Procalcitonin and CGRP-1 mrna expression in various human tissues.

Procalcitonin (PCT) is a highly sensitive and specific marker of systemic bacterial infection and sepsis. In contrast to its diagnostic significance, the cellular sources of plasma procalcitonin remain to be clarified. Two forms of PCT mRNAs originate from calcitonin/calcitonin gene-related peptide gene (CALC-I gene) along with mRNA for calcitonin gene-related peptide-I (CGRP-I). Reverse transcription polymerase chain reaction with newly designed primers detecting different PCT mRNAs and CGRP-I mRNA was used to identify tissues that might contribute to PCT production. Our study indicates that a variety of human tissues (13 of the 16 analyzed overall) express PCT-I, PCT-II, and/or CGRP-I mRNAs, with the highest levels detected for liver, testis, lung, prostate, kidney, and small intestine. Various tissues differ in the proportions of PCT-I, PCT-II, and CGRP-I mRNA expression levels. Thus we demonstrate the complexity of tissue-specific regulation of CALC-I gene expression and suppose a variety of tissues as a potential source of CALC-I-encoded peptides.

Model of statoconia accumulation in gravireceptors of mollusks.

The kinetics of formation and accumulation of statoconia are different for Aplysia californica and Biomphalaria glabrata. In Aplysia californica, the fast growth of statoconia number occurs after the critical size (approximately 45 micrometers) of statocyst is reached; then the increase of statoconia number is proceeding with the nonmonotonic rate during the life of an animal. In Biomphalaria the growth of statoconia number occurs only in the initial phase. Then long-term evolution of statoconia in the absence of their generation is the result of their growth in the cyst lumen. In the case of Aplysia californica it is not clear whether a temporal change of the statoconia size distribution (SSD) is caused by statoconia growth in the cyst lumen similar to that in Biomphalaria (Model 1) or statoconia growth takes place in supporting cells until their release into the cyst lumen occurs. (Model 2). This problem is of practical importance because the majority of experiments related to the development of molluscan gravireceptors in altered gravity dealt with an initial phase of statoconia evolution in Aplysia californica and Biomphalaria glabrata. The purpose of the present work is the application of mathematical modeling to the analysis of mechanisms of statoconia formation by supporting cells.

[Procalcitonin as monocytic marker for early diagnosis in septic abortion]. / Procalcitonin als monozytärer Marker für die Frühdiagnostik bei septischem Abort.

BACKGROUND: In search of sensitive and specific markers of systemic infection procalcitonin (PCT) recently was promoted to the focus of clinical research. Little is known about the biology of PCT and until now no data have been presented about clinical importance of PCT in obstetric patients. PATIENT AND METHODS: Daily PCT values in a 17 year old patient with septic abortion were compared with established markers of systemic inflammation. Cultivated monocytes were analyzed by the means of indirect immunofluorescence for intracellular distribution of PCT. Additionally, PCT release into culture medium was examined. RESULTS: PCT values in comparison with established inflammation markers was demonstrated in the patient with septic abortion. Indirect immunofluorescence studies revealed the presence of PCT within monocytes. In the supernatants of monocyte cultures PCT was detectable under control conditions. Stimulation with lipopolysaccharide resulted in the increased PCT concentrations both in the supernatants of healthy and patient monocyte cultures. CONCLUSIONS: In the given patient PCT was superior to other inflammation markers with regard to early and progression diagnosis. Peripheral blood monocytes appear to be a potential site of inflammation-induced PCT production. For the first time intracellular distribution pattern and release of PCT from human monocytes was described. DISCUSSION: Based on the presented data broad clinical studies devoted to PCT evaluation in obstetric patients seem to be promising. As till now the interpretation of increased PCT values depended on empirical knowledge, extensive studies of the potential production site as well as its biological significance should be performed, too.

Lipopolysaccharide induces distinct alterations in the microtubule cytoskeleton of monocytes.

