hundred and eight GBM cases were studied by tissue microarray immunohistochemical preparation. Expression of HIF-1α, VEGF and PDGF-C was observed in 184 (88.5%), 131 (63%) and 160 (76.9%) tumor cases, respectively. The numbers of vessels were quantified by CD34, PDGF-C, VEGF and CD105 staining, and were in median 20, 16, 5 and 6, respectively. The GBMs that showed positive or negative expression for HIF-1α showed a median vascular density of 30 and 14, respectively, for CD34 (P < 0.015). Positive expression for HIF-1α was correlated with VEGF and PDGF-C expression in tumors (P < 0.001). There was a significant correlation between VEGF and PDGF-C expression in the cytoplasm of GBM tumor cells (P < 0.0001). We showed that VEGF expression in tumor cells was correlated with its expression in blood vessels (P < 0.0001). Endothelial cells with PDGF-C and VEGF positive expression were also positive for CD105 and their nuclei for Ki-67, confirming the neoangiogenic and proliferative influence of VEGF and PDGF-C. VEGF nuclear staining in tumor cells (P = 0.002) as well as nuclear staining for HIF-1α and VEGF (P = 0.005) correlated with survival. In summary, our present findings of the concomitant upregulation of PDGF-C with VEGF in GBM tumor cells and vessels further reinforce the benefit of using combined anti-angiogenic approaches to potentially improve the therapeutic response for GBM.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Immunogenicity of a bovine herpes virus I peptide expressed in tandem copies in attenuated Salmonella.

A live system to release heterologous antigens using an attenuated Salmonella strain was developed. We transformed Salmonella typhimurium LVR03 (S. LVR03) with a recombinant pTECH2 vector encoding 0, 1, 2, and 4 tandem copies of an imunogenic peptide of bovine herpes virus-1 (BoHV-1) glycoprotein D (gD). The system used yielded peptides fused to the non-toxic C fragment of the tetanus toxin (TetC), which has been shown to have adjuvant properties. Inoculation of BALB/c mice with the transformed Salmonella strains gave rise to a mild self-limited infection, with primary replication of bacteria occurring in Peyer's patches, even when the bacteria was administered intranasally. Humoral and cellular immune responses directed against the BoHV-1 antigens were evaluated after oral or intranasal administration of the recombinant bacteria. The results showed that the S. LVR03-dimer vaccine induced specific humoral (IgG in serum and IgG(1) and IgA in saliva), and cellular immune responses (lymphoproliferation and lymphokine secretion), against not only the selected peptide and whole gD, but also against BoHV-1, when administered intranasally. This is the first time Salmonella has been used as an expression vector to induce immunity against BoHV-1. This work demonstrates the feasibility of using this antigen-release system and encourages future experimentation with a bovine experimental model.

[The bacillus Calmette-Guérin as immunomodulator in bladder cancer]. / Il bacilo de Calmette-Guérin como inmunomodulador en el cáncer de vejiga.

The bacillus Calmette-Guérin (BCG) is regarded as the most successful immunotherapy against superficial bladder carcinoma recurrences to date. BCG intravesical therapy for superficial bladder cancer has shown its efficacy and advantage over classical therapeutic strategies. This efficacy is based on complex and long lasting immune activation. The initial step is the binding of mycobacteria to the urothelial lining, which depends on the interaction of a fibronectin attachment protein on the bacteria surface with fibronectin in the bladder wall. Granulocytes and other immunocompetent mononuclear cells became attracted to the bladder wall and a cascade of proinflammatory cytokines sustains the immune response. In the bladder wall a largely TH1 based cytokine milieu and granuloma-like cellular foci are established. Within this scenario, the most important effector mechanisms might be the direct antitumor activity of interferons and the cytotoxic activity of NK cells. Current treatment consists of an induction phase of 6 weeks and a maintenance dose schedule of 3 weeks every three months up to 36. The majority of patients present adverse events related to dose administration due to bladder inflammatory response and on only a few occasions, there are mayor complications like granulomatous prostatitis. Among all the neoplasms only in superficial bladder cancer the BCG is proved to be effective.

