Hantavirus pulmonary syndrome and rodent reservoirs in the savanna-like biome of Brazil's southeastern region.

This paper describes the diversity of rodent fauna in an area endemic for hantavirus cardiopulmonary syndrome (HCPS) in Brazil, the population dynamics and the relationship of rodents with hantavirus in the Cerrado (savanna-like) biome. Additionally, an analysis is made of the partial S segment sequences of the hantaviruses obtained from serologically confirmed human HCPS cases and from rodent specimens. Rodents were collected during four campaigns. Human serum samples were collected from suspected cases of HCPS at hospitals in the state of Minas Gerais. The samples antibody-reactive by ELISA were processed by RT-PCR. The PCR product was amplified and sequenced. Hantavirus was detected only in Necromys lasiurus, the wild rodent species most prevalent in the Cerrado biome (min-max: 50-83·7%). All the six human serum samples were hantavirus seropositive and five showed amplified PCR products. The analysis of the nucleotide sequences showed the circulation of a single genotype, the Araraquara hantavirus. The environmental changes that have occurred in the Cerrado biome in recent decades have favoured N. lasiurus in interspecific competition of habitats, thus increasing the risk of contact between humans and rodent species infected with hantavirus. Our data corroborate the definition of N. lasiurus as the main hantavirus reservoir in the Cerrado biome.

The strategies to reduce iron deficiency in blood donors randomized trial: design, enrolment and early retention.

BACKGROUND AND OBJECTIVES: Repeated blood donation produces iron deficiency. Changes in dietary iron intake do not prevent donation-induced iron deficiency. Prolonging the interdonation interval or using oral iron supplements can mitigate donation-induced iron deficiency. The most effective operational methods for reducing iron deficiency in donors are unknown. MATERIALS AND METHODS: 'Strategies To Reduce Iron Deficiency' (STRIDE) was a two-year, randomized, placebo-controlled study in blood donors. 692 donors were randomized into one of two educational groups or one of three interventional groups. Donors randomized to educational groups either received letters thanking them for donating, or, suggesting iron supplements or delayed donation if they had low ferritin. Donors randomized to interventional groups either received placebo, 19-mg or 38-mg iron pills. RESULTS: Iron deficient erythropoiesis was present in 52·7% of males and 74·6% of females at enrolment. Adverse events within 60 days of enrolment were primarily mild gastrointestinal symptoms (64%). The incidence of de-enrolment within 60 days was more common in the interventional groups than in the educational groups (P = 0·002), but not more common in those receiving iron than placebo (P = 0·68). CONCLUSION: The prevalence of iron deficient erythropoiesis in donors enrolled in the STRIDE study is comparable to previously described cohorts of regular blood donors. De-enrolment within 60 days was higher for donors receiving tablets, although no more common in donors receiving iron than placebo.

Prevalence of myopia in a population with malocclusions.

AIM: The purpose of this study was to investigate the prevalence of myopia among a paediatric population with malocclusions. MATERIALS AND METHODS: A total of 322 consecutive patients of the department of Orthodontics and Gnathology, Dental Clinic, University of L'Aquila, were enlisted for the study and 292 were selected according to the exclusion criteria. Pretreatment diagnostic data, which included radiographic cephalometric and dental cast evaluation, were recorded and presence of myopia was assessed through an ophthalmological examination. Differences in the prevalence of myopia by sex and malocclusion were analysed by using Pearson's chi-square and Fisher's exact tests. RESULTS: According to the sagittal malocclusion, patients were classified as Class I (N=162), Class II division 1 (N=75), Class II division 2 (N=38), or Class III (N=12). No gender influence was found for myopia or malocclusion. No differences were recorded when analysing the influence of sex on the prevalence of myopia in classes of malocclusion. A statistical significant higher prevalence was found for subjects showing myopia in Class II division 1 malocclusion, while no other significant differences were found for prevalence in the other classes of malocclusions. DISCUSSION: Few studies investigated a possible relationship between the ocular and stomatognathic system, and no data are available in the scientific literature. A higher prevalence of myopia was found in patients with Class II division 1: as expected no other significant association was found. CONCLUSION: The findings of the present study suggest a possible association between myopia and Class II, but further studies are needed to confirm and explain this observation.

