MR-based proton density fat fraction (PDFF) of the vertebral bone marrow differentiates between patients with and without osteoporotic vertebral fractures.

The bone marrow proton density fat fraction (PDFF) assessed with MRI enables the differentiation between osteoporotic/osteopenic patients with and without vertebral fractures. Therefore, PDFF may be a potentially useful biomarker for bone fragility assessment. INTRODUCTION: To evaluate whether magnetic resonance imaging (MRI)-based proton density fat fraction (PDFF) of vertebral bone marrow can differentiate between osteoporotic/osteopenic patients with and without vertebral fractures. METHODS: Of the 52 study patients, 32 presented with vertebral fractures of the lumbar spine (66.4 ± 14.4 years, 62.5% women; acute low-energy osteoporotic/osteopenic vertebral fractures, N = 25; acute high-energy traumatic vertebral fractures, N = 7). These patients were frequency matched for age and sex to patients without vertebral fractures (N = 20, 69.3 ± 10.1 years, 70.0% women). Trabecular bone mineral density (BMD) values were derived from quantitative computed tomography. Chemical shift encoding-based water-fat MRI of the lumbar spine was performed, and PDFF maps were calculated. Associations between fracture status and PDFF were assessed using multivariable linear regression models. RESULTS: Over all patients, mean PDFF and trabecular BMD correlated significantly (r = - 0.51, P < 0.001). In the osteoporotic/osteopenic group, those patients with osteoporotic/osteopenic fractures had a significantly higher PDFF than those without osteoporotic fractures after adjusting for age, sex, weight, height, and trabecular BMD (adjusted mean difference [95% confidence interval], 20.8% [10.4%, 30.7%]; P < 0.001), although trabecular BMD values showed no significant difference between the subgroups (P = 0.63). For the differentiation of patients with and without vertebral fractures in the osteoporotic/osteopenic subgroup using mean PDFF, an area under the receiver operating characteristic (ROC) curve (AUC) of 0.88 (P = 0.006) was assessed. When evaluating all patients with vertebral fractures, those with high-energy traumatic fractures had a significantly lower PDFF than those with low-energy osteoporotic/osteopenic vertebral fractures (P < 0.001). CONCLUSION: MR-based PDFF enables the differentiation between osteoporotic/osteopenic patients with and without vertebral fractures, suggesting the use of PDFF as a potential biomarker for bone fragility.

Donor-matched comparison of chondrogenic progenitors resident in human infrapatellar fat pad, synovium, and periosteum - implications for cartilage repair.

Purpose: There is a clinical need to better characterize tissue sources being used for stem cell therapies. This study focuses on comparison of cells and connective tissue progenitors (CTPs) derived from native human infrapatellar fatpad (IPFP), synovium (SYN), and periosteum (PERI). Materials and Methods: IPFP, SYN, PERI were harvested from twenty-eight patients undergoing arthroplasty. CTPs were quantitatively characterized using automated colony-forming-unit assay to compare total nucleated cell concentration-[Cell], cells/mg; prevalence-(PCTP), CTPs/million nucleated cells; CTP concentration-[CTP], CTPs/mg; proliferation and differentiation potential; and correlate outcomes with patient's age and gender. Results: [Cell] did not differ between IPFP, SYN, and PERI. PCTP was influenced by age and gender: patients >60 years, IPFP and SYN had higher PCTP than PERI (p < 0.001) and females had higher PCTP in IPFP (p < 0.001) and SYN (p = 0.001) than PERI. [CTP] was influenced by age: patients <50 years, SYN (p = 0.0165) and PERI (p < 0.001) had higher [CTP] than IPFP; patients between 60 and 69 years, SYN (p < 0.001) had higher [CTP] than PERI; patients >70 years, IPFP (p = 0.006) had higher [CTP] than PERI. In patients >60 years, proliferation potential of CTPs differed significantly (SYN>IPFP>PERI); however, differentiation potentials were comparable between all three tissue sources. Conclusion: SYN and IPFP may serve as a preferred tissue source for patients >60 years, and PERI along with SYN and IPFP may serve as a preferred tissue source for patients <60 years for cartilage repair. However, the heterogeneity among the CTPs in any given tissue source suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.

