Analysis of the qPCR Assay Efficacy as Molecular Diagnostic in Patients with Chemotherapy and/or Antibiotic Therapy

Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.

Evaluation of hydroxyurea genotoxicity in patients with sickle cell disease / Avaliação da indução de genotoxicidade pela hidroxiureia em pacientes com doença falciforme

ABSTRACT Objective To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. Methods The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. Results Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. Conclusion In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.

RESUMO Objetivo Avaliar o efeito da indução de danos ao DNA em células monocelulares do sangue periférico de pacientes com doença falciforme, genótipos SS e SC, tratados com hidroxiureia. Métodos Os sujeitos da pesquisa foram divididos em dois grupos: um de 22 pacientes com doença falciforme genótipos SS e SC tratados com hidroxiureia, e o outro controle, composto por 24 pacientes com doença falciforme que não eram tratados com o fármaco. As amostras de sangue periférico foram submetidas ao isolamento de células mononucleares do sangue periférico para avaliação da genotoxicidade pelo ensaio de micronúcleo citoma com bloqueio da citocinese, tendo sido quantificados os biomarcadores de danos ao DNA - micronúcleos, pontes nucleoplasmáticas e brotamento nuclear. Resultados Os pacientes com doença falciforme tratados com hidroxiureia apresentaram média de idade de 25,4 anos, enquanto aqueles com doença falciforme não tratados com hidroxiureia tiveram média de idade de 17,6 anos. A dose média de hidroxiureia utilizada pelos pacientes foi de 12,8mg/kg/dia, por período médio de 44 meses. A frequência média de micronúcleos por 1.000 células de 8,591±1,568 foi observada no Grupo Hidroxiureia e de 10,040±1,003 no Grupo Controle. Adicionalmente, a frequência média de pontes nucleoplasmáticas por 1.000 células e brotamento nuclear por 1.000 células para o Grupo Hidroxiureia e Controle foi de 0,4545±0,1707 versus 0,5833±0,2078, e de 0,8182±0,2430 versus 0,9583±0,1853, respectivamente. Não houve diferença estatisticamente significativa entre os grupos. Conclusão Na população estudada de pacientes com doença falciforme com tratamento em dose padrão de hidroxiureia, não houve evidência de indução de danos ao DNA.

Analysis of corneal esthesia in patients undergoing photorefractive keratectomy / Análise da estesia corneal em pacientes submetidos à ceratectomia fotorrefrativa

ABSTRACT Purpose: To quantitatively analyze corneal esthesia in patients undergoing photorefractive keratectomy (PRK) surgery. Methods: Forty-five patients selected for PRK in one eye underwent corneal esthesia using a Cochet-Bonnet esthesiometer preoperatively and 30 and 90 days postoperatively. Patients with a refractive diopter error of 4 or greater received intraoperative 0.02% mitomycin C for 20 s. Results: Twenty-four (53.3%) of the 45 eyes received intraoperative 0.02% mitomycin. Decreased sensitivity was observed on postoperative day 30. By postoperative day 90, corneal esthesia had normalized but remained 14.9% lower than preoperative levels. In the mitomycin group, no recovery of corneal esthesia to normal sensitivity levels was observed. The mean esthesiometer level was 39.2 mm on postoperative day 90 (P<0.001). Conclusions: The results of the present study demonstrate recovery of corneal esthesia to normal levels at 90 days postoperatively in patients who did not receive mitomycin C. In patients administered mitomycin C, a 23.59% reduction in the corneal touch threshold was observed compared with preoperative levels indicating a failure of recovery to normal levels.

RESUMO Objetivo: Análise quantitativa da estesia corneal em pacientes submetidos à cirurgia refrativa (PRK). Métodos: Estudo prospectivo, longitudinal e intervencionista, analisando 45 olhos com estesiômetro de Cochet Bonnet no período pré-operatório, no 30º dia após a cirurgia e no 90º dia após cirurgia refrativa. Os pacientes com erro refracional maior ou igual a 4 dioptrias, foram submetidos ao uso de mitomicina 0,02%, por período de 20 segundos no intraoperatório. Resultados: Observou-se diminuição da sensibilidade corneal no 30º dia em todos os olhos, retornando a níveis próximo ao normal no 90º dia, apresentando redução média final de 14,9%. Mitomicina C foi utilizada em 24 (53,3%) dos 45 olhos examinados. No grupo que recebeu mitomicina C, não houve recuperação da estesia normal (média de 39,2 mm) (p<0,001), após 90 dias de cirurgia. Conclusão: Com base nos resultados obtidos, verificamos que ocorreu recuperação da estesia corneal próximo ao normal 90 dias após a cirurgia, porém inferior aos valores iniciais, no grupo sem o uso de mitomicina C intraoperatória. No entanto, no grupo submetido ao uso de mitomicina, não houve a recuperação da sensibilidade corneal a níveis normais, mesmo após o período de 90 dias.