Prevalence of astigmatism in a paediatric population with malocclusions.

AIM: The purpose of this study was to investigate the prevalence of astigmatism among a paediatric population. MATERIALS AND METHODS: A total of 322 consecutive patients of the department of Orthodontics and Gnathology, Dental Clinic, University of L'Aquila, were enlisted for the study and 176 were selected according to the exclusion criteria. Pre- treatment diagnostic data, which included radiographic cephalometric and dental cast evaluation, were recorded and presence of astigmatism was assessed through an ophthalmological examination. Differences in the prevalence of astigmatism by sex and malocclusion were analysed by using the chi-square (Pearson's chi-square test) and Fisher's exact tests. RESULTS: According to the sagittal malocclusion, patients were classified as Class I (N=122), Class II Division 1 (N=26), Class II Division 2 (N=9), or Class III (N=19); according to the transverse malocclusion patients were classified into cross-bite (N=39) and no cross-bite (N=137) groups; after ophthalmological examination astigmatism was detected in 32 patients (18.18%). Statistically significant correlations were found between astigmatism and cross-bite (p < 0.0001), while no associations were found with other malocclusions. No gender influence was found for astigmatism or malocclusion. DISCUSSION: Few study investigated a possible relationship between the ocular and stomatognathic system, and no data are available in the scientific literature. A higher prevalence of astigmatism was found in patients with cross-bite: as expected no other significant association was found. The relationship between astigmatism and cross-bite could be either related to a specific skeletal pattern, which could induce visual alterations, or to the effect of abnormal visual input on the postural system, which could induce stomatognathic alterations. CONCLUSION: The findings of the present study suggest a possible association between astigmatism and cross-bite, but future studies are needed to confirm and explain this observation.

Prevalence of hyperopia and strabismus in a paediatric population with malocclusions.

AIM: The purpose of this study was to investigate the prevalence of hyperopia and strabismus among a paediatric population with malocclusions. MATERIALS AND METHODS: From a total of 322 consecutive patients observed at the department of Orthodontics and Gnathology, Dental Clinic, University of L'Aquila, 176 were selected for the study according to the exclusion criteria. Pretreatment diagnostic data, which included radiographic, cephalometric and dental cast evaluation, were recorded and presence of hyperopia or strabismus was assessed through an ophthalmological examination. Differences in incidence rates of strabismus or hyperopia by sex and malocclusion were analysed by using the chi-square (Pearson's chi-square test) and Fisher's exact tests. RESULTS: According to the sagittal malocclusion, patients were classified as Class I (122), Class II division 1 (26), Class II Division 2 (9), or Class III (19); after ophthalmological examination, hyperopia was detected in 8.6% of patients, while strabismus was present in 3.6% of them. No gender influence was found for hyperopia, strabismus or malocclusion. DISCUSSION: Few studies investigated a possible relationship between the ocular and stomatognathic system, and no data are available in the scientific literature; the present study did not highlight differences in the prevalence of hyperopia or strabismus in the three Angle classes malocclusion. CONCLUSION: The findings of the present study did not suggest a possible association between hyperopia or strabismus and sagittal malocclusion, but future studies are needed to confirm and explain this observation.

Rehabilitative treatment in peripheral artery disease: protocol application and follow-up.

AIM: Peripheral arterial disease (PAD) is a chronic figure suitable to be treated at the II stage to prevent the extreme developments both of the critical limb ischemia and the amputation, as well. The aim of this study was to establish a rehabilitation program (pharmacological and physical) focused not only on the improvement of the flow but also on the metabolic rebalancing in the claudicant limb. METHODS: The study enrolled 222 patients, (125 non-diabetics and 97 diabetics): 54 II A and 168 II B stage; 172 patients (131 II B and 41 II A; 104 non-diabetics and 68 diabetics) were submitted to iv. L-propionil carnytine (Lpc) and physical training on treadmill or exercise bike and 50 patients to iv. therapy alone. Instrumental (Rheoscreen, Oximetry, ABI, walking distance measurement) and clinical checks (questionnaire - Appendix 1) were performed at days: T0, T45,T 90,T180, T230 and during the follow up stated at T 90,T180,T360 from T 230 (end of DH). RESULTS: A significant increasing of the walking distance has been reached in the group undergoing the rehabilitation program. Treadmill: non-diabetics +261.48% at 0% and +122.53% at slope 10% (T230) further increasing to +502.31% at 0% and +289.42% at slope 10% (T360); diabetics: + 158.49% at T0 and + 98.26% at slope 10% (T230) further increased to +287.74% at 0% and +197.39% at 10% (T360) in comparison with the group which had only iv. Lpc : non-diabetics +141.63% at 0% and +104.08% at slope 10% (T230) further increased to +202.064% at 0% and +155.10% at slope 10% (T360); diabetics: +109.124% at T0 and +100% at slop 10% (T230) further increased to +171.08% at 0% and +140% at 10% (T360) . Exercise bike: non-diabetics: +170.27% at T230 in comparison T0 increased to +305.4% at T360; diabetics: +166.66 at T230 reaching +288.88% at T 360. CONCLUSION: Our rehabilitative program gives not only good results at the end of the treatment but mainly stable, with the chance to reach further improving of both walking distance and quality of life, particularly in those patients which observe constantly the physical training.

