Depletion of chloramphenicol in trout after a hypothetic therapeutic treatment.

The study was intended to evaluate the depletion of chloramphenicol (CAP) in rainbow trout (about 300-550 g body weight), after 10 days treatment with fish feedstuff containing chloramphenicol. A total of 60 animals were separated in two groups: one was fed with CAP containing feedstuff in order to have a dosage of about 80 mgkg(-1)day(-1), while a second group of fishes was fed with feedstuff not containing any CAP formulation (negative controls). The treatment was maintained for 10 days. After this period, groups of 2-5 animals were sacrificed at different withdrawal times up to a maximum of 31 days. Muscle tissues of each group of animals were then analysed for quantitative residual CAP determination both by enzyme linked immunoassay (ELISA) and liquid chromatography coupled to mass spectrometry (HPLC/MSMS). The methods applied were in house validated according to the guidelines laid down by the European Decision 657/2002/EC. Results and considerations are presented.

Videolaparoscopic appendectomy: the current outlook.

BACKGROUND: Mini-invasive techniques have revolutionized surgery, but the superiority of laparoscopic access for appendectomy is widely debated. The authors analyze their monocentric experience with 1,347 laparoscopic appendectomies. METHODS: Between October 1991 and December 2002, all the patients with an indication for appendectomy underwent surgery (301 emergency and 1,046 interval appendectomies) using the laparoscopic approach. RESULTS: For 1,248 patients, appendectomy was performed laparoscopically, whereas for 99 patients (7.3%), it was converted to an open procedure because of technical reasons (90 patients, 6.7%) or intraoperative complications (9 patients, 0.6%). For 59 patients (4.4%), the appendectomy was associated with another procedure. Histology showed "acute" alterations in 261 of the 301 emergency surgeries and in 148 of the 1,046 elective operations. Postoperative complications arose in 37 patients (2.7%), with 5 patients (0.3%) requiring invasive treatment. The mean postoperative stay was 30 h. CONCLUSIONS: Laparoscopic appendectomy offers unquestionable advantages, but it is not yet considered the "gold standard" for appendiceal pathology. Many centers reserve it for selected patients (e.g., obese patients and women suspected of having other pathologies). No randomized trials or metaanalyses have definitively proved its superiority.

An unusual complication of gastric banding: recurrent small bowel obstruction caused by the connecting tube.

Laparoscopic adjustable gastric banding (LAGB) is a widely performed surgical procedure for morbid obesity. The application of this mini-invasive approach has given the benefits of shorter hospital stay, less postoperative pain and quicker functional recovery. LAGB complications are related either to the access-port, such as port-site infection or tubing disconnection, or to the band, such as band slippage, pouch dilatation, or intragastric migration. We report a case of recurrent small bowel obstruction caused by the connecting tube around a jejunal loop, in a woman who had under-gone LAGB 3 years before. The diagnosis was difficult to establish because the clinical history and examination were non-specific. A 3-dimensional CT scan was needed to explain the cause of the recurrent abdominal pain, and the small bowel loop was freed from the connecting tube at laparoscopy.

Impact of excess weight and estrogen receptor gene polymorphisms on clinical course of homozygous beta thalassemia.

In homozygous beta thalassemic patients we examined the role played by the interaction of ER gene polymorphisms with adverse environmental factors. A total of 108 homozygous beta thalassemic patients, 60 prepubertal mean age 9,5 +/- 3,7 years (27 M, 33 F) and 48 pubertal mean age 22,2 +/- 5,4 years (21 M, 27 F), regularly treated with red cell transfusion and iron chelation therapy were segregated on the basis of their XbaI and PvuII ER gene polymorphisms. Body mass index (BMI), lipidic pattern and blood pressure values were evaluated in each group. No significant differences were observed between patients segregated by their PvuII ER genotypes. Prepubertal and pubertal patients of both sexes lacking XbaI site showed BMI, HDL, LDL cholesterol significantly different than the other patients. In addition, triglyceride levels and blood pressure values were significantly higher in pubertal patients of both sexes lacking XbaI site than in other patients. ER XbaI polymorphism appear to influence nutritional factors, metabolic status and blood pressure and could be considered additional risk factors for later cardiac involvement in beta thalassemic patients.

[Inherited disorders of bilirubin metabolism]. / Disordini ereditari del metabolismo della bilirubina.