Surgical Results of Trabeculectomy and Ahmed Valve Implantation Following a Previous Failed Trabeculectomy in Primary Congenital Glaucoma Patients

PURPOSE: To compare the surgical results of trabeculectomy and Ahmed glaucoma valve implantation after a previous failed trabeculectomy. METHODS: A retrospective comparative case series review was performed on 31 eye surgeries in 20 patients with primary congenital glaucoma who underwent trabeculectomy or Ahmed glaucoma valve implantation after a previous failed trabeculectomy with mitomycin C. RESULTS: The preoperative mean intraocular pressure was 25.5 mmHg in the trabeculectomy group and 26.9 mmHg in the Ahmed glaucoma valve implantation group (p = 0.73). The 48-month postoperative mean intraocular pressure was 19.6 mmHg in the trabeculectomy group and 20.2 mmHg in the Ahmed glaucoma valve implantation group (p = 0.95). The 12-month trabeculectomy success rate was 69%, compared with 64% for Ahmed glaucoma valve implantation, and the 48-month success rates were 42% and 36% for trabeculectomy and valve implantation, respectively. The success rates following the entire follow-up period were not significantly different between the two groups (p > 0.05 by log rank test). Postoperative complications occurred in 25% of the trabeculectomy-operated eyes and 9% of the Ahmed-implanted eyes (p = 0.38). CONCLUSIONS: There was no significant difference in surgical outcome between the trabeculectomy and Ahmed glaucoma valve implantation groups, neither of which had favorable results. However, the trabeculectomy group demonstrated a higher prevalence of adverse complications such as post-operative endophthalmitis.

Efficacy of mitomycin C in reducing recurrence of anterior urethral stricture after internal optical urethrotomy

PURPOSE: To determine the efficacy of mitomycin C in reducing the recurrence of anterior urethral stricture after internal optical urethrotomy (IOU). MATERIALS AND METHODS: This was a randomized controlled trial conducted in the Department of Urology at the Institute of Kidney Diseases Peshawar from March 2011 to December 2013. A total of 151 patients who completed the study were divided into two groups by the lottery method. Group A (cases) comprised 78 patients in whom mitomycin C 0.1% was injected submucosally in the stricture after conventional IOU. Group B (controls) comprised 73 patients in whom IOU only was performed. Self-clean intermittent catheterization was not offered in either group. All patients were regularly followed up for 18 months. Recurrence was diagnosed by use of retrograde urethrogram in all patients and flexible urethroscopy in selected cases. Data were collected on a structured pro forma sheet and were analyzed by SPSS. RESULTS: The mean age of the patients in group A was 37.31+/-10.1 years and that in group B was 40.1+/-11.4 years. Recurrence of urethral stricture was recorded in 11 patients (14.1%) in group A and in 27 patients (36.9%) in group B (p=0.002). The mitomycin group also showed a delay in recurrence compared with the control group (p=0.002). CONCLUSIONS: Recurrence of urethral stricture is high after optical urethrotomy. Mitomycin C was found to be highly effective in preventing the recurrence of urethral stricture after IOU.

Ação do anestésico tópico diluído e da mitomicina sobre a sintomatologia e re-epitelização corneana no pós-operatório da ceratectomia fotorrefrativa / The action of topic diluted anesthetic and mitomycin on the symptomatology and corneal re-epithelialization at postoperative photorefractive keratectomy

