RESUMO
Toxoplasma gondii (RH strain) tachyzoites were transfected with a plasmid containing a fusion of the chloramphenicol acetyl transferase and the Herpes simplex virus-2 thymidine kinase coding regions and transgenic parasites obtained by chloramphenicol selection. CTK11, a single high expressing clone was isolated based on immunofluorescence and contained approximately five integrated copies of the fusion sequence. Lysates prepared from this clone displayed thymidine kinase activity of 2.9 pmol min(-1) microg(-1) protein, whereas thymidine kinase activity was not detected in lysates from the parental RH strain. Growth of CTK11 tachyzoites was fully inhibited in 5 microM ganciclovir and thymidine and in 2.5 microM 5-bromo-2'-deoxyuridine. While the inhibitory effects of ganciclovir were lethal, low concentrations of thymidine (10 microM) were largely reversible. Asynchronously growing CTK11 tachyzoites were found to contain major G1 (1 N) and S phase (1 N+) distributions as determined by relative propidium iodide fluorescence and with reference to the haploid (1 N) DNA content of a T. gondii sporozoite population. CTK11 tachyzoites blocked 4 h in 10 microM thymidine exhibited mean fluorescence consistent with a 1 N complement of DNA indicating growth was arrested in G1. Following the removal of excess thymidine, parasites immediately entered S phase, thus confirming the late G1 block. Parasites with a 2 N complement of DNA (G2 + M) first appear at 2 h post-release, while 1 N (G1) parasites re-appear at 3 h suggesting the length of S phase is < or = 2 h and that of G2 + M is < or = 1 h. Within 7 h, parasites had transited G2 + M and much of G1 and re-entered S of the subsequent cell cycle--a time consistent with the doubling of these parasites in culture. Thus, the CTK11 tachyzoite cell cycle is similar to those of higher eukaryotic cells and is characterized by major G1 and S phases and a relatively short G2 + M.
Assuntos
Timidina Quinase/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/genética , Animais , Antimetabólitos/farmacologia , Bromouracila/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Protozoário/metabolismo , Eletroporação , Citometria de Fluxo , Fase G1 , Fase G2 , Ganciclovir/farmacologia , Vetores Genéticos , Herpesvirus Humano 2/enzimologia , Herpesvirus Humano 2/genética , Mitose , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Seleção Genética , Timidina/farmacologia , Timidina Quinase/metabolismo , Toxoplasma/citologia , Toxoplasma/enzimologia , Uridina/análogos & derivados , Uridina/farmacologiaRESUMO
Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.
Assuntos
Ciclo Celular/fisiologia , Toxoplasma/crescimento & desenvolvimento , Animais , Divisão Celular , DNA de Protozoário/análise , Citometria de Fluxo , Imunofluorescência , Fase G1 , Fase G2 , Expressão Gênica , Mitose , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S , Toxoplasma/citologia , Toxoplasma/genéticaRESUMO
Some patients with cancer and others with benign lesions which obstruct the central airways (larynx, trachea, major bronchi) can be treated with a laser. Ninety-nine patients were considered for treatment during the first 18 months of experience with a YAG (yttrium aluminum garnet) laser at Henry Ford Hospital; 55 patients were treated 82 times. Results were satisfactory (surgery was avoided) in eight of ten patients with benign lesions. Satisfactory results (doubling of airway size with relief of dyspnea/drainage of obstructive pneumonia) were obtained in 12 of 13 patients with bronchogenic carcinoma managed initially with the laser, and in 22 of 32 (69 percent) patients with recurrent malignancies. There were five minor and seven major complications, including two deaths. We conclude that laser treatment can relieve central airways obstruction with its associated symptoms of dyspnea and infection. Avoidance of complications requires a skillful approach, careful anesthetic management, and availability of back-up posttreatment intensive care.
Assuntos
Carcinoma Broncogênico/cirurgia , Terapia a Laser , Neoplasias Pulmonares/cirurgia , Anestesia Endotraqueal/efeitos adversos , Carcinoma Broncogênico/mortalidade , Humanos , Doenças da Laringe/cirurgia , Lasers/efeitos adversos , Neoplasias Pulmonares/mortalidade , Recidiva Local de Neoplasia/cirurgia , Pneumonia/etiologia , Edema Pulmonar/etiologia , Doenças da Traqueia/cirurgia , Fístula Traqueoesofágica/etiologiaRESUMO
Ninety-seven consecutive peripheral lung lesions were evaluated by biplane fluoroscopically guided flexible fiberoptic bronchoscopy and analyzed to define features that predict diagnostic yield. The overall diagnostic accuracy was 56 percent (63 percent for malignant and 38 percent for benign lesions). The most important characteristic associated with a positive cyto- or histopathologic diagnosis was size of the lesion; the yield was 28 percent when the diameter was less than 2.0 cm compared to 64 percent if the diameter was greater than or equal to 2.0 cm (P = 0.0035). The diagnostic yield was similar for lesions located in the outer and middle third of the lung if the diameter was greater than 2.0 cm; inner one-third lesions were correctly diagnosed more frequently, related in part to the larger size of these lesions. There was no significant difference in diagnostic yield for the following: segmental location, greatest distance from carcina on either the posteroanterior or lateral radiograph, or radiographic characteristics of the lesion. We conclude that biplane fluoroscopically guided flexible fiberoptic bronchoscopy is a reasonable diagnostic procedure for peripheral lesions greater than or equal to 2.0 cm in diameter, but that alternative procedures should be used for lesions under 2.0 cm in diameter.