Variation in the helminth community structure of Thrichomys pachyurus (Rodentia: Echimyidae) in two sub-regions of the Brazilian Pantanal: the effects of land use and seasonality.

The Pantanal is a large ecosystem located in South America. This preserved area is seasonally flooded due to abundant rainfall during the summer and the subsequent overflow of the Paraguai River. In this paper, we examine the helminth community structure in the wild rodent Thrichomys pachyurus during the wet and dry seasons in two locations of the preserved and cattle ranching areas in the Southern Pantanal. We identified 12 species of helminth, and, although we did not find any differences in species richness between locations within the Pantanal, we found that richness was higher during the wet season. Helminth species were largely aggregated in both farm locations and during seasons. The most common helminth species were more abundant during the dry season than during the wet season, which may have been due to the increased habitat availability and rodent population increase. The intensity of the infection also followed the same pattern for most helminths. The trichostrongylids (Heligmostrongylus crucifer, H. almeidai and Pudica cercomysi) were dominant at both farm locations. The land use of each area was not correlated with helminth diversity. However, species composition of the helminth community of T. pachyurus differed between locations and may be correlated with environmental differences between the habitats. The seasonality of the Pantanal was highly correlated with helminth parasitism in T. pachyurus.

Genetic diversity of Hepatozoon spp. in rodents from Brazil.

Hepatozoon spp. are Apicomplexan protozoa that parasitize a wide diversity of vertebrate hosts. In Brazil, few studies have reported the occurrence of Hepatozoon spp. in rodent species. Additionally, an evaluation of the population structure and distribution of Hepatozoon species over several Brazilian biomes has not yet been performed. The present work aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from 31 genera sampled in five Brazilian biomes. Samples were submitted to PCR assays for Hepatozoon spp. targeting two regions of the 18S rRNA gene. Infection by Hepatozoon spp. was detected in 195 (42.2%) rodents comprising 24 genera. Phylogenetic analyses of 18S rRNA sequences grouped all sequences in the clade of Hepatozoon spp. previously detected in rodents and reptiles, apart from those detected in domestic/wild carnivores. These data raise two non-exclusive hypotheses: (i) rodents play an important role as intermediate or paratenic hosts for Hepatozoon infections in reptiles; and (ii) rodents do not seem to participate in the epidemiology of Hepatozoon infections of domestic/wild canids and felids in Brazil. TCS analyses performed with available 18S rRNA Hepatozoon sequences detected in rodents from Brazil showed the occurrence of six haplotypes, which were distributed in two large groups: one from rodents inhabiting the coastal region of Brazil and Mato Grosso state, and another from rodents from the central region of the country. A wide survey of the South American territory will help to elucidate the evolutionary history of Hepatozoon spp. parasitizing Rodentia in the American continent.

Variation in the prevalence and abundance of acanthocephalans in brown-nosed coatis Nasua nasua and crab-eating foxes Cerdocyon thous in the Brazilian Pantanal.