Jaundice in an infant or older child may reflect accumulation of either unconjugated or conjugated bilirubin and could be related to inherited bilirubin disorders. Three grades of inherited unconjugated hyperbilirubinemia are recognised in humans. This spectrum of disorders is distinguished primarily on the basis of the plasma bilirubin level, the response to phenobarbital administration, and the presence or absence of bilirubin glucoronides in bile. The enzyme responsible for the conjugation of bilirubin is the bilirubin uridine-diphosphate-glucuronosyltransferase (UGT). Mutations in the gene encoding bilirubin-UGT (UGT1A1), lead to complete or partial inactivation of the enzyme causing the rare autosomal recessively inherited conditions, Crigler-Najjar syndrome type 1 (CN-1) and type 2 (CN-2). Gilbert syndrome (GS) is due to an insertional mutation at homozygous state of the TATAA element (seven TA repeats) of UGT1A1 producing a reduced level of expression of the gene. The association of GS with haemolytic anemias, e.g., Hereditary Spherocytosis (HS) or Congenital Dyserythropoietic Anemia type 2 (CDA 2), increase the hyperbilirubinemia level and the risk of cholelithiasis. Forms of chronic conjugated hyperbilirubinemia are Dubin-Johnson syndrome, Rotor syndrome, Alagille syndrome or arteriohepatic dysplasia, Wilson disease or hepatolenticular degeneration. Liver or liver cell transplantation is the therapy in some cases.

Heavy metals affect the circulating haemocyte number in the shrimp Palaemon elegans.

Environmental contamination by heavy metals produced by either anthropogenic or natural activities represents a threat to many species of aquatic animals worldwide. This study investigates the effect of short-term (96 h) exposure to dissolved heavy metals on the number of circulating haemocytes in the shrimp, Palaemon elegans (Rathke). Changes in haemocyte counts were determined in relation to time of exposure and with heavy metal concentration, relating the results to toxicity. It was found that immersion in artificial seawater containing Hg, Cd, Cu, Cr, Zn or Pb caused a decrease in the haemocyte count during the first 8 h exposure, although the haemocyte number returned to the initial (time 0) levels over the following 16 h immersion. In each case, the decrease in circulating haemocyte count induced by these metals was significantly different from the controls. The greatest decrease in haemocyte numbers (haemocytopenia) was induced by Pb, followed, in descending order, by Zn, Hg, Cr, Cu and Cd. The lethal level of haemocytopenia for the shrimps, defined as the number of haemocytes ml remaining in moribund animals (i.e. threshold of mortality) was found to be significantly lower than the levels tolerated by surviving shrimps (i.e. the limit of survival). The percentage of haemocytes remaining in the circulation at the threshold of mortality as a function of the number at time 0 was 56.6 +/- 8.8%. By contrast, the equivalent value for the threshold of survival was 63.7 +/- 12.4%. Importantly, the percentage decrease in haemocyte counts tolerated by P. elegans appears to vary with the metal. Animals treated with Pb or Zn survived with a lower number of circulating haemocytes than animals exposed to the other heavy metals.

Hematological and molecular analysis of beta-thalassemia and Hb Lepore in Campania, Italy.

This epidemiological study was based on a hematological and a molecular analysis of 310 heterozygous beta thalassemic and 75 carriers of Hb Lepore out of 3,000 microcythemic subjects from the Campania region of Italy. The molecular analysis of beta chains and the deltabeta hybrid gene has shown different beta chain defects, but only the Hb Lepore-Boston-Washington type in association with haplotypes I and V. The prevalence and distribution of these molecular defects in Campania show that they are linked to historical events and to the geographical characteristics of this region.

Clinical and molecular evaluation of non-dominant hereditary spherocytosis.

About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or beta-spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC)(n) microsatellite located in the non-coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the beta-spectrin gene were utilized for the detection of de novo mutations influencing beta-spectrin mRNA stability. They were also screened for the presence of alpha-spectrin(LEPRA) as well as for the mutation -108T-->C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty-five patients showed ankyrin de novo mutations and 10 HS subjects had beta-spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for alpha-spectrin(LEPRA) and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.

Heavy metal toxicity and differential effects on the hyperglycemic stress response in the shrimp Palaemon elegans.