Objetivo: Avaliar a ação do anestésico tópico proximetacaína diluído, sobre a sintomatologia e re-epitelização corneana no pós-operatório de ceratectomia fotorrefrativa (PRK). Métodos: Estudo prospectivo, comparativo e duplo cego. Os pacientes foram divididos em dois grupos: Com (n=32) e Sem (n=24) o uso de mitomicina C 0,02% no intraoperatório (indicada para ametropia igual ou acima que 4 dioptrias). Cada paciente recebeu dois frascos de colírio: um com a lágrima artificial carmelose sódica 0,5% como placebo e o outro com a mesma acrescida do anestésico proximetacaína a 0,125%. A escolha do olho a receber um ou outro colírio foi aleatória. Cada paciente foi questionado, no 1º, 2º e 3º pós-operatórios, sobre o tipo de sintoma apresentado em cada olho e sua intensidade (medida por meio da Escala Analógica Visual de Dor). O estado da re-epitelização corneana de cada olho foi avaliado no 1º e 5º dias pós-operatórios. Resultados: Os escores da intensidade da sintomatologia foram significativamente menores nos olhos que utilizaram anestésico, tanto para os pacientes do grupo que recebeu mitomicina como para os pacientes do grupo que não. A diminuição da sintomatologia foi significativamente maior nos olhos que receberam concomitantemente o anestésico e a mitomicina. Ocorreu retardo da re-epitelização corneana em apenas 3 casos do grupo que utilizou mitomicina. Conclusão: O anestésico tópico diluído na concentração e posologia propostas neste estudo mostrou-se seguro e não tóxico ao epitélio cornenano, proporcionando alívio significante da sintomatologia pós-operatória do PRK. Quando associados, o anestésico tópico diluído e a mitomicina apresentaram uma ação sinérgica nesse alívio.

Objective: To evaluate the effects of diluted topic anesthetic proximetacaine on the symptoms and corneal re-epithelialization in postoperative photorefractive keratectomy (PRK). Methods: A prospective, comparative and double-blind Study. Patients were divided into two groups: With (n=32) and Without (n=24) the use of Mitomycin C at 0.02% (indicated for eyes with 4 or more diopters). Each patient received two bottles of eye drops one had artificial tear carmellose sodium 0.5%, and other had the same along with 0.125% proximetacaine. The choice of which eye would receive one or another was random. Each patient was asked, at the 1st, 2nd and 3rd postoperative days, about the type of presenting symptom on each eye and its intensity (assessed by Pain Visual Analogue Scale). The corneal re-epithelialization of each eye was evaluated at the first and fifth postoperative days. Results: The scores were significantly lower in the eyes in which anesthetic was used for both groups. The decrease in symptomatology was significantly superior in the eyes that received concomitantly the diluted anesthetic and the mitomycin when compared to the eyes that received only the anesthetic. There were only three cases of corneal re-epithelialization retardation in mitomycin group. Conclusion: The diluted proximetacaine, at the concentration and dosage proposed in this study, is safe and non-toxic to the corneal epithelium, providing significant relief for PRK postoperative symptoms. When combined, the diluted symptoms.

Efeitos de algumas drogas sobre a proliferação de fibroblastos de pterígio primário in vitro / Effects of some drugs in the fibroblastic proliferation in primary pterygium in vitro

OBJETIVO: Este estudo tem por objetivo observar a inibição da proliferação celular in vitro em pterígios primários utilizando mitomicina C, ciclofosfamida e metotrexato. MÉTODOS: Os pterígios foram retirados de 7 pacientes com idade entre 30 e 60 anos e foram submetidos à cultura de suas células epiteliais. Foi então verificado o efeito de drogas sobre as células: ciclofosfamida, metotrexato e mitomicina. As células foram observadas por 5 dias ao microscópio para avaliar a proliferação celular e os experimentos foram repetidos 5 vezes. RESULTADOS: Quando a mitomicina foi utilizada observou-se importante inibição da proliferação celular. Quando a ciclofosfamida foi utilizada houve também inibição do crescimento, 50% após 24 horas de cultura após a exposição da droga aumentando nos dias subsequentes. Nenhum efeito foi observado quando o metotrexato foi utilizado. CONCLUSÃO: Os efeitos de inibição da proliferação celular pela mitomicina C já eram esperados, porém a ciclofosfamida também apresentou-se bastante eficaz. A ação inibitória da ciclofosfamida sobre a proliferação fibroblástica in vitro nos leva a acreditar que ela possa ser usada para prevenir a recorrência do pterígio depois da excisão. Entretanto, testes em animais e posteriormente em humanos se fazem necessários para se chegar a essa conclusão.

