Whole body hyperthermia by extracorporeal circulation in spontaneously breathing sarcoma patients: hemodynamics and oxygen metabolism.

PURPOSE: This phase I study was performed to evaluate the feasibility and toxicity of a new method of extracorporeal perfusion-induced whole body hyperthermia (WBHT) in patients with advanced sarcoma avoiding the need of intubation and general anesthesia. METHODS: One double-lumen femoral venous access was inserted by Seldinger's technique to obtain WBHT (41.8°C for 120 minutes) via an extracorporeal circuit. No concomitant chemotherapy was applied. Up to 4 treatments of WBHT were performed under moderate sedation in 6 spontaneously breathing patients. Invasive hemodynamic monitoring was performed by use of a pulmonary artery catheter. RESULTS: After their first WBHT session, 2 patients were excluded from further treatment due to transient liver toxicity or catheter-related complication, so a total of 12 cycles remained for analyses. In all patients, conscious sedation resulted in sufficient spontaneous respiration without the need for mandatory ventilation. Median time to reach the target temperature was 84 minutes (range 60-142). Hemodynamic changes revealed the expected hyperdynamic state: heart rate, cardiac index, and stroke volume index significantly increased (p<0.05), whereas blood pressure and systemic and pulmonary vascular resistance index significantly decreased (p<0.05). A net fluid balance of 5822±1766 mL as well as norepinephrine (mean; 0.062 µg·kg¹·min⁻¹) were necessary to maintain the mean arterial blood pressure >60 mmHg. CONCLUSION: Our data demonstrate the feasibility of this method of extracorporeal WBHT without mandatory ventilation. Hemodynamic side effects in spontaneously breathing patients during perfusion-induced WBHT seem less severe than those observed in radiant heat WBHT.

Prevalence of osteoporosis in men in a VA rehabilitation center.

OBJECTIVE: To evaluate the prevalence of previously unrecognized osteoporosis in men admitted for long-term rehabilitation nursing home care. DESIGN: Cross-sectional study. METHODS: A total of 1179 consecutive admissions to a VA rehabilitation center were reviewed. Men who were already diagnosed with osteoporosis, had confounding medical illness, were unable to complete the study, or who declined to participate were excluded. A total of 153 patients were enrolled and 106 were evaluated. Measurements included dual-energy X-ray absorptiometry of the hip and lumbar spine, biochemical and hormonal studies, and functional evaluation. RESULTS: A total of 33 patients (31.1%) had osteoporosis (T-score at any site lumbar spine, total hip, or femoral neck <-2.5). Patients with osteoporosis were significantly older than those without: 68.4 +/- 13.2 years versus 62.7 +/- 12.1 years (P <.05), respectively. Body mass index (BMI) and weight were lower in men with osteoporosis: 23.4 +/- 3.9 kg/m2 versus 28.7 +/- 7.08 kg/m2 and 72.6 +/- 14.4 kg versus 90.3 +/- 23.8 kg, respectively (both, P <.001). There were no differences in use of medications thought to affect bone metabolism or functional status, or in hormonal and metabolic measurements. Hip and spine bone density were correlated (r = 0.3, P <.05). Multivariate analysis showed that hip bone density was independently associated with BMI. CONCLUSION: Hip osteoporosis is common in this unscreened population, suggesting that screening should be more widely performed in veterans admitted to rehabilitation units. These data suggest that nutritional status could impact osteoporosis risk.