Agricultural and industrial activities cause heavy metal pollution of the aquatic environment. The sensitivity of crustaceans to heavy metals is well documented. However, the hormonal and metabolic target of physiological functions affected by sublethal toxicity and stress responses have been scarcely investigated. Exposure of Palaemon elegans to increasing concentrations of heavy metals dissolved in artificial sea water resulted in an order of toxicity tested by LC(50) for 96 h in intact and eyestalkless animals in which Hg is the most toxic, followed by Cd, Cu, Zn, and Pb. Eyestalkless animals were found to be more sensitive than intact individuals. Heavy metals affect the blood glucose levels, yet manipulative stress does not. The intermediate sublethal concentrations of Hg, Cd, and Pb produced significant hyperglycemic responses within 3 h, while the highest concentrations elicited no hyperglycemia in 24 h. In contrast, animals exposed to Cu and Zn showed hyperglycemia even at high concentrations. This difference in response between Cu or Zn and the nonessential heavy metals Cd, Hg, or Pb can probably be explained by the physiological roles of the former in crustaceans and by tolerance adaptations. Involvement of the crustacean hyperglycemic hormone (cHH) was tested by routine bioassay on eyestalkless individuals; each group was injected with a two-eyestalk-equivalent extract from control animals or from shrimp exposed to high concentrations of Cd, Hg, Pb, or low concentrations of Cu or Zn. All showed a hyperglycemic response within 2 h. In contrast, extracts of eyestalk removed from animals that had developed a full hyperglycemic reaction after exposure to low concentrations of Hg, Cd, Pb, or high concentrations of Cu and Zn were depleted of cHH as shown by the attenuation of the response after injection of the extracts into eyestalkless animals. This generalized and predictable sublethal response can be used as a quantitative physiological biomarker for water quality monitoring assessment.

Frequent de novo monoallelic expression of beta-spectrin gene (SPTB) in children with hereditary spherocytosis and isolated spectrin deficiency.

This report represents an attempt to define the rate of beta-spectrin de novo mutations affecting mRNA accumulation in patients with hereditary spherocytosis (HS). 19 HS children with haematologically normal parents and varying degrees of spectrin deficiency were studied. 13 of the 19 cases who were heterozygous at the genomic level for polymorphisms in the beta-spectrin coding region were further studied. However, in an analysis of reverse-transcripted amplified cDNA from the regions of the polymorphisms, seven patients appeared to be homozygous, suggesting the occurrence of de novo mutational events affecting expression of one beta-spectrin allele. We conclude that in HS patients with isolated spectrin reduction and normal parents the apparently recessive pattern of inheritance may frequently be associated with de novo monoallelic expression of beta-spectrin.

High frequency of de novo mutations in ankyrin gene (ANK1) in children with hereditary spherocytosis.

OBJECTIVE: To evaluate the frequency of de novo monoallelic expression of the ANK1 gene in hereditary spherocytosis individuals appearing as recessive. STUDY DESIGN: We studied 40 unrelated children with spherocytosis and their normal parents. The genomic distribution of the ankyrin (AC)n dinucleotide repeats was evaluated in the patients showing combined ankyrin and spectrin deficiency. To search for the absence of mRNA derived from one of the two ANK1 genes, cDNA from the heterozygous patients was amplified using polymerase chain reaction. This was analyzed for the (AC)n dinucleotide repeats. RESULTS: Thirty-three hereditary spherocytosis subjects had variable degrees of combined ankyrin and spectrin reduction; 19 were found to be heterozygous for the AC repeat lengths and were further studied. In 12, we found a cDNA polymerase chain reaction product from one ankyrin gene alone. These findings strongly suggested the nonexpression of one of the two ANK1 genes because of the de novo mutational events. CONCLUSION: The de novo loss of an ankyrin allele expression is a frequent cause of hereditary spherocytosis in children with normal parents. Therefore the category of genuinely recessive hereditary spherocytosis cases is further reduced compared with spherocytosis cases because of de novo mutations. The determination of the (AC)n microsatellite polymorphisms appears as a helpful and reliable tool for the discrimination between these two categories.

Molecular characterization of G6PD deficiency in Southern Italy: heterogeneity, correlation genotype-phenotype and description of a new variant (G6PD Neapolis).

We report on the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty-one unrelated G6PD-deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A-, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A- and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467(Pro-->Arg) substitution responsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.

Apparently normal ankyrin content in unsplenectomized hereditary spherocytosis patients with the inactivation of one ankyrin (ANK1) allele.

The study of erythrocyte membrane protein concentration by polyacrylamide gel electrophoresis (PAGE) is the first step in approaching the primary molecular defect in hereditary spherocytosis (HS). Normal or greater than normal protein 2.1 levels were found in ten unrelated HS patients showing the inactivation of one ankyrin allele. Erythrocyte membranes from the same patients, once splenectomized, showed a homogeneous degree of protein 2.1 reduction. Thus protein 2.1 levels could misleadingly appear normal due to the high number of circulating reticulocytes. To calculate the true ankyrin level using PAGE and consequently to avoid mistakes in studying a mutated gene, a simple equation, based on the number of reticulocytes, was developed.