A model for preservation of thymocyte-depleted thymus.

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

A model for preservation of thymocyte-depleted thymus

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

BLM germline and somatic PKMYT1 and AHCY mutations: Genetic variations beyond MYCN and prognosis in neuroblastoma.

Neuroblastoma (NB) is the most common extra cranial solid tumor of childhood and often lethal in childhood. Clinical and biologic characteristics that are independently prognostic of outcome in NB are currently used for risk stratification to optimally the therapy. It includes age at diagnosis, International Neuroblastoma Staging System tumor histopathology and MYCN amplification. However, even in patients with theoretically good prognosis, such as localized tumor and non-amplified MYCN, either disease progress or recurrence may occur. Potential genetic determinants of this unfavorable behavior are not yet fully clarified. The presence of elevated expression of AHCY, PKMYT1, and BLM has accompanied poor prognosis MYCN-amplified neuroblastoma patients. Considering the potential implication of these genes on the clinical management of NB, we hypothesize that the identification of genetic variations may have significant impact during development of the recurrent or progressive disease. Using targeted DNA sequencing, we analyzed the mutation profiles of the genes PKMYT1, AHCY, and BLM in tumor samples of five patients with MYCN amplified and 15 MYCN non-amplified NB. In our study, BLM germline variants were detected in two patients with MYCN-non-amplified neuroblastoma. Our data allow us to hypothesize that, regardless of MYCN status, these mutations partially abolish BLM protein activity by impairing its ATPase and helicase activities. BLM mutations are also clinically relevant because BLM plays an important role in DNA damage repair and the maintenance of genomic integrity. We also found a novel variant in our cohort, PKMYT1 mutation localized in the C-terminal domain with effect unknown on NB. We hypothesize that this variant may affect the catalytic activity of PKMYT1 in NB, specifically when CDK1 is complexed to cyclins. The prognostic value of this mutation must be further investigated. Another mutation identified was a nonsynonymous variant in AHCY. This variant may be related to the slow progression of the disease, even in more aggressive cases. It affects the maintenance of the catalytic capacity of AHCY, leading to the consequent functional effects observed in the NB patients studied. In conclusion, our hypothesis may provide that mutations in BLM, AHCY and PKMYT1 genes found in children with MYCN-amplified or MYCN-non amplified neuroblastomas, may be associated with the prognosis of the disease.

Repetitive sequences in the ribosomal intergenic spacer of Trypanosoma cruzi.

A fragment of Trypanosoma cruzi ribosomal intergenic spacer (IGS) located at 6.7 kb from the 3' end of the 24S rRNA gene was analyzed. This IGS fragment is characterized by the presence of three types of repetitive elements (designated Spacer Repetitive Elements, SRE), short direct repeats (5-6 bp) and chi-like recombinational sequences. SRE elements are composed of relatively short repeats (43-145 bp) which show variabilities consisting of nucleotide changes, insertions and deletions. SRE-1 element (145 bp) has a short oligo(dA) tail at the end of the repeat and can be found flanked by other SRE elements. SRE elements are species-specific, suggesting that probes based on them may be diagnostic for Trypanosoma cruzi.

DNA polymorphisms of apolipoprotein B and AI-CII-AIV genes in a Brazilian population: a preliminary report.

Possible associations between coronary heart disease (CHD) and restriction fragment length polymorphisms (RFLPs) in the apo AI-CII-AIV cluster and the apo B gene were investigated in a Brazilian population consisting of 46 patients with CHD and 24 individuals without evidence of CHD. A preliminary genetic analysis of SstI RFLP in the apo AI-CII-AIV cluster showed a significantly higher frequency of the rare SstI allele (S2) in CHD patients as compared with controls. No significant differences were found in the frequencies of PstI RFLP in the apo AI-CII-AIV cluster or XbaI and EcoRI RFLPs in the apo B gene between CHD patients and controls. Moreover, no association was seen between the RFLPs studied and myocardial infarction or plasma cholesterol or triglyceride levels.

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report.

