The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model.

OBJECTIVE: To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. METHODS: After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaP scaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. RESULTS: The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the +P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P < 0.0001). CONCLUSIONS: The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the -P group integrated well with the recipient bone. The study stresses the importance of long-term in vivo studies to assess the efficacy of cartilage repair techniques.

Diverse styles of submarine venting on the ultraslow spreading Mid-Cayman Rise.

Thirty years after the first discovery of high-temperature submarine venting, the vast majority of the global mid-ocean ridge remains unexplored for hydrothermal activity. Of particular interest are the world's ultraslow spreading ridges that were the last to be demonstrated to host high-temperature venting but may host systems particularly relevant to prebiotic chemistry and the origins of life. Here we report evidence for previously unknown, diverse, and very deep hydrothermal vents along the approximately 110 km long, ultraslow spreading Mid-Cayman Rise (MCR). Our data indicate that the MCR hosts at least three discrete hydrothermal sites, each representing a different type of water-rock interaction, including both mafic and ultramafic systems and, at approximately 5,000 m, the deepest known hydrothermal vent. Although submarine hydrothermal circulation, in which seawater percolates through and reacts with host lithologies, occurs on all mid-ocean ridges, the diversity of vent types identified here and their relative geographic isolation make the MCR unique in the oceans. These new sites offer prospects for an expanded range of vent-fluid compositions, varieties of abiotic organic chemical synthesis and extremophile microorganisms, and unparalleled faunal biodiversity--all in close proximity.

Bubbles and Dust: Experimental Results of Dissolution Rates of Metal Salts and Glasses From Volcanic Ash Deposits in Terms of Surface Area, Chemistry, and Human Health Impacts.

Explosive volcanic eruptions lead to ash deposition and subsequent leaching of contaminants into soils or surface water, impacting flora and fauna, including human health. This study determined the control of ash surface area and chemical composition on ash dissolution rates. Fresh, unhydrated ash samples from four contrasting volcanoes were analyzed in the laboratory. Column leachate tests were used to compare leaching rates over a range of basaltic to andesitic ashes as a function of time and surface area, to analyze the effects of ash deposition. It was found that surface area, measured both geometrically and by multipoint Brunauer-Emmett-Teller analysis, generally increases for a short time, gradually decreases, then increases over the rest of the leaching experiment, due to area to mass ratio fluctuations. After the column leachate tests, postleaching water analyses for elemental compositions were conducted by inductively coupled plasma-mass spectrometry and ion chromatography. Steady state dissolution rates initially decayed rapidly due to the smallest size fraction of ash (dust), which provides a large area of fresh leachable surfaces as well as the rapid dissolution of highly soluble metal salts. Some of the dissolved concentrations of elements relevant to human and ecosystem health such as F, Cd, Se, As, and Cr rose above World Health Organization (WHO) drinking water standards within an hour of experimental leaching. In nature, however, safe consumption standards are further dependent upon bioaccumulation and chronic exposure. As such, individual and recurring ash deposition events have applications to emergency response and preparedness in volcanic regions.

Sulfur mass-independent fractionation in subsurface fracture waters indicates a long-standing sulfur cycle in Precambrian rocks.

The discovery of hydrogen-rich waters preserved below the Earth's surface in Precambrian rocks worldwide expands our understanding of the habitability of the terrestrial subsurface. Many deep microbial ecosystems in these waters survive by coupling hydrogen oxidation to sulfate reduction. Hydrogen originates from water-rock reactions including serpentinization and radiolytic decomposition of water induced by decay of radioactive elements in the host rocks. The origin of dissolved sulfate, however, remains unknown. Here we report, from anoxic saline fracture waters ∼2.4 km below surface in the Canadian Shield, a sulfur mass-independent fractionation signal in dissolved sulfate. We demonstrate that this sulfate most likely originates from oxidation of sulfide minerals in the Archaean host rocks through the action of dissolved oxidants (for example, HO· and H2O2) themselves derived from radiolysis of water, thereby providing a coherent long-term mechanism capable of supplying both an essential electron donor (H2) and a complementary acceptor (sulfate) for the deep biosphere.

Genetic variability for pathogenicity, isozyme, ribosomal DNA and colony color variants in populations of Rhynchosporium secalis.