Host infection by parasites is influenced by an array of factors, including host and environmental features. We investigated the relationship between host sex, body size and age, as well as seasonality on infection patterns by acanthocephalan in coatis (Procyonidae: Nasua nasua) and in crab-eating foxes (Canidae: Cerdocyon thous ) from the Brazilian Pantanal wetlands. Between 2006 and 2009, we collected faecal samples from these hosts and analyzed for the presence of acanthocephalan eggs. Prevalence, abundance and intensity of eggs of acanthocephalans were calculated. Egg abundance was analyzed using generalized linear models (GLM) with a negative binomial distribution and models were compared by Akaike criteria to verify the effect of biotic and abiotic factors. Prevalence of acanthocephalans was higher in the wet season in both host species but did not differ between host sexes; however, adult crab-eating foxes showed higher prevalence of acanthocephalan eggs than juveniles. In contrast, prevalence of acanthocephalan eggs found in coatis was higher in coati juveniles than in adults. Host age, season and maximum temperature were the top predictors of abundance of acanthocephalan eggs in crab-eating foxes whereas season and host sex were predictors of egg abundance in coatis. The importance of seasonality for abundance of acanthocephalan was clear for both host species. The influence of host-related attributes, however, varied by host species, with host gender and host age being important factors associated with prevalence and parasite loads.

Variables that modulate the spatial distribution of Trypanosoma cruzi and Trypanosoma evansi in the Brazilian Pantanal.

An evaluation was made on how the landscape and cattle ranching affect the transmission cycles and the patterns of tripanosomatid infection (Trypanosoma cruzi and Trypanosoma evansi) of small wild mammals in the Pantanal. This region comprises a large natural environment with a multiplicity of habitats, wide variety of biodiversity besides the presence of livestock. T. cruzi and T. evansi infections were evaluated by parasitological and serological methods in one preserved and one cattle ranching area. The diversity of the small mammal fauna showed to be the same in the two studied areas, however, their relative abundance was different. Distinct enzootiological scenarios of both trypanosomatids could be observed. Transmission of T. cruzi occurred mainly in forested areas, in the two study areas, while T. evansi occurred dispersed among all habitats studied in the unpreserved area. The arboreal rodent Oecomys mamorae, the most abundant species in both areas, displayed high T. cruzi and T. evansi serum prevalence and parasitemias. Also, the caviomorph rodent Thrichomys pachyurus was shown to be an important host due to its expressive relative abundance, prevalence of infection by both trypanosomatid species and a broad range use of habitats. The role of the small mammal fauna in the transmission cycle of both trypanosomes species seems to be distinct according to land use since we found a broad range of T. evansi infected hosts in the preserved area in contrast to cattle ranching area and a half number of the rodents species infected with T. cruzi in unpreserved in comparison to protect area. The present study showed that cattle ranching in this study area did not enhance overall prevalence of T. cruzi infection among small wild mammals. Together with the observation that small mammals diversity in FA is similar to RN area suggest that ranching activity may also not necessarily conduct to biodiversity loss or risk of Chagas disease.

Mapping of the distribution of Trypanosoma cruzi infection among small wild mammals in a conservation unit and its surroundings (Northeast-Brazil).

Maps are a useful tool that permits correlation of landscapes with hotspots of parasite transmission. Here, they were used as a tool for geovisualization to evaluate variables involved in the transmission of Trypanosoma cruzi among small wild mammals in an area endemic for Chagas disease, the "Serra da Capivara" National Park (PARNA) and its surroundings in Piauí State, Northeast Brazil. The implementation of a Geographical Information System (GIS) allowed the observation that a previously noted aggregated distribution of Triatoma sordida and Triatoma brasiliensis, T. cruzi prevalence and infection pattern of small wild mammals was directly or indirectly influenced by the local relief and human action. Small mammalian species diversity was higher in mesic refugia inside the park and in its buffer zone and lower in the disturbed area by anthropic activities. Didelphis albiventris was more abundant in the areas affected by human action. Thrichomys laurentius demonstrated to be an eclectic species and a competent reservoir of T. cruzi, being infected in all study areas. Small wild mammals infected with the TCII genotype of T. cruzi were localized only in the buffer zone of PARNA while TCI infected specimens were found in both areas, inside the PARNA and its buffer zone. The impact of biodiversity loss on the transmission cycle of T. cruzi in the wild environment was discussed.

Small mammal populations of an agroecosystem in the Atlantic Forest domain, southeastern Brazil.

