Evaluation of the effect of allopurinol as a protective factor in post ischemia and reperfusion inflammation in Wistar rats

PURPOSE: To investigate the potential protective effect of allopurinol on reperfusion injury by determining the inflammatory response through the measurement of tumor necrosis factor-alpha (TNF-alpha). METHODS: Sixty rats were distributed into two groups: control and allopurinol and each group was divided into three subgroups, ischemia for two hours, ischemia for three hours and ischemia simulation. Allopurinol group rats received 100mg/kg dose of allopurinol, whereas control group rats received an equivalent dose of saline. Clamping of the infrarenal aorta was performed for two or three hours depending on the subgroup. Ischemia simulation subgroups did not suffer ischemia, just aortic dissection, and maintenance for three hours. After 72 hours of reperfusion, blood was collected by cardiac puncture for TNF-alpha measurement. RESULTS: Allopurinol reduced TNF-alpha significantly (p <0.001) when compared to the matching control subgroups (control X allopurinol in ischemia for two hours and for three hours). CONCLUSION: Allopurinol reduced the concentrations of serum TNF-alpha when used at different times of ischemia followed by reperfusion, which might indicate reduction of the inflammation provoked by the reperfusion injury.

Effect of allopurinol on the kidney function, histology and injury biomarker (NGAL, IL 18) levels in uninephrectomised rats subjected to ischaemia-reperfusion injury

PURPOSE: To investigate whether allopurinol exerts a protective effect on kidneys by measuring new kidney injury biomarkers (NGALp, NGALu, KIM 1 and IL 18) and analysing the renal function and histology in uninephrectomised rats subjected to ischaemia-reperfusion injury. METHODS: Thirty two Wistar rats were randomly allocated to four groups: Sham (S): laparotomy; Control (C): laparotomy and ischaemia-reperfusion in the left kidney; Control Allopurinol (CA): laparotomy and allopurinol at a dose of 100mg·kg 1·d 1; and Allopurinol (A): laparotomy ischaemia-reperfusion in the left kidney and allopurinol at a dose of 100mg·kg 1·d 1. The NGALp, NGALu, KIM 1, IL 18 and creatinine levels and the kidney histology were analysed. The significance level was established as p<0.05. RESULTS: Creatinine level increased in all the groups, with A ≈ C > S ≈ CA. The NGALp, NGALu and IL 18 levels exhibited similar behaviour in all the groups. KIM 1 was higher in group A than C and showed intermediate values in groups S and CA. Severity of injury in the left kidney was greater in groups C and A compared to S and CA. CONCLUSION: Allopurinol did not exert protective or damaging effects on the kidneys of rats subjected to ischaemia-reperfusion injury. .

prophylactic role of ginger on the pulmonary injury induced by azathioprine on adult male rats [light and scanning electron microscopic study]

Azathioprine is one of the most commonly prescribed immunosuppressive drugs. It is commonly used in the treatment of the immune-mediated diseases. Pulmonary toxicity has been reported as one of its side effects. Ginger [Zingiber officinal roscoe], is a well-known spice plant that has been used traditionally in a wide variety of diseases as cardiovascular disorders, diabetes mellitus and gastrointestinal health. A study of the possible protective role of ginger on the azathioprine induced pulmonary injury in the adult male albino rats using light and scanning electron microscopy. Thirty male adult albino rats were used in the study. They were subdivided into three groups. Each group includes 10 rats. First group was considered as a control, Second group was given azathioprine in a dose of 25 mg/kg body weight twice daily for two days. The third group was given ginger 250 mg orally per kg body weight daily for 5 consecutive days then given azathioprine drug orally for another two days in the same dose as the second group. At the end of the experiment the animals were anaethetized, sacrified and specimens of the lung tissues were extracted and processed to he examined with light and scanning electron microscopy. In group 11, there were thickness of interalveolar septa, lymphocytic cellular infiltration and narrowing of alveolar sacs by light microscopy while in scanning electron microscopy there was thickening of interalveolar septa with narrowing of alveolar sacs, loss of microvilli and laceration of type 11 pneumocytes, disorganized cilia of terminal bronchioles. In group 111 the effect of azathioprine was less than that of group 11 there was less narrowing of the alveolar spaces, less cellular infiltration both type 1 and type 11 pneumocytes and Clara cells were nearly similar to those of control. Administration of ginger prior to azathioprine therapy had a protective effect against the pulmonary injury induced by azathioprine on adult male rats

Enhancement of radionuclide induced cytotoxicity by 2-deoxy-D-glucose in human tumor cell lines.