Samples of Rhynchosporium secalis were collected from two experimental barley populations known to carry a diverse array of alleles for resistance to this fungal pathogen. Classification of 163 isolates for four putative isozyme systems, a colony color dimorphism and 20 ribosomal DNA restriction fragment length variants revealed 49 different multilocus phenotypes (haplotypes). The six most common haplotypes differed significantly in pathogenicity. Genetic analyses of the data indicated that effective population sizes of the fungus were very large, that the effects of genetic drift were small, and that negligible recombination occurred in the populations studied. Frequency dependent selection was suggested as an explanation for the maintenance of variation in pathogenicity in the fungus.

Differentiation of isolates of Discula umbrinella (Teleomorph: Apiognomonia errabunda) from beech, chestnut, and oak using randomly amplified polymorphic DNA markers.

Genetic variation in 30 isolates of Discula umbrinella derived from beech, chestnut, and oak was assessed using randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphic markers. Polymerase chain reaction amplifications with 17 primers produced 134 different DNA fragments. Three RAPD fragments were subsequently used for Southern hybridization. By these techniques up to four different individuals could be detected in the same leaf. The presence of several individuals within a single leaf indicates a finely tuned balance between the endophyte and its host. Cluster analysis of all arbitrary primed amplified DNA fragments showed that the isolates could be placed into four groups corresponding to their host origin. The high percentage of private RAPD variants within groups is consistent with low gene flow.

Mortality associated with multiple gestation in Malawi.

BACKGROUND: Multiple gestation is associated with increased maternal, perinatal, and infant mortality. The prevalence of multiple gestation varies widely with the highest rates reported among populations in Africa. There have been few population-based studies of the impact of multiple gestation on pregnancy outcomes in sub-Saharan Africa. METHODS: Data from a 1987-1990 prospective study of the effect of malaria chemoprophylaxis among pregnant women on birthweight and mortality of their infants in a rural area of Malawi were used to estimate the prevalence of multiple gestation and to quantify the risk of mortality associated with multiple gestation compared with single gestation. RESULTS: There were 88 (2.2%) multiple gestations among 4049 women. Mortality was high; only 38% of mothers were known to have all their infants survive to 1 year, compared with 74% in singleton gestations. The increased mortality associated with multiple gestation was due to two factors: a higher frequency of low birthweight and a fourfold increase in perinatal mortality among the infants with birthweights > or = 2500 g and among infants with unknown birthweight. We estimated that multiple gestation contributes to 5.5% of the perinatal, 1.2% of the postperinatal, and 11.5% of the maternal deaths in this population. CONCLUSION: Multiple gestation in Malawi contributed to increased perinatal and maternal mortality, but did not increase the risk of mortality after the perinatal period.

PIP: Data from a 1987-1990 prospective study of the effect of malaria chemoprophylaxis among pregnant women on birth weight and mortality of their infants in a rural area of Malawi were used to estimate the prevalence of multiple gestation and to quantify the risk of mortality associated with multiple gestation compared with single gestation. There were 88 (2.2%) multiple gestations among 4049 women enrolled at their first antenatal clinic visit at 1 of 4 antenatal clinics in the Mangochi District Malaria Research Project from 1987 to 1990. 87 had twin pregnancies and 1 had triplets. 25 (28%) of the 88 women delivered at home vs. 1659 (42%) of the 3962 women with singleton pregnancies (p 0.016). Mortality was high among the 177 infants of the 88 multiple gestations. In 20 (23%) of the pregnancies all of the infants were either fetal deaths or died within the 1st year. One of the infant pairs died in 29 (33%) of the pregnancies; 7 of the survivors were lost to follow-up. Only 33 (38%) of mothers were known to have all their infants survive to 1 year, compared with 74% in singleton gestations. Multiple gestation infants had higher rates of perinatal and postperinatal deaths and loss to follow-up than singleton infants (p 0.0001, 0.004, and 0.04, respectively). The risk of death for an infant of a multiple gestation was more than twice the risk for an infant of a singleton gestation. The increased mortality associated with multiple gestation was caused by 2 factors: a higher frequency of low birth weight and a 4-fold increase in perinatal mortality among the infants with birth weights or = 2500 g and among infants with unknown birth weight. It was estimated that multiple gestation contributed to 5.5% of perinatal, 1.2% of the postperinatal, and 11.5% of the maternal deaths in this population. Multiple gestation in Malawi contributed to increased perinatal and maternal mortality but did not increase the risk of mortality after the perinatal period.

