Consensus of experts from the Spanish Pharmacogenetics and Pharmacogenomics Society and the Spanish Society of Medical Oncology for the genotyping of DPYD in cancer patients who are candidates for treatment with fluoropyrimidines.

5-Fluorouracil (5-FU) and oral fluoropyrimidines, such as capecitabine, are widely used in the treatment of cancer, especially gastrointestinal tumors and breast cancer, but their administration can produce serious and even lethal toxicity. This toxicity is often related to the partial or complete deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme, which causes a reduction in clearance and a longer half-life of 5-FU. It is advisable to determine if a DPD deficiency exists before administering these drugs by genotyping DPYD gene polymorphisms. The objective of this consensus of experts, in which representatives from the Spanish Pharmacogenetics and Pharmacogenomics Society and the Spanish Society of Medical Oncology participated, is to establish clear recommendations for the implementation of genotype and/or phenotype testing for DPD deficiency in patients who are candidates to receive fluoropyrimidines. The genotyping of DPYD previous to treatment classifies individuals as normal, intermediate, or poor metabolizers. Normal metabolizers do not require changes in the initial dose, intermediate metabolizers should start treatment with fluoropyrimidines at doses reduced to 50%, and poor metabolizers are contraindicated for fluoropyrimidines.

Impact of NADPH oxidase functional polymorphisms in acute myeloid leukemia induction chemotherapy.

Efficacy and toxicity of anthracycline treatment in acute myeloid leukemia (AML) is mediated by reactive oxygen species (ROS). NADPH oxidase is the major endogenous source of ROS and a key mediator of oxidative cardiac damage. The impact of NADPH oxidase polymorphisms (CYBA:rs4673, NCF4:rs1883112, RAC2:rs13058338) was evaluated in 225 adult de novo AML patients. Variant alleles of NCF4 and RAC2 were related to higher complete remission (P=0.035, P=0.016), and CYBA homozygous variant showed lower overall survival with recessive model (P=0.045). Anthracycline-induced cardiotoxicity was associated to NCF4 homozygous variant (P=0.012) and CYBA heterozygous genotype (P=0.027). Novel associations were found between variant allele of CYBA and lower lung and gastrointestinal toxicities, and a protective effect in nephrotoxicity and RAC2 homozygous variant. Moreover, RAC2 homozygous variant was related to delayed thrombocytopenia recovery. This study supports the interest of NADPH oxidase polymorphisms regarding efficacy and toxicity of AML induction therapy, in a coherent integrated manner.

A systematic review and meta-analysis of the impact of WT1 polymorphism rs16754 in the effectiveness of standard chemotherapy in patients with acute myeloid leukemia.

The polymorphism rs16754 of the WT1 gene has been described as a possible prognostic marker in different acute myeloid leukemia (AML) cohorts; however, it is not supported by all the studies. We performed the first meta-analysis evaluating the effect of this polymorphism upon the effectiveness of standard AML therapy. Fourteen cohort studies were included (3618 patients). Patients with the variant allele showed a significant higher overall survival (OS) at 5 years (OR:1.24, 95% CI: 1.06-1.45, P=0.007, with dominant model). WT1 did not influence complete remission, but a higher disease-free survival was observed with the variant allele. In the subgroup analysis, Caucasians, pediatric and patients treated with idarubicin and etoposide carrying the variant allele showed consistent results in OS, whereas patients with cytogenetically normal AML did not show differences. To verify the effect of this polymorphism upon other outcomes, studies in larger and multiracial populations are needed.

Influence of ABCB1 polymorphisms upon the effectiveness of standard treatment for acute myeloid leukemia: a systematic review and meta-analysis of observational studies.

