Do community-level predictors of pneumococcal carriage continue to play a role in the conjugate vaccine era?

This paper examined whether previously identified community-level factors (high proportion of crowded households and/or persons below the poverty level) remained associated with childhood pneumococcal carriage in the heptavalent pneumococcal conjugate vaccine (PCV7) era. Using logistic regression, individual factors were used to develop base models to which community-level factors were added to evaluate impact on pneumococcal carriage within two paediatric study cohorts from Massachusetts (urban Boston, outside Boston). Six years after introduction of universal childhood PCV7 vaccination, we found no consistent evidence that census tract characteristics (e.g. population size and density, age and race distribution, percent participating in group childcare, parental education, percent lacking in-unit plumbing, poverty, and community stability) affected odds of pneumococcal carriage when added to individual predictors (e.g. younger age, current respiratory tract infections, and attendance in group childcare). How community-level factors influence pneumococcal carriage continues to change in the era of increasing immunization coverage.

Variable virulence among isolates of Ascosphaera apis: testing the parasite-pathogen hypothesis for the evolution of polyandry in social insects.

The queens of many eusocial insect species are polyandrous. The evolution of polyandry from ancestral monoandry is intriguing because polyandry undermines the kin-selected benefits of high intracolonial relatedness that are understood to have been central to the evolution of eusociality. An accumulating body of evidence suggests that polyandry evolved from monoandry in part because genetically diverse colonies better resist infection by pathogens. However, a core assumption of the "parasite-pathogen hypothesis", that there is variation in virulence among strains of pathogens, remains largely untested in vivo. Here, we demonstrate variation in virulence among isolates of Ascosphaera apis, the causative organism of chalkbrood disease in its honey bee (Apis mellifera) host. More importantly, we show a pathogen-host genotypic interaction for resistance and pathogenicity. Our findings therefore support the parasite-parasite hypothesis as a factor in the evolution of polyandry among eusocial insects.

Clonal replacement among 19A Streptococcus pneumoniae in Massachusetts, prior to 13 valent conjugate vaccination.

As part of an ongoing study of the response of the Streptococcus pneumoniae population to conjugate vaccination, we applied multi-locus sequence typing (MLST) to 291 isolates sampled from nasopharyngeal carriage in Massachusetts children. We found 94 distinct sequence types (STs), including 19 that had not been previously recorded, and a xpt allele containing a large insertion. Comparison with a similar sample collected in 2007 revealed no significant overall difference in the ST composition (p=0.51) suggesting that the population has reached a new equilibrium following the introduction of 7 valent vaccination in 2000. Within serotypes, a large and statistically significant increase (p=0.014 Fisher's Exact test) was noted in the prevalence of the major multiresistant clone ST 320, which is apparently outcompeting ST 199 among serotype 19A strains. This sample will be used as a baseline to study the future evolution of the pneumococcal population in Massachusetts following introduction of vaccines with higher valency.

Comparison of gene expression profile between human chondrons and chondrocytes: a cDNA microarray study.

OBJECTIVE: The chondron is a basic unit of articular cartilage that includes the chondrocyte and its pericellular matrix (PCM). This current study was designed to investigate the effects of the chondron PCM on the gene expression profile of chondrocytes. DESIGN: Chondrons and chondrocytes were enzymatically isolated from human articular cartilage, and maintained in pellet culture. Pellets of chondrons or chondrocytes were collected at days 1, 3 and 5 for cDNA microarray analysis. RESULTS: In comparison with chondrocytes alone, chondrons had 258 genes, in a broad range of functional categories, either up- or downregulated at the three time points tested. At day 1, 26 genes were significantly upregulated in chondrons and four downregulated in comparison to chondrocytes. At day 3, the number of upregulated chondron genes was 97 and the number downregulated was 43. By day 5, there were more downregulated genes (56) than upregulated genes (32) in chondrons. Upregulation of a group of heat shock proteins (HSPA1A, HSPA2 and HSPA8) in chondrons was validated by real time reverse transcription polymerase chain reaction (RT-PCR). Genes related to chondrocyte hypertrophy and dedifferentiation such as SSP1 and DCN were downregulated in chondrons as compared to the expression in chondrocytes. CONCLUSION: The presence of the PCM in chondrons has a profound influence on chondrocyte gene expression. Upregulation of the heat shock protein 70 may contribute to the robustness and active matrix production of chondrons. The intact PCM may further stabilize the phenotype of chondrocytes within chondrons.

Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells.

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l(-1) YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 microg ml(-1), which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q(hTPO) (the specific rate of hTPO production). The supplementation of YH in SFM increased q(hTPO) by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.

A neocartilage ideal for extracellular matrix macromolecule immunolocalization.