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein alpha (C/EBP alpha) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBP alpha factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBP alpha factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBP alpha factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBP alpha factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

[Experimental neuropathy induced by DDT: analysis of the nerves by microdissection of the fibers]. / Neuropatia experimental por DDT: análise de nervo por microdissecção de fibras.

The genito-femoral nerves of ten albinic rats chronically intoxicated with DDT (5 mg/kg day during 180 days) was studied by teased fibers examination. It was found 85,37% types A + B fibers, 13,63% type C and 1% of types D, E, F and G. It is concluded that there occurs an axonal degeneration of myelin induced by chronic DDT intoxication.

[Insecticide poisoning. Microdissection of nerves and muscle histochemistry in 10 cases]. / Intoxicações por inseticidas. Microdissecção de nervos e histoquímica de músculos en 10 casos.

A sural nerve and a muscle biopsy study of patients with chronic insecticides poisoning, with teased fiber preparations, routine pathologic studies of nerves and histochemistry of muscle is reported. The sural nerves of ten patients were studied and a teased fiber preparation was done in nine. The tenth patient had only fibrosis and no myelin was found. The sural nerves were abnormal in all patients and the teased fiber preparation resulted in preponderance of type C, D and large amount of G type fibers, according to Dyck's classification. These fibers had enlargement of the axon and myelin sheath, also seen in routine sections. The muscle biopsy with routine and histochemistry methods was done in 8 cases; in 6 there was found signs of denervation; the remaining cases were normal, but these were proximal muscles. The authors conclude that the process primarily interfere with the functions of the axons, with distal axonal degeneration and a dying back phenomen.

[Neurologic involvement caused by insecticides. Study of chronic manifestations in 13 cases]. / Comprometimento neurologico por inseticidas. Estudo das manifestações cronicas em 13 casos

The clinical study of 13 patients with chronic neurological manifestation induced by insecticides (organophosphorades and organochlorades compounds) is reported. Twelve patients were male and farmers and one was a saleslady. The authors adopted five conditions for diagnosis: frequent contact with insecticides, synaptomatology directly related to the compounds exposition, clinical and laboratorial exclusion of others pathologies, quickly improvement of the symptoms when the patients were away from their original environment and high levels of insecticides in blood and urine determination. The clinical data was not uniform and the manifestation include pure motor neuropathy, mixed sensory-motor neuropathy, mielopathy and cranial nerves palsies. No relationship could be established between the insecticides (type, frequency of number of exposition) to the clinical picture. The insecticides determination was positive (moderated or elevated levels) in all patients and in 40% of controls (traces). Eletromyographic studies showed a neuropathic pattern in the majority of cases and reduced motor nerve conduction velocities. The authors believe that insecticides determination has a relative value and others criterion must be used in the diagnosis of chronic illness caused by insecticides. They think that others factors can be related with the symptomatology (hypersensibility, malnutrition, previous sensibilization or cumulative effect).

[Behcet's syndrome with neurologic involvement. Report of a case in a pregnant women]. / Doença de Behçet com envolvimento neurologico. Relato de um caso em gestante

A case of Behcet's disease in a pregnant patient is reported. The patient showed central nervous system involvement as well eyes, mouth, external genitalia and skin lesions. The neurological signs were cranial nerves palsies and pyramidal tract involvement. The cerebrospinal fluid showed hypercytosis and hyper-gammaglobulin. The patient was treated with sistemic dexamethasone and eye drops of atropine and fluorometolone. The delivery was by elective cesarean section at 8th and a half month of pregnancy. In the examination of 6 month-old the infant was normal. The patient improved but remained with important visual loss. The authors think that the pregnancy helped the partial recovery and the good evolution; similar cases have been reported in the literature. A rapid review about the central nervous system involvement and treatment of Behçet's disease is made.

[Meningitis caused by primary lymphoma of the central nervous system. Report of a case]. / Meningite por linfoma primitivo do sistema nervosos central. Relato de um caso.