Hypothesis: dysregulation of immunologic balance resulting from hygiene and socioeconomic factors may influence the epidemiology and cause of glomerulonephritis worldwide.

Glomerular diseases show diverse epidemiological characteristics throughout the world, which has been suggested to be caused by differences in genetics of the underlying populations or environmental exposure to the putative antigens or agents that either trigger or induce the disease. Recently, an alteration in immune balance of the T helper 1 (T(H)1) and T helper 2 (T(H)2) subsets has been implicated as a mechanism to explain the relative increase in allergic diseases in industrialized nations. According to the Hygiene Hypothesis, overcrowding and poor hygiene early in life may protect from atopic diseases because exposure to microbes predisposes in favor of a T(H)1-dominant response. Conversely, dominance of the T(H)2 subset would be responsible for the increasing incidence of allergies. We present the hypothesis that this imbalance may help explain the predilection for membranoproliferative glomerulonephritis (GN) and mesangial proliferative GN to be associated with developing and/or poor nations, whereas immunoglobulin A nephropathy and minimal change disease are observed more commonly in industrialized nations. The implication of the Hygiene Hypothesis is that clinical expression of immune-mediated renal disease would depend on the prevailing T(H)1/T(H)2 balance, rather than the etiologic agent, and it may help explain the epidemiological pattern of glomerular diseases worldwide.

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.

Antiproliferative, antiangiogenic and proapoptotic activity of h-R3: A humanized anti-EGFR antibody.

The epidermal growth factor receptor (EGFR) proto-oncogene is frequently overexpressed in tumors of epithelial origin. This event is thought to be causative for tumor development and progression and henceforth associated with poor prognosis. The recent considerable interest in developing EGFR-targeting agents resulted in derivation of the monoclonal, humanized, neutralizing antibody h-R3, which binds to the extracellular domain of EGFR with high affinity and strongly inhibits EGFR-dependent cellular transformation. Thus, treatment of A431 squamous cell carcinoma cells with h-R3 in either 2-dimensional or 3-dimensional culture resulted in appreciable antimitotic effects through induction of the G1 arrest. Although h-R3 does not appear to have a direct proapoptotic activity in this setting, it inhibits production of the vascular endothelial growth factor (VEGF) by A431 cells both in vitro and in vivo. In the latter case, h-R3 treatment (0.25-1 mg/mouse; every other day per 2 weeks) not only significantly reduced VEGF mRNA expression of A431 tumors growing subcutaneously in SCID mice but also resulted in reduction of the overall microvascular density (MVD), disappearance of dilated "mother vessels," as well as in suppression of tumor growth followed by regression of established tumors. This apparent antiangiogenic activity of h-R3 was associated with reduction in Ki67-positive tumor cell fraction and (unlike in vitro) also with an elevated apoptotic index, the latter indicative of a cytotoxic mode of action in vivo. Taken together, h-R3 is a promising new antagonist of the EGFR oncogene, the anticancer properties of which are associated with combined and potent antiproliferative, antiangiogenic and proapoptotic activity.

Regulation of estrogen target genes and growth by selective estrogen-receptor modulators in endometrial cancer cells.