Microtubules are obligate functional elements of almost all eukaryotic cells. They are involved in a broad range of essential cellular functions and structural changes of this system may trigger cell death. Recently, we have reported that lipopolysaccharides inhibit in vitro microtubule formation due to exclusion of microtubule-associated proteins. The distinct epitopes of lipopolysaccharides responsible for these effects and the in vivo relevance of these data are unknown. Therefore, this study was conducted to elucidate the effects of lipid A, the biologically active motif of lipopolysaccharides, on microtubule formation in vitro and to prove whether lipopolysaccharides affect the microtubule architecture of cultured human monocytes in vivo. Despite a dose- and pH-dependent inhibition of microtubule formation by lipopolysaccharides, inhibition of microtubule assembly could be mimicked by lipid A. Near-infrared two-photon microscopy revealed that human peripheral blood monocytes accumulate lipopolysaccharides. A vesicular distribution pattern of lipopolysaccharides within the monocytes was observed. Confocal laser scanning microscopy demonstrated alterations in the microtubule architecture of monocytes after incubation with lipopolysaccharides. Lipid A seems to be responsible for the observed crosstalk between lipopolysaccharides and microtubule proteins. Furthermore, our data indicate that lipopoly-saccharides may affect the microtubule architecture in human monocytes after intracellular accumulation directly. Therefore, we conclude, that the microtubule cytoskeleton is an essential intracellular target for sepsis-relevant bacterial components such as lipopolysaccharides.

Otoliths developed in microgravity.

Little is known about mechanisms that regulate the development of the otoliths in the gravity-sensing organs. Several reported experiments suggest that the growth of the otoliths is adjusted to produce a test mass of the appropriate weight. If this is the case, larger than normal otoliths would be expected in animals reared in reduced gravity and a reduced mass, relative to 1-g controls, would be expected in animals reared at elevated g. In gastropod mollusks, the gravity-sensing organ is the statocyst, a spherical organ whose wall is made largely of sensory receptor cells with motile cilia facing the lumen. Dense statoconia in the cyst lumen interact with cilia of receptor cells at the bottom of the cyst and action potentials in their axons carry information on direction and magnitude of gravity and linear acceleration. In the marine mollusk, Aplysia californica, larvae reared at 2 to 5-g, the volume of statoconia was reduced in a graded manner, compared to 1-g control animals. In the statocyst of the fresh-water pond snail, Biomphalaria glabrata, reared in space in the Closed Equilibrated Biological Aquatic System (CEBAS), the number and total volume of statoconia was increased approximately 50%, relative to ground-reared controls. Lychakov found the utricular otolith to be 30% larger in space-reared Xenopus, whereas we found the saccular otolith to be significantly larger in newt larvae reared in space. In cichlid fish reared on a centrifuge, the saccular otolith was smaller than in 1-g controls. Here, we demonstrate that the otoliths of late-stage embryos of the swordtail fish, Xiphophorus helleri, reared in space on STS-89 and STS-90 (Neurolab) were significantly larger than those of ground-controls reared in functionally identical hardware.

Stimulation of mouse macrophage antigen presentation by cocaine.

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.

Molecular aspects and natural source of procalcitonin.

The search for sensitive and specific markers of systemic infection has shown that procalcitonin levels are increased in sepsis, and, consequently, this plasma protein has come into the focus of clinical research. Human procalcitonin is encoded by the Calc-l gene, which gives rise to two alternatively spliced transcripts. Despite systemic investigation of the Calc-l gene and mechanisms of the tissue-specific regulation and alternative splicing, little is known about the biology of procalcitonin and the cells which express this protein during inflammation. Here we focus on the molecular and biochemical properties of the molecule and summarize the known biological functions of procalcitonin. We report on the structure of the Calc-l gene, the amino acid conservation of procalcitonin in different species, and the consensus sequences of the protein with regard to sites relevant for posttranslational modification, spatial distribution, and homologies to other cytokines. We discuss aspects of intracellular location of procalcitonin and demonstrate that it has the characteristics of a secreted protein.

HepG2 human hepatoma cells express multiple cytokine genes.