This study reports 2 years of the population dynamics and reproduction of a small mammal community using the removal method. The study was conducted in a rural area of the Atlantic Forest, in Sumidouro, Rio de Janeiro State, Brazil. The population sizes, age structure and reproduction were studied for the four most common species in the study area. The overall diversity was 1.67 and ranged between 0.8 to 1.67. The species richness was 13 considering the whole study. The most abundant species were the rodents Nectomys squamipes (n = 133), Akodon cursor (n = 74), Oligoryzomys nigripes (n = 25) and the marsupials Didelphis aurita (n = 58) and Philander frenatus (n = 50). Seven other rodents were captured once: Necromys lasiurus, Akodon montensis, Sooretamys angouya, Oecomys catherine, Oxymycterus judex, Euryzygomatomys spinosus and Trinomys iheringi. There were higher peaks for diversity and species richness during the winter (dry) months, probably due to higher food availability. The marsupials had a seasonal reproduction with highest population sizes at the end of the rainy seasons. Nectomys squamipes reproduced mostly during rainy periods. Akodon cursor reproduced predominantly in the winter with the highest population peaks occurring during this season. The analysis of the population dynamics of the rodent species indicated that no species behaved as an agricultural pest, probably due to the heterogeneous landscape of high rotativity of vegetable cultivation. Rodent populations were more susceptible to the removal procedure than marsupial ones.

Ocular correction effects on EMG activity of stomatognathic muscles in children with functional mandibular lateral- deviation: a case control study.

AIM: This study was conducted in order to determine the ocular correction effects on electromyography activity of anterior temporal, masseter, sternocleidomastoid and anterior digastric muscles in children with functional mandibular lateral-deviation. METHODS: This study was performed on 32 subjects, aged between 8 and 12 years with functional lateral deviation of mandible and oculo-extrinsic muscular tone disorders. After complete ophthalmologic evaluation and ocular correction prescription, the children were randomly divided into two groups: study and control. In the study group (16 subjects) EMG activity at rest was recorded in the following conditions: with eyes closed; with eyes open; with ocular correction modified through electromyographic control. In the control group (16 subjects) EMG activity at rest was recorded in the following conditions: with eyes closed; with eyes open; with conventional ocular correction. Data were acquired in 15-seconds trial using a SEMG K7, while the subjects maintained rest dental position. RESULTS/STATISTICS: Both groups presented a significative correlation between value of lateral-deviation and the degree of ocular-extrinsic muscular tone disorders (r=0.69). In addition, a significant statistical increase of EMG activity at rest with eyes open in both groups was observed in the anterior temporal left and right. A significant decrease of EMG activity with open eyes was observed only with ocular correction upon electromyographic control (study group). The findings, expressed as Symmetry Index, showed a significant increase of muscles imbalance right/left with conventional methods of ocular correction (p < 0.0001). CONCLUSION: The significant worsening in EMG activity, mainly observed with conventional corrective lenses, could have an important consequence in clinical diagnostic and therapeutic behaviour because anterior temporal muscles are important in postural position of the mandible. Instead EMG corrective lenses could improve tonus and balance of stomatognathic muscles and, therefore, support the functionality of orthodontic treatment. Moreover, the data suggest an interesting new tool in order to reach an interdisciplinary approach to complex growth disorders represented by functional lateral deviation of mandible and ocular-extrinsic muscular tone disorders.

Trypanosoma cruzi infection in wild mammals of the National Park 'Serra da Capivara' and its surroundings (Piaui, Brazil), an area endemic for Chagas disease.

We studied the prevalence of Trypanosoma cruzi infection among eight species of wild small mammals (n=289) in an area where human cases of infection/disease have occurred. Dogs (n=52) and goats (n=56) were also surveyed. The study was carried out inside a biological reserve, the National Park 'Serra da Capivara' and its surroundings in Piaui State, Brazil. The marsupial Didelphis albiventris and the caviomorph rodent Trichomys apereoides were found to be the most important reservoirs in the study area. Trichomys apereoides was the most abundant species (80%) and D. albiventris the most frequently infected (61%). Both T. cruzi I and T. cruzi II genotypes were isolated from these species. One specimen of Tr. apereoides displayed a mixed T. cruzi I/zymodeme 3 infection. Serum prevalence among dogs suggests that they may be involved in the maintenance of the parasite in the peridomestic environment, in contrast to goats, which are not apparently of any epidemiological importance. The distinct distribution and patterns of infection observed in the study areas suggest that even in the same biome, epidemiological studies or determination of control measures must take into account ecological peculiarities.