The ABCB1 gene encodes for P-glycoprotein (P-gp), an efflux pump for a variety of xenobiotics. The role of ABCB1 polymorphisms in acute myeloid leukemia (AML) outcomes of standard chemotherapy (cytarabine plus anthracyclines) remains controversial. A systematic search was made of studies evaluating the association between ABCB1 polymorphisms 1236C>T, 2677G>T/A and 3435C>T and effectiveness variables. We found seven cohort studies (1241 patients) showing a significantly higher overall survival (OS) among carriers of the variant allele of 1236C>T at year 4 (odds ratio (OR): 1.47, 95% confidence interval (CI): 1.07-2.01), 2677G>T/A at years 4-5 (OR: 1.37, 95% CI: 1.01-1.86) and 3435C>T at years 3 (OR: 1.41, 95% CI: 1.03-1.94) and 4-5 (OR: 1.42, 95% CI: 1.05-1.91). In the subgroup analysis according to ethnicity, Caucasians carrying variant allele showed consistent results in OS. ABCB1 influence upon complete remission could not be demonstrated. Future studies based on larger populations and multiethnic groups should help clarify the effect of P-gp polymorphisms upon other outcomes.

A surgical model for isolating the pig liver in vivo for gene therapy.

Several studies report results that suggest the need of vascularization blocking for efficient gene transfer to the liver, especially in nonviral gene therapy. In this study, we describe a surgical strategy for in vivo isolation of the pig liver, resulting in a vascular watertight organ that allows the evaluation of several gene injection conditions. The hepatic artery and portal, suprahepatic and infrahepatic cava veins were dissected. Then, liver vascularization was excluded for 5-7 min. In that time, we first injected 200 ml saline solution containing the p3c-eGFP plasmid (20 µg/ml) simultaneously through two different catheters placed in the portal and cava veins, respectively. Vital constants were monitored during the surgery to assess the safety of the procedure. Basal systolic/diastolic blood pressures were 92.8/63.2 mm Hg and dropped to 40.7/31.3 mm Hg at the end of vascular exclusion; the mean basal heart rate was 58 bpm, reaching 95 bpm when the blood pressure was low. Oxygen saturation was maintained above 98% during the intervention, and no relevant changes were observed in the ECG tracing. Peak plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were observed after 24 h (151 and 57 IU, respectively). These values were higher, but not relevant, in 60 ml/s injection than in 20 ml/s injection. Efficiency of gene transfer was studied with simultaneous (cava and portal veins) injection of eGFP gene at flow rates of 20 and 60 ml/s. Liver tissue samples were collected 24 h after injection and qPCR was carried out on each lobe sample. The results confirmed the efficiency of the procedure. Gene delivery differed between 20 ml/s (9.9-31.0 eGFP DNA copies/100 pg of total DNA) and 60 ml/s injections (0.6-1.1 eGFP DNA copies/100 pg of total DNA). Gene transcription showed no significant differences between 20 ml/s (15,701.8-21,475.8 eGFP RNA copies/100 ng of total RNA) and 60 ml/s (12,014-36,371 eGFP RNA copies/100 ng of total RNA). The procedure is not harmful for animals and it offers a wide range of gene delivery options because it allows different perfusion ways (anterograde and retrograde) and different flow rates to determine the optimal conditions of gene transfer. This strategy permits the use of cell therapy and viral or non-viral liver gene therapy, especially appropriated to a wide variety of inherited or acquired diseases because of the liver's ability to produce and deliver proteins to the bloodstream.

Comparative antitumor effect among GM-CSF, IL-12 and GM-CSF+IL-12 genetically modified tumor cell vaccines.