Evaluation of rhBMP-2/collagen/TCP-HA bone graft with and without bone marrow cells in the canine femoral multi defect model.

Recombinant human bone morphogenetic protein-2, when applied to an absorbable type 1 bovine collagen sponge (rhBMP-2/ACS) is an effective therapy in many bone grafting settings. Bone marrow aspirate (BMA) has also been used as a source of transplantable osteogenic connective tissue progenitors. This study was designed to characterize the performance of a scaffold comprising rhBMP-2/ACS in which the sponge wraps around tri-calcium phosphate hydroxyapatite granules (rhBMP-2/ACS/TCP-HA) and to test the hypothesis that addition of BMA will improve the performance of this construct in the Canine Femoral Multi Defect Model. In each subject, two sites were grafted with rhBMP-2/ACS/TCP-HA scaffold loaded with BMA clot and two other sites with rhBMP-2/ACS/TCP-HA scaffold loaded with wound blood (WB). After correction for unresorbed TCP-HA granules, sites grafted with rhBMP-2/ACS/TCP-HA+BMA and rhBMP-2/ACS/TCP-HA+WB were similar, with mean percent bone volumes of 10.9 %±1.2 and 11.2 %±1.2, respectively. No differences were seen in quantitative histomorphometry. While bone formation using both constructs was robust, this study did not support the hypothesis that the addition of unprocessed bone marrow aspirate clot improved bone regeneration in a site engrafted with rhBMP-2/ACS/TCP-HA+BMA. In contrast to prior studies using this model, new bone formation was greater at the center of the defect where TCP-HA was distributed. This finding suggests a potential synergy between rhBMP-2 and the centrally placed ceramic and cellular components of the graft construct. Further optimization may also require more uniform distribution of TCP-HA, alternative cell delivery strategies, and a more rigorous large animal segmental defect model.

A new retrotransposable human L1 element from the LRE2 locus on chromosome 1q produces a chimaeric insertion.

We have found a 2 kilobase insertion containing a rearranged L1 element in the dystrophin gene of a muscular dystrophy patient. We cloned the precursor of this insertion, the second known active human L1 element. The locus, LRE2, has one allele derived from the patient which matches the insertion sequence exactly. LRE2 has a perfect 13-15 bp target site duplication, two open reading frames, and an unusual 21 bp truncation of the 5' end, suggesting that a slightly truncated element can still retrotranspose. It differs from LRE1 by approximately 0.7%. There is an L1 element at LRE2 on approximately 66% of human chromosomes 1q, and the element is absent from chimpanzee and gorilla genomes. These data demonstrate that multiple active L1 elements exist in the human genome, and that a readthrough transcript of an active element is capable of retrotransposition.

Primary Cells Isolated from Human Knee Cartilage Reveal Decreased Prevalence of Progenitor Cells but Comparable Biological Potential During Osteoarthritic Disease Progression.

BACKGROUND: Current decisions on cellular therapies for osteoarthritis are based primarily on clinical experience or on assumptions about preferred cell sourcing. They have not been informed by rigorous standardized measurements of the chondrogenic connective-tissue progenitors (CTP-Cs) or their intrinsic diversity of chondrogenic potential. The goal of this study was to quantitatively define the CTP-Cs resident in cartilage of different grades of osteoarthritis and to compare their concentration, prevalence, and biological potential. METHODS: Twenty-three patients who had varus malalignment of the knee and were scheduled to undergo elective total knee arthroplasty for idiopathic osteoarthritis and who had grade 1-2 osteoarthritis on the lateral femoral condyle and grade 3-4 osteoarthritis on the medial femoral condyle were recruited for study of the cartilage removed during surgery. CTP-Cs were assayed by a standardized colony-forming-unit assay using automated image-analysis software based on ASTM standard test method F2944-12. RESULTS: Cell concentration was significantly greater (p < 0.001) in grade 3-4 cartilage than in grade 1-2 cartilage. The prevalence of CTP-Cs varied widely, but it trended lower in grade 3-4 cartilage than in grade 1-2 samples (p = 0.078). The biological performance of CTP-Cs from grade 1-2 and grade 3-4 cartilage was comparable. Increased cell concentration was a significant predictor of decreased CTP-C prevalence (p = 0.002). CONCLUSIONS: Although grade 3-4 cartilage showed fewer CTP-Cs than grade 1-2 cartilage, the range of biological performance was comparable, which suggests that either may be used as a source for potent CTP-Cs. However, the biological reason for the heterogeneity of CTP-Cs in cartilage and the biological implications of that heterogeneity are not well understood and require further study. CLINICAL RELEVANCE: In order to improve the efficacy of cartilage cell therapy procedures, it is key to characterize the quality and quantity of the cells and progenitors being administered. Additionally, understanding the heterogeneity in order to select appropriate subsets of populations will improve the rigor of decisions concerning cell sourcing and targeting for pharmacological and cellular therapies.