Although cytokines are known to be involved in the regulation of a variety of hepatocellular functions, hepatocytes themselves are generally considered only targets but not producers of these important mediators. In order to investigate whether cells of hepatocellular linages are a potential source of various regulatory cytokines we have estimated the multiple cytokine gene expression in the culture of well differentiated human HepG2 hepatoma cells using RT-PCR. Our findings demonstrate that HepG2 cells express mRNAs for interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), macrophage colony-stimulating factor (M-CSF), oncostatin-M (OSM), intercellular adhesion molecule (ICAM-1), interleukin 4 (IL-4), IL-5, IL-7, IL-10, IL-11, IL-12 and IL-6 receptor (IL-6R). At the same time the expression of IL-1, IL-2, IL-3, IL-6, CD40 ligand and IL-2R genes was not detected. It was concluded that hepatocytes are potential producers of a variety of cytokines, some of them being able to regulate hepatocellular functions directly, while others are important regulators of leukocyte activity. Thus, on the one hand, hepatocytes may express autoregulatory cytokines and on the other hand, influence the functions of other liver cells like Kupffer, Ito or endothelial cells. Due to their large amount, liver parenchymal cells could be an important source of sytemically acting pro- and anti-inflammatory and other regulatory cytokines.

Clinical observations during virtual reality therapy for specific phobias.

Virtual environments offer a new method of providing exposure therapy to patients with specific phobias. Although the stimuli (three-dimensional computer simulations) is new, the method of systematically desensitizing the patient to phobic stimuli until habituation occurs is a concept that was formally introduced by Joseph Wolpe over 40 years ago. Our article discusses some of the clinical observations that have been made during nearly 500 virtual reality exposure therapy sessions with patients and research participants who have come to our center in the past 15 months with a fear of flying or driving.

Production of otoconia in the endolymphatic sac in the Japanese red-bellied newt, Cynops pyrrhogaster: light and transmission electron microscopic study.

The formation of otoconia in the endolymphatic sac (ES) of the larval newt, Cynops pyrrhogaster, has been studied by light and transmission electron microscopy. Some of the epithelial cells of the ES contain an abundance of swollen vesicles, Golgi complexes, rough endoplasmic reticula and ribosomes at the late larval stages 50 and 51, approximately 26-30 days after eggs are laid. Five days later, at stage 52, crystals are present in the vacuoles between the epithelial cells. Serial sections indicate that these vacuoles actually form small canals which lie in the wall and join the lumen of the ES. Reconstruction of the ES shows that several canals are contained in the ES wall. At stage 56, about 72 days after eggs are laid, a large number of otoconia are present in the ES lumen, while the otoconia disappear from the canals. It appears that the otoconia are first produced in the canals and then released to the lumen. Some epithelial cells of the ES are thought to expel the organic and inorganic material to the canals to form the otoconia in situ. The process of formation of the otoconia in the ES is discussed.

Development of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogaster.

The development and maturation of the endolymphatic sac (ES) and duct (ED) were studied in the newt Cynops pyrrhogaster. The ES first appears as an oval capsule at the dorsal-medial tip of the otic vesicle at stage 39, about 11 days after oviposition. The ES consists of polymorphous epithelial cells with a minimum of cytoplasm. The intercellular space (IS) between the epithelial cells is narrow and has a smooth surface. At stage 44, the size of the ES increases as many vacuoles in the IS become filled. At stage 46, 18 days after oviposition, the ES elongates markedly and a slit-like lumen is found in the ES. The epithelium contains a few cell organelles which are scattered in the cytoplasm. The vacuoles in the IS are fused, which expands the IS. Two days later (stage 48), floccular material (endolymph) is present in the expanded lumen. The IS dilates and has a wide and irregular appearance. At stage 50, approximately 26 days after oviposition, the ES extends and expands significantly and crystals (otoconia) can now be seen in the widened lumen of the ES. The cytoplasm of the cuboidal epithelial cells contains an abundance of vesicles surrounded by ribosomes and Golgi complexes. Intercellular digitations are formed in the expanded IS. At stage 54, the ES forms a large bellow-like pouch. Numerous otoconia accumulate in the lumen. Free floating cells and cell debris can be seen in the lumen at this stage. The epithelial cells contain numerous cytoplasmic organelles which are evenly distributed in the cytoplasm. Granules are found in the apical and lateral cytoplasm. The IS is loose and displays a labyrinthine appearance. The primitive ED first appears as a connection between the ES and the saccule but no lumen is present inside at stage 39. At stage 46, a narrow lumen is formed in the ED, which corresponds to the formation of the ES lumen. At stage 50, as the ED extends, floccular material is seen in the lumen. At stage 54, the ED bears numerous microvilli on its luminal surface. Otoconia and endolymph are present in the ED. Tight junctions between the epithelial cells are formed at stage 46. A fully developed intercellular junctional complex is produced at stage 54. Based on the development of the ES and ED, the maturation of function of the ES and ED are discussed.