Genetically modified cells have been shown to be one of the most effective cancer vaccine strategies. An evaluation is made of the efficacy of both preventive and therapeutic antitumor vaccines against murine melanoma, using C57BL/6 mice and irradiated B16 tumor cells expressing granulocyte and macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12) or both. Tumor was transplanted by the injection of wild-type B16 cells. Tumor growth and survival were measured to evaluate the efficacy of vaccination. Specific humoral response and immunoglobulin G (IgG) switch were evaluated measuring total IgG and IgG1 and IgG2a subtypes against tumor membrane proteins of B16 cells. In preventive vaccination, all treated groups showed delayed tumor growth. In addition, the group vaccinated to express only GM-CSF achieved 100% animal survival (P<0.005). Vaccination with GM-CSF+IL-12-producing B16 cells yielded lesser results (60% survival, P<0.005). Furthermore, all surviving animals remained disease-free after second tumor implantation 1 year later. The therapeutic vaccination strategies resulted in significantly delayed tumor growth, mainly using B16 cells producing GM-CSF+IL-12 cytokines, with 70% tumor growth inhibition (P<0.001)-although none of the animals reached overall survival. The results obtained suggest that the GM-CSF+IL-12 combination only increases the efficacy of therapeutic vaccines. No differences in classical regulatory T cells were found among the different groups.

Charcot neuroarthropathy triggered and complicated by osteomyelitis. How limb salvage can be achieved.

BACKGROUND: Charcot neuroarthropathy is a severe complication in the feet of patients with diabetes, which can lead to a major amputation. Osteomyelitis and surgery for osteomyelitis have been reported as trigger mechanisms of developing Charcot neuroarthropathy. However, the development of acute Charcot neuroarthropathy triggered by osteomyelitis during conservative antibiotic treatment is not well outlined in the medical literature. CASE REPORTS: Two patients apparently developed mid and rear foot Charcot neuroarthropathy, which was clinically suspected while being treated with antibiotics for osteomyelitis. One of them presented osteomyelitis of the navicular bone and subsequently developed acute Charcot neuroarthropathy of the tarsometatarsal joints. The other presented calcaneal osteomyelitis with pathological fracture and developed Charcot neuroarthropathy of the transverse tarsal joint. No offloading had been implemented in either case. A major amputation had been indicated in both cases in their teaching hospitals. Limb salvage was achieved in both cases by means of surgery, culture-guided post-operative antibiotics, intraosseus instillation of super-oxidized solution, bed rest before placing a total contact cast and stabilization of the unstable foot with a total contact cast with an opening for checking the healing course and to detect any complications. The mechanisms of the development of acute Charcot neuroarthropathy in a patient with osteomyelitis are discussed. CONCLUSIONS: Osteomyelitis in the feet of patients with diabetes and neuropathy may trigger the development of acute Charcot neuroarthropathy. Fractures and dislocated joints may subsequently become infected from the index focus, producing a severe infected and unstable foot that may require a major amputation. Limb salvage can be achieved in specialized departments.

Does osteomyelitis in the feet of patients with diabetes really recur after surgical treatment? Natural history of a surgical series.

AIMS/HYPOTHESIS: The aim of this study was to determine the rate of recurrence, reulceration and new episodes of osteomyelitis and the duration of postoperative antibiotic treatment in a prospective cohort of patients with diabetes who underwent conservative surgery for osteomyelitis. METHODS: The prospective cohort included patients with diabetes and a definitive diagnosis of osteomyelitis who were admitted to the Diabetic Foot Unit (Surgery Department, La Paloma Hospital, Las Palmas de Gran Canaria, Spain) and underwent surgical treatment from 1 November 2007 to 30 May 2010. RESULTS: Eighty-one patients were operated on for osteomyelitis during the study period. Seven patients were lost to follow-up at different stages of the study. The median duration of follow-up was 101.8 weeks (quartile 1 = 56.6, quartile 3 = 126.7). Forty-eight patients (59.3%) underwent conservative surgery, 32 (39.5%) had minor amputations and there was one (1.2%) major amputation. Twenty patients (24.7%) required reoperation because of persistent infection. Postoperative antibiotic treatment over a median period of 36 days was provided. Wound healing was achieved by secondary intention for a median of 8 weeks. Sixty-five patients were available for follow-up after healing. The percentage of recurrence, reulceration, and new episodes of osteomyelitis was 4.6% (3/65), 43% (28/65) and 16.9% (11/65), respectively. Mortality during follow-up (excluding in-hospital deaths and patients lost to follow-up) was 13% (9/69). CONCLUSION: A low rate of recurrence of osteomyelitis after surgical treatment for osteomyelitis was achieved. Despite new episodes, our approach to managing this cohort of patients with diabetes and foot osteomyelitis achieved 98.8% limb salvage.