Variability in subjective review of umbilical cord blood colony forming unit assay.

BACKGROUND: The goal of this study was to quantitatively evaluate the reproducibility of current manual counting methods of colony forming units (CFUs) from umbilical cord blood samples METHODS: Fresh and reconstituted frozen cells from 10 cord blood samples were cultured under standard conditions. The number of BFU-Es, CFU-GMs, and CFU-GEMMs were counted by three expert reviewers using the standard microscope method and manually traced CFUs on digital images of cell cultures. RESULTS: The mean colony count based on the traced digital images was 82 (22% CV) and 52 (15% CV) for the fresh and frozen samples, respectively. This was significantly greater than that observed using the microscope, 61 (13% CV) for fresh and 43 (16% CV) for frozen. The difference was mainly due to the reviewers observing more CFU-GMs in the digital images than through the microscope review. All three reviewers agreed on the presence of a colony 72% of the time based on the digital review in both fresh and frozen samples. Reviewer agreement with respect to colony type in the fresh samples was 38% (22%CV), 25% (51%CV), and 6% (115%CV) for BFU-Es, CFU-GMs, and CFU-GEMMs, respectively. Reviewer agreement increased for BFU-Es and CFU-GMs in the frozen samples where fewer colonies were present. CONCLUSIONS: Although this study showed marked variability between reviewers, the analysis of manually traced digital images has the potential to improve inter-observer variation when compared to current methods by identifying features that lead to discrepancies in colony counting and providing cases with consensus results. © 2016 International Clinical Cytometry Society.

Sugar transport and potassium permeability in yeast plasma membrane vesicles.

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained. The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal. The yield of vesicles sealed to K+ was maximal at pH 4 and accounted for about one third of the total vesicle volume. The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K+. As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO22+. Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane. After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.

The molecular basis of genetic disease.

The pace of localization and characterization of genes affected in human genetic disorders is quickening. Many important genes were localized or characterized recently: genes for in cystic fibrosis, NF-2, Marfan's syndrome and xeroderma pigmentosum, to name a few. Also, in the past 15 months, the CFTR gene affected in cystic fibrosis has been isolated, the first disease gene to be isolated without use of previous cytogenetic clues, such as deletions or translocations in sporadic cases. Other examples should follow, although we have been disappointed to date by the difficulties encountered in the isolation of Huntington's disease gene which was localized a number of years ago to distal chromosome 4p. It is still very difficult to isolate a disease gene without critical cytogenetic information. New improved techniques for finding the desired expressed sequences in a large cloned segment of human DNA are needed. Our ability to find mutant alleles of a given sequence has expanded greatly with the recent technical advances in denaturing gradient gel electrophoresis, chemical cleavage, and single-stranded conformational electrophoresis. One would predict that information derived from the human genome project will have a major impact upon the isolation of further disease genes. As whole regions of human chromosomes or indeed entire chromosomes are physically mapped and cloned as continuous, overlapping YACs (yeast artificial chromosomes), isolation of disease genes will become easier and easier.(ABSTRACT TRUNCATED AT 250 WORDS)

ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half-transporter (ALDP) involved in the import of very long-chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false-negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X-ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X-ALD ( http://www.x-ald.nl). In this review we report a detailed analysis of all 406 X-ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X-ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients.

