(Table 1) Hepatic EROD activity and and retinoid concentrations in the liver, and plasma thyroid hormones of northern fulmars (Fulmarus glacialis) from Bjørnøya

We investigated whether the hepatic cytochrome P450 1A activity (measured as 7-ethoxyresorufin-O-deethylase (EROD)) and plasma thyroid hormone and liver retinoid concentrations were explained by liver and blood levels of halogenated organic contaminants (HOCs) in free-ranging breeding northern fulmars (Fulmarus glacialis) from Bjornoya in the Norwegian Arctic. Hepatic EROD activity and liver levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were positively correlated, suggesting that hepatic EROD activity is a good indicator for dioxin and dioxin-like HOC exposure in breeding northern fulmars. There were not found other strong relationships between HOC concentrations and hepatic EROD activity, plasma thyroid or liver retinoid concentrations in the breeding northern fulmars. It is suggested that the HOC levels found in the breeding northern fulmars sampled on Bjornoya were too low to affect plasma concentrations of thyroid hormones and liver levels of retinol and retinyl palmitate, and that hepatic EROD activity is a poor indicator of polychlorinated biphenyl (PCB) and pesticide exposure.

Induction of ovulation in rabbit does using purified nerve growth factor and camel seminal plasma

The presence of an ovulation-inducing factor (OIF) in the seminal plasma (SP) of several species with spontaneous and induced ovulation, including the rabbit, has been documented. Recent studies have demonstrated that the OIF in the SP of camels (SPCAM) is a nerve growth factor (β-NGF). The aim of this study was to determine if purified β-NGF from mouse submandibular glands or SPCAM could provoke ovulation induction in the rabbit doe. A total of 35 females were synchronized with 25 IU of equine chorionic gonadotropin (Serigan, Laboratorios Ovejero, Spain) and allocated into 4 groups. Forty-eight hours later (Day 0), does were given a single dose (IM) of 1 mL of saline solution (SS; n = 8); 1 mL of gonadorelin (GnRH; Inducel, Laboratorios Ovejero, Spain; n = 9); 24 µg of β-NGF (2.5S-NGF; Promega, USA; n = 10); or 1 mL of centrifuged raw camel SP (SPCAM; 127 pg mL–1 NGF; n = 8). After treatment, an empty catheter was introduced through the vagina to simulate the nervous/mechanical stimulus of coitus (4 animals per group). Plasma LH concentrations were determined in blood samples taken 30 min before treatment and at 0, 30, 60, 90, and 120 min after injection. Progesterone concentrations were assessed at 0 and 120 min and every 2 days until Day 6 after treatment. Concentrations of β-NGF in camel SP and hormone determinations were made by enzyme immunoassay. Ovulation rate (OR) was determined after euthanasia on Day 7.

Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen

Angiotensin produced systemically or locally in tissues such as the brain plays an important role in the regulation of blood pressure and in the development of hypertension. We have established transgenic rats [TGR(ASrAOGEN)] expressing an antisense RNA against angiotensinogen mRNA specifically in the brain. In these animals, the brain angiotensinogen level is reduced by more than 90% and the drinking response to intracerebroventricular renin infusions is decreased markedly compared with control rats. Blood pressure of transgenic rats is lowered by 8 mmHg (1 mmHg = 133 Pa) compared with control rats. Crossbreeding of TGR(ASrAOGEN) with a hypertensive transgenic rat strain exhibiting elevated angiotensin II levels in tissues results in a marked attenuation of the hypertensive phenotype. Moreover, TGR(ASrAOGEN) exhibit a diabetes insipidus-like syndrome producing an increased amount of urine with decreased osmolarity. The observed reduction in plasma vasopressin by 35% may mediate these phenotypes of TGR(ASrAOGEN). This new animal model presenting long-term and tissue-specific down-regulation of angiotensinogen corroborates the functional significance of local angiotensin production in the brain for the central regulation of blood pressure and for the pathogenesis of hypertension.