OBJECTIVE: This study aims at observing the inhibition of cell proliferation in primary pterygium by the use in vitro of mitomicyn C, cyclophosphamide, methotrexate. METHODS: Pterigyum was removed from seven pacients between 30 and 60 years and were submitted to culture of epithelial cells. Later the effect of drugs was tested on the cells: cyclophsphamide, methotrexate, mitomicyn. The cells were observed for five days under the microscope to assess cellular proliferation, and the experiments were repeated five times RESULTS: When mitomicyn was used, a marked inhibition of cellular proliferation was observed. When cyclophosphamide was used there also was inhibition of cellular proliferation, 50% within 24 hs of the culture exposition to the drug increasing in the following days CONCLUSION: The inhibition effects in the cellular proliferation by the use of mitomicyn C was already expected, but the use of cyclophosphamide was also very effective. The cyclophsphamide inhibitory action on fibroblastic proliferation in vitro lead us to believe that it may be used to prevent pterygium recurrence after incision. However, tests in animals and later in humans are necessary.

The use of mitomycin C in caustic esophagitis in rats

PURPOSE: To evaluate the topical effects of mitomycin C (MMC) in rats, with or without esophageal dilation, in different moments after esophageal caustic injury with NaOH10%. METHODS: Forty eight Wistar rats were divided into six groups: "GS" infusion of 0.9% saline solution in the esophagus; "CG" infusion of 0.9% saline solution in the esophagus, with temporary ligation of the organ; "NTG" induction of a caustic lesion without treatment; "GmmcD0" MMC applied immediately after the caustic injury; "GmmcD14" MMC applied 14 days after the caustic injury; "Gdil+mmcD14" esophageal dilation and application of MMC 14 days after caustic injury. We performed contrast esophagograms of four animals from each group, seven and 21 days after the caustic injury. On day 28, all animals were sacrificed, and histopathological analyses were performed on the esophageal specimens. RESULTS: The contrast images showed total stenosis in NTG and GmmcD0, improving to partial stenosis in GmmcD0. In GmmcD14 and Gdil+mmcD14, two animals of each group improved to partial stenosis. By histopathological analysis, NTG and GmmcD14 presented intermediate damage and GmmcD0 and Gdil+mmcD14 severe damage. CONCLUSION: The use of mitomycin C had beneficial effects specially when applied immediately after the induction of esophageal lesions.

Potent in vitro interference of fleroxacin in DNA-binding, unwinding and ATPase activities of Bloom helicase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.</p><p><b>METHODS</b>DNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra.</p><p><b>RESULTS</b>The DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands.</p><p><b>CONCLUSION</b>FLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.</p>

Apoptose em pólipos nasais eosinofílicos submetidos in vitro ao tratamento com mitomicina C / Apoptosis in eosinophilic nasal polyps treated in vitro with mitomycin C

A etiopatogênese da polipose nasal eosinofílica ainda não foi esclarecida. Os eosinófilos constituem as principais células do infiltrado inflamatório e estão relacionados com a perpetuação do processo inflamatório na rinossinusite crônica com pólipos nasais. OBJETIVO: Avaliar a ação in vitro da mitomicina no índice apoptótico de pólipos nasais eosinofílicos. MATERIAL E MÉTODO: Estudo prospectivo experimental autopareado com amostra de biópsia de 15 pacientes com polipose nasal eosinofílica. Cada fragmento foi dividido em dois grupos. No grupo experimental aplicou-se mitomicina por cinco minutos, na dosagem de 400 µg/ml. O grupo controle foi submetido às mesmas manipulações, mas utilizando-se somente meio de cultura. Os fragmentos contidos nos dois primeiros compartimentos, controle e experimento, foram imediatamente submetidas ao preparo para histopatologia. O outro par de amostra, contendo controle e experimento, foi incubado por 12 horas. Após 12 horas, os fragmentos foram retirados para exame histopatológico. O índice apoptótico foi determinado pela morfometria na coloração hematoxilina-eosina e pela análise da fragmentação do DNA. RESULTADO: A comparação do dois grupos demonstrou diferença significativa (p < 0,001) no índice apoptótico das culturas incubadas por 12 horas. CONCLUSÃO: A mitomicina induz in vitro o aumento do índice apoptótico dos eosinófilos dos pólipos nasais eosinofílicos.