A neocartilage construct readily amenable to microscopy and biomechanical studies is described. Porcine articular cartilage was digested with a mixture of dispase and collagenase for chondrons or pronase and collagenase for chondrocytes. Chondrons or chondrocytes plated in 96-well plates were fixed and immunolabeled in situ for fluorescence microscopy at days 4 and 11. Collagen types I and II, aggrecan, and MMP-13 expression was assayed by semiquantitative RT-PCR. Cell numbers were analyzed by MTT assay. Chondrons and chondrocytes produced neocartilage that could be handled with minimal tearing on day 3 and none on day 11. Some cell division occurred between days 4 and 7. In both cultures, chondrocytes were surrounded by a thin rim of type VI collagen and osteopontin. Type II collagen, keratan sulfate, and tenascin were abundant throughout. At day 3, cells were rounded but by day 11 flattened cells were visible in the substratum. Continued synthesis of aggrecan and type II collagen mRNA indicated maintenance of the chondrocyte phenotype. The neocartilage was easy to immunolabel in situ without the need for sectioning, and individual cells were readily observed by microscopy. The versatility of these constructs makes them ideal for microscopy and for biomechanical studies.

Effects of cloned gene dosage on the response of recombinant CHO cells to hyperosmotic pressure in regard to cell growth and antibody production.

The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (mu) and increased specific antibody productivity (q(Ab)) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in q(Ab) and less reduction in mu at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and q(Ab), indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G(1)-phase arrest, suggesting that the increase of cell population in G(1)-phase may contribute in part to enhanced q(Ab) at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in q(Ab) at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage.

Management of febrile children in the age of the conjugate pneumococcal vaccine: a cost-effectiveness analysis.

OBJECTIVES: The optimal practice management of highly febrile 3- to 36-month-old children without a focal source has been controversial. The recent release of a conjugate pneumococcal vaccine may reduce the rate of occult bacteremia and alter the utility of empiric testing and treatment. The objective of this study was to determine the cost-effectiveness of 6 different management strategies of febrile 3- to 36-month-old children at current and declining rates of occult pneumococcal bacteremia. METHODS: A cost-effectiveness (CE) analysis was performed to compare the strategies of "no work-up," "clinical judgment," "blood culture," "blood culture + treatment," "complete blood count (CBC) + selective blood culture and treatment," and "CBC and blood culture + selective treatment." A hypothetical cohort of 100 000 children who were 3 to 36 months of age and had a fever of >/=39 degrees C and no source of infection was modeled for each strategy. Our main outcome measures were cases of meningitis prevented, life-years saved compared with "no work-up," total cost (1999 dollars), and incremental CE ratios. RESULTS: When compared with "no work-up," the strategy of "CBC + selective blood culture and treatment" using a white blood cell (WBC) cutoff of 15 x 10(9)/L prevents 48 cases of meningitis, saves 86 life-years per 100 000 patients, and is less costly at the current rate of bacteremia (1.5%). Using the strategy of "CBC + selective blood culture and treatment" with a lower WBC cutoff of 10 x 10(9)/L costs an additional $72 300 per life-year saved. If the rate of bacteremia declines to 0.5%, then the incremental CE ratio of "clinical judgment" compared with "no work-up" is $38 000 per life-year saved; however, strategies that include empiric testing or treatment result in CE ratios greater than $300 000 per life-year saved. CONCLUSIONS: "CBC + selective blood culture and treatment" using a WBC cutoff of 15 x 10(9)/L is cost-effective at the current rate of pneumococcal bacteremia. If the rate of occult bacteremia falls below 0.5% with widespread use of the conjugate pneumococcal vaccine, then strategies that use empiric testing and treatment should be eliminated.

Domain and genomic sequence analysis of bdellin-KL, a leech-derived trypsin-plasmin inhibitor.

Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR and 5' rapid amplification of cDNA ends. The cDNA showed an open reading frame of 155 amino acids comprising one signal peptide and two separated domains. The C-terminal domain consists of distinct internal repeats, including HHEE and HHDD. The bdellin-KL sequence, from the constructed genomic library of Korean leech, was determined for the 2109 bases comprising the open reading frame and flanking regions (3' and 5'). The promoter region contains potential regulatory sequence motifs, including TATA, CAAT, and GC boxes. To characterize the properties of each domain, an N-terminal fragment was prepared by limited proteolysis of the intact protein. The inhibitory activity of the region was as potent as that of the intact protein. This suggests that the compact domain plays an important part in the inhibitory action of bdellin-KL. The C-terminal domain was revealed to have binding affinity to ions such as Ca(2+), Zn(2+), Fe(3+), and Fe(2+) without an influence on the inhibitory activity. This study demonstrates that bdellin-KL may be a novel bifunctional protein with two distinct domains.