Effects of anti-myeloid antibodies on the generation of hematopoietic colony-forming units in long-term bone marrow culture.

We have prepared and characterized several monoclonal antibodies (MoAbs), PM-18 (CD 15),AML-2-23 (CD 14), and AML-1-99 (no cluster designation) reactive with antigens expressed on myeloid cells. Previous studies using complement-dependent lysis have determined the reactivity of these MoAbs with hematopoietic cells in vitro. PM-81 and AML-2-23 react with variable percentages of CFU-GM but not BFU-E or CFU-Mix. AML-1-99 reacts with greater than 90% of CFU-GM. CFU-E, and CFU-Mix. In order to determine the reactivity of these MoAbs with the bone marrow-derived precursors of in vitro colony-forming cells we have performed complement-dependent lysis and fluorescence activated cell sorting of bone marrow cells followed by long-term culture of surviving or sorted cells. Bone marrow cells from four normal subjects were subjected to various combinations of MoAbs and complement and assayed for residual colony-forming cells. Total surviving cells were then placed in flasks which contained a monolayer of irradiated bone marrow-derived adherent cells previously obtained from allogeneic donors. The cultures supported production of non-adherent colony-forming cells for up to 6 weeks as determined by serial in vitro colony-forming assays in methylcellulose. Cultures treated with one, two or three MoAbs and complement demonstrated variable reductions in colony-forming cells at the initiation of the experiments. However, cumulative production of colony-forming cells in anti-MoAb-treated cultures was usually at least as great as in control cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of placental malaria infection on perinatal mortality in rural Malawi.

Perinatal deaths (fetal or infant deaths from the 28th week of pregnancy up to the seventh day after birth) occur as a result of adverse conditions during pregnancy, labor, and delivery, or in the first few days of life. Placental malaria infection is known to increase the risk of delivery of a low birth weight infant, thus, potentially increasing the risk of perinatal and infant mortality. To better understand the relationship among the adverse events in pregnancy, including placental malaria infection, adverse conditions in labor, and birth weight to perinatal mortality, we investigated the perinatal mortality among a cohort of infants born to rural Malawian women for whom placental malaria infection status and birth weight were documented. Among the 2,063 mother-singleton infant pairs, there were 111 perinatal deaths (53.8 perinatal deaths per 1,000 births). The risk of perinatal death increased as birth weight decreased. Risk factors identified for perinatal mortality among all infants excluding birth weight included abnormal delivery (cesarean section, breech, or vacuum extraction), a history of a late fetal or neonatal death in the most recent previous birth among multiparous women, reactive maternal syphilis serology, nulliparity, and low socioeconomic status. Placental malaria infection was not associated with increased perinatal mortality, but was associated with lower perinatal mortality among normal birth weight (> or = 2,500 g) infants (odds ratio = 0.35, 95% confidence interval = 0.14, 0.92). Interventions to address these risk factors could have a substantial impact on reducing perinatal mortality in this population.

Prospective assessment of mortality among a cohort of pregnant women in rural Malawi.

Maternal mortality has recently received attention as a neglected public health problem in many developing countries where mortality rates are estimated to be 8-200 times those in developed countries. Most maternal mortality estimates in sub-Saharan Africa have used retrospective methods because of the lack of large population-based studies. The Mangochi Malaria Research Project, a trial of antimalarial chemoprophylaxis in pregnant women, provided an opportunity to examine prospectively mortality among the study women. Among 4,053 monitored pregnant women, 27 women were known to have died during pregnancy, labor, delivery and the one-year follow-up period. Three women died during the antenatal period and 12 died within six weeks of delivery for an estimated maternal mortality rate of 370 per 100,000 pregnant women; this rate was consistent with rates reported from retrospective surveys in Malawi. Twelve women died between three and 10 months after delivery, and the mortality rate in this nonmaternal period was estimated to be 341 per 100,000. Mortality rates in the maternal and nonmaternal periods were surprisingly similar. Human immunodeficiency virus type-1 (HIV-1) infection and anemia were strongly associated with death in the nonmaternal period. Mortality among infants of mothers who died was 3.7 times higher than the rate of death among infants born to mothers who survived. This study highlights that for rural Malawian women, pregnancy and delivery are risky periods, that the death of the mother adversely affects the survival of her children, and that HIV and anemia are important contributors to nonmaternal mortality in reproductive-age women. Strategies to reduce mortality among women of child-bearing age in sub-Saharan Africa must focus on decreasing the complications of pregnancy and delivery, and address important preventable causes of death, such as anemia and HIV infection.

