Sequential cytokine dynamics in chronic rejection of rat renal allografts: roles for cytokines RANTES and MCP-1.

Chronic rejection, the most important cause of long-term graft failure, is thought to result from both alloantigen-dependent and -independent factors. To examine these influences, cytokine dynamics were assessed by semiquantitative competitive reverse transcriptase-PCR and by immunohistology in an established rat model of chronic rejection lf renal allografts. Isograft controls develop morphologic and immunohistologic changes that are similar to renal allograft changes, although quantitatively less intense and at a delayed speed; these are thought to occur secondary to antigen-independent events. Sequential cytokine expression was determined throughout the process. During an early reversible allograft rejection episode, both T-cell associated [interleukin (IL) 2, IL-2 receptor, IL-4, and interferon gamma] and macrophage (IL-1 alpha, tumor necrosis factor alpha, and IL-6) products were up-regulated despite transient immunosuppression. RANTES (regulated upon activation, normal T-cell expressed and secreted) peaked at 2 weeks; intercellular adhesion molecule (ICAM-1) was maximally expressed at 6 weeks. Macrophage products such as monocyte chemoattractant protein (MCP-1) increased dramatically (to 10 times), presaging intense peak macrophage infiltration at 16 weeks. In contrast, in isografts, ICAM-1 peaked at 24 weeks. MCP-1 was maximally expressed at 52 weeks, commensurate with a progressive increase in infiltrating macrophages. Cytokine expression in the spleen of allograft and isograft recipients was insignificant. We conclude that chronic rejection of kidney allografts in rats is predominantly a local macrophage-dependent event with intense up-regulation of macrophage products such as MCP-1, IL-6, and inducible nitric oxide synthase. The cytokine expression in isografts emphasizes the contribution of antigen-independent events. The dynamics of RANTES expression between early and late phases of chronic rejection suggest a key role in mediating the events of the chronic process.

Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase.

Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.

A splice variant of alpha 6 integrin is associated with malignant conversion in mouse skin tumorigenesis.

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.

Estudo da viabilidade para introduzir na rotina testes de diagnóstico para infecção respiratória aguda

Para avaliar os benefícios da comunicação rápida ao clínico do diagnóstico de vírus respiratórios, foi analisado a viabilidade econômica de 2 testes, com o tempo de entrega de resultado em 2 horas para teste rápido e 48 horas para Biologia Molecular. As amostras coletadas foram processadas utilizando técnicas convencionais e os testes disponíveis no mercado local. Foram escolhidos dois testes rápidos pelo método de imunocromatografia para quatro parâmetros analíticos: Influenza A, Influenza H1N1, Influenza B e Vírus Sincicial Respiratório (RSV) e em Biologia Molecular um teste de RT-PCR multiplex com 25 patógenos entre vírus e bactérias. O tipo de amostra utilizada foi swab e lavado de nasofaringe. A população escolhida para o estudo foi paciente adulto, em tratamento de câncer, que necessita de uma resposta rápida já que a maioria se encontra com comprometimento do sistema imune por doença ou por tratamento. O estudo foi transversal, realizado entre os anos de 2012 e 2013, para avaliar a viabilidade econômica da introdução de testes de diagnóstico da infecção respiratória aguda de etiologia viral a partir de amostras de nasofaringe em pacientes com câncer atendidos no Centro de Atendimento de Oncologia Intercorrência (CAIO ), do Instituto do Câncer do Estado de São Paulo (ICESP), hospital público que atende exclusivamente Sistema Único de Saúde (SUS) e Hospital A.C. Camargo, que atende tanto a pacientes do SUS como da rede privada. O estudo incluiu 152 pacientes em tratamento para qualquer tipo de câncer, predominantemente do sexo feminino (81 mulheres e 70 homens) com idades entre 18-86 anos. Para participar do estudo o paciente era consultado e o critério para escolha do paciente foi ser portador de câncer, com história de febre (ainda que referida) acompanhada de tosse ou dor de garganta, tosse e sintomas respiratórios agudos, atendidos por protocolo padronizado que inclui avaliação na admissão, seguimento e manejo antimicrobiano. Para a avaliação econômica os pacientes foram classificados de acordo com o estado geral de saúde, se apresentavam bom estado de estado de saúde poderiam receber alta e faziam uso da medicação em casa evitando 5 dias de internação se recebessem algum resultado para Influenza ou RSV, no entanto os pacientes que apresentavam outro vírus, resultado negativo ou o estado geral era ruim permaneciam internados por 7 dias em observação e cuidados com medicação adequada. Foram realizadas análises econômicas em dois âmbitos: o sistema de saúde publico e o privado considerando o fator diminuição de dias de internação. A analise de Custo-benefício foi eficiente no Sistema privado mas inadequada para o SUS assim como, qualquer outra medida monetária já que os valores de reembolso do SUS estão defasados do custo de qualquer internação. A análise de Custo-efetividade que olha para outros fatores além do monetário foi efetiva nos dois sistemas que enfrentam falta de leitos além da condição de saúde do paciente de evitar a ingestão desnecessária de antibióticos, evitar os gastos do acompanhante, perda de dias de trabalho e estudo. Não houve correspondência de resultados dos testes rápidos com o multiplex de Biologia Molecular

