Infections in lung and heart transplant recipients : Studies on the impact of bronchoscopy in the diagnosis of respiratory infections and detection of viral infections in blood and bronchoalveolar lavage fluid

Infection is a major cause of mortality and morbidity after thoracic organ transplantation. The aim of the present study was to evaluate the infectious complications after lung and heart transplantation, with a special emphasis on the usefulness of bronchoscopy and the demonstration of cytomegalovirus (CMV), human herpes virus (HHV)-6, and HHV-7. We reviewed all the consecutive bronchoscopies performed on heart transplant recipients (HTRs) from May 1988 to December 2001 (n = 44) and lung transplant recipients (LTRs) from February 1994 to November 2002 (n = 472). To compare different assays in the detection of CMV, a total of 21 thoracic organ transplant recipients were prospectively monitored by CMV pp65-antigenemia, DNAemia (PCR), and mRNAemia (NASBA) tests. The antigenemia test was the reference assay for therapeutic intervention. In addition to CMV antigenemia, 22 LTRs were monitored for HHV-6 and HHV-7 antigenemia. The diagnostic yield of the clinically indicated bronchoscopies was 41 % in the HTRs and 61 % in the LTRs. The utility of the bronchoscopy was highest from one to six months after transplantation. In contrast, the findings from the surveillance bronchoscopies performed on LTRs led to a change in the previous treatment in only 6 % of the cases. Pneumocystis carinii and CMV were the most commonly detected pathogens. Furthermore, 15 (65 %) of the P. carinii infections in the LTRs were detected during chemoprophylaxis. None of the complications of the bronchoscopies were fatal. Antigenemia, DNAemia, and mRNAemia were present in 98 %, 72 %, and 43 % of the CMV infections, respectively. The optimal DNAemia cut-off levels (sensitivity/specificity) were 400 (75.9/92.7 %), 850 (91.3/91.3 %), and 1250 (100/91.5 %) copies/ml for the antigenemia of 2, 5, and 10 pp65-positive leukocytes/50 000 leukocytes, respectively. The sensitivities of the NASBA were 25.9, 43.5, and 56.3 % in detecting the same cut-off levels. CMV DNAemia was detected in 93 % and mRNAemia in 61 % of the CMV antigenemias requiring antiviral therapy. HHV-6, HHV-7, and CMV antigenemia was detected in 20 (91 %), 11 (50 %), and 12 (55 %) of the 22 LTRs (median 16, 31, and 165 days), respectively. HHV-6 appeared in 15 (79 %), HHV-7 in seven (37 %), and CMV in one (7 %) of these patients during ganciclovir or valganciclovir prophylaxis. One case of pneumonitis and another of encephalitis were associated with HHV-6. In conclusion, bronchoscopy is a safe and useful diagnostic tool in LTRs and HTRs with a suspected respiratory infection, but the role of surveillance bronchoscopy in LTRs remains controversial. The PCR assay acts comparably with the antigenemia test in guiding the pre-emptive therapy against CMV when threshold levels of over 5 pp65-antigen positive leukocytes are used. In contrast, the low sensitivity of NASBA limits its usefulness. HHV-6 and HHV-7 activation is common after lung transplantation despite ganciclovir or valganciclovir prophylaxis, but clinical manifestations are infrequently linked to them.

Cancer incidence, mortality, and pregnancy outcome among women treated for cervical intraepithelial neoplasia

Cervical cancer develops through precursor lesions, i.e. cervical intraepithelialneoplasms (CIN). These can be detected and treated before progression to invasive cancer. The major risk factor for developing cervical cancer or CIN is persistent or recurrent infection with high-risk human papilloma virus (hrHPV). Other associated risk factors include low socioeconomic status, smoking, sexually transmitted infections, and high number of sexual partners, and these risk factors can predispose to some other cancers, excess mortality, and reproductive health complications as well. The aim was to study long-term cancer incidence, mortality, and reproductive health outcomes among women treated for CIN. Based on the results, we could evaluate the efficacy and safety of CIN treatment practices and estimate the role of the risk factors of CIN patients for cancer incidence, mortality, and reproductive health. We collected a cohort of 7 599 women treated for CIN at Helsinki University Central Hospital from 1974 to 2001. Information about their cancer incidence, cause of death, birth of children and other reproductive endpoints, and socio-economic status were gathered through registerlinkages to the Finnish Cancer Registry, Finnish Population Registry, and Statistics Finland. Depending on the endpoints in question, the women treated were compared to the general population, to themselves, or to an age- and municipality-matched reference cohort. Cervical cancer incidence was increased after treatment of CIN for at least 20 years, regardless of the grade of histology at treatment. Compared to all of the colposcopically guided methods, cold knife conization (CKC) was the least effective method of treatment in terms of later CIN 3 or cervical cancer incidence. In addition to cervical cancer, incidence of other HPV-related anogenital cancers was increased among those treated, as was the incidence of lung cancer and other smoking-related cancers. Mortality from cervical cancer among the women treated was not statistically significantly elevated, and after adjustment for socio-economic status, the hazard ratio (HR) was 1.0. In fact, the excess mortality among those treated was mainly due to increased mortality from other cancers, especially from lung cancer. In terms of post-treatment fertility, the CIN treatments seem to be safe: The women had more deliveries, and their incidence of pregnancy was similar before and after treatment. Incidence of extra-uterine pregnancies and induced abortions was elevated among the treated both before and after treatment. Thus this elevation did not occur because they were treated rather to a great extent was due to the other known risk factors these women had in excess, i.e. sexually transmitted infections. The purpose of any cancer preventive activity is to reduce cancer incidence and mortality. In Finland, cervical cancer is a rare disease and death from it even rarer, mostly due to the effective screening program. Despite this, the women treated are at increased risk for cancer; not just for cervical cancer. They must be followed up carefully and for a long period of time; general health education, especially cessation of smoking, is crucial in the management process, as well as interventions towards proper use of birth control such as condoms.