ATP induces Ca(2+) signaling in human chondrons cultured in three-dimensional agarose films.

OBJECTIVE: In vivo, chondrocytes are surrounded by an extracellular matrix, preventing direct cell-to-cell contact. Consequently, intercellular communication through gap junctions is unlikely. However, signaling at a distance is possible through extracellular messengers such as nitric oxide (NO) and nucleotides and nucleosides, adenosine triphosphate (ATP), uridine triphosphate (UTP), or adenosine diphosphate (ADP). We hypothesized that chondrons, chondrocytes surrounded by their native pericellular matrix, increase their intracellular calcium concentration ([Ca(2+)]ic) in response to ATP and other signaling molecules and that the source of Ca(2+) is from intracellular stores. The objectives of this study were to determine if chondrons in a 3-D gel respond to ATP by increasing [Ca(2+)]ic through a purinoceptor mechanism and to test whether chondrons in whole tissue samples would respond to ATP in a similar fashion. DESIGN: Human chondrons, cultured in a three-dimensional agarose gel or in whole cartilage loaded with Fura-2AM, a calcium sensitive dye, were stimulated with 1, 5 and 10 microM ATP. A ratio-imaging fluorescence technique was used to quantitate the [Ca(2+)]ic. RESULTS: ATP-stimulated chondrons increased their [Ca(2+)]ic from a basal level of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca(2+)]ic in response to ATP, indicating the source of Ca(2+) was not extracellular. ATP-induced calcium signaling was inhibited in chondrons pre-treated with suramin, a generic purinoceptor blocker. In addition, UTP and adenosine 5'-O-(3-thiotriphosphate) (ATPgammas) induced a calcium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca(2+)]ic, substantiating that the P2Y2 purinoceptor was dominant. Chondrons in whole cartilage increased [Ca(2+)]ic in response to ATP. CONCLUSIONS: We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca(2+)]ic via P2Y2 receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca(2+) in a signaling response.

Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp.

Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat. Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase. When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C. The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration. Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase.

Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification.

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 microM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) with increasing MTX concentration up to 0.32 microM. Compared with the parental clone (hu17), the q(Ab) of hu17 resistant at 0.32 microM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 microM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 microM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.

Effect of hypoosmotic pressure on cell growth and antibody production in recombinant Chinese hamster ovary cell culture.

To determine the response of recombinant Chinese hamster ovary (rCHO) cells subjected to hypoosmotic pressure, rCHO cells (CS13*-1.0) producing a chimeric antibody were cultivated in the hypoosmolar medium resulting from NaCl subtraction. At hypoosmotic pressure, CS13*-1.0 cells displayed decreased specific growth rate (mu) and increased specific antibody productivity (q (Ab)).When the medium osmolality was decreased from 300 mOsm kg(-1)(physiological osmolality) to 150 mOsm kg(-1), mu was decreased by 68% and q (Ab) was increased by 128%. To understand the mechanism of enhanced q (Ab) resulting from hypoosmotic pressure, cellular responses of cells in the exponential phase of growth were observed at the transcription level. Total cytoplasmic RNA content per cell at 150 mOsm kg(-1) was increased by 140%, compared with that at 300 mOsm kg(-1). On a per mug RNA basis, immunoglobulin (Ig) mRNA levels at 150 mOsm kg(-1) were comparable to those at 300 mOsm kg(-1), indicating that hypoosmotic pressure did not lead to the preferential transcription of Ig mRNAs. Taken together, the data obtained here suggest that the increase in total RNA pool is primarily responsible for the enhanced q (Ab) of CS13*-1.0 cells subjected to hypoosmotic pressure.

Morphological selection of parental Chinese hamster ovary cell clones exhibiting high-level expression of recombinant protein.

To facilitate the establishment of recombinant Chinese hamster ovary (rCHO) cell lines with dihydrofolate reductase (dhfr)-mediated gene amplification, a primary selection method based on morphology of parental CHO clones has been developed. Morphology of parental clones that were made by transfecting various plasmids encoding thrombopoietin (TPO) and its analogs and humanized antibodies into dhfr-deficient (dhfr-) CHO cells was not uniform. Morphology of many parental clones exhibiting high-level expression of the introduced gene was similar to that of nontransfected dhfr- CHO cells. On the other hand, most parental clones with low-level expression experienced noticeable morphological changes such as bipolar fibroblast-like morphology. In case of selection of parental clones with TPO expression level higher than 200 ng/mL, morphological selection improved selection efficiency by 3.5-fold compared with random selection. Furthermore, when subjected to methotrexate for gene amplification, parental clones that were selected based on morphology elevated the expression level as much as those that were selected randomly. Taken together, morphological selection of parental clones can facilitate the establishment of rCHO cell lines expressing recombinant proteins.