Ultrastructure of the endolymphatic sac in the larva of the japanese red-bellied newt Cynops pyrrhogaster.

The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the "floccular" vesicle and the "granular" vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain floccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.

The rat heme oxygenase-1 gene is transcriptionally induced via the protein kinase A signaling pathway in rat hepatocyte cultures.

Heme oxygenase-1 (HO-1) is the inducible form of the rate-limiting enzyme of heme degradation; it regulates the cellular content of heme. To investigate the role of the cAMP-dependent protein kinase (PKA) signaling pathway on hepatic HO-1 gene expression, primary rat hepatocyte cultures were treated with the PKA-stimulating agents dibutyryl-cAMP (Bt2cAMP), forskolin, and glucagon. HO-1 mRNA levels were induced by these agents in a time-dependent manner with a transient maximum after 6 hr of treatment. The induction of HO-1 was dose dependent, reaching a maximum at concentrations of 250 muM Bt2cAMP and 50 nM glucagon, respectively. The accumulation of HO-1 mRNA correlated with increased levels of HO-1 protein as determined by Western blot analysis. The Bt2cAMP-dependent induction of HO-1 mRNA expression was prevented by pretreatment with the PKA inhibitor KT5720 but not with the protein kinase G inhibitor KT5823. HO-1 mRNA induction by CdCl2 and heme was differentially affected by Bt2cAMP. Up-regulation of the HO-1 gene by Bt2cAMP occurred on the transcriptional level as determined by nuclear run-off assay and blocking of the Bt2cAMP-dependent induction of HO-1 mRNA by actinomycin D. Treatment with Bt2cAMP increased the half-life of HO-1 mRNA from 4.7 to 5.5 hr. Taken together, the results of the current study demonstrate that HO-1 gene expression is induced by activation of the cAMP signal transduction pathway via a transcriptional mechanism in primary rat hepatocyte cultures.

Basic issues in the use of virtual environments for mental health applications.

In order for Virtual Environments (VE) to be efficiently developed in the areas of clinical psychology and neuropsychology, a number of basic theoretical and pragmatic issues need to be considered. The current status of VE's in these fields, while provocative, is limited by the small number of controlled studies that have been reported which apply this technology to clinical populations. This is to be expected considering it's relatively recent development, expense, and the lack of familiarity with the technology by mainstream researchers in these fields. In spite of this, some work has emerged which can begin to provide a basic foundation of knowledge which could be useful for guiding future research efforts. Although much of the work does not involve the use of fully immersive head mounted displays (HMD's), studies reporting PC-based flatscreen approaches are providing valuable information on issues necessary for the reasonable and measured development of VE/mental health applications. In light of this, the following review will focus on basic issues that we see as important for the development of both HMD and non-HMD VE applications for clinical psychology, neuropsychological assessment, and cognitive rehabilitation. These basic issues are discussed in terms of decision-making for choosing to develop and apply a VE for a mental health application. The chapter covers the issues involved with choosing a VE approach over already existing methods, deciding on the "fit" between a VE approach and the clinical population, level of presence, navigation factors, side effects, generalization, and general methodological and data analysis concerns.

The effects of immersiveness on physiology.