The etiopathogenesis of eosinophilic nasal polyps is yet to be explained. Eosinophils are key components in the inflammatory infiltrate and are related to the perpetuation of the inflammatory process in chronic rhinosinusitis with nasal polyps. OBJECTIVE: This paper aims to evaluate the in vitro action of mitomycin upon the apoptotic index of nasal polyps. MATERIALS AND METHODS: This is a self-paired prospective experimental study using biopsy fragments from 15 patients with eosinophilic nasal polyps. Biopsy fragments were divided into two groups. In the case group, the fragments were treated with 400 µg/ml of mitomycin for five minutes. The control group fragments were treated with culture medium. The pair of fragments contained in the two first compartments - control and case - were immediately sent to the histopathologist. The other pair of samples containing control and case fragments was incubated for 12 hours. The fragments were then taken to the histopathologist for testing. The apoptotic index was determined by the morphometry in hematoxylin and eosin staining and DNA fragmentation analysis (TUNEL reaction). RESULTS: The comparison between the two groups showed a statistically significant difference (p < 0,001) in the apoptotic index of the 12-hour incubated cultures. CONCLUSION: Mitomycin acts in vitro upon the eosinophilic nasal polyps inducing the rise of the eosinophilic apoptotic index.

Comparative Study of Encapsulated Blebs Following Ahmed Glaucoma Valve Implantation and Trabeculectomy with Mitomycin-C

PURPOSE: To compare the histopathologic and morphologic findings of encapsulated blebs following Ahmed glaucoma valve implantation and primary standard trabeculectomy with mitomycin-C. METHODS: We reviewed the records of patients with otherwise uncontrollable glaucoma who had undergone Ahmed glaucoma valve implantation or trabeculectomy with mitomycin-C. Five eyes that underwent Ahmed valve implantation and three eyes that underwent trabeculectomy needed surgical revision of the initial surgery due to encapsulated bleb development with total loss of function. The surgically removed encapsulated blebs were analyzed macroscopically and microscopically. RESULTS: Removal of the encapsulated bleb was performed at a mean follow-up time of 26.6 +/- 19.4 weeks in the Ahmed valve implantation group and 12.0 +/- 11.4 weeks in the trabeculectomy group. The fibrotic wall of the encapsulated blebs had an overall thickness of 2.48 +/- 0.42 mm in the Ahmed valve implantation group and 1.62 +/- 0.37 mm in the trabeculectomy group. Macroscopically, the coconut flesh-like smooth surface was split into two layers, and the wall of the capsule was thicker in the Ahmed valve implantation group than in the trabeculectomy group. Histopathologically, the fibrotic capsule was composed of an inner fibrodegenerative layer and an outer fibrovascular layer, and there were no histopathological differences between the two groups. CONCLUSIONS: The fibrotic capsule wall was thicker in the Ahmed valve group, but there were no differences in histological findings between the two groups.

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.

Effect of DRB/alpha-Amanitin on localization of Nrf2 in A549 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.</p><p><b>METHODS</b>A549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.</p><p><b>RESULTS</b>The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.</p><p><b>CONCLUSION</b>DRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.</p>