Prenatal diagnosis of hemoglobinopathies by DNA analysis.

Restriction site polymorphisms are normal inherited variations in DNA that can be readily detected by restriction endonuclease analysis. Currently, 17 such polymorphisms are recognized within a 60 kb (kilobase) stretch of DNA which includes the beta-globin gene complex. Because of their proximity to the beta-globin gene, often these restriction site polymorphisms can be used to predict inheritance of beta-globin variants that produce disease. For example, restriction site polymorphisms can be used for prenatal diagnosis for the large majority of couples at risk of having a child with beta-thalassemia. When each member of such a couple is heterozygous at one or more of these 17 sites, family studies are usually successful in determining which forms of the polymorphism are co-inherited with the beta-thalassemia genes in that particular family. Subsequently, study of fetal DNA isolated from amniocytes obtained by midtrimester amniocentesis or from chorionic villi obtained by first trimester chorion biopsy will reveal which DNA polymorphisms that fetus has inherited. By deductive reasoning one can then predict which beta-globin genes it has co-inherited. Because of the general nature of these polymorphisms, which are related to the beta-globin gene and its variants only because of their proximity on chromosome 11, they are potentially useful in the prenatal diagnosis of any beta-chain hemoglobinopathy. Some hemoglobinopathies (including alpha-thalassemia, sickle cell anemia, and some cases of beta-thalassemia) can be detected directly by DNA analysis. In these cases in utero diagnosis does not need to rely on restriction site polymorphisms, which require preliminary family studies and are not applicable in all at risk pregnancies. Recently, genetic probes, which are necessary for detecting restriction site polymorphisms, have been isolated for sequences of several genes whose protein products are important in blood coagulation. These include probes for all three genes whose polypeptide products combine to form the fibrinogen molecule as well as probes for the prothrombin, Factor IX, Factor VIII, and antithrombin III genes. Defects in these genes are expected to be the causes of afibrinogenemia, prothrombin deficiency, hemophilia B, hemophilia A, and antithrombin III deficiency, respectively. From experience with other genes, it is expected that restriction site polymorphisms within and/or flanking these genes will be found.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and structure-activity relationships of oxamic acid and acetic acid derivatives related to L-thyronine.

Aryloxamic acids 7 and 23, (arylamino)acetic acids 29, arylpropionic acids 33, arylthioacetic acids 37, and (aryloxy)acetic acid 41 related to L-triiodothyronine (L-T3) were prepared and tested in vitro for binding to the rat liver nuclear L-T3 receptor and the rat membrane L-T3 receptor. The structure-activity relationships for these compounds are described, with 7f, 23a, 29c, 33a, 37b, and 41 showing excellent potency (IC50's of 0.19, 0.16, 1.1, 0.11, 3.5, and 0.10 nM, respectively) to the nuclear receptor and significantly lower binding affinity to the membrane receptor (IC50's > 5 microM). Some of these compounds, especially in the oxamic acid series 7 and 23, showed an unprecedented potency for methyl-substituted derivatives such as 7f and 23a. Compounds 7f and 23a showed good lipid lowering effects in rats with ED50's of 20 and 5 micrograms/kg po, respectively, and a lack of cardiac side effects in rats at doses as high as 10 and 25 mg/kg po, respectively.

Cardiac beta-adrenoceptor binding characteristics with age following adrenal demedullation.