The effects of varying levels of immersion in virtual reality environments on participant's heart rate, respiration rate, peripheral skin temperature, and skin resistance levels were examined. Subjective reports of presence were also noted. Participants were presented with a virtual environment of an airplane flight both as seen from a two-dimensional computer screen and as seen from within a head-mounted display. Subjects were randomly assigned to different order of conditions presented, but all subjects received both conditions. Differences between the non-phobics' physiological responses and the phobic's response when placed in a virtual environment related to the phobia were noted. Also noted were changes in physiology based on degree of immersion.

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica.

To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

The structure of the statocyst of the freshwater snail Biomphalaria glabrata (Pulmonata, Basommatophora).

The structure of the statocyst of the freshwater snail Biomphalaria glabrata has been examined by light and electron microscopy. The two statocysts are located on the dorsal-lateral side of the left and right pedal ganglion. The statocysts are spherical, fluid-filled capsules with a diameter of approximately 60 microm for young and 110 microm for adult snails. The wall of the cyst is composed of large receptor cells and many smaller supporting cells. The receptor cells bear cilia which are evenly distributed on the apical surface. The cilia have the typical 9+2 internal tubule configuration. Striate rootlets originate from the base of the basal body and run downward into the cytoplasm. Side-roots arise from one side of the basal body and a basal foot from the other. For each receptor cell, the basal foot always points to the periphery of the surface, indicating that the receptor cell is non-polarized. The receptor cells contain cytoplasmic organelles such as mitochondria, ribosomes, rough and smooth endoplasmic reticulum, compact Golgi bodies and multivesicular bodies. Supporting cells bearing microvilli are interposed between the receptor cells. The junction complex between the supporting cells and the receptor cells is composed of adherens and septate junctions, while between supporting cells only the adherens junctions are present. The static nerve arises from the lateral side of the cyst and contains axons in which parallel neurotubules and mitochondria are found. The axons arise directly from the base of the receptor cells without synapse. In the cyst lumen there are unattached statoconia. The statoconia have a plate-like or concentric membranous ring structure. Based on the morphology, the function of the statocyst in Biomphalaria is discussed.

Development of the statocyst in the freshwater snail Biomphalaria glabrata (Pulmonata, Basommatophora).

The development of the statocyst of the freshwater snail Biomphalaria glabrata has been examined from embryo to adult. Special emphasis was put on the growth of the statoconia in the statocysts. In the statocysts of embryonic snails (90-120 h after oviposition) there is not a single statolith but an average of 40-50 statoconia per statocyst. The number of statoconia increases to 385-400 when the snails reach a shell diameter of 4 mm and remains relatively constant thereafter, irrespective of shell size. Small statoconia are found in supporting cells, which suggests that the statoconia are produced within these cells. The average diameter of statoconia and the total mass of statoconia increase with increasing shell diameter. The average number of large statoconia (diameter > 7 microm) per statocyst continues to increase from 2 to 10 mm animals while the number of small ones (diameter < 4 microm) initially rises and then decreases after 4 mm. These results demonstrate continuous growth of the statoconia in the cyst lumen of Biomphalaria. The single statoconia vibrate in a regular pattern in vivo, indicating beating of the statocyst cilia. The statoconia sink under the influence of gravity to load and stimulate receptor cells which are at the bottom. The length of cilia and the size of statocyst gradually increase as the animal grows. However, the increase in the volume of the statocyst is relatively small compared with the increase in body weight during normal development.

Development of gravity-sensing organs in altered gravity conditions: opposite conclusions from an amphibian and a molluscan preparation.

NASA: Researchers examined early otolith development in microgravity using fertilized eggs of the Japanese newt, Cynops pyrrhogaster in space flight. Ground experiments examined statocyst, embryonic statolith volume, and statoconia in the post-metamorphic marine mollusk Aplysia californica reared at 1-g and 2-5.7-g. Results indicate that exposure to hypergravity decreased the otolith mass to compensate for increased weight in Aplysia. In the Cynops, there was no compensatory difference in otolith mass, though otoconia production in the endolymphatic system was enhanced.^ieng