Triggering mechanisms of neuroarthropathy following conservative surgery for osteomyelitis.

BACKGROUND: The purpose of this study was to raise awareness and stimulate discussion of the possible triggering factors of Charcot neuroarthropathy by presenting the case of one patient who had both undergone surgery and was suffering from osteomyelitis. CASE REPORT: We have extracted one case from our data set for a patient who underwent conservative surgery for osteomyelitis and subsequently developed acute Charcot in the midfoot. We present the clinical findings, photographs and X-ray studies. Preoperative X-ray showed irregular severe bone destruction in the fourth metatarsal head and a fracture of the fourth metatarsal bone. No signs of midfoot Charcot neuroarthropathy were found in this preoperative X-ray. The third and fourth metatarsal bones were both removed and the surgical wound was left open to heal by second intention. Histopathological study confirmed osteomyelitis in the bone sample. Twenty-five days after surgery, the surgical wound showed no signs of infection and healing progressed in a satisfactory way. However, the foot was swollen, erythematous and warm. Skin temperature was two degrees higher than the contralateral foot. X-ray was taken and acute neuroarthropathy of the tarso-metatarsal joints was diagnosed. CONCLUSIONS: Charcot neuroarthropathy appears to have been triggered by bone infection and/or surgery. We believe that the pivotal factor in the development of acute Charcot neuroarthropathy in this case was the weight bearing in the deformed foot so soon after the operation. Immobilization of the foot is critical as it serves to decrease the inflammation which has a key role in the development of Charcot neuroarthropathy.

Are diabetic foot ulcers complicated by MRSA osteomyelitis associated with worse prognosis? Outcomes of a surgical series.

AIMS: The aim of this study was to compare the outcomes of surgical treatment of osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA) with cases caused by methicillin-sensitive Staphylococcus aureus (MSSA). METHODS: We abstracted data of a series of 185 consecutive patients with diabetes and foot osteomyelitis undergoing surgery within the first 12 h after admission at a single hospital. Bone infection was confirmed by histopathological studies. Only cases where Staphylococcus aureus was isolated from bone specimens were included in this analysis. We analysed several variables between the two groups: MRSA vs. MSSA. RESULTS: MRSA bone infection was associated with higher body temperature (P = 0.02) and white blood cell count (P = 0.02) than MSSA. Patients with MRSA infections underwent a greater number of surgical procedures (P = 0.03). Limb salvage was achieved in 93.6% of the patients, with no statistically significant difference in limb salvage rates between MRSA and MSSA-related osteomyelitis. CONCLUSIONS: From our experience, where treatment is based on early and aggressive surgical treatment, MRSA bone infections are not associated with worse prognosis.

Outcomes of surgical treatment of diabetic foot osteomyelitis: a series of 185 patients with histopathological confirmation of bone involvement.

AIMS/HYPOTHESIS: We analysed the factors that determine the outcomes of surgical treatment of osteomyelitis of the foot in diabetic patients given early surgical treatment within 12 h of admission and treated with prioritisation of foot-sparing surgery and avoidance of amputation. METHODS: A consecutive series of 185 diabetic patients with foot osteomyelitis and histopathological confirmation of bone involvement were followed until healing, amputation or death. RESULTS: Probing to bone was positive in 175 cases (94.5%) and radiological signs of osteomyelitis were found in 157 cases (84.8%). Staphylococcus aureus was the organism isolated in the majority of cultures (51.3%), and in 35 cases (36.8%) it proved to be methicillin-resistant. The surgical treatment performed included 91 conservative surgical procedures, which were defined as those where no amputation of any part of the foot was undertaken (49.1%). A total of 94 patients received some degree of amputation, consisting of 79 foot-level (minor) amputations (42.4%) and 15 major amputations (8%). Five patients died during the perioperative period (2.7%). Histopathological analysis revealed 94 cases (50.8%) of acute osteomyelitis, 43 cases (23.2%) of chronic osteomyelitis, 45 cases (24.3%) of acute exacerbation of chronic osteomyelitis and three remaining cases (1.6%) designated as 'other'. The risks of failure in the case of conservative surgery were exposed bone, the presence of ischaemia and necrotising soft tissue infection. CONCLUSIONS/INTERPRETATION: Conservative surgery without local or high-level amputation is successful in almost half of the cases of diabetic foot osteomyelitis. Prospective trials should be undertaken to determine the relative roles of conservative surgery versus other approaches.