Preparation and characterization of immunoliposomes for targeting of antiviral agents

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia)

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

剪切流动法研究表面固定化配基与目标分子的相互作用力

基于剪切流动腔技术,以微球作为受力载体,设计了一套可用于研究表面固定化配基与目标分子特异性相互作用力的实验和分析方法,并以人免疫球蛋白G (human IgG)和羊抗人免疫球蛋白G(goat anti-human IgG)分别作为模型配基和模型目标分子进行了研究.基于平面Poiseuille层流模型设计了流场参数,以数值计算结果验证了设计的合理性.使用牛血清白蛋白(BSA)作为非特异性对照,判断微球与基片表面的结合力来自配基和目标分子的生物特异性相互作用,并由进一步的目标分子灭活对比实验确认了这一结论.实验观察到微球与基片表面的结合力受到配基面密度的影响,说明发生结合的是多对而非单对蛋白质分子.将95%的微球被剥离时对应的壁面剪切率设定为临界剪切率,由大量实验结果拟合得到了临界剪切率与配基面密度间的定量关系.在受力分析模型中,考虑到多分子的结合,以及分子键位置不同造成的力臂长度的差异,最终计算得到单对配基与目标分子的平均结合力约为342pN.

Study of Interaction Force between Antigen and Antibody Using Flow Chamber Method

A parallel plate flow chamber was used to study the interaction force between human IgG (immobilized on a chip surface as ligand) and goat anti-human IgG (immobilized on microspheres surface as receptor). First, it was demonstrated that the binding force between the microspheres and the chip surface came from the bio-specific interaction between the antigen and the antibody. Secondly, it was obtained that the critical shear rate to detach microspheres from the chip surface increases with the ligand surface concentration. Finally, two models to estimate the antigen-antibody bond strength considering bonds' positions were proposed and analyzed.

HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

Carcinoma epidermóide invasivo de pênis: subexpressão dos fragmentos C3 e C4A/B do sistema complemento detectado no plasma pela plataforma proteômica ClinProt/MALDI/TOF