OBJECTIVE: Tamoxifen has mixed agonist/antagonist activities, leading to tissue-specific estrogen-like actions and endometrial cancer. The purpose of this study was to evaluate the effects of antiestrogens on the growth of estrogen receptor (ER)-positive ECC-1 endometrial cancer cells in vitro and in vivo. METHODS: We performed growth studies and luciferase assays using ERE-tK and AP-1 reporters. ERalpha protein expression was measured by Western blot after antiestrogen treatments. We investigated the actions of antiestrogens on the transcription of the pS2 gene in situ measured by Northern blot and the actions of antiestrogens on the VEGF protein secreted by ELISA. ERalpha, ERbeta, EGFR, and HER2/neu mRNAs were determined by RT-PCR. Last, ECC-1 tumors were developed by inoculation of cells into ovariectomized athymic mice and treated with estradiol (E2), tamoxifen, raloxifene, and a combination. RESULTS: E2 induced cell proliferation while antiestrogens did not. E2 and raloxifene down regulated ERalpha protein; in contrast, 4OHT did not. ICI182,780 completely degraded the receptor. ECC-1 cells express ERbeta at insignificant levels. Luciferase assays did not show any induction in ERE- nor AP-1-mediated transcription by antiestrogens. E2 caused a concentration-dependent increase in pS2 mRNA but antiestrogens did not. E2 increased VEGF expression in a dose-dependent manner and antiestrogens blocked E2 action. E2 down regulated HER2/neu while 4OHT and raloxifene did not change HER2/neu levels compared to control. In addition, EGFR mRNA was down regulated by E2 but raloxifene did not change it. Tamoxifen and raloxifene did not promote tumor growth in vivo. However, raloxifene (1.5 mg daily) only partially blocked E2-stimulated growth. CONCLUSION: Tamoxifen and raloxifene are antiproliferative agents and antiestrogens in ECC-1 endometrial cells in vitro and in vivo. The observation that selective estrogen-receptor modulators do not down regulate EGFR and HER2/neu mRNA may provide a potential role for these oncogenes in the development of raloxifene- or tamoxifen-stimulated endometrial cancer. The ECC-1 cell line could provide important new clues about the evolution of drug resistance to tamoxifen and raloxifene.

CA 125 and vascular endothelial growth factor in the differential diagnosis of epithelial ovarian tumors.

Tumor markers have been investigated in differentiation of benign and malignant tumors. We analyzed CA 125 and vascular endothelial growth factor (VEGF) levels in serum and cyst fluid in patients with epithelial ovarian tumors. Serum and tumor cyst fluid of 50 patients with ovarian epithelial tumors (7 malignant, 3 bordeline and 40 benign) were assayed for VEGF by ELISA and CA 125 levels by chemoluminescence. CA 125 serum levels were significantly higher in patients with malignant and borderline tumors than in patients with benign cysts (p = 0.0005). CA 125 cyst fluid contents were comparable for malignant, borderline and benign ovarian tumors (p = 0.39). Significantly higher levels of VEGF were present in cyst fluid for malignant and borderline tumors compared with benign cysts (p < 0.0001); however, serum levels of VEGF were similar among all patients (p = 0.25). The CA 125 serum levels correlated with matched VEGF cyst fluid levels (r = 0.44, p = 0.0015). Serum CA 125 and cystic VEGF were good methods to differentiate benign and malignant epithelial ovarian tumors. Patients with elevated intracystic VEGF levels presented significantly higher CA 125 serum levels, although the CA 125 intracystic content overlapped. The angiogenesis and enhancement of vascular permeability induced by VEGF represents a new hypothesis for the release of the CA 125 antigen into the circulation in patients with ovarian epithelial neoplasm.

Inflammatory infiltrate, VEGF and FGF-2 contents during corneal angiogenesis in STZ-diabetic rats.

Angiogenesis is a key mechanism that influences several physiological and pathological processes, including wound healing. During the past decades, many groups have shown that controlling angiogenesis might be an answer to overcome pathological situations when this process is out of control. Many altered metabolic states exert considerable influence on the development of angiogenesis. We have chosen diabetes as a model of a progressive metabolic disease with many associated conditions, including an alteration of wound healing dynamics described elsewhere. To evaluate the growth of newly formed blood vessels during diabetes, we induced corneal angiogenesis through silver nitrate cauterization in streptozotocin-induced diabetic rats, always comparing to control non-diabetic or insulin-treated diabetic rats. Computer-aided analysis showed that both the percentage of area taken by vessels on the cornea and their average length were decreased in diabetic animals; furthermore, this diminishment was prevented by insulin treatment in previously diabetic rats. Immunohistochemical staining of neutrophils and macrophages (EDI clone) did not show any differences on number of migrating cells in the cornea. Immunolocalization of vascular endothelial growth factor and basic fibroblast growth factor did not differ considerably among groups either. These results support previous findings that angiogenesis is decreased due to the development of diabetes mellitus but contrasts to descriptions from other investigators in regard to the inflammatory infiltrate and production of growth factors. In our experimental conditions, the cause of the decreased angiogenesis in diabetic rats remains for further elucidation.