effect of hydroxyurea in the management of Pediatric Sickle cell patients

Low levels of fetal hemoglobin [HbF] in sickle cell disease [SCD] patients [pts] are associated with a variety of vaso-acciusive complications and an increased risk of early death. Raising HbF levels can reduce the effect of the disease. Hydroxyurea [HU] reduces the production of HbS containing red cells and favours the production of HbF containing red cells. It has been used successfully in the management of adults with SCD and there is growing data on its efficacy and safety in pediatric age group. This study reviews our clinical experience with HU in the treatment of pediatric pts with SCD attending the Pediatric Hematology Clinic of Cairo University. Sixty SCD pts from 2001 to 2007 who received HU over the period of 6 years [yrs] and who continued therapy for at least 6 months were included. Four pts were excluded because of noncompliance to treatment. Response to HU was assessed both clinically and by laboratory findings. Pts were considered responders if they showed >/= 50% improvement in clinical and laboratory data. These data included number of blood transfusions/yr, vasoocciusive crisis [VOC]/yr requiring admission to the emergency unit, hospital admissions/yr. Laboratory data included Hb [g/dl], MCV [fl], HbF%, total leucocytic count [TLC], x10[3]ml absolute neutrophil count [ANC], platelets x10[3]ml and reticulocytic count [%] and serum ferritin level [ng/ml]. Fifty six pts [44 children and 12 adults [>18 yrs] were included. Their mean age was 14.05 +/- 5.3 yrs [range of 6-28 yrs]. Thirty seven were females and 19 males. Twenty four [42.9%] were sickle-beta thalassemia while 32 [57.1%] were homozygous sickle cell anemia [SS]. The main indications for starting HU therapy were frequent VOC, transfusion dependency and acute chest syndrome [91%, 86% and 16% respectively]. Other indications included hepatic crisis [5%], bone infarction [7%], sequestration crisis [5%] and pulmonary hypertension in one case. HU was started in a dose of 15mg/kg/day with careful monthly monitoring for side effects. There was no attempt to achieve maximum tolerated dose. Dose increase or decrease was done depending on clinical and laboratory response with a maximum dose of 30mg/kg/day. The mean dose of HU was 15.8mg/kg/day [range 10-30 mg/kg/day] and the mean duration of therapy was 3.25 yrs [range of 0.5-6 yrs]. Forty four pts [78.6%] were found to be responders. There was a significant [p<0.05] improvement in all clinical parameters. Responders showed a significant decrease in TLC, ANC, reticulocytic count and serum ferritin [P values 0.002, 0.019, 0.000, 0.001 respectively]. Significant increase in HbF and MCV [p=0.000, 0.001 respectively] was also observed. HU toxicity was defined by>3 fold increase in ALT, platelet count<80,000 p1, ANC<1500 or increase in serum creatinine>50%above baseline. Twenty pts [35.7%] showed signs of HU toxicity: elevated ALT [n=9], neutropenia [n=7], thrombocytopenia [n=1], unexplained jaundice [n=1] and both neutropenia and jaundice [n=2]. Thirteen pts continued therapy with reduction of the dose or temporary stopping of HU while 7 stopped HU. It was noticed that all pts who developed hepatotoxicity were HCV positive p=0.036]. It was also shown that hepatotoxicity was significantly higher among those receiving Deferiprone with HU [n=20] [p=0.001]. There was no relation between response to HU and patients' age, sex, spleen status or phenotype. HU provides the best available strategy to achieve clinical and hematological improvement in SCD in pediatrics, but requires periodic monitoring of blood count and ALT levels especially for HCV positive pts and those on Deferiprone therapy

Effects of mitomycin C on infiltration of polymorphonuclear leukocytes after epithelial scrape injury in the mouse cornea / Efeito da mitomicina C na infiltração de leucócitos polimorfonucleares após lesão epitelial em córnea de camundongo

PURPOSE: To determine whether mitomycin C (MMC) alters appearance and disappearance of polymorphonuclear leucocytes (PMN) in the cornea stroma, using an epithelial scrape injury in eye mouse model. METHODS: Twenty-mice underwent mechanical epithelium debridement in the central cornea using 20 percent ethanol. After the scrape, the right eye received 0.02 percent MMC for one minute, while the left eye received physiological saline. The animals were sacrificed on days 1, 2, 5, and 14 after surgery, and corneal whole mounts were prepared for histology. PMN distribution was analyzed in digitized microscope images. Cell division in the cornea was determined by immunohistochemical detection of bromodeoxyuridine (BrdU), which was injected intraperitoneally before the mice were sacrificed. RESULTS: Epithelial scrape injury triggered infiltration of PMNs into the corneal stroma. An analysis of PMN distribution revealed that there was no difference between eyes treated with and without MMC at all time points. BrdU labeling showed that 0.02 percent MMC for one minute blocked keratocyte proliferation completely. CONCLUSION: MMC treatment regimen, which is common in clinical practice, inhibits keratocyte proliferation during wound healing, but when used at 0.02 percent for one minute, it does not affect PMN infiltration into the corneal stroma, and subsequent movement toward the injury site, or the disappearance of PMNs from the stroma, in the mouse epithelial injury model.

OBJETIVO: O objetivo do estudo foi determinar se a mitomicina C (MMC) altera o aparecimento dos leucócitos polimorfonucleares (PMN) no estroma corneano após abrasão epitelial central, utilizando olhos de camundongo como modelo. MÉTODOS: Vinte camundongos foram submetidos à abrasão epitelial em córnea central utilizando etanol a 20 por cento. Após a lesão, o olho direito recebeu MMC a 0,02 por cento por 1 minuto, enquanto o olho esquerdo recebeu solução salina. Os animais foram sacrificados em 1, 2, 5 e 14 dias após a cirurgia e a córnea foi preparada para histologia. A distribuição dos PMN foi analisada e digitalizada em imagens microscópicas. A divisão celular na córnea foi detectada pela imuno-histoquímica da bromodeoxirudina (BrdU), injetada intraperitonialmente duas horas antes dos animais serem secrificados. RESULTADOS: A lesão epitelial gerou infiltração de PMN no estroma da córnea. A análise da distribuição dos PMNs revelou que não houve diferença estatisticamente significante entre os olhos tratados e não tratados com MMC, em todos os tempos estudados. O estudo com BrdU mostrou que a MMC quando utilizada a 0,02 por cento por um minuto bloqueou completamente a proliferação de ceratócitos. CONCLUSÃO: O tratamento com MMC, que é utilizada comumente na prática clínica, inibe a proliferação dos ceratócitos durante a cicatrização corneana, porém quando utilizada a 0,02 por cento por um minuto, não altera a infiltração dos PMNs dentro do estroma corneano após lesão epitelial em córneas de camundongos.

