Air-guided manual deep lamellar keratoplasty

PURPOSE: To evaluate the efficacy of a new modified technique of deep lamellar keratoplasty (DLK). METHODS: Nine eyes of eight patients with keratoconus of moderate degree were included. All patients underwent DLK with manual dissection from a limbal side port after an air bubble injection in the anterior chamber. The patients underwent a complete ophthalmologic examination 6 months after the suture removal, evaluating best-corrected visual acuity, corneal thickness, endothelial cell count, and topographic astigmatism. RESULTS: One case (11.1%) was converted to penetrating keratoplasty because of microperforation. In the eight successful cases, 7 eyes (77.8%) achieved 20/30 or better visual acuity 6 months after suture removal. Mean postoperative pachymetry was 604.76 microm (SD 46.76). Specular microscopy 6 months after suture removal revealed average endothelial cell count of 2273/mm2 (SD 229). CONCLUSIONS: This modified DLK technique is a safe and effective procedure and could facilitate, after a short learning curve, this kind of surgery with a low risk of conversion to penetrating keratoplasty.     

Immunomodulatory and antimetastatic action of propolis and related polyphenolic compounds

The effect of polyphenolic compounds isolated from propolis and propolis itself was investigated on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of CBA mouse. Metastases in the lung were generated by intravenous injection of tumor cells (2 x 10(5)). A water-soluble derivative of proplis (WSDP), caffeic acid (CA), caffeic acid phenethyl ester (CAPE) and quercetin (QU) were given to mice per os before tumor cells inoculation. Tested compounds significantly decreased the number of tumor nodules in the lung. According to the results obtained the antitumor activity of tested compounds can be related to the immunomodulatory properties of the compounds, their cytotoxicity to tumor cells, and their capacity to induce apoptosis and necrosis. The experimental data support that WSDP, CA, CAPE and QU could be potentially useful in the control of tumor growth in experimental models.     

Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer

Tankyrase promotes telomere elongation by interaction with the telomeric protein binding factor TRF1, a negative regulator of telomere extension. We measured tankyrase mRNA by real-time RT-PCR in 66 breast cancers and in paired normal tissues. Results were compared with hTERT mRNA expression. The levels of tankyrase in breast cancers were significantly higher in comparison to normal tissues (P<0.0001) and significantly related to the status of progesterone receptors. No relationship was found between tankyrase and hTERT mRNA expression in breast cancers. According to our results, tankyrase expression appeared up regulated in breast cancers.     

N-methyl-3,4-methylenedioxyamphetamine-induced hepatotoxicity in rats: oxidative stress after acute and chronic administration

BACKGROUND: The underlying mechanisms of N-methyl-3,4-methylenedioxyamphetamine--MDMA--induced hepatotoxicity are still unknown. The aim of this study was to evaluate hepatic oxido-reductive status in the rats liver after the single and repeated administration of MDMA. METHODS: MDMA was dissolved in distilled water and administered in the doses of 5 mg, 10 mg, 20 mg, and 40 mg/kg. The animals from the acute experiment were treated per os with the single dose of the appropriate solution, through the orogastric tube. The animals from the chronic experiment were treated per os, with the doses of 5, 10, or 20 mg/kg of MDMA every day during 14 days. The control groups were treated with water only. Eight hours after the last dose, the animals were sacrificed, dissected, their livers were rapidly removed, frozen and stored at -70 degrees C until the moment of analysis. The parameters of oxidative stress in the crude mitochondrial fractions of the livers were analyzed. RESULTS: Superoxide dismutase (SOD) activity increased in the livers of the animals that were treated with single doses of MDMA. Chronically treated animals showed the increased SOD activity only after the highest dose (20 mg/kg). The content of reduced glutathione decreased in both groups, but the depletion was much more expressed after the single administration. Lipid peroxidation index increased in dose-dependent manner in both groups, being much higher after the single administration. CONCLUSION: The increased index of lipid peroxidation and the decreased reduced glutathione levels suggested that MDMA application induced the state of oxidative stress in the liver. These changes were much more expressed after the single administration of MDMA.     

Rapid site-directed mutagenesis of chemokines and their purification from a bacterial expression system

Chemokines are a family of small chemoattractant cytokines implicated in the recruitment and migration of leukocytes from the blood into tissues during disease and routine immune homeostasis. Although there are many similarities in the structure and function of certain chemokines, the importance of many residues in the function of these proteins is yet to be determined, and studies from related chemokines have shown that similar sequences may play different roles in each protein. The migration-inducing capacity of many chemokines is thought to involve the cell surface glycosaminoglycan (GAG), heparan sulphate (HS), which may assist in the formation of an immobilised chemokine gradient within inflamed tissues. To examine the heparan sulphate binding ability of the CC chemokine monocyte chemoattractant protein (MCP)-3 and its importance in chemotactic migration, we have identified and mutated conserved basic residues within the mature MCP-3 protein to the neutral amino acid alanine using a novel inverse polymerase chain reaction (I-PCR) method that rapidly generates essentially 100% mutational efficiency due to decreased requirements for template DNA and an alkaline denaturation step; this increased mutational efficiency reduces both screening time and sequencing costs. We also describe an optimised method for the expression of soluble, correctly folded MCP-3 in a bacterial system using nickel affinity columns and reverse-phase fast protein liquid chromatography (RP-FPLC), and achieve purified yields of up to 0.4 mg/l of initial culture medium after 5 h of induction. These optimised methods could work equally well for any small circular plasmid (< or =4.5 kb) incorporating a polyhistidine tag.     

