Expression of Brachyury during development of the dendrobatid frog Colostethus machalilla.

The expression of Brachyury (Bra) during development of Colostethus machalilla was analyzed with a polyclonal antibody. The observed molecular mass of Bra was of 48 kDa, as in Xenopus laevis. During cleavage, low levels of Bra were expressed. In contrast, in the blastula Bra became up-regulated, and Bra protein was present in a wide ring of surface cells. The surface expression of Bra disappeared in the gastrula, and a new ring of Bra-positive nuclei was detected in deep cells around the closing blastopore. The C. machalilla external and internal rings of Bra-positive nuclei apparently mark the prospective mesoderm in the blastula and gastrula, respectively. The two Bra expression rings were dissociated in time in the fairly slow developing embryos of this frog. Brachyury expression in the notochord became visible only after the blastopore closed, in contrast with X. laevis. In addition, Bra expression in the notochord indicated that dorsal convergence and extension occurred after blastopore closure. The C. machalilla Bra-positive notochord was originally exposed on the gastrocoel roof, in agreement with a superficial component of the prospective mesoderm.     

Seroprevalence of human herpesvirus-8 in blood donors from different geographical regions of Argentina, Brazil, and Chile

Human herpesvirus-8 (HHV-8) causes Kaposi's sarcoma (KS) and lymphoproliferative disorders in both HIV-infected and uninfected patients. HHV-8 has a worldwide occurrence but infection rates vary according to a combination of geographic and behavioral risks. The main transmission route seems to be sexual, nevertheless, nasal secretions, saliva, blood, and organ graft have been proposed. HHV-8 was postulated as a new infectious agent for screening in blood donors. The aim of this study was to evaluate the prevalence of antibodies against HHV-8 antigens in blood donors of South America. Serum samples from 2,470 blood donors from Argentina, Brazil, and Chile corresponding to five geographic regions were studied by indirect immunofluorescence assay (IFA). Seroprevalence rate was 3.7% (92/2,470; 95% CI 2.9-4.5) in the entire blood donor population distributed as follows: Argentina, 4.0% (Buenos Aires city, 4.3%; Bahia Blanca, 2.4%; and Córdoba, 4.0%), Campinas (Brazil), 2.8%; and Santiago de Chile, 3.0%. There was no difference (P>0.05) between men and women or age related, except in Brazil where positive cases were 30-49-year-old males. The present study, which includes different geographical areas of multiple countries from South America, has not been done before. The results show similar prevalence rates among the studied zones corresponding to low-prevalence regions. South America is a large sub-continent with a wide spectrum of population and geographical characteristics, thus, more HHV-8 prevalence studies should be necessary to establish possible regional differences.     

Two new mutations and three novel polymorphisms in the<Emphasis Type="Italic"> RB1</Emphasis> gene in Ecuadorian patients

RB1 is the gene responsible for retinoblastoma, the most common malignant intraocular tumor of infancy and early childhood. There are no reports about this gene in Ecuadorian populations, and only a few studies have been published in Latin America about this subject. There is a spectrum of more than 370 mutations described in the RB1 gene mutation database (http://www.d-lohmann.de/Rb/mutations.html), and alterations have been found in 25 of the 27 exons. During the exon-by-exon analysis of 31 tumor and blood samples from Ecuadorian patients, we found two new mutations and three novel polymorphisms. One of the polymorphisms is located in intron 26 where no alterations of the gene have been described previously. The polymorphisms were found in all of the patients tumor samples, but not in normal population, suggesting there might be a relationship between these polymorphisms and the development of retinoblastoma in the Ecuadorian population.The nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY243567, AY260472, AY260473, AY273783     

Nonmyeloablative stem cell transplantation is an effective therapy for refractory or relapsed hodgkin lymphoma: results of a spanish prospective cooperative protocol.

We report the results of reduced-intensity conditioning allogeneic stem cell transplantation (allo-RIC) in patients with advanced Hodgkin lymphoma (HL). Forty patients with relapsed or refractory HL were homogeneously treated with an RIC protocol (fludarabine 150 mg/m(2) intravenously plus melphalan 140 mg/m(2) intravenously) and cyclosporin A and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Twenty-one patients (53%) had received >2 lines of chemotherapy, 23 patients (58%) had received radiotherapy, and 29 patients (73%) had experienced treatment failure with a previous autologous stem cell transplantation. Twenty patients (50%) were allografted in resistant relapse, and 38 patients received hematopoietic cells from an HLA-identical sibling. Five patients (12%) died from early transplant-related mortality (before day +100 after allo-RIC). One-year transplant-related mortality was 25%. Acute GVHD developed in 18 patients (45%). Chronic GVHD developed in 17 (45%) of the 31 evaluable patients. The response rate 3 months after the allo-RIC was 67% (21 [52%] complete remissions and 6 [15%] partial remissions). Eleven patients received donor lymphocyte infusions (DLIs) for disease relapse. The response rate after DLI was 54% (3 complete remissions and 3 partial remissions). Overall survival (OS) and progression-free survival (PFS) were 48% +/- 10% and 32% +/- 10% at 2 years, respectively. Refractoriness to chemotherapy was the only adverse prognostic factor for both OS (63% +/- 12% versus 35% +/- 13%; P = .05) and PFS (55% +/- 16% versus 10% +/- 9%; P = .006). For patients with failure of a prior autologous hematopoietic stem cell transplantation, results were especially good for those who experienced late relapses (>/=12 months: 2-year OS and PFS were 75% +/- 16% and 70% +/- 18%, respectively). These data suggest that allo-RIC is feasible in heavily pretreated HL patients and has an acceptable early transplant-related mortality. Results are better in patients allografted in sensitive disease. Both responses observed after the development of GVHD and DLI may suggest a graft-versus-HL effect. Allo-RIC has to be considered an effective therapeutic approach for patients who have had treatment failure with a previous autologous hematopoietic stem cell transplantation.     

Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations

The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were >/=0.12 micro g/ml (range, 0.12 to 32 micro g/ml), and the ciprofloxacin MICs for 28 matched control isolates were /=0.5 micro g/ml and the vast majority of those for which nalidixic acid MICs were >/=32 micro g/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).     

Specific IgE determination to Ani s 1, a major allergen from Anisakis simplex, is a useful tool for diagnosis.

BACKGROUND: The most serious limitation of the serodiagnosis of parasitoses is the occurrence of cross-reactions. OBJECTIVE: The possible use of Anisakis simplex major allergen Ani s 1 for diagnosis. PATIENTS AND METHODS: Forty-nine non-fish-allergic patients with Anisakis simplex hypersensitivity, 21 patients without allergic episodes suffering intestinal anisakiasis with obstruction of the intestinal lumen, and 10 unrelated sera as a control were included in this study to determine specific immunoglobulin (Ig)E and IgG to Anisakis simplex major allergen Ani s 1 by immunoblotting. RESULTS: Eighty-six percent of patients with Anisakis simplex hypersensitivity showed specific IgE directed to Ani s 1. Identical result was obtained for IgG detection in this group. Among patients with intestinal anisakiasis, 86% showed specific IgE, but only 29% had specific IgG (P < 0.001, two-tailed Fisher exact test). One of the 10 control subjects was positive both for IgE and IgG (P < 0.001). CONCLUSIONS: Determination of specific IgE directed to Anisakis simplex major allergen Ani s 1 is a useful tool for the diagnosis of hypersensitivity and intestinal anisakiasis. Further, measurement of specific IgG directed to Anisakis simplex major allergen Ani s 1 is only valid for Anisakis simplex allergy.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

Interaction between α- and β-adrenoceptors in rat pineal adenosine cyclic 3′,5′-monophosphate phosphodiesterase activation

Summary The adrenergic regulation of the low-Km pineal cAMP phosphodiesterase (PDE) activity was studied in adult female rats. PDE activity showed a transient enhancement (up to 42%) during the process of degeneration of pineal sympathetic nerve terminals that followed superior cervical ganglionectomy (SCGx), thus confirming the neural modulation of the enzyme. Treatment with isoproterenol (0.3–5.0 mg/Kg) increased significantly PDE activity within 2 hours. Phenylephrine induced a significant increase of pineal PDE only at a 10 mg/Kg dose, while at a lower dose (1 mg/Kg) it potenciated the stimulatory effect of isoproterenol. Treatment of pineal organ cultures with 100 M propranolol inhibited norepinephrine (NE)-induced PDE activity while 100 M phentolamine had no significant effect. Propranolol at doses unable to alter thein vitro NE-induced stimulation of pineal PDE activity (1 M), antagonized such NE effect when used in combination with 1 uM phentolamine. At equimolecular concentrations (1 M) the mixed --adrenergic agonist NE was more effective than the -adrenergic agonist isoproterenol to increase pineal PDEin vitro. These resultsSupported by grant no. 6638, Consejo Nacional de Investigaciones Cientificas y Técnicas de la RepÚblica Argentina (CONICET), Argentina.Established Investigator, CONICET.     

Immediate repair of urethroperineal fistulas after abdominoperineal amputation by means of a cutaneo-muscular flap of the gluteus maximus. Apropos of 2 successfully treated cases

Although a rare complication, the development of a urethro-perineal urinary fistula immediately after abdomino-perineal amputation is a difficult problem to solve. The fragility of the membranous urethra immediately in contact with the exenterated pelvic cavity which takes several weeks to fill in makes any attempt at isolated direct suture very hazardous. Secondary repair is also frequently difficult and the "functional prognosis is far from being always favourable". The major handicap resulting from the perpetuation of this type of fistula has led us to propose an attempt at immediate repair as soon as it is diagnosed with filling of the pelvi-perineal cavity by a cutaneo-muscular flap taken from gluteus maximus. In the two cases in which this treatment was performed, the fistula was cured and a good quality functional result was obtained.