1. The role of adrenal medullary catecholamines in the regulation of cardiac function becomes more important when adrenergic neural influences in the heart are decreased. Since adrenergic nervous input to the heart decreases with age, it would be expected that catecholamine influence on cardiac neuroeffector junction would increase. 2. Fischer-344 rats of 6-, 12- and 24-months were adrenal demedullated or sham-operated and the animals were killed at the end of two weeks. beta-Adrenoceptors were studied in the membrane preparations from the ventricles of rat hearts. [125I]-iodopindolol was used as the radioligand. The density of beta-receptors (Bmax), dissociation constant (KD) and the ratio of cardiac beta-adrenoceptor subtypes were studied. The relative percentages of beta-receptor subtypes were determined by use of ICI 89,406 (beta 1-selective antagonist) and ICI 118,551 (beta 2-selective antagonist). 3. In 24-month-old animals which were adrenal demedullated, hydrocortisone replacement was employed for one week; the animals were killed one week later. 4. The data indicate that there was a diminution of the Bmax following adrenal demedullation at all ages but that the ratios of beta 1: beta 2-adrenoceptors remain the same as in the controls (67:33). The effect of adrenal medullary catecholamines on cardiac beta-receptor binding characteristics did not seem to be influenced by age.

Usefulness of a CACA repeat polymorphism in genotype assignments in Duchenne/Becker muscular dystrophy.

RFLP analysis in Duchenne/Becker muscular dystrophy (D/BMD) has been limited by the lack of informative marker loci at the 3' end of the dystrophin gene. Recently a CACA repeat polymorphism was described in the 3' untranslated end of the dystrophin gene which we have found helpful in genotype assignments of D/BMD families when an RFLP approach is required. The CACA repeat marker has 2 common alleles (1 and 2) that are easily visualized by a nonradioactive PCR method followed by polyacrylamide gel electrophoresis. We present 2 families which demonstrate the use of this polymorphism. Since 35-50% of females are heterozygous, this locus is a useful marker in RFLP analysis of D/BMD families.

Practical guide to the diagnosis of thalassemia. Council of Regional Networks for Genetic Services (CORN).

Thalassemias occur in individuals of all ethnic backgrounds and are among the most common genetic diseases worldwide. The diagnosis of thalassemia can easily be part of primary medical practice. Here we outline a practical approach to the detection of thalassemias in three common clinical settings. The first involves any patient with a low mean corpuscular volume (MCV) with or without anemia. The second is a neonatal screening result indicating possible presence of thalassemia. Finally, evaluation for thalassemia should be considered in the context of family planning or pregnancy in patients whose ethnicity indicates origin from high risk geographic areas. We also review the various types of the thalassemia syndromes and provide an overview of general therapeutic considerations.

Identification of carriers of Duchenne muscular dystrophy: value of molecular analysis.

Currently, molecular methods are the most accurate diagnostic tools for carrier detection and prenatal diagnosis of Duchenne muscular dystrophy (DMD). This report illustrates the value of molecular diagnosis as opposed to previous diagnostic methods, the need for frequent re-evaluations as new methodologies develop, and the necessity for in-depth genetic counseling. In Family 1, the proposita was predicted to be a carrier by an indirect assay (abnormal in vitro muscle ribosomal protein synthesis). DNA analysis using restriction fragment length polymorphisms (RFLPs) indicated that she was not a carrier. She gave birth to a predicted non-affected male, who inherited the gene in question. In Family 2 the proposita, an obligate carrier, was initially evaluated by RFLP analysis. Two pregnancies were monitored by first trimester chorionic villous sampling. Re-evaluation indicated that all affected individuals, including one of the embryos, carried a deletion of the dystrophin gene. The identification of an RFLP within the region containing the deletion allowed unambiguous determination of the carrier status of all individuals.

Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors.

Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be induced to express a bone phenotype in vitro. The number of osteoblastic progenitors can be estimated by counting the colony-forming units which express alkaline phosphatase (CFU-APs). This study was undertaken to test the hypothesis that human aging is associated with a significant change in the number or prevalence of osteoblastic progenitors in the bone marrow. Four 2-ml bone marrow aspirates were harvested bilaterally from the anterior iliac crest of 57 patients, 31 men (age 15-83) and 26 women (age 13-79). A mean of 64 million nucleated cells was harvested per aspirate. The mean prevalence of CFU-APs was found to be 55 per million nucleated cells. These data revealed a significant age-related decline in the number of nucleated cells harvested per aspirate for both men and women (P = 0.002). The number of CFU-APs harvested per aspirate also decreased significantly with age for women (P = 0.02), but not for men (P = 0.3). These findings are relevant to the harvest of bone marrow derived connective tissue progenitors for bone grafting and other tissue engineering applications, and may also be relevant to the pathophysiology of age-related bone loss and post-menopausal osteoporosis.

Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation.

Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8-80 years, with no systemic illness, yielded a mean of 92 +/- 65 x 10(6) nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 +/- 28 alkaline phosphatase-positive colonies per 10(6) nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D3 synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.

Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume.

UNLABELLED: Bone marrow contains osteoblast progenitor cells that can be obtained with aspiration and appear to arise from a population of pluripotential connective-tissue stem cells. When cultured in vitro under conditions that promote an osteoblastic phenotype, osteoblast progenitor cells proliferate to form colonies of cells that express alkaline phosphatase and, subsequently, a mature osteoblastic phenotype. We evaluated the number of nucleated cells in bone-marrow samples obtained with aspiration from the anterior iliac crest of thirty-two patients without systemic disease. There were nineteen male patients and thirteen female patients; the mean age was forty-one years (range, fourteen to seventy-seven years). The prevalence and concentration of the osteoblast progenitor cells also were determined, by placing the bone-marrow-derived cells into tissue-culture medium and counting the number of alkaline phosphatase-positive colony-forming units. In order to assess the effect of aspiration volume, two sequential experiments were performed. In the first experiment, aspiration volumes of one and two milliliters were compared. In the second experiment, aspiration volumes of two and four milliliters were compared. The mean prevalence of alkaline phosphatase-positive colony-forming units in the bone-marrow samples was thirty-six per one million nucleated cells (95 per cent confidence interval, 28 to 47); a mean of 2400 alkaline phosphatase-positive colony-forming units was obtained from a two-milliliter aspirate. There was a significant difference among the patients with respect to the number of alkaline phosphatase-positive colony-forming units in these bone-marrow samples (p < 0.001). Seventy per cent of this variation in the prevalence was due to variation among patients, and 20 per cent was due to variation among aspirates. The number of alkaline phosphatase-positive colony-forming units in the aspirate increased as the aspiration volume increased. However, contamination by peripheral blood also increased as the aspiration volume increased. An increase in the aspiration volume from one to four milliliters caused a decrease of approximately 50 per cent in the final concentration of alkaline phosphatase-positive colony-forming units in an average sample. CLINICAL RELEVANCE: On the basis of these data, we recommend that, when bone marrow is obtained with aspiration for use as a bone graft, the volume of aspiration from any one site should not be greater than two milliliters. A larger volume decreases the concentration of osteoblast progenitor cells because of dilution of the bone-marrow sample with peripheral blood. We estimate that four one-milliliter aspirates will provide almost twice the number of alkaline phosphatase-positive colony-forming units as will one four-milliliter aspirate. In addition, these data confirm that humans differ significantly from one another with respect to the cellularity of bone marrow and the prevalence of osteoblast progenitor cells. Additional studies are necessary to determine if the number or prevalence of alkaline phosphatase-positive colony-forming units in bone marrow is a determining factor in the efficacy of an autogenous bone or bone-marrow graft and to ascertain how the number and function of alkaline phosphatase-positive colony-forming units may change as a function of factors such as age, menopausal status, and selected diseases.

The natural selection of altruistic traits.

Proponents of the standard evolutionary biology paradigm explain human "altruism" in terms of either nepotism or strict reciprocity. On that basis our underlying nature is reduced to a function of inclusive fitness: human nature has to be totally selfish or nepotistic. Proposed here are three possible paths to giving costly aid to nonrelatives, paths that are controversial because they involve assumed pleiotropic effects or group selection. One path is pleiotropic subsidies that help to extend nepotistic helping behavior from close family to nonrelatives. Another is "warfare"-if and only if warfare recurred in the Paleolithic. The third and most plausible hypothesis is based on the morally based egalitarian syndrome of prehistoric hunter-gatherers, which reduced phenotypic variation at the within-group level, increased it at the between-group level, and drastically curtailed the advantages of free riders. In an analysis consistent with the fundamental tenets of evolutionary biology, these three paths are evaluated as explanations for the evolutionary development of a rather complicated human social nature.