The case of a 17 years-old woman, with clinical findings of meningites, without identification of a pathogenic agent is reported. The pacient received treatment for tuberculous and fungi meningitis. In the clinical course were found neoplastic cells in the spinal fluid. Despite the radiologic investigation, the original site of the tumor was not found in life. In the post-mortem examination, a small tumor was discovered in the 4th ventricle. The hystologic examination revealed a primary central nervous system lymphoma, diffuse infiltration of the meninges by tumor cells and proliferation into the central nervous system through the Virchow-Robin space. The clinical course, laboratory investigation and anatomical findings of the tumor are presented. A short revision about the pathogenesis of neoplastic meningites and primary central nervous system lymphoma is made.

[Myasthenia gravis with the electrographic response of a myasthenic syndrome. Report of a case]. / Miastenia grave com resposta eletrográfica de síndrome miastênica: relato de um caso.

It is reported a case of a 61 years-old man with progressive asthenia, disfagia, disphonia and diplopia, of variable intensity during the day, who had a very good response to anticolinesterasic drugs and corticosteroids. The repetitive stimulation tests at low frequency, resulted in large increment (maximum 275%) of the basal voltage after exercise. At high frequency he also had a large increment. Radiological and laboratory investigation three times in a seven-year period was normal, without evidence of any neoplasia. Muscle biopsy showed a type II muscle fiber atrophy. The repetitive stimulation tests repeated three times, was typical of myasthenia gravis in one occasion and in the other two, typical of myasthenic syndrome (increment of 418%). A discussion about other cases with similar findings is made after a review of the literature.

[Dupuytren's and Ledderhose's diseases associated with chronic use of anticonvulsants. Case report]. / Doenças de Dupuytren e de Ledderhose associadas ao uso crônico de anticonvulsivantes. Relato de caso.

We present the case of a patient that after chronic use of anticonvulsant drugs without proven epilepsy, developed Dupuytren's and Ledderhose's diseases. We discuss the most frequent predisposing factors, and their relationship with chronic use of anticonvulsants, particularly phenobarbitone.

Neurofibromatosis, stroke and basilar impression. Case report.

Neurofibromatosis type 1 (NF1) can virtually affect any organ, presenting most frequently with "cafe au lait" spots and neurofibromas. Vasculopathy is a known complication of NF1, but cerebrovascular disease is rare. We report the case of a 51-year-old man admitted to the hospital with a history of stroke four months before admission. On physical examination, he presented various "cafe au lait" spots and cutaneous neurofibromas. Neurologic examination demonstrated right-sided facial paralysis, right-sided hemiplegia, and aphasia. Computed tomography scan of head showed hypodense areas in the basal ganglia and centrum semiovale. Radiographs of cranium and cervical spine showed basilar impression. Angiography revealed complete occlusion of both vertebral and left internal carotid arteries, and partial stenosis of the right internal carotid artery. A large network of collateral vessels was present (moyamoya syndrome). It is an uncommon case of occlusive cerebrovascular disease associated with NF1, since most cases described in the literature are in young people, and tend to spare the posterior cerebral circulation. Basilar impression associated with this case may be considered a pure coincidence, but rare cases of basilar impression and NF1 have been described.

[Stroke in young adults: analysis of 164 patients]. / Acidente vascular cerebral em pacientes jovens: análise de 164 casos.

We retrospectively analyzed the epidemiological features of 164 out-clinic patients with a first-onset stroke between 15 and 49 years old. Ischemic stroke occurred in 141 patients, hemorrhagic stroke in 16 patients, and venous thrombosis in 7 patients. Forty-eight percent of ischemic strokes were atherothrombotic, but no etiology was found in 32% of patients with ischemic stroke. Systemic arterial hypertension was the most frequent etiology in the hemorrhagic stroke group. The most frequent risk factors were systemic arterial hypertension, smoking, hypercholesterolemia, alcoholism and diabetes mellitus. Although stroke in young adults deserves some specific etiological investigation, we found that ordinary risk factors such as hypertension, tobacco use, hypercholesteremia and diabetes were prevalent in our population. It seems that prevention campaigns should be the target of our work.

[Correlation between diagnostic hypothesis and result of cranial computed axial tomography]. / Correlação entre hipótese diagnóstica e laudo de tomografia axial computadorizada craniana.