Osmoprotective effect of glycine betaine on thrombopoietin production in hyperosmotic Chinese hamster ovary cell culture: clonal variations.

When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO). However, the relative decrease in q(TPO) varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production.

Epidemiology of acute viral respiratory tract infections in Korean children.

OBJECTIVE: Viruses are the most common causes of respiratory tract infection in children. We investigated the aetiologies and the epidemiological features of acute viral respiratory tract infections in Korean children. METHODS: We tried to isolate respiratory syncytial virus (RSV) and parainfluenza virus from January 1994, influenza virus from February 1995, and adenovirus from April 1996 through August 1998, and identified the isolated viruses by indirect immunofluorescence (IF) staining in the children hospitalized with acute respiratory tract infections (ARTI). RESULTS: Virus was identified in 360 of 1389 (25.9%) nasopharyngeal aspirates cultured. Of a total of 392 viruses, 164 (41.8%) RSV, 90 (23%) parainfluenza virus, 66 (16.8%) influenza A virus, 54 (13.8%) adenovirus, and 18 (4.6%) influenza B virus were cultured, including cases in mixed viral infections. The male to female ratio of the culture-positive patients was 2:1, and the proportions of the patients aged >6 months, 6-11 months, 1, 2, 3, 4, 5, 6-7, 8-9, and >10 years were 22.5, 29.5, 25.7, 9.5, 3.8, 3.8, 1.7, 1.7, 1.2, and 0.6%, respectively. The major clinical diagnosis was bronchiolitis for RSV, croup for parainfluenza virus, and pneumonia for adenovirus and influenza virus. Infections by RSV, parainfluenza virus, and influenza virus occurred in annual epidemics, and infections by adenovirus occurred annually with or without epidemics. There were somewhat larger epidemics by adenovirus and influenza virus in May to July 1996 and March to June 1997, respectively. CONCLUSIONS: Viral agents are one of the main aetiologies and the main causes of admission in Korean children with ARTI.

Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines.

When three recombinant Chinese hamster ovary (rCHO) cell lines, CHO/dhfr-B22-4, CS13-1.00*, and CS13-0.02*, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality. However, due to a simultaneous suppression of cell growth at elevated osmolality, no enhancement in the maximum foreign protein titer was made in batch cultures. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved foreign protein production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures were carried out in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all three rCHO cell lines. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled rCHO cell lines to grow at 557 to 573 mOsm/kg, whereas they could not grow in the absence of glycine betaine. However, effect of glycine betaine inclusion in hyperosmolar medium on foreign protein production differed among rCHO cell lines. CHO/dhfr-B22-4 cells retained enhanced specific human thrombopoietin (hTPO) productivity in the presence of glycine betaine, and thereby the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg). On the other hand, enhanced specific antibody productivity of CS13-1.00* and CS13-0.02* at elevated osmolality was decreased significantly in the presence of glycine betaine. As a result, the maximum antibody titer at 557 mOsm/kg was similar to that obtained in the control culture with physiological osmolality. The mRNA contents per cell determined by northern blot hybridization correlated with q in all three rCHO cell lines, indicating that transcriptional regulation is responsible in part for q enhancement at hyperosmolality in the absence as well as the presence of glycine betaine. Taken together, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO cell lines.

ATP release by mechanically loaded porcine chondrons in pellet culture.

OBJECTIVE: To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS: Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS: Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION: The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

The use of electric bed heaters and the risk of clinically recognized spontaneous abortion.

We conducted a prospective cohort study to evaluate the relation of spontaneous abortion and electric bed heater use during the first trimester of pregnancy. Compared with non-users, rates of spontaneous abortion were lower for women who used electric bed heaters. The adjusted odds ratio and 95% confidence interval (CI) for the two major devices used, electric blankets (N = 524) and waterbeds (N = 796), were, respectively, 0.8 (95% CI = 0.5-1.1) and 0.9 (95% CI = 0.7-1.2). An increase of risk with increasing intensity (setting-duration combination) of use was not observed. Users of electric blankets at low settings for most of the night (N = 171) had lower risks of spontaneous abortion than non-users (adjusted odds ratio = 0.5; 95% CI = 0.3-1.0). Twenty women who used electric blankets at a high setting for 1 hour or less had an adjusted odds ratio of 3.0 (95% CI = 1.1-8.3), but we found no spontaneous abortions among the few women (N = 13) who used a high setting for 2 or more hours. We found that exposure rankings of the magnetic field time-weighted average and a rate of change metric did not correspond monotonically to the pattern of spontaneous abortion risks and that electric blankets contribute less to overnight time-weighted average magnetic fields than has been thought.