Efavirenz versus Nevirapina : comparação da efectividade e do perfil de segurança, a longo prazo, no tratamento da infecção por vírus da imunodeficiência humana tipo 1

Tese de mestrado, Doenças Infecciosas Emergentes, Universidade de Lisboa, Faculdade de Medicina, 2016

Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

Kinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl-dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C-terminus to kleisin's N-terminus. Our findings imply that proteolysis-independent release of cohesin from chromatin is mediated by Wapl-dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.

Expression of biomineralization-related ion transport genes in Emiliania huxleyi

Biomineralization in the marine phytoplankton Emiliania huxleyi is a stringently controlled intracellular process. The molecular basis of coccolith production is still relatively unknown although its importance in global biogeochemical cycles and varying sensitivity to increased pCO2 levels has been well documented. This study looks into the role of several candidate Ca2+, H+ and inorganic carbon transport genes in E. huxleyi, using quantitative reverse transcriptase PCR. Differential gene expression analysis was investigated in two isogenic pairs of calcifying and non-calcifying strains of E. huxleyi and cultures grown at various Ca2+ concentrations to alter calcite production. We show that calcification correlated to the consistent upregulation of a putative HCO3- transporter belonging to the solute carrier 4 (SLC4) family, a Ca2+/H+ exchanger belonging to the CAX family of exchangers and a vacuolar H+-ATPase. We also show that the coccolith-associated protein, GPA is downregulated in calcifying cells. The data provide strong evidence that these genes play key roles in E. huxleyi biomineralization. Based on the gene expression data and the current literature a working model for biomineralization-related ion transport in coccolithophores is presented.

Statistical Hurdle Models for Single Cell Gene Expression: Differential Expression and Graphical Modeling

Thesis (Ph.D.)--University of Washington, 2016-06

Real-time PCR in the microbiology laboratory

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

Endothelin axis expression is markedly different in the two main subtypes of renal cell carcinoma

BACKGROUND. The endothelin axis has been implicated in cancer growth, angiogenesis, and metastasis, but to the authors' knowledge the expression of endothelin genes has not been defined in renal cell carcinoma (RCC). METHODS. Tissue specimens were harvested from both normal and tumor-affected regions at the time of radical nephrectomy from 35 patients with RCC (22 with clear cell RCC [ccRCC] and 13 with papillary RCC [PRCC]). Real-time reverse transcriptase-polymerase chain reaction analysis determined the expression profile of the preproendothelins (PPET-1, PPET-2, and PPET-3), the endothelin receptors (ETA and ETB), and the endothelin-converting enzymes (ECE-1 and ECE-2). RESULTS. PPET-1 was found to be up-regulated in ccRCC tumor specimens and down-regulated in PRCC tumor specimens. ETA was significantly down-regulated in PRCC tumor specimens. ECE-1 was expressed in all tissue specimens at comparable levels, with moderate but significant elevation in normal tissue specimens associated with PRCC. Of the other genes, PPET-2 and ETB were expressed in all tissue specimens and no differences were observed between tumor subtypes or tumor-affected and normal tissue specimens, whereas PPET-3 and ECE-2 were present in all tissue specimens but were barely detectable. CONCLUSIONS. The endothelin axis was expressed differently in the two main subtypes of RCC and appeared to match macroscopic features commonly observed in these tumors (i.e., high expression of PPET-I in hypervascular ccRCC contrasted against low PPET-1 and ETA expression in hypovascular PRCC). The presence of ECE-1 mRNA in these tissue specimens suggested that active endothelin ligands were present, indicating endothelin axis activity was elevated in ccRCC compared with normal kidney, but impaired in PRCC. The current study provided further evidence that it is not appropriate to consider ccRCC and PRCC indiscriminately in regard to treatment. (C) 2004 American Cancer Society.