The use of computed axial tomography (CT) in the investigation of neurologic disorders is attractive for its disponibility in the health services. However, the indications of the exam and the correlation with the clinical features has not been frequently studied. We study correlation between the requests of CT and the findings reported by the radiologist, in 367 exams performed from 07/1995 to 07/ 1996. The mean age was 31.7 +/- 22.9 years. The CT were requested in decrescent order of frequency by the Services of Neurology (36.2%), Emergency room (17.4%), Pediatric Neurology (16.9%) and Internal Medicine (5.9%). The CT was more indicated in cases of seizures (30%), headache (26.2%), motor impairment (20.2%) and reduction of conscience level (16.9%). The main hypothetic diagnosis were "to discard anatomic lesions" (9.0%), not specified stroke (8.2%) and neurocisticercosis (8.2%). The result of the CT was normal in 50.4% of the exams specially those requested in cases of headache (94.4%), seizures (71.4%) and "to discard anatomic lesions"(66.7%). The more frequently CT abnormalities were hydrocephalus (5.4%), ischemic stroke (5.4%) and neoplasm (3.5%) The greatest rates of correlation were among those to discard anatomic lesions (66,7%), hydrocephalus (50%), ischemic stroke (50%) and hematoma (50%). We concluded that CT is more helpful if more clinical data is provided in the request form, so aiding the radiologist in the final report.

Neuro-Behçet: report of three clinically distinct cases.

We report three patients who collectively have very representative clinical forms of neuro-Behçet and different neurological findings. The first case, male, 49 years old, presents symptoms similar to multiple sclerosis. The second case, male 15 years old, presents with parenchymatous compromise and an association with antiphospholipid antibody. And the third case, female 25 years old, presents an acute meningitis. Neuro-Behçet must always be included as a differential diagnosis of neurological disorders that have any difficulties in establishing a definite diagnosis.

[Headache incidence in a hospital community]. / Incidência de cefaléia em uma comunidade hospitalar.

The purpose was to describe the main features of headache incidence in a hospital community, its frequency and the most requested medical investigation. Due to the stressful work environment, hospital is considered to hold a high-risk population. Interviews and questionnaires were utilized. Of a 1006 files, which were randomly filled out, 987 could be analyzed. Of all, 38.5% were from headache sufferers. By using a table of pain symptoms taken from the International Headache Society classification as a pattern, headaches were assigned as migraine, tension-type and other. The mean age was 31.18 and the frequency in females was higher than in males, at any type. Family occurrence in first-degree relatives was 76.8%. Frontal location, medium intensity and pulsation were the most described features. Stress was the most frequently mentioned trigger factor. A physician was consulted only by 41.3%. Cranium X-ray was the most frequently requested exam.

Uncommon allele in apo AI-CIII-AIV gene cluster in a family with congenital generalized lipodystrophy.

Congenital generalized lipodystrophy is a rare inherited disease. One of its features is a disturbance in lipid metabolism characterized by hypercholesterolemia and hypertriglyceridemia. A brother and a sister with congenital generalized lipodystrophy, an 8-year old male and a 12-year old female were studied. The mother and a 6-year old brother were healthy. The genetic analysis of Sstl RFLP of the apo AI-CIII-AIV gene cluster showed the presence of the rare Sstl allele (S2) in the patients but not in the healthy mother and brother. As this uncommon allele has been reported to be related to high plasma triglyceride levels, this association could be relevant in explaining in part the hypertriglyceridemia observed in these patients.

Long chain omega-3 fatty acids: micronutrients in disguise.

Considerable information has accumulated to show that DHA and EPA have unique roles that differ from other n-3 fatty acids and the n-6 fatty acids, with increasing understanding of the mechanisms through which these fatty acids reduce risk of disease. DHA and EPA regulate hepatic lipid and glucose metabolism, but are present in foods of animal origin, which are generally high in protein with variable triglycerides and low carbohydrate. Biological activity at intakes too low to provide significant amounts of energy is consistent with the definition of a vitamin for which needs are modified by life-stage, diet and genetic variables, and disease. Recent studies reveal that DHA may play a central role in co-coordinating complex networks that integrate hepatic glucose, fatty acid and amino acid metabolism for the purpose of efficient utilization of dietary protein, particularly during early development when the milk diet provides large amounts of energy from fat.