Differential neuroendocrine expression of multiple brain-derived neurotrophic factor transcripts.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin with important growth-promoting properties. We report here the first characterization of a BDNF gene in an amphibian, Xenopus laevis, and demonstrate that environmental factors can activate this gene in a promoter-specific fashion. The Xenopus BDNF gene contains six promoter-specific 5'-exons and one 3'-protein-encoding exon. We examined the expression of promoter-specific transcripts in Xenopus neuroendocrine melanotrope cells. These cells make a good model to study how environmental factors control gene expression. In animals placed on a black background melanotrope cells more actively produce and release alphaMSH than in animals on a white background. BDNF is cosequestered and coreleased with alphaMSH and stimulates biosynthesis of proopiomelanocortin (POMC), the precursor protein for alphaMSH. Our analysis of the expression of the BDNF transcripts revealed that there is differential use of some BDNF promoters in melanotrope cells, depending on the adaptation state of the frog. During black-background adaptation, stimulation of expression of BDNF transcript IV preceded that of the POMC transcript, suggesting the BDNF gene is an effector gene for POMC expression. The possible mechanisms regulating expression of the various transcripts are discussed on the basis of the potential calcium- and cAMP-responsive elements in the promoter region of exon IV. Finally, we show that the upstream open reading frames of BDNF transcripts I and IV markedly decrease BDNF translation efficiency, giving the first indication for a functional role of untranslated BDNF exons.

Both 5' and 3' flanks regulate Zebrafish brain-derived neurotrophic factor gene expression.

BACKGROUND: Precise control of developmental and cell-specific expression of the brain-derived neurotrophic factor (BDNF) gene is essential for normal neuronal development and the diverse functions of BDNF in the adult organism. We previously showed that the zebrafish BDNF gene has multiple promoters. The complexity of the promoter structure and the mechanisms that mediate developmental and cell-specific expression are still incompletely understood. RESULTS: Comparison of pufferfish and zebrafish BDNF gene sequences as well as 5' RACE revealed three additional 5' exons and associated promoters. RT-PCR with exon-specific primers showed differential developmental and organ-specific expression. Two exons were detected in the embryo before transcription starts. Of the adult organs examined, the heart expressed a single 5' exon whereas the brain, liver and eyes expressed four of the seven 5' exons. Three of the seven 5' exons were not detectable by RT-PCR. Injection of promoter/GFP constructs into embryos revealed distinct expression patterns. The 3' flank profoundly affected expression in a position-dependent manner and a highly conserved sequence (HCS1) present in 5' exon 1c in a dehancer-like manner. CONCLUSIONS: The zebrafish BDNF gene is as complex in its promoter structure and patterns of differential promoter expression as is its murine counterpart. The expression of two of the promoters appears to be regulated in a temporally and/or spatially highly circumscribed fashion. The 3' flank has a position-dependent effect on expression, either by affecting transcription termination or post-transcriptional steps. HCS1, a highly conserved sequence in 5' exon 1c, restricts expression to primary sensory neurons. The tools are now available for detailed genetic and molecular analyses of zebrafish BDNF gene expression.

Insert-containing neurotrophins in teleost fish and their relationship to nerve growth factor.

Some teleost species express neurotrophins not known in other vertebrates, namely neurotrophin-6 (NT-6) and neurotrophin-7 (NT-7). Mature proteins of both genes are closely related to nerve growth factor (NGF). We have cloned zebrafish NGF (zNGF) and show that genomic organization and transcript structure of zNGF, zNT-7, and mouse NGF are highly similar. This suggests the vital importance of hitherto unrecognized untranslated regions and principal features of gene structure, retained in species separated 410 mya. Aiming to clarify the relation between NT-6 and NT-7, we have identified partial NGF and NT-6/7 sequences of additional teleost species and a complete set of neurotrophins in two pufferfish genomes (fugu and tetraodon). Interestingly, this includes neurotrophin-4/5, hitherto not described in any fish species. Identification of only one NT-6/7-like gene in pufferfish and salmon, phylogenetic analysis and a strikingly high identity of an untranslated sequence of zNT-7 and the pufferfish NT-6/7 genes strongly suggest that these genes have evolved from a common ancestor after a single "fish specific" duplication of NGF. Evidence for sub- and neofunctionalization is provided.

A novel BDNF gene promoter directs expression to skeletal muscle.

BACKGROUND: Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF) is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. RESULTS: The cloning and analysis of three additional zebrafish (Danio rerio) BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes) and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. CONCLUSION: The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are expressed in an independently regulated and cell-specific fashion. An initial structural and functional analysis has shown that the regions controlling zebrafish BDNF gene expression have been cloned and identified. They can now be subjected to detailed molecular and genetic analyses to identify the cellular mechanisms by which the transcription factors that act on these regions control BDNF gene expression.