Response of plasma and gastrointestinal melatonin, plasma cortisol and activity rhythms of European sea bass (Dicentrarchus labrax) to dietary supplementation with tryptophan and melatonin.

Melatonin is an effective antioxidant, immunostimulant, gonadal maturating regulator and antistress indoleamine that may be potentially useful for fish farmers. We have explored two possible ways of increasing plasma melatonin levels through the diet: direct melatonin supplementation (ME diet) and supplementation with the melatonin precursor tryptophan (TRP diet). To this end, a group of sea bass was fed a commercial diet (STD diet) at a regular time for 16 days, after which plasma, intestine, and bile samples were taken at four different time points: 120 min before, and 15, 180 and 480 min after feeding. Locomotor activity, intestinal and biliary melatonin, and plasma melatonin, serotonin and cortisol levels were measured. This same sampling process and analyses were also carried out after feeding sea bass TRP diet or ME diet for 1 week. Our results show that melatonin, but not tryptophan supplementation of the diet increases plasma, intestine and bile levels of melatonin. Plasma serotonin levels, on the other hand, were increased by dietary tryptophan, but not by melatonin, confirming the availability of supplemented tryptophan for serotonin synthesis. Both treatments were equally effective in reducing the high cortisol levels observed with the STD diet.

Demand-feeding rhythms and feeding-entrainment of locomotor activity rhythms in tench (Tinca tinca).

Tench (Tinca tinca) has been described as a strictly nocturnal species whose locomotor activity rhythms, albeit strongly synchronised by light, have an endogenous nature. Aside from light, a number of other environmental factors, such as mealtime, can act as circadian system synchronisers in fish; however, there is a scarcity of information on tench feeding rhythms. This study describes daily self-feeding rhythms in tench, and analyses the role of feeding time on synchronisation of locomotor activity rhythms. Tench were able to operate string sensor-activated self-feeders, and they displayed a strictly nocturnal behavior, both under indoor and outdoor conditions. Locomotor activity remained strictly nocturnal irrespective of whether tench were fed only during the scotophase (D-feeding) or the photophase (L-feeding). However, no statistically significant differences were detected between both groups in terms of food intake or growth performance. Furthermore, unlike L-feeding, D-feeding elicited a clear anticipatory activity (FAA). When tench were given the possibility of feeding at both times of the day, they showed a clear preference for D-feeding. Finally, in fish exposed to constant darkness (DD), feeding time acted as a true zeitgeber and FAA was observed. When animals were fasted under DD conditions, locomotor activity free-run and 6 out of 12 individuals yielded significant results in the periodogram analysis. Under DD, fish resynchronised when regular food was resumed, with some tench displaying FAA. The obtained results indicated the existence of a feeding-entrainable oscillator (FEO) in tench.

Sudden neonatal death from congenital cystic adenomatoid malformation.

We report a case of congenital cystic adenomatoid malformation (CCAM) of the lungs resulting in sudden death immediately after birth. The case is extremely unusual because of the diffuse bilateral involvement. The extensive involvement of both lungs could explain the abrupt onset of the symptoms and the ineffectiveness of resuscitation attempts. The presence of cartilage as a part of the malformation adds interest to the case, since it is seldom found in this malformation and to the best of our knowledge has been reported only exceptionally in a type II CCAM.