Assuntos
Broncoscópios , Pneumopatias/diagnóstico , Neoplasias Pulmonares/diagnóstico , Adulto , Idoso , Estudos de Avaliação como Assunto , Feminino , Tecnologia de Fibra Óptica , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Patients presenting with inoperable non-small cell carcinoma of the lung and major symptomatic bronchial obstruction were treated initially with debulking of the airways by YAG laser, followed by conventional external-beam radiotherapy. The former method was used to minimize postobstructive pneumonitis or respiratory failure (or both) that often complicates major brochial obstruction and also to lessen the burden of tumor to be treated by radiotherapy. The preliminary results of 19 patients treated in this manner are reported, emphasizing the impact of this combined method on morbidity and mortality.
Assuntos
Carcinoma/cirurgia , Terapia a Laser , Neoplasias Pulmonares/cirurgia , Adenocarcinoma/radioterapia , Adenocarcinoma/cirurgia , Idoso , Carcinoma/radioterapia , Carcinoma Broncogênico/radioterapia , Carcinoma Broncogênico/cirurgia , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/cirurgia , Terapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/radioterapia , Masculino , Pessoa de Meia-Idade , Complicações Pós-OperatóriasRESUMO
Flexible fiberoptic bronchoscopy was performed in 101 patients with sarcoidosis, and the endoscopic findings were analyzed and correlated with radiographic stages and diagnostic bronchial biopsy. Endoscopic findings included bronchostenosis (26% of patients), mucosal nodularity (64%), hypervascularity (38%), and mucosal edema (55%). The only correlation between these findings and the radiographic stage was the presence of mucosal nodularity observed in 73% of stage I (14/19 patients), decreasing to only 48% in stage III (15/31 patients). Bronchial biopsy yield was 58% for stage I, 62% for stage II, and 46% for stage III, with overall yield of 57%. However, the addition of bronchial biopsies increased the diagnostic yield of the bronchoscopic procedure from 73% for transbronchial biopsies alone to 88% when the two procedures were combined. In the presence of endobronchial stenosis, mucosal nodularity and hypervascularity, bronchial biopsy yield was 91% vs 37% when these were absent. We conclude that the endoscopic characteristics, with the exception of nodularity, do not correlate with radiographic stages. Bronchial biopsy yield is higher in those patients with mucosal nodularity, increased vascularity, and bronchostenosis. Bronchial mucosal biopsy also improved the overall diagnostic yield when it is obtained in conjunction with transbronchial lung biopsy.
Assuntos
Brônquios/patologia , Broncoscopia , Sarcoidose/patologia , Biópsia , Brônquios/irrigação sanguínea , Edema/patologia , Tecnologia de Fibra Óptica , Humanos , Mucosa/patologia , Radiografia , Sarcoidose/diagnóstico por imagemRESUMO
Coccidians represent a large class of important intracellular parasites that traverse multiple developmental stages that are distinct and required to complete the life cycle. The biochemical details underlying the regulation of transformation from one developmental form to the next are limited and the study of such details presents unique obstacles. However, the genetic program is critical and may provide a basis for understanding the biology of these organisms in addition to the opportunity to suppress development and infection. We provide a basic overview of several strategies, including previously unpublished results, used by this laboratory to isolate stage-specific genes from Eimeria bovis. Additionally, we have included detailed discussions that summarize the associated advantages and disadvantages of each as applied to coccidia and potentially to other parasites in the phylum Apicomplexa. Given that the purification of sufficient quantities of high-quality RNA is vital, we have included detailed protocols for the isolation of RNA from various parasite stages. Also included is a detailed protocol to apply mRNA differential display to investigate stage-specific developmental regulation.