The efficacy of targeted radiotherapy can be enhanced by selective delivery of radionuclide to the tumors and/or by differentially enhancing the manifestation of radiation damage in tumors. Our earlier studies have shown that the 2-deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolytic ATP production, selectively enhances the cytotoxicity of external beam radiation in tumor cells. Therefore, it is suggested that 2-DG may also enhance the cytotoxic effects of radionuclides selectively in tumor cells, thereby improving the efficacy of radionuclide therapy. In vitro studies on breast carcinoma (MDA-MB-468) and glioma (U-87) cell lines, has been carried out to verify this proposition. Clonogenicity (macrocolony assay), cell proliferation, cytogenetic damage (micronuclei formation) and apoptosis were investigated as parameters of radiation response. Mean inactivation dose D (dose required to reduce the survival from 1 to 0.37), was 48 MBq/ml and 96 MBq/ml for 99 mTc, treated MDA-MB-468 and U-87, respectively. The dose response of growth inhibition, induction of micronuclei formation and apoptosis observed under these conditions, were correlated well with the changes in cell survival. Presence of 2-DG (5 mM) during radionuclide exposure (24 hrs), reduced the survival by nearly 2 folds in MDA-MB-468 (from 48.5 MBq to 18.5 MBq) and by 1.6 folds in U-87 cells (from 96 MBq to 66 Mbq). These results clearly show that the presence of 2-DG during radionuclide exposure, significantly enhances the cytotoxicity, by increasing mitotic as well as interphase death. Further studies to understand the mechanisms of radio-sensitization by 2-DG and preclinical studies using tumor-bearing animals, are required for optimizing the treatment schedule.

Glycolytic inhibitor, 2-deoxy-D-glucose, does not enhance radiation-induced apoptosis in mouse thymocytes and splenocytes in vitro.

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production selectively enhances radiation-induced damage in cancer cells by inhibiting the energy (ATP) dependent postirradiation DNA and cellular repair processes. A reduction in radiation induced cytogenetic damage has been reported in normal cells viz., peripheral blood lymphocytes and bone marrow cells. Since induction of apoptosis plays a major role in determining the radiosensitivity of some most sensitive normal cells including splenocytes and thymocytes, we investigated the effects of 2-DG on radiation induced apo tosis in these cells in vitro. Thymocytes and splenocytes isolated from normal Swiss albino mouse were irradiated with Co60 gamma-rays and analyzed for apoptosis at various post-irradiation times. 2-DG added at the time of irradiation was present till the termination of cultures. A time dependent, spontaneous apoptosis was evident in both the cell systems, with nearly 40% of the cells undergoing apoptosis at 12 hr of incubation. The dose response of radiation-induced apoptosis was essentially similar in both the cell systems and was dependent on the incubation time. More than 70% of the splenocytes and 60% of the thymocytes were apoptotic by 12 hr following an absorbed dose of 2 Gy. Presence of 2-DG marginally reduced the fraction of splenocytes undergoing apoptosis at all absorbed doses, while no change was observed in thymocytes. Presence of 2-DG did not significantly alter either the level or the rate of induction of spontaneous apoptosis in both these cell systems. These results are consistent with the earlier findings on radiation-induced cytogenetic damage in human PBL in vitro and mouse bone marrow cells and lend further support to the proposition that 2-DG does not enhance radiation damage in normal cells, while radiosensitizing the tumors and hence is an ideal adjuvant in the radiotherapy of tumors.

Terapia genica antitumoral mediante el uso de genes suicidas / Cancer gene therapy using suicide genes