Efficacy of chemoprophylaxis in preventing Plasmodium falciparum parasitaemia and placental infection in pregnant women in Malawi.

73 pregnant women in Malawi were given weekly antimalarial chemoprophylaxis under observation and were monitored for Plasmodium falciparum parasitaemia and placental infection. 3 of 19 women (16%) who were parasitaemic at the time they began chemoprophylaxis were infected with chloroquine-resistant P. falciparum. After clearance of initial infections, 25% of the 73 women became parasitaemic while taking prophylaxis and 56% had evidence of active or past placental infection at the time of delivery. None of the women who were parasitaemic at the time of enrollment, and only 11% of those who had breakthrough parasitaemias while taking prophylaxis, had a history of fever and signs or symptoms that they recognized as malaria. Although the density of P. falciparum infection and rates of placental infection appeared to be lower among women taking regular chloroquine prophylaxis, this drug did not prevent P. falciparum infection among pregnant women.

High Genetic Similarity Among Populations of Phaeosphaeria nodorum Across Wheat Cultivars and Regions in Switzerland.

ABSTRACT Phaeosphaeria nodorum was sampled from nine wheat fields across a 30-km transect representing three geographical regions in Switzerland to determine the scale of genetic differentiation among subpopulations. Three different wheat cultivars were sampled three times to determine whether differences in host genotype correlated with differences among corresponding pathogen populations. Seven restriction fragment length polymorphism (RFLP) loci and one DNA fingerprint were assayed for each of the 432 isolates in the collection. DNA fingerprints differentiated 426 unique genotypes. Though absolute differences were small, five RFLP loci exhibited significant differences in allele frequencies across the nine sub-populations. Gene diversity within all subpopulations was high (H(T) = 0.51), but only 3% of the total genetic variation was distributed among the nine subpopulations. When subpopulations were grouped according to geographical region or host cultivar, less than 1% of the genetic variation was distributed among groups, suggesting widespread gene flow and the absence of pathogen adaptation to specific wheat cultivars. Tests for gametic equilibrium within subpopulations and across the entire Swiss population supported the hypothesis of random mating.

Gene Flow and Sexual Reproduction in the Wheat Glume Blotch Pathogen Phaeosphaeria nodorum (Anamorph Stagonospora nodorum).

ABSTRACT Restriction fragment length polymorphisms (RFLPs) were used to characterize the genetic structures of three field populations of Phaeosphaeria nodorum from Texas, Oregon, and Switzerland. Data from seven nuclear RFLP loci were used to estimate gene diversity and genetic distances and to make indirect measures of gene flow between populations. Three of the seven RFLP loci differed significantly in allele frequencies across populations. On average, 96% of the total gene diversity was found within populations. There was little evidence for population subdivision, suggesting that gene flow was not restricted among populations. Based on an average population differentiation of 0.04, we estimated that the exchange of 11 migrants among populations per generation would be needed to account for the present level of population subdivision. Genotype diversity based on DNA fingerprints was at a maximum for the Swiss population, whereas populations in Texas and Oregon had lower genotype diversities. Many multilocus haplotypes were found in each population. Ninety-five percent of RFLP allele pairs were in gametic equilibrium. The data were consistent with random mating within each population.

Regulation of acetyl-CoA carboxylase by ADP-ribosylation.

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.

Identification of apple cultivars using RAPD markers.

Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.

Phorbol esters, but not insulin, promote depletion of cytosolic protein kinase C in rat adipocytes.

The tumor-promoting phorbol esters have insulinomimetic effects in several tissues. Employing two different assay systems, we have compared the effects of phorbol ester and insulin on the activity and intracellular distribution of the Ca++ and phospholipid dependent protein kinase (protein kinase C) in isolated rat adipocytes. Phorbol ester leads to a prompt depletion of kinase activity from the cytosolic fraction and appearance of activity in membrane extracts; neither of these effects is mimicked by insulin. These results, taken together with other data, emphasize important divergences between the actions of these agonists and suggest that changes in protein kinase C activity or intracellular distribution are not a necessary concomitant of the cascade of insulin action.