Correlation of thyroid and growth hormones to chromosomal instability in Egyptian Fanconi anemia patients.

OBJECTIVE: Fanconi anemia (FA) is a rare inherited genomic instability syndrome and usually associated with endocrine dysfunctions. We aimed to assess the diagnostic standards of chromosomal instability in FA and to correlate the breakage frequency with the severity of endocrinal dysfunctions. METHODS: Twenty seven FA patients were randomly selected from Hematology Unit of Mansoura University Children's Hospital; their mean age 8.8 yr. Sixteen normal children matched for age and sex were used as controls. Cytogenetic studies included peripheral blood lymphocyte cultures using phytohemagglutinin to obtain chromosomal spreads. Chromosomal breakage was induced by (i) Diepoxybutane 0.1 mug/ml. (ii) Mitomycin C 0.1 microg/ml. (iii) Irradiation of cultures to four radiation doses; 75, 150, 300 and 400 rads (rad1, rad2, rad3 and rad4 respectively). Chromosomal aberrations were scored from the previous 6 cultures besides a culture for spontaneous chromosomal breakage; then mean chromosomal breakage was calculated for the seven cultures. Endocrinal evaluation included quantitative determination of thyroid stimulating hormone (TSH) and tetraiodothyronine (T4), serum growth hormone (GH), insulin like growth factor-1 (IGF-1) and insulin levels. RESULTS: Chromosomal breakage was found to be significantly higher in patients than control when induced by Diepoxybutane (p = 0.003), Mitomycin (p = 0.001), rad3 (p = 0.043) and rad4 (p = 0.001). Mean chromosomal breakage was significantly negative correlated to head circumference (r = -0.57) and GH level (r = -0.50), with no significant correlation to other hormonal parameters. Mitomycin and rad4 were found more accurate than DEB test for diagnosis of FA in suspected cases. CONCLUSION: Correction of the frequently associated hormonal dysfunction (reduced GH and T4) should be considered in the treatment discipline of FA patients to improve their final height.

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

Mitomicina C colírio como tratamento para granuloma piogênico em cavidade anoftámica / Mitomicyn C eye drops as alternative treatment for pyogenic granuloma in anophthalmic socket

OBJETIVO: Avaliar a eficácia do uso tópico de colírio de mitomicina C 0,02 por cento (MMC 0,02 por cento) no tratamento de pacientes com granuloma piogênico em cavidade anoftálmica. MÉTODOS: Seis pacientes portadores de granuloma piogênico em cavidade anoftálmica foram submetidos ao tratamento com mitomicina C 0,02 por cento instilada quatro vezes ao dia durante ciclos quinzenais e intervalo de 15 dias entre os ciclos. Os pacientes foram acompanhados semanalmente. RESULTADOS: Foram acompanhados seis pacientes dos quais um apresentou resolução do granuloma piogênico ao final do primeiro ciclo de uso da MMC 0,02 por cento. Em dois pacientes a MMC 0,02 por cento foi eficaz na resolução da lesão após a segunda semana de tratamento (primeiro ciclo). Dois pacientes apresentaram resolução completa do granuloma piogênico na quinta semana de uso da MMC 0,02 por cento (início do segundo ciclo). Este fármaco foi ineficaz em apenas um paciente que persistiu com a lesão após dois ciclos de tratamento. Não foram observadas complicações decorrentes do uso de MMC 0,02 por cento em nenhum dos pacientes. CONCLUSÃO: A MMC 0,02 por cento tópica mostrou-se eficaz no tratamento de granuloma piogênico em cavidade anoftálmica, entretanto, diante de casuística pequena, torna-se necessário estudo mais amplo, comparando a eficácia da MMC 0,02 por cento com drogas já utilizadas no tratamento desta lesão, tais como os esteróides e antimetabólicos.