O carcinoma epidermóide de pênis (CEP) representa 95% das neoplasias penianas e afeta quase sempre pacientes não circuncidados estando muitas vezes associado à falta de higiene local adequada e à fimose. No Brasil a sua incidência é de 2,7 % porém em algumas áreas do país pode chegar a 17% dos casos diagnosticados por ano. O tumor pode ocorrer em qualquer parte do órgão sexual masculino e o tipo de estadiamento empregado é controverso. A classificação de Broders é a mais utilizada. Estudos sugerem a relação entre o desenvolvimento do carcinoma de pênis com a infecção por HPV (Papiloma Vírus Humano). O método de avaliação dos linfonodos inguinais permanece controverso sendo difícil a diferenciação entre linfadenomegalia inflamatória reacional e metastática. O exame físico não é um preditor confiável do comprometimento linfonodal pois pacientes com linfonodos palpáveis podem não apresentar metástases. Há poucas publicações sobre os mecanismos moleculares envolvidos na gênese e progressão do CEP. Apesar de vários marcadores terem sido avaliados, atualmente a aplicação clínica destes é limitada. A maior parte dos marcadores estudados requer procedimentos invasivos para obtenção do tecido tumoral. Existe a necessidade de encontrar através de uma técnica pouco invasiva marcadores tumorais circulantes capazes de diferenciar portadores de CEP com e sem envolvimento metastático. Neste tipo de neoplasia, a descoberta de biomarcadores que avaliem o prognóstico é relevante, pois o exame físico não é um indicador confiável do comprometimento linfonodal e da sobrevida.Os objetivos foram 1) revisar e discutir a epidemiologia, a etiologia, os diversos tipos de abordagem cirúrgica e as controvérsias no tratamento cirúrgico do câncer de pênis 2) investigar através da plataforma ClinProt/ MALDI / TOF a presença de marcadores plasmáticos capazes de discriminar indivíduos saudáveis de pacientes afetados por carcinoma epidermóide de pênis (CEP) 3) avaliar a importância destes marcadores na evolução da doença. Foram coletados e analisados pela plataforma ClinProt / MALDI / TOF o plasma de 36 indivíduos saudáveis e 25 pacientes com CEP invasivo, submetidos a tratamento cirúrgico entre junho de 2010 e junho de 2011, nos serviços de urologia do Instituto Nacional de Câncer e do Hospital Mário Kröeff (Rio de Janeiro). Nossos resultados apontaram para um conjunto de dois peptídeos (A = m / z 1897,22 + -9 Da e B = m / z 2021,99 + -9 Da) que foram capazes de diferenciar pacientes com CEP de indivíduos controles. Esses peptídeos foram posteriormente identificados como fragmentos C3 e C4 A/B do sistema complemento. A validação cruzada, utilizando toda casuística apresentou 62,5% e 86,76% de sensibilidade e de especificidade, respectivamente, com uma alta sensibilidade (100%) e especificidade (97%) nos pacientes que morreram pela doença. Além disso, os pacientes com envolvimento ganglionar obtiveram uma sensibilidade e uma especificidade de 80 % e 97%, respectivamente. Ficou demonstrado que à medida que a doença progride mais subexpressos está o conjunto de peptídeos quando comparados com indivíduos saudáveis. Estes resultados podem ser úteis como ferramentas para a avaliação do prognóstico destes pacientes.

Population structure and history in East Asia

Archaeological, anatomical, linguistic, and genetic data have suggested that there is an old and significant boundary between the populations of north and south China. We use three human genetic marker systems and one human-carried virus to examine the north/south distinction. We find no support for a major north/south division in these markers; rather, the marker patterns suggest simple isolation by distance.

一种多皱软磺酸在抗HIV-1病毒中的应用

本发明涉及抗HIV-1病毒活性的化合物，具体地说是一种多皱软磺酸在抗HIV-1病毒中的应用，多皱软磺酸的结构式为，以所述多皱软磺酸为抑制HIV-1病毒活性的有效成分，该化合物在细胞水平的活性检测中，对1型人类免疫缺陷病毒 (Human Immunodeficiency Virustype I，HIV-1)表现出很好的抑制活性，而对宿主细胞的毒性非常小，作为药物先导化合物具有良好的应用前景。

兔抗人外周血单核细胞、兔抗猴脾脏淋巴细胞免疫血清的制备及其在分离人和猕猴胚胎内细胞团中的应用

采用梯度离心的方法用淋巴细胞分离液从浓缩的人白细胞悬液中分离人外周血单核细胞，从猕猴新 鲜脾脏中研磨分离猕猴脾脏淋巴细胞；分别将人外周血单核细胞和猕猴脾脏淋巴细胞作为免疫原，每次通过耳 缘静脉注射4×10s个细胞免疫日本大耳白兔，每周免疫一次，共免疫3次，制备兔抗人外周血单核细胞和兔抗 猕猴脾脏淋巴细胞免疫血清。体外培养人和猕猴胚胎至囊胚，采用制备的免疫血清，分离人和猕猴囊胚内细胞 团，用于建立人和猕猴胚胎干细胞系。结果如下：(1)用半微量细胞毒实验法测得兔抗人外周血单核细胞免疫 血清和兔抗猕猴脾脏淋巴细胞免疫血清的效价分别为1：320和1：640；(2)两种免疫血清成功地裂解了15个人 囊胚和33个猕猴囊胚的滋养层细胞，分离出了内细胞团，表明免疫血清的制备取得了成功，为建立人和猕猴 胚胎干细胞系奠定了基础。

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Au and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000 ng mL(-1), with detection limits 8 (thermal initiation) and 12 ng mL(1) (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode. (C) 2004 Elsevier B.V. All rights reserved.

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb3+ chelate. N,N.N-1,N-1-12,6-bis(3'-aminomethyl-1'-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb3+ (BPTA-Tb3+), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2- aminoethylamino)-ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric quantum methods show that the nanoparticles are spherical and uniform in size, 45 +/- 3 nm in diameter, strongly fluorescent with fluorescence yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle's surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human alpha-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of (x-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml(-1) to about 100 ng ml(-1) with the detection limit of 0.10 ng ml(-1). The coefficient variations (CVs) of the method are less than 9.0%. and the recoveries are in the range of 84-98% for human serum sample measurements. (C) 2004 Elsevier B.V. All rights reserved.