Assuntos
Eimeria/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/isolamento & purificação , Animais , Bovinos , Células Cultivadas , Primers do DNA , DNA Complementar , Eimeria/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA de Protozoário/genética , DNA Polimerase Dirigida por RNARESUMO
We tested the virulence in mice of Toxoplasma gondii RH strain tachyzoites containing various copies of the chloramphenicol acetyl transferase-herpes simplex virus thymidine kinase fusion sequence (CAT-HSTK). Tachyzoite isolates containing >/=five copies of the fusion sequence were not lethal to female CD-1 outbred or BALB/c inbred mice, at doses up to 10(6) parasites, while the parental RH strain caused 100% mortality within 2 weeks at doses as low as 10 parasites. Mice infected with CTK11, an isolate containing five copies of the fusion sequence, showed no overt symptoms of disease and were protected from lethal challenge with the parental RH strain. The CTK11 isolate showed no difference in growth rate, the rate of host cell invasion, or extracellular viability in cell culture compared with parental RH parasites, demonstrating that the CAT-HSTK fusion protein does not affect the normal viability of this isolate. B11, B11C, and D1 isolates contained one or two copies of the CAT-HSTK coding sequence, were not sensitive to thymidine in cell culture, and caused 100% mortality in CD-1 outbred mice in <12 days. A fourth isolate, D1C, contained seven copies of the CAT-HSTK fusion sequence and was sensitive to exogenous thymidine (50% inhibitory concentration = 5.5 microM). Mice infected with D1C showed no symptoms of disease and survived beyond 90 days, thus correlating increased CAT-HSTK gene copies with thymidine sensitivity in cell culture and attenuated virulence in mice. BALB/c mice containing a targeted disruption of the gamma interferon gene (gko) were also susceptible to infection with CTK11 parasites but could be rescued by administration of subcutaneous thymidine once each day for 5 or 10 days following infection. These results suggest that the attenuation of CAT-HSTK(+) isolates in mice is directly due to active thymidine kinase that likely alters the pyrimidine biosynthetic pathway in these parasites.
Assuntos
Simplexvirus/enzimologia , Timidina Quinase/fisiologia , Toxoplasma/patogenicidade , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Timidina/farmacologia , Timidina Quinase/genética , Toxoplasma/crescimento & desenvolvimento , VirulênciaRESUMO
The development of superior vena cava syndrome in sarcoidosis is uncommon, as is the development of lymph node enlargement after the pulmonary component of the disease is well established. A search for other etiologic factors is indicated.
Assuntos
Sarcoidose/complicações , Veia Cava Superior , Feminino , Humanos , Doenças Linfáticas/complicações , Doenças Linfáticas/diagnóstico por imagem , Doenças do Mediastino/complicações , Doenças do Mediastino/diagnóstico por imagem , Pessoa de Meia-Idade , Radiografia , Síndrome , Doenças Vasculares/etiologia , Veia Cava Superior/diagnóstico por imagemRESUMO
We mutagenized RH delta hxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40 degrees C. These isolates grew normally at 34 degrees C, but showed variable growth at temperatures between 34 and 39 degrees C. The inability to grow at 40 degrees C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (congruent with 85%) than that observed for RH delta hxgprt (congruent with 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degrees C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40 degrees C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear division.
Assuntos
Ciclo Celular/genética , Mutagênese , Toxoplasma/genética , Animais , Linhagem Celular , DNA de Protozoário/análise , Etilnitrosoureia , Feminino , Fibroblastos/parasitologia , Citometria de Fluxo , Humanos , Camundongos , Temperatura , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , VirulênciaRESUMO
Tachyzoites (VEG strain) that emerge from host cells infected with Toxoplasma gondii sporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following approximately 20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker, BAG1. Within 24 h of the growth shift, 2% of the population expressed BAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. Spontaneous BAG1 expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to express BAG1 earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >10(4) parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressed BAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.
Assuntos
Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/parasitologia , Animais , Diferenciação Celular , Divisão Celular , Feminino , Proteínas de Choque Térmico/biossíntese , Interferon gama/deficiência , Interferon gama/genética , Camundongos , Camundongos Knockout , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/biossíntese , Toxoplasma/patogenicidade , VirulênciaRESUMO
We studied 46 patients, 26 with central visible tumors and 20 with peripheral nodules (in these, biplane fluoroscopic guidance was used), to determine the optimal number of forceps biopsies necessary to establish a diagnosis of carcinoma with the flexible fiberoptic bronchoscope. Overall yield was 96% (25 of 26) for central tumors and 75% (15 of 20) for peripheral nodules, using the combination of forceps biopsies and brushings. The combination of cytologic examinations plus one forceps biopsy produced a 92% diagnostic accuracy for central visible tumors. However, for central tumors the maximal diagnostic yield was not achieved until the fourth forceps biopsy, and for peripheral lesions accuracy continued to increase through the sixth forceps biopsy, and for peripheral lesions accuracy continued to increase through the sixth forceps biopsy in this study. Theoretically, as many as 10 biopsies may be necessary to maximize diagnostic yield for peripheral carcinomas.