Elevated blood pressures in mice lacking endothelial nitric oxide synthase

Nitric oxide produced in endothelial cells affects vascular tone. To investigate the role of endothelial nitric oxide synthase (eNOS) in blood pressure regulation, we have generated mice heterozygous (+/−) or homozygous (−/−) for disruption of the eNOS gene. Immunohistochemical staining with anti-eNOS antibodies showed reduced amounts of eNOS protein in +/− mice and absence of eNOS protein in −/− mutant mice. Male or female mice of all three eNOS genotypes were indistinguishable in general appearance and histology, except that −/− mice had lower body weights than +/+ or +/− mice. Blood pressures tended to be increased (by approximately 4 mmHg) in +/− mice compared with +/+, while −/− mice had a significant increase in pressure compared with +/+ mice (≈18 mmHg) or +/− mice (≈14 mmHg). Plasma renin concentration in the −/− mice was nearly twice that of +/+ mice, although kidney renin mRNA was modestly decreased in the −/− mice. Heart rates in the −/− mice were significantly lower than in +/− or +/+ mice. Appropriate genetic controls show that these phenotypes in F2 mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL/6J) and are linked either to the eNOS locus or to an unlinked chromosomal region containing the renin locus. Thus eNOS is essential for maintenance of normal blood pressures and heart rates. Comparisons between the current eNOS mutant mice and previously generated inducible nitric oxide synthase mutants showed that homozygous mutants for the latter differ in having unaltered blood pressures and heart rates; both are susceptible to lipopolysaccharide-induced death.

Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure

Potent antiretroviral therapy can reduce plasma HIV RNA levels below the threshold of detection for periods of a year or more. The magnitude of HIV RNA reduction in the lymphoid tissue in patients with suppression of HIV RNA levels in plasma beyond 6 months has not been determined. We evaluated levels of HIV RNA and DNA and characterized resistance mutations in blood and inguinal lymph node biopsies obtained from 10 HIV-infected subjects who received 36–52 weeks of indinavir (IDV)/zidovudine (ZDV)/lamivudine (3TC), IDV, or ZDV/3TC. After 1 year of therapy, viral RNA levels in LN of individuals remained detectable but were log10 = 4 lower than in subjects on the triple drug regimen with interruption of therapy or in those treated with ZDV/3TC alone, who had viral loads in their lymph nodes indistinguishable from those expected for untreated patients. In all cases viral DNA remained detectable in lymph nodes and peripheral blood mononuclear cells (PBMC). When plasma virus suppression was incomplete, lymph node and PBMC cultures were positive and drug resistance developed. These studies indicate that pronounced and sustained suppression of plasma viremia by a potent antiretroviral combination is associated with low HIV RNA levels in the lymph nodes 1 year after treatment. Conversely, the persistence of even modest levels of plasma virus after 1 year of treatment reflects ongoing viral replication, the emergence of drug resistance, and the maintenance of high burdens of virus in the lymph nodes.

Cardiolipin is a normal component of human plasma lipoproteins

Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean ± SD) was 14.9 ± 3.7 μg/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 ± 1.7 μg/ml), phosphatidylethanolamine (64 ± 20 μg/ml), or choline-containing phospholipid (1,580 ± 280 μg/ml). Based on studies of fasting blood donors, CL (≥94%) was recovered in very low density, low density, and high density lipoproteins (11 ± 5.3%, 67 ± 11.0%, and 17 ± 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e.g., β2-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

Blood-borne tissue factor: Another view of thrombosis

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused ≈70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VIIai, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor Xa formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.

Cohort study of plasma natriuretic peptides for identifying left ventricular systolic dysfunction in primary care

Objectives: To determine whether blood natriuretic peptide concentrations are helpful in identifying or excluding left ventricular systolic dysfunction in stable survivors of acute myocardial infarction.

Chronic elevation of plasma thioredoxin: Inhibition of chemotaxis and curtailment of life expectancy in AIDS

Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like and chemokine-like activities. We show here that, although plasma Trx levels are unrelated to survival of HIV-infected individuals with CD4 cell counts above 200/μl blood, survival is significantly impaired (P = 0.003) when plasma Trx is chronically elevated in HIV-infected subjects with CD4 T cell counts below this level (i.e., with Centers for Disease Control (CDC)-defined AIDS). Relevant to the mechanism potentially underlying this finding, we also present data from experimental studies in mice showing that elevated plasma Trx efficiently blocks lipopolysaccharide (LPS)-induced chemotaxis, an innate immune mechanism that is particularly crucial when adaptive immunity is compromised. Thus, we propose that elevated plasma Trx in HIV-infected individuals with low CD4 T cell counts directly impairs survival by blocking pathogen-induced chemotaxis, effectively eliminating the last (innate) barrier against establishment of opportunistic and other infections in these immunodeficient individuals.

Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.

Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.

Avaliação sistemática do uso do Dried Blood Spot para determinação de elementos químicos em sangue capilar visando estudos de biomonitoramento no Brasil

A biomonitorização humana ou biomonitoramento (BH) é definido como a medida periódica de determinada substância química ou seu metabólito em fluidos biológicos, principalmente sangue e urina, de uma população com o objetivo de avaliar a exposição e os riscos à saúde. Tal método tem se tornado comum em países desenvolvidos, porém ainda é uma prática pouco utilizada no Brasil. Isso ocorre pela dificuldade de coleta, armazenamento e transporte das amostras, principalmente em regiões sem infraestrutura e de difícil acesso. Diante disso, alguns procedimentos alternativos de coleta de amostra vêm sendo propostos. Um destes procedimentos é o Dried Blood Spot (DBS) ou coleta e armazenamento de amostra em papel-cartão. Este método oferece uma série de vantagens sobre os procedimentos de coleta convencionais, principalmente por reduzir consideravelmente o volume de amostra coletada. Entretanto, pouco se sabe sobre a estabilidade dos analitos após a deposição da amostra no cartão e do risco de contaminação da amostra pelo substrato sólido. Além disso, procedimentos de extração dos analitos do papel, para posterior quantificação, ainda não estão totalmente estabelecidos. Neste sentido, o presente trabalho avaliou de forma sistemática o procedimento de coleta de sangue por DBS visando sua futura aplicação em programas de biomonitoramento no Brasil para determinação dos elementos químicos As, Cd, Cu, Hg, Mn, Pb, Se e Zn por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para isso, no estudo foram utilizadas três diferentes marcas comerciais de cartão coletor: Whatman 903(TM), Munktell(TM), e DMPK-C(TM). Todas as marcas de cartão apresentaram baixas concentrações dos elementos químicos. Após a deposição da amostra no papel cartão verificou-se que a concentração dos elementos químicos manteve-se estável por um período de pelo menos 60 dias (temperatura ambiente e ao abrigo da luz). Foi otimizado o método de extração dos analitos do substrato, com melhor condição obtida após a imersão do papel (corte circular de diâmetro de 1/2´´) por 60 minutos em solução extratora (0,5% v/v HNO3 e 0,01% v/v Triton(TM) X-100) na proporção de 1:50 v/v, seguida de 10 segundos de agitação por vortex. Após a extração, a solução resultante contendo os analitos foi diretamente injetada no ICP-MS. Cabe também destacar que não foram observadas diferenças estatísticas nas concentrações dos elementos químicos com coleta de sangue da veia do antebraço (sangue venoso) ou do dedo (sangue capilar). Os resultados obtidos no presente estudo, devem contribuir para a implementação deste procedimento em análises de elementos químicos (biomonitoramento da população brasileira), principalmente considerando as dificuldades de coleta, armazenamento e transporte de amostras clínicas em nosso país por sua extensão territorial. Além disso, este procedimento pode facilitar estudos com populações vulneráveis e que vivem em áreas remotas e de difícil acesso.

Detecção de DNA de HPV no plasma para potencial identificação precoce de recidiva de câncer do colo de útero

