Laryngeal chondronecrosis after radiation therapy in a dog.

A 5-year-old pug presented with a soft tissue swelling on the ventral neck and moderate stridor with associated respiratory effort. This patient received hypofractionated radiotherapy for metastatic upper lip mast cell tumour and to the submandibular lymph nodes 6 months before presentation. Oral examination showed moderate elongation of the soft palate, stage III laryngeal collapse with only the right laryngeal saccule mildly everted and exuberant pale epiglottal and left pharyngeal mucosa. Staphylectomy, resection of the epiglottal mucosa and left arytenoid lateralisation were performed. One day after surgery, temporary tracheostomy was performed after respiratory distress due to the severe laryngeal and pharyngeal oedema. A third oral exam showed pale and redundant caudal pharyngeal mucosa obstructing the rima glottis, soft and collapsible arytenoid cartilage with pale mucosa and bilateral everted laryngeal saccules. Permanent tracheostomy was elected and laryngeal cartilage biopsies were taken. Histologic diagnosis showed cartilage necrosis and abundant tissue oedema. The patient was euthanased 1 week later.

Simultaneous bilateral contracture of the infraspinatus muscle.

A case of bilateral fibrotic contracture of the infraspinatus muscles in a five-year-old Belgian Shepherd dog is described. The dog was presented with progressive forelimb lameness with postural and gait abnormalities three months after an episode of overexertion. When walking, the lower part of both forelimbs swung in a lateral arc causing a circumduction movement and in the standing position, the dog showed elbow adduction with external rotation of the distal part of both front limbs. Orthopaedic examination revealed bilateral atrophy of both infraspinatus and supraspinatus muscles and restriction in the range of motion of both shoulders, especially when attempting abduction and flexion. No specific findings were observed in the shoulder or elbow radiographs but hyperechogenic areas were evident in the ultrasonographic examination of both infraspinatus muscles. A diagnosis of fibrotic contracture of both infraspinatus muscles was established and bilateral tenectomy of the insertion tendons of the infraspinatus muscles was performed. Complete recovery of the animal was achieved after the surgery, which was confirmed in a long-term follow-up (10 months). In conclusion, physical examination and ultrasonography allowed a proper diagnosis of the condition, and tenectomy of the infraspinatus muscles resulted in a complete recovery of the patient even with bilateral involvement.

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages.

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K(m) = 0.24 +/- 0.01 mM and V(max) of 8.5 +/- 0.12 pmol.microg protein(-1) min(-1)), pH-insensitive and largely Na(+)-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na(+). These results strongly implicate system y(+)L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y(+)LAT-1 but not y(+)LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y(+)LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

[Common variable immunodeficiency in children]. / Inmunodeficiencia común variable en la edad pediátrica.

UNLABELLED: Common variable immunodeficiency (CVID) is one of the more frequent primary immunodeficiencies (PID), after IgA deficiency, and affects a heterogeneous group of patients of various ages and with autosomal recessive inheritance. Our objective is to present the group of children diagnosed with CVID treated in our Hospital Infantil Vall d'Hebron and comment on the diagnostic problems that can arise. Sixteen boys and girls were diagnosed between the ages of 7 months and 15 years. The diagnosis is based on low immunoglobulins and a clinical picture of infection. Differential diagnosis in the paediatric age must consider mainly other PIDs: transient hypogammaglobulinaemia of infancy, X chromosome-linked agammaglobulinaemia (XLA), X chromosome-linked hyper IgM syndrome (X-HIM), IgG subclass deficiency and IgA deficiency (IgAD). Other processes that evolve with recurrent respiratory infections, such as cystic fibrosis, must also be discarded. CONCLUSIONS: These patients present a high incidence of respiratory infections and bronchiectasias. We also observe associated allergic and autoimmune processes. Early diagnosis is indispensable to initiate suitable treatment and avoid the consequences of both respiratory and digestive infections.

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Parkinson's disease, smoking, and family history. EUROPARKINSON Study Group.

There is growing evidence that both genetic and environmental factors play a role in the etiology of Parkinson's disease (PD). The hypothesis of an interaction between genetic and environmental risk factors has been little explored, and never using a population-based case-control study design. Our objective was to investigate the possible interaction between smoking and family history in the etiology of PD, as part of a collaborative population-based case-control study. We included 149 nondemented PD patients ascertained in three European prevalence surveys using a two-phase design. Each patient was matched by age (+/-2 years), gender, and center to three controls drawn from the same populations (n=375). Presence of PD among first-degree relatives and smoking history were assessed through an interview for 127 cases and 306 controls. In the overall sample we found suggestive evidence that family history and ever-smoking interact in determining the risk of PD (P=0.09), with individuals exposed to both risk factors having the highest risk (OR=10.0; 95% CI=2.0-49.6). Analyses were repeated after stratification into two age-groups (cutoff: 75 years). In older patients, the joint exposure to both risk factors was associated with a significant increase in the risk of PD (OR=17.6; 95% CI=1.9-160.5). Among younger subjects, the OR for joint exposure was not significant. In conclusion, our findings suggest that smoking and family history interact synergistically on a multiplicative scale in determining the risk of PD in individuals older than 75 years.