Coevolution of host and pathogen populations in the Hordeum vulgare-Rhynchosporium secalis pathosystem.

Isolates of Rhynchosporium secalis collected from two experimental barley populations were scored for putative isozyme, colony color, and virulence loci. Allelic frequencies, multilocus haplotype frequencies, and multilocus genetic structure differed in the two populations of R. secalis; haplotypes also differed widely from each other in virulence. The average virulence of isolates collected from the more resistant host population was greater than the average virulence of the isolates collected from the less resistant host population; also the least virulent haplotype, which made up 19% of the pathogen population collected from the less resistant host population, accounted for only 0.3% of the isolates collected from the more resistant host population. It was concluded that the genetic systems of the barley host and fungal pathogen interacted in a complementary fashion and that the genetic structures of both the host and pathogen populations were shaped by coevolutionary processes featuring interactions among loci affecting many different traits, including interactions among host resistance genes and pathogen virulence genes.

Factors associated with inadequate prenatal care during the second pregnancies among African-American women.

A longitudinally linked data set for Georgia was used to identify characteristics, including previous prenatal care use and complications at the first birth, associated with prenatal care use in the second pregnancy among 8,224 African-American women. More than 70% of the women who were < 25 years of age at their first birth (younger women) and almost 40% of women who were > or = 25 years at their first birth received inadequate care with at least one of their first two births. Women who received inadequate care in their first pregnancy were more likely to receive inadequate care in their second pregnancy than women who received adequate care in their first pregnancy. Younger women with a history of a stillbirth, neonatal death, or vacuum extraction were less likely to receive inadequate care in their subsequent pregnancy. Although this study was not able to evaluate the content of prenatal care, it suggested that many African-American women may not receive sufficient care to prevent adverse pregnancy outcomes. Women who receive inadequate care in their first pregnancy must be targeted for interventions that help them overcome economic, situational, or attitudinal barriers to receiving adequate care in their next pregnancy.

Does inadequate prenatal care contribute to growth retardation among second-born African-American babies?

The authors evaluated the relation between adequacy of prenatal care and risk of delivery of full term small-for-gestational-age (SGA) infants. Data were derived from maternally linked birth certificates for 6,325 African-American women whose first two pregnancies ended in singleton, full term live births in Georgia from 1989 through 1992. The authors used stratified analysis to assess the effect of prenatal care on the risk of having an SGA baby in the second pregnancy among women with and without an SGA baby in their first pregnancy. The group of women with a history of SGA birth may be more likely to include persons for whom SGA delivery is related to factors, such as genetics, that are not amenable to intervention by prenatal care. Inadequate prenatal care was not associated with the risk of SGA delivery among women who had previously delivered an SGA baby. In unadjusted analyses, inadequate prenatal care was associated with an increased risk of delivering a full term SGA baby in the second pregnancy among women whose first baby was not SGA (risk ratio = 1.28; 95% confidence interval: 1.05, 1.55). The association did not persist when data were adjusted for confounding variables (odds ratio = 1.11; 95% confidence interval: 0.89, 1.38). Regardless of outcome in the first pregnancy, adequate prenatal care did not reduce the risk of full term SGA birth among second pregnancies in this population.

Constructing reproductive histories by linking vital records.

Certificates of 1,449,287 live births and fetal deaths filed in Georgia from 1980 through 1992 were linked to create chronologies that, excluding induced abortions and ectopic pregnancies, constituted the reproductive experience of individual women. The authors initially used a deterministic method (whereby linking rules were not based on probability theory) to link as many records as possible, knowing that some of the linkages would be incorrect. They subsequently used a probabilistic method (whereby evaluation of linkages was developed from probability theory) to evaluate each linkage, and they broke those that were judged to be incorrect. Of the 1.4 million records, 38% did not link to another record. From the remaining records, 369,686 chains of two or more events were constructed. The longest chain included 12 events. Of the chains, 69% included two events; 22% included three events. Longer chains tended to have lower scores for probable validity. The probability-based evaluation of chains affected 3.0% of the records that had been in chains at the end of the deterministic linkage. A greater percentage of records in longer chains were affected by the evaluation. Unfortunately, the small subset of records that were the most difficult to link tended to overrepresent groups with the greatest risk of adverse pregnancy outcomes. Researchers contemplating a similar linkage can anticipate that, for the majority of records, linkage can be accomplished with a relatively straightforward, deterministic approach.