Interleukin-1beta increases the functional expression of connexin 43 in articular chondrocytes: evidence for a Ca2+-dependent mechanism.

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, whereas in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 (Cx43) and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of Interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of Cx43 and by an enhanced Cx43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of Cx43 could be prevented by intracellular Ca2+ chelation but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of Cx43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism; its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.

Ca2+ oscillations and intercellular Ca2+ waves in ATP-stimulated articular chondrocytes.

Cytosolic Ca2+ oscillations are known to occur in many cell types stimulated with agonists linked to the phosphoinositide signaling pathway. Trains of repetitive short-lasting Ca2+ spikes could be induced in articular chondrocytes by extracellular ATP, an agonist potently effective in stimulating cartilage resorption. The mechanism of these Ca2+ oscillations was studied by computerized video imaging on primary cultures of articular chondrocytes. Few cycles of oscillatory activity could be evoked in the absence of extracellular Ca2+, while, for oscillations to be sustained, Ca2+ influx was required. Thapsigargin irreversibly blocked Ca2+ oscillations, thus demonstrating the crucial involvement of intracellular stores in triggering the rhythmic activity. Apart from activating intracellular Ca2+ release, extracellular ATP also induced a noncapacitive Ca2+ influx in these cells. This ATP-mediated influx modulates both the oscillation frequency and intracellular stores refilling. In monolayers of confluent cells, Ca2+ oscillations spread from cell to cell in the form of intercellular waves. Propagating waves could also be observed in the absence of extracellular Ca2+, demonstrating that Ca2+ itself is not required for signal coordination. These results demonstrate that complex spatiotemporal pathways of Ca2+ oscillations and intercellular Ca2+ waves could be activated in articular chondrocytes during degenerative diseases.

Dual mechanism of intercellular communication in HOBIT osteoblastic cells: a role for gap-junctional hemichannels.

Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast-like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell-to-cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18alpha-glycyrrhetinic acid (18-GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signaling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate (InsP3) from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18-GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested.

Mechanism of mechanically induced intercellular calcium waves in rabbit articular chondrocytes and in HIG-82 synovial cells.

Intercellular communication through gap junctions allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Intercellular Ca2+ signaling was investigated with digital fluorescence video imaging in primary cultures of articular chondrocytes and in HIG-82 synovial cells. In both cell types, mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+, mechanical stimulation induced communicated Ca2+ waves similar to controls; however, the number of HIG-82 cells recruited decreased significantly. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte and HIG-82 cell, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Treatment of cells with thapsigargin and with the phospholipase C (PLC) inhibitor U73122 blocked mechanically induced signal propagation. These results provide evidence that in chondrocytes and in HIG-82 synovial cells, mechanical stimulation activates PLC, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighboring cells. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate tissue responses in joint physiology.

[Ca2+]i oscillations in rat chromaffin cells: frequency and amplitude modulation by Ca2+ and InsP3.

Rat chromaffin cells in primary culture exhibit oscillations of cytosolic Ca2+ concentration, sustained by the rhythmic discharge of Ca2+ from specialized intracellular stores. Each Ca2+ spike starts from a discrete region of the cell (pacemaker), and then propagates across the entire cytosol. Spike initiation and propagation, governing the oscillation frequency and amplitude respectively, appeared to be controlled by different mechanisms. The pacemaker was found to be directly activated by increases of cytosolic Ca2+ concentration obtained by either K+ depolarization or nicotinic stimulation. On the other hand, muscarinic or B2 stimulation was required for an efficient spreading to occur, thus suggesting a key role of InsP3 in the signal propagation. The pacemaker displayed an autonomous activity, as documented by the presence of local Ca2+ discharges, which were not necessarily accompanied by spreading to the rest of the cell. This uncoupling could be stimulated by the selective increase of the pacemaker firing rate, due to the rise of the intracellular Ca2+ concentration. Modulation of Ca2+ spike amplitude by treatments affecting either the pacemaker or the spreading phase might be related to quantal Ca2+ release from functionally discrete stores.

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.