Células tumorales transducidas con vectores retrovirales portanto el gen de la timidina kinasa del virus herpes simplex-1 (HSV-tk), son capaces de transformar la droga antiherpética ganciclovir (GCV) en un metabolito tóxico para células en división. Esta terapia suicida aumenta su eficiencia debido a un efecto "bystander" que induce la muerte de células no transducidas, vecinas a células modificadas. El mecanismo del mencionado efecto no se conoce totalmente, pero existe evidencia que asigna un rol preponderante al sistema inmune, para lograr una completa erradicación tumoral. En este trabajo estudiamos la efectividad del sistema en tres líneas celulares: un melanoma humano y uno murino, y un glioma de rata. Los tumores fueron estabelecidos por inyección de células tumorales s.c. en ratones nude y C57BI/6, e intracerebralmente por esterotaxis en ratas Sprague Dawley, respectivamente. Animales tratados fueron co-inyectados con células productoras de retrovirus expresando HSV-tk y posterior administración i.p. de GCV. En experimentos in vivo a corto plazo, se observó inhibición total o parcial del crecimiento tumoral en todos los modelos. En experimentos de supervivencia a largo plazo con células C6, el 50 por ciento de los animales sobrevivió más de 75 dias (p < 0 0001) y fue capaz de rechazar un desafio con células parentales C6 inyectadas en el hemisferio contralateral. El análisis histológico e inmunohistoquímico mostró la presencia de un infiltrado inflamatorio compuesto por linfocitos T, macrófagos y polimorfonucleares. Estos resultados demuestran que el uso de genes suicidas puede ser una herramienta de enorme importancia en el tratamiento de tumores de cerebro y de metástasis cerebrales.

Characterization of primed lymphocytes on the basic of sensitivity of chromatin to DNase I.

Certain qualitative criteria for primed lymphocytes in the expression of cytotoxic function have been studied. Unlike normal lymphocytes, primed lymphocytes expressed cytotoxicity even when DNA synthesis and new gene expression were inhibited by hydroxyurea (HU) and bromodeoxyuridine (BU) respectively. Such differential cytotoxic expression in presence of HU and BU by primed lymphocytes might have their basis in conformational change within the chromatin. Chromatin from primed lymphocytes was more susceptible to DNase I digestion than virgin lymphocytes indicating exposition of more DNase I sensitive sites in primed state. The result suggest the presence of more ready to act sites for the polymerases in the genomic material of primed lymphocytes even at quiescent state.

Cellular accumulation and biological activity of hematoporphyrin derivative(L) in comparison with photofrin II.

Hematoporphyrin derivative, a drug used in the photodynamic therapy of solid tumours was synthesized in the laboratory and was called Hpd(L). Physico-chemical and biological properties of this drug have been compared with Photofrin II, the commercially available drug. Both Hpd(L) and Photofrin II possess similar properties qualitatively. Quantitatively, Hpd(L) was half as active as Photofrin II in its efficacy in causing photodynamic cytotoxicity or in the optical densities at the absorption peaks. These differences could be due to the differences in the compositions. Hpd(L) is a non-purified complex mixture of a number of porphyrin derivatives whereas Photofrin II is a relatively purer compound consisting of di- and tri-hematoporphyrins linked through ether or ester bonds. In vitro cellular uptake and retention of these drugs has been found to be a passive process not involving energy expenditure. pH and temperature of the incubation media have been found to profoundly influence these processes, while a complex relation seems to exist between physiological state of a cell and accumulation of these photosensitizers.

The role of initial 5-fluorouracil trabeculectomy in primary glaucoma.

Sixty-eight patients with primary glaucoma involving 68 eyes were divided into two groups: Group I eyes were subjected to trabeculectomy (n = 38) and Group II eyes underwent trabeculectomy followed by subconjunctival injections of 5-fluorouracil (35 mg) (n = 30). After one year follow-up, Group I eyes exhibited reduction of mean intra-ocular tension from 45.7 mm Hg (pre-operative) to 16 mm Hg; optic disc cupping remained unchanged and 24/38 eyes (63%) were found to have field defects (19/38 i.e. 50% had preoperative field defects.) Group II eyes showed a reduction of mean intra-ocular pressure from 47.3 mmHg to 9.3 mmHg after one year. Mean cup disc ratio was lowered from 0.50:1 to 0.46:1 and 17/30 eyes (57%) which had field defects initially continued to exhibit the same. Complications in Group I and II eyes were shallow anterior chamber [8/38 eyes (21%) from Group I and 8/30 eyes (26%) from Group II], posterior synechiae formation in 10/38 eyes (26%) and 8/30 eyes (26%) and cataract progression in 13/38 eyes (34%) and 12/30 eyes (40%) respectively; only Group II eyes had transient superficial keratitis in 9/30 eyes (30%) and thin blebs in 6/30 eyes (20%). The use of 5-fluorouracil after trabeculectomy for primary glaucoma resulted in lowering of intra-ocular pressure, eliminated the need for antiglaucoma medications post-operatively, reduced the galucomatous cup size, and prevented progression of field loss without having a significantly increased complication rate.