Heteromeric amino acid transporters explain inherited aminoacidurias.

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed amino acid transporter) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.

Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection.

BACKGROUND: A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. MATERIALS AND METHODS: To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. RESULTS: No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). CONCLUSIONS: Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.

Gas phase intramolecular proton transfer in cationized glycine and chlorine substituted derivatives (M-Gly, M = Na+, Mg2+, Cu+, Ni+, and Cu2+): existence of Zwitterionic structures?

The intramolecular proton transfer in cationized glycine and chlorine substituted derivatives with M = Na+, Mg2+, Ni+, Cu+, and Cu2+ has been studied with the three parameter B3LYP density functional method. The coordination of metal cations to the oxygens of the carboxylic group of glycine stabilizes the zwitterionic structure. For all monocations the intramolecular proton transfer occurs readily with small energy barriers (1-2 kcalmol(-1)). For the dication Mg2+ and Cu2+ systems, the zwitterionic structure becomes very stable. However, whereas for Mg2+, the proton transfer process takes place spontaneously, for Cu2+ the reaction occurs with an important energy barrier. The substitution of the hydrogens of the amino group by chlorine atoms decreases the basicity of nitrogen, which destabilizes the zwitterionic structure. For monosubstituted glycine complexed with Na+, the zwitterionic structure still exists as a minimum, but for disubstituted glycine no minimum appears for this structure. In contrast, for Mg2+ complexed to mono- and disubstituted glycine, the zwitterionic structure remains the only minimum, since the enhanced electrostatic interaction with the dication overcomes the destabilizing effect of the chlorine atoms.

[Results of surgery of the endolymphatic sac]. / Resultados de la cirugía del saco endolinfático.

Surgery of the endolymphatic sac is controversial. Some consider it a placebo and others consider it the surgical treatment of choice in Ménière's disease. We studied the medical records of 87 patients who underwent surgery between 1978 and 1996. Simple decompression was practiced in 89% and a House shunt in 27.6%. The 1-year results were improvement or recovery from vertigo in 65.4%, no improvement in 25%, and reoperation in 9.5%. Tinnitus improved in only 11.9% and hearing loss improved in 9.5% and worsened in 19%. We reviewed the cases of 50 patients with a 5-year follow-up(60%). Vertigo improved in 72%,hearing loss worsened in 72%, and tinnitus remained unchanged in 78%. In view of the good results, scant complications and simplicity of the surgical procedure, we consider endolymphatic saccule surgery to be the first choice in Ménière's disease that is unresponsive to medical treatment.

Molecular biology of mammalian plasma membrane amino acid transporters.

Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

Targeted integration of a recombinant globin gene adeno-associated viral vector into human chromosome 19.

Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno-associated viral (rAAV) vectors for this purpose. A vector designated rHS32A gamma*3'RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head-to-tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated gamma globin gene expression. Inducible expression of the non-structural Rep proteins of wild-type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32A gamma*'3'RE into human chromosome 19 was documented.

Smoking and Parkinson's disease. An age-dependent risk effect? The EUROPARKINSON Study Group.

We studied the association between smoking and Parkinson's disease (PD) through a case-control study. Several studies have shown an inverse association between smoking and PD. This association has been interpreted as spurious by some investigators, and as real and causal by others. Several other studies did not confirm the inverse association. We included 193 prevalent cases of PD ascertained in five European prevalence surveys that followed a two-phase design of screening and clinical examination. Each case was matched by center, age (+/- 2 years), and gender to three control subjects drawn from the same populations (N = 579). Information on smoking was obtained through direct or proxy interview. Overall, there was no association between ever smoking and PD (odds ratio = 1.1; p = 0.6). Analyses stratified by age showed that ever smoking was associated with a decreased risk of PD in the younger individuals (odds ratio = 0.4; p = 0.03) and with a significant trend of increasing risk with advancing age (p = 0.003). The risk of PD in relation to smoking is strongly modified by age; smoking may be protective in the younger cases but not in the older cases. This finding may explain the conflicting results from previous studies.

Comparative effect of verapamil, cyclosporin A and SDZ PSC 833 on rhodamine 123 transport and cell cycle in vinblastine-resistant Chinese hamster ovary cells overexpressing P-glycoprotein.

The product of the mdr1 gene, P-glycoprotein (P-gp), represents a common mechanism of cellular resistance to a wide variety of structurally and functionally unrelated drugs. A range of structurally different P-gp inhibitors, such as verapamil, cyclosporin A and SDZ PSC 833, have been shown to modify multidrug resistance (MDR). We used flow cytometry to investigate in vitro modulation of P-gp-dependent efflux of rhodamine 123 (Rh123). The capacity to modulate the MDR phenotype of vinblastine-resistant Chinese hamster ovary (CHO) cells was assessed by analyzing the concentration of modulator needed to decrease the Rh123 mean fluorescence intensity by 50%. We found that the cyclosporin derivative SDZ PSC 833 was significantly more effective than cyclosporin A and verapamil, either in the presence or absence of fetal calf serum-supplemented media. This study indicates that analysis of Rh123 efflux modulation can be used to determine the optimal doses of MDR inhibitors in vitro and suggests that more than one modulator is needed to measure P-gp function, since verapamil had no effect on Rh123 modulation when MDR cells were used.