O câncer do colo do útero constitui a terceira neoplasia maligna mais comum na população feminina, com aproximadamente 520 mil novos casos e 260 mil óbitos por ano e origina-se a partir da infecção genital persistente pelo Papiloma Vírus Humano (HPV) oncogênico. Os principais HPVs considerados de alto risco oncogênico são os tipos HPV-16 e 18, responsáveis por cerca de 70% de todos os casos de cânceres cervicais (CC) no mundo. Pacientes com CC apresentam taxa de recidiva variando de 8% a 49%. Dentro de dois anos de seguimento, 62% a 89% das recidivas são detectadas. Atualmente, os testes usados para detecção de recidiva são a citopatologia da cúpula vaginal e exames de imagem, porém ainda não estão disponíveis testes específicos. O DNA livre-circulante (cf-DNA) representa um biomarcador não-invasivo facilmente obtido no plasma e soro. Vários estudos mostram ser possível detectar e quantificar ácidos nucléicos no plasma de pacientes com câncer e que as alterações no cfDNA potencialmente refletem mudanças que ocorrem durante a tumorigênese. Essa ferramenta diagnóstica não-invasiva pode ser útil no rastreio, prognóstico e monitoramento da resposta ao tratamento do câncer. Portanto, o desenvolvimento e a padronização de testes laboratoriais não invasivos capazes de identificar marcadores tumorais e diagnosticar precocemente a recidiva da doença aumentam a chance de cura através da utilização dos tratamentos preconizados. Sendo assim, este estudo tem o objetivo de detectar o DNA de HPV no plasma de pacientes com CC para avaliar sua potencial utilidade como marcador precoce de recidiva. Um fragmento de tumor e sangue de pacientes com CC, atendidas no ICESP e HC de Barretos, foram coletados antes do tratamento. Entraram no estudo 137 pacientes nas quais o tumor foi positivo para HPV-16 ou 18, sendo 120 amostras positivas para HPV-16 (87,6%), 12 positivas para HPV-18 (8,8%) e cinco positivas para HPV-16 e 18 (3,6%). A média de idade das pacientes deste estudo foi de 52,5 anos. Plasma de 131 pacientes com CC da data do diagnóstico e de 110 pacientes do seguimento foram submetidas ao PCR em Tempo Real HPV tipo específico. A presença do DNA de HPV no plasma pré-tratamento foi observada em 58,8% (77/131) com carga viral variando de 204 cópias/mL a 2.500.000 cópias/mL. A positividade de DNA no plasma pré-tratamento aumentou com o estadio clínico do tumor: I - 45,2%, II - 52,5%, III - 80,0% e IV - 76,9%, (p=0,0189). A presença do DNA de HPV no plasma pós-tratamento foi observada em 27,3% (30/110). A média de tempo das recidivas foi de 3,1 anos (2,7 - 3,5 anos). O DNA de HPV foi positivo até 460 dias antes do diagnóstico clínico da recidiva. As pacientes com DNA de HPV no plasma apresentaram pior prognóstico, tanto sobrevida como o tempo livre de doença, em relação às que foram negativas. Nas pacientes com CC a presença de HPV no plasma de seguimento pode ser um marcador precoce útil para o monitoramento da resposta terapêutica e detecção de pacientes com risco aumentado de recidiva e progressão da doença.

Elevated plasma levels of human urotensin-II immunoreactivity in congestive heart failure

Human urotensin-II (hU-II) is the most potent endogenous cardiostimulant identified to date. We therefore determined whether hU-II has a possible pathological role by investigating its levels in patients with congestive heart failure (CHF). Blood samples were obtained from the aortic root, femoral artery, femoral vein, and pulmonary artery from CHF patients undergoing cardiac catheterization and the aortic root from patients undergoing investigative angiography for chest pain who were not in heart failure. Immunoreactive hU-II (hU-II-ir) levels were determined with radioimmunoassay. hU-II-ir was elevated in the aortic root of CHF patients (230.9 +/- 68.7 pg/ml, n = 21; P < 0.001) vs. patients with nonfailing hearts (22.7 +/- 6.1 pg/ml, n = 18). This increase was attributed to cardiopulmonary production of hU-II-ir because levels were lower in the pulmonary artery (38.2 +/- 6.1 pg/ml, n = 21; P < 0.001) than in the aortic root. hU-II-ir was elevated in the aortic root of CHF patients with nonischemic cardiomyopathy (142.1 +/- 51.5 pg/ml, n = 10; P < 0.05) vs. patients with nonfailing hearts without coronary artery disease (27.3 +/- 12.4 pg/ml, n = 7) and CHF patients with ischemic cardiomyopathy (311.6 +/- 120.4 pg/ml, n = 11; P < 0.001) vs. patients with nonfailing hearts and coronary artery disease (19.8 +/- 6.6 pg/ml, n = 11). hU-II-ir was significantly higher in the aortic root than in the pulmonary artery and femoral vein, with a nonsignificant trend for higher levels in the aortic root than in the femoral artery. The findings indicated that hU-II-ir is elevated in the aortic root of CHF patients and that hU-II-ir is cleared at least in part from the microcirculation.