Optimising restriction enzyme cleavage of DNA derived from archival histopathological samples: an improved HUMARA assay

AIMS: Our aim was to evaluate and optimise methylation-sensitive restriction enzyme assays for use in formalin-fixed, paraffin-embedded tissue (FFPET) samples, in order to improve the application of the HUMARA X-chromosome inactivation assay to FFPET samples. METHODS: We extracted DNA from normal male colon and thyroid FFPET. Several DNA clean-up procedures and restriction enzyme buffer compositions were tested for two methylation-sensitive enzymes, HhaI and HpaII and a non-methylation-sensitive isoschizomere, MspI. RESULTS: By including both a non-methylation-sensitive control enzyme and DNA from male archival specimens in our experiments, we were able to detect even subtle degrees of incomplete digestion. We showed that FFPET-derived DNA is a poor substrate for restriction enzymes, especially for methylation-sensitive restriction endonucleases. An optimised DNA clean-up protocol and restriction enzyme buffer-mix allowed us to achieve complete digestion. CONCLUSIONS: The combination of multiple, rigorous controls, DNA clean-up and restriction buffer optimisation increases the reliability of HUMARA-based X-chromosome inactivation analysis of FFPET samples. Analogous approaches are likely to allow optimisation of other restriction enzyme-based assays of FFPET samples.     

Molecular characterization of 22q11 deletion in a three-generation family with maternal transmission

Haploinsufficiency of chromosome 22q11.2 is a well-established cause of both the DiGeorge anomaly and the velocardiofacial syndrome. This condition shows a continuous spectrum of phenotypic manifestations with a considerable inter- and intrafamilial variability. We report on a three-generation family with four members sharing the same 3 Mb long deletion but showing different phenotypic expression. In the first generation, the deleted patient has hypernasal speech and suffers from recurrent psychotic episodes. Two of her offspring inherited the deletion. One of these, a male, has hypernasal speech, low-set ears, hypocalcemia, severe development delay, and tetralogy of Fallot. The other, a female, has hypernasal speech, minor facial anomalies, and very mild mental retardation. Her daughter has tetralogy of Fallot, velopharyngeal insufficiency, and mild facial anomalies. This family is an example of the widely variable phenotypic expressivity of the 22q11.2 deletion. There is no correlation between the size of the deletion and the phenotypic manifestations. Genetic background and/or environmental factors could explain the different phenotypes observed in the affected members of the family.     

Recurrent parastomal hernia and middle incisional hernia: surgical treatment through midline incision with wide prosthetic mesh

Parastomal hernia is a frequent complication of stoma surgery. The results of parastomal hernia repair however are poor, showing an high incidence of postoperative recurrences. In the last years, hernia repair with prosthetic mesh has given better postoperative results. The parastomal hernia, however, is associated with middle incisional hernia. The authors review the problem of surgical repair of parastomal hernia and report a case of recurrent parastomal hernia associated to middle incisional hernia. The technique of surgical repair using, through midline incision, one, wide, prosthetic polypropylene mesh, in sublay position, according to Rives' technique, is described. The mesh has been incised in a trasverse direction for the stoma crossing. At 6 years follow-up the patient does not show postoperative recurrence. According literature and the authors' results, the parastomal hernia might be considered an incisional hernia and, therefore, a sing of diffuse abdominal wall disease. The Rives' surgical technique might be the gold standard for treatment of parastomal hernia, even if not associated to incisional hernia. The more complexity of Rives' technique compared to local fascial mesh repair is compensated by the result of total abdominal wall reinforcement.     

Analgesic effect of caffeine and clomipramine: a possible interaction between adenosine and serotonin systems

The goals of this study were to determine whether the nonselective adenosine receptor antagonist caffeine exerts an analgesic effect and to investigate the time-dependent influence of the selective serotonin reuptake inhibitor clomipramine on the action of caffeine. Results suggest a possible interaction between serotonin and adenosine systems, which may contribute to the analgesic action of drugs. Therefore, the hot-plate and formalin tests were employed in order to measure the response to painful thermic and chemical stimuli. Results have shown that caffeine (1.67, 16.7 and 67 mg kg(-1), i.p.) exerts a direct dose-dependent analgesic action. When caffeine (1.67 and 16.7 mg kg(-1)) was combined with clomipramine (3 mg kg(-1) i.p.), an enhanced analgesic effect was obtained. However, the same combinations were ineffective in a subacute model. In this model, clomipramine was administered for 14 days and the respective dose of caffeine was added on the last day. Therefore, it can be concluded that the serotonin system interacts with the analgesic action of caffeine and that a long-term use of clomipramine probably triggers subsensitivity of adenosine receptors.