PURPOSE: To evaluate the efficacy of mitomycin C eye drops 0.02 percent concentration (0.02 percent MMC) in pyogenic granuloma in anophthalmic socket. METHODS: Six patients who presented pyogenic granuloma were treated with 0.02 percent MMC four times a day in fifteen-day cycles with intervals of fifteen days between each cycle. The patients were observed every week. RESULTS: Among the six studied patients, one presented a complete resolution of the pyogenic granuloma after a ten-day treatment with 0.02 percent MMC that is, before the end of the first cycle. In two patients, the treatment was successful after two weeks using 0.02 percent MMC (one complete cycle). In other two patients, the resolution of the lesion occurred after five weeks that is, at the beginning of the second cycle. 0.02 percent MMC was not successful in only one patient whose lesion persisted after two cycles of the treatment. None of the patients presented complications after the use of 0.02 percent MMC. CONCLUSION: The topical use of 0.02 percent MMC proved efficacy in the treatment of anophthalmic socket pyogenic granuloma. A small number of patients took part in this study, therefore another randomized and comparative research is necessary in order to compare the efficacy of 0.02 percent MMC with other drugs such as corticosteroids and antimetabolites.

Efeito da mitomicina C em polipose nasossinusal eosinofílica, in vivo: dosagem de IL5 e GM-CSF, RT-PCR / Effect of mitomocin C in eosinophilic nasal polyposis, in vivo: concentration of IL5 and GM-CSF, RT-PCR

A polipose nasossinusal eosinofílica (PNS) é manifestação de uma doença inflamatória crônica na mucosa do nariz e nos seios paranasais caracterizada por infiltração de granulócitos eosinófilos. O fator responsável pela eosinofilia e manutenção dessas células com a perpetuação do processo inflamatório e formação polipóide é objeto constante de estudos. As citocinas como IL5 (interleucina 5) e GM-CSF (fator estimulador de colônia granulócito macrófago) aumentam a sobrevida dos eosinófilos e prolongam a sua presença no tecido polipóide, diminuindo o índice de apoptose eosinofílica. OBJETIVO: Avaliar o efeito da mitomicina C - MMC - por meio de aplicação tópica em pacientes portadores de PNS eosinofílica quanto à presença de IL5 e GM-CSF. CASUÍSTICA E MÉTODOS: Quinze pacientes portadores de PNS eosinofílica foram submetidos à aplicação tópica de MMC na concentração de 0,5mg/ml, 1ml, durante cinco minutos, na cavidade nasal direita, e submetidos à biópsia para RT-PCR 24hs após. O grupo-controle foi a cavidade nasal esquerda. O perfil de citocinas foi analisado para IL5 e GM-CSF. RESULTADOS: A comparação dos resultados de GM-CSF pré e pós-uso de MMC quando usamos o teste t pareado apresenta p=0,041. A comparação para IL5 resulta em p < 0,001. CONCLUSÃO: O uso de MMC em pacientes com PNS mostra redução com significância estatística par GM-CSF e importante significância para IL5.

Eosinophilic nasosinusal polyposis is a chronic inflammatory infection with elevated infiltration of eosinophils, which presents high rate of recurrence after surgical treatment. The continuous inflammatory process that leads to the formation of polyps requires constant clinical treatment. Contributing to the maintenance of eosinophilia are cytokines IL5 (interleukin-5) and GM-CSF (granulocyte macrophages colony-stimulating factor), which show up in elevated concentrations. These oligoproteins diminish the rate of apoptosis and prolong the survival of eosinophils. AIM: By diminishing these cytokines, the action of Mitomycin C (MMC), an antineoplasic drug which inhibits the synthesis of DNA, was studied. In a recent study the power of this drug to cause apoptosis in eosinophils, in vitro, of nasal polyps was verified. METHODOLOGY: A biopsy of the nasal polyps was undertaken in 15 patients carriers of eosinophilic nasosinusal polyposis 24 hours after applying 0.5 mg/ml of MMC during five minutes. RT-PCR (reverse transcription of polymerase chain reaction) for IL5 and GM-CSF was the method used to obtain the results. RESULTS: The comparison of the results of GM-CSF pre- and post-application of MMC, when the paired T-test was used, showed p=0.041 and for IL5 we found p<0.001. CONCLUSION: Topic use of MMC in patients with eosinophilic nasosinusal polyposis shows statistically significant reduction for GM-CSF and significant and important reduction for IL5.