Gene transfer into hematopoietic cells.

Transfer of a gene into stem cells with subsequent lineage-specific gene expression is a desired goal with many potential therapeutic applications. Retroviral vectors developed from murine leukemia viruses reproducibly transfer genes into murine stem cells, but are inefficient at gene insertion into stem cells of larger animals or man. A growing knowledge of stem cell biology suggests that this inefficiency reflects the quiescent state of stem cells, even when incubated in the presence of multiple cytokines and low expression of the receptor for amphotropic retroviral vectors. Alternative vector systems are being explored in an effort to overcome these barriers to stem cell-targeted gene transfer. Our work has shown that recombinant adeno-associated virus vectors, which have the potential for transducing quiescent cells, transfer, express and integrate a globin gene linked to its normal regulatory elements in human erythroid cells, but only at very high multiplicities of infection. The integrated genome was stable and the encoded globin gene was expressed at levels equivalent to a normal globin gene.

Genotypic analysis of cutaneous T-cell lymphoma: a comparative study of Southern blot analysis with polymerase chain reaction amplification of the T-cell receptor-gamma gene.

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.

CD34+ cell positive selection from mobilized peripheral blood by an indirect immunomagnetic method: effect of the type of mobilization and assessment of tumor depletion ability.

Mobilized peripheral blood has been shown to be a suitable source of hematopoietic progenitor cells for autologous transplantation in oncologic patients. However, tumor cell contamination can potentially occur, although to a lesser extent than in the bone marrow. CD34+ cell positive selection has been developed as a system for the ex vivo purging of remaining tumor cells when used in mobilized peripheral blood. This method has shown a lower purification potential than that obtained with bone marrow or cord blood. The reason for this is not clear, but different groups have tried to improve the purity and yield of the positive selection on mobilized peripheral blood by predepletion of nonlymphoid cell populations, since they can induce nonspecific binding. The present study was designed to test an indirect immunomagnetic CD34+ cell selection method to make it reproducible, feasible, and effective for purging mobilized peripheral blood. Twenty-nine samples from mobilized peripheral blood were tested. The median starting CD34+ percentage was 0.8 (0.3%-4.2%), and the median final purity was 87% (32.7%-99.7%), with a median yield of 44.8% (15%-83.5%). The highest purity was reproducibly achieved when the starting percentage of CD34+ cells was higher than 0.65% (median purity 93.7, range 81%-99.7%, CV 6%) on samples obtained from patients primed with chemotherapy alone or chemotherapy plus recombinant human granulocyte-colony stimulating factor. No relation was found between the content of contaminating nucleated cells and the final CD34+ cell purity. This method showed a depletion capacity, assessed by PCR on samples contaminated with K562 leukemic cells, of about 3 logs. These results indicate that this indirect immunomagnetic method can produce high purity CD34+ cell fractions from mobilized peripheral blood with a high efficiency of tumor depletion.

Presentation of a PCR-nuclease protection strategy for minimal residual disease monitoring in B-ALL.

Methods for detecting residual malignant cells in patients suffering from lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcome. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some strategies have been developed for several groups. Up to now the most specific and sensitive methodology, which consists of designing leukemia-specific oligonucleotides, requires sequencing of the complementary determining region III-DNA for each particular patient and is too laborious to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, consisting of: (i) amplification of DNA segments corresponding to the complementarity determining region III of the immunoglobulin heavy chain genes from samples at disease diagnosis; (ii) isolation of the disease-specific single-stranded DNA; (iii) labeling of the single-stranded DNA to generate specific probes; (iv) hybridization to amplified DNA from samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and specificity of this approach compares with those of the above mentioned specific oligonucleotide strategy in detecting residual malignant B cells. Moreover, this strategy is much less tedious and could be used by most hospital laboratories.

[Long term outcome of phonatory fistulae]. / Resultados a largo plazo de las fístulas fonatorias.

Since January 1983 to January 1990 we have operated 84 Phonatory fistuloplasties (P.F.). The aim of this paper is an intend to know the long term results, farther than 3 years follow-up. We have practiced 70 primary P.F. and 14 secondary. The technique used have been plain puncture and Algaba's procedure. Myotomy of the cricopharyngeus muscle and Herrmann' flap (skin-platysma-fascia) was sometimes associated. The early success of the primary P.F. has been 95 percent and the late one 48 percent. In the secondary P.F. 54 percent and 29 percent, respectively. The most frequent causes of failure have been: enlargement, accidental loss of the prothesis and spontaneous closure and unhappy patient and fibrosis periprosthesis as well. Two factors have to be taken into account in late failures; the immediate postoperative positive psychological feeling and the fact that every patient uses the esophageal voice satisfactorily.