Adamantylamide dipeptide as effective immunoadjuvant in rabbits and mice.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis.

BACKGROUND: The aim of this study was to evaluate the concentration of vascular endothelial growth factor (VEGF) in follicular fluid and in granulosa cell cultures in relation to the degree of apoptosis in granulosa cells from patients with different types of ovarian response to controlled ovarian hyperstimulation. METHODS: We studied 30 women who underwent controlled ovarian hyperstimulation and oocyte retrieval. Group A comprised patients with 1-4 follicles (n = 10), group B patients with 5-14 follicles (n = 10) and group C patients with >15 follicles (n = 10). RESULTS: Mean (+/-SD) VEGF concentrations in follicular fluid were 1232 +/- 209, 813 +/- 198 and 396 +/- 103 pg/ml for groups A, B and C respectively (P > 0.01). Concentrations of VEGF in granulosa cell supernatant were 684 +/- 316, 1101 +/- 295 and 1596 +/- 227 pg/ml respectively (P < 0.05). Percentages of apoptotic cells in granulosa cells culture was 55.02 +/- 7.5, 23.98 +/- 4.4 and 14.2 +/- 2.3% respectively (A versus B, P < 0.01, A versus C, P < 0.006, B versus C, NS). CONCLUSIONS: Our findings showed that in patients with decreased ovarian response to controlled ovarian hyperstimulation, follicular fluid VEGF concentration is elevated, the concentration from granulosa cells culture supernatant is decreased and the percentage of apoptotic granulosa cells is increased, while opposite findings occurred in patients with normal or hyper-responses.

Angiogenesis modulators expression in culture cell lines positives for HPV-16 oncoproteins.

Altered angiogenesis response is observed in patients with cervical cancer. In this study we examined whether Human Papilloma Virus (HPV) positive epithelial cells are able to produce angiogenic modulators. When added to human umbilical vein endothelial cells (HUVEC) the media conditioned by HPV-16 positive cells was able to induce proliferation, whereas a contrary effect was observed for media derived from non-tumorigenic keratinocytes. The analyses of angiogenesis modulator's mRNA levels result in a decrease of the antiangiogenic factors TSP-1 and 2 in HPV-16 positive cells. In contrast the expression of the pro-angiogenic molecules: bFGF, IL-8, TGF-beta, TNFalpha, and VEGF were higher in these cells as compared to control keratinocytes. Furthermore the pattern of VEGF isoforms observed in the cells positive for the viral genome point to a preferential induction of the VEGF(189) isoform. We therefore conclude that cervical cancer cells expressing HPV-16 genome are able to contribute to the pro-angiogenic response that might support tumor growth and invasion of the surrounding tissues.

Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

Like other types of pre-malignant lesions and carcinoma, angiogenesis is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular endothelial cell growth factor (VEGF) is known to be one of the most important inducers of angiogenesis and is upregulated in carcinoma of the cervix. Human Papilloma Virus 16 (HPV-16) has been etiologically linked to human cervical cancer, and the major oncogenic proteins encoded by the viral genome, E6 and E7, are involved in the immortalization of target cells. Because several oncogenes including mutant ras, EGF receptor, ErbB2/Her2, c-myc and v-src upregulate VEGF expression, we asked whether HVP-16 E6 oncoprotein could act in a similar fashion. We found that HPV-16 E6-positive cells generally express high levels of VEGF message. Furthermore, co-expression of the VEGF promoter-Luc (luciferase) reporter gene with E6 in both human keratinocytes and mouse fibroblast showed that E6 oncoprotein upregulates VEGF promoter activity, and does so in a p53 independent manner. An E6 responsive region which comprises four Sp-1 sites, between -194 and -50 bp of the VEGF promoter, is also necessary for constitutive VEGF transcription. Taken together, our results suggest the possibility that the HPV oncoprotein E6 may contribute to tumor angiogenesis by direct stimulation of the VEGF gene.