Reevaluation of the body mass estimate for the KNM-ER 5428 Homo erectus talus.

OBJECTIVES: In this study, we reexamined the body mass estimate for the Homo erectus specimen KNM-ER 5428 based on talus dimensions. Previous estimates of >90 kg for this fossil are large in comparison to body mass estimates for other H. erectus specimens. MATERIALS AND METHODS: The study sample consisted of tali and femora of 132 modern cadaver males from a documented body mass skeletal collection. We recorded the talus trochlear mediolateral (TTML) breadth and femoral head diameter (FHD) for each modern human specimen, and obtained KNM-ER 5428's TTML values from the literature. We developed regression formulae based on TTML using the body mass estimated from FHD for the entire human sample and for known body masses from a normal-BMI subsample, and then used these formulae to calculate body mass for KNM-ER 5428. In addition, we examined the range of body masses for individuals with TTML measurements comparable to KNM-ER 5428. RESULTS: The body masses of normal-BMI individuals with a TTML ≥32.3 mm (the smaller of the two fossil measurements from the literature) ranged between 60.3 and 86.2 kg and averaged 72.3 kg. The body masses of normal-BMI individuals with a TTML ≥33.7 mm (the larger measurement) ranged between 63.5 and 86.2 kg with a mean of 73.6 kg. The correlations between TTML and body mass are moderate. Revised body mass point estimates for KNM-ER 5428 ranged between 69.2 and 81.6 kg based on TTML, and average 70.5 and 76.0 kg. DISCUSSION: Results suggest previously published body mass estimates of KNM-ER 5428's are too large. Its body mass was likely between 70 and 76 kg rather than >90 kg.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

The effect of ontogeny on estimates of KNM-WT 15000's adult body size.

The Homo erectus specimen KNM-WT 15000 has played a critical role in our understanding of body size evolution. New interpretations suggest that KNM-WT 15000 had a younger age-at-death and a more rapid ontogenetic trajectory than previously suggested. Recent fossil discoveries and new interpretations suggest a wide range of body size and shape variation in H. erectus. Based on these new insights, we argue that KNM-WT 15000's adult stature and body mass could have been much smaller than has been traditionally presented in the literature. Using chimpanzee and modern human growth trajectories, we bracketed the range of possibilities for KNM-WT 15000's adult body size between 160.0 and 177.7 cm (5'3″-5'10″) for stature and 60.0 and 82.7 kg (132-182 lbs.) for body mass. These estimates put KNM-WT 15000 near the mean rather than among the largest known H. erectus specimens.

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield.

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 µU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets.

One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

UNLABELLED: Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. METHODS: We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). RESULTS: Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. CONCLUSIONS: A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation.

Human hepatocyte transplantation is gaining acceptance for the treatment of liver diseases. However, the reagents used to isolate hepatocytes from liver tissue are not standardized and show lot-to-lot variability in enzyme activity and endotoxin contamination. For clinical application, highly purified reagents are preferable to crude digest preparations. A purified tissue dissociating enzyme (TDE) preparation (CIzyme(TM) purified enzymes) was developed based on the enzyme compositions found in a superior lot of collagenase previously used by our group for human hepatocyte isolation. The performance of this enzyme preparation was compared to collagenase type XI on 110 liver cases by assessing hepatocyte yield, viability, and seven other functional assays that included plating efficiency, basal and induced CYP450 activities, phase II conjugation activity, and ammonia metabolism. No statistically significant difference was observed between these TDEs when they were used to isolate hepatocytes from liver resections or organ donor tissue on 54 hepatocyte isolations with type XI enzyme and 56 isolations using CIzyme(TM). These results show that a highly purified and defined TDE preparation can be formulated that provides excellent performance with respect to viability, yield, and functional activity of the isolated cells. In addition to reproducible formulation, these purified enzyme products have only 2-3% of the endotoxin of crude enzyme preparations. These results show that purified enzymes such as CIzyme(TM) will be a safe and effective for the isolation of human hepatocytes for clinical transplants.

The effect of unerupted permanent tooth crowns on the distribution of masticatory stress in children.

Human mothers wean their children from breast milk at an earlier developmental stage than do ape mothers, resulting in human children chewing solid and semi-solid foods using the deciduous dentition. Mechanical forces generated by chewing solid foods during the post-weaning period travel through not only the deciduous teeth, but also the enamel caps of the developing permanent teeth within the maxilla and mandible, which are not present in the adult face. The effects of mechanical stress propagating through these very stiff structures have yet to be examined. Based on a heuristic model, we predicted that the enamel of the embedded developing teeth would act to reduce stresses in the surrounding bony elements of the juvenile face. We tested this hypothesis by simulating occlusal loading in a finite element (FE) model of a child's cranium with a complete set of deciduous teeth and the first permanent molars embedded in the bony crypt in the maxilla. We modeled bone and enamel with appropriate material properties and assessed the effect of embedding high-stiffness enamel structures on stress distribution in the juvenile face. Against expectation, the presence of unerupted enamel caps does not affect the magnitude or location of stresses in the juvenile face. Our results do not support the hypothesis that the unerupted secondary teeth act to moderate stresses in the juvenile face.

Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

A preliminary 3D computed tomography study of the human maxillary sinus and nasal cavity.

Despite centuries of investigation, the function of the maxillary sinus (MS) and underlying patterns governing its form remain elusive. In this study, we articulate a methodology for collecting volumetric data for the MS and nasal cavity (NC) from computed tomography (CT) scans and report details for a small sample of 39 dried human crania of known ecogeographic provenience useful for assessing variation in MS size and shape. We use scaling analyses to preliminarily test the hypothesis that volumes of the nasal cavity (NCV) and maxillary sinus (MSV) are inversely correlated such that the NC covaries with size of the face, whereas the MS "fills in" the leftover space [proposed by Shea: Am J Phys Anthropol 47 (1977):289-300]. Against expectation, MSV is not significantly correlated with NCV or any cranial size variable. NCV, on the other hand, scales isometrically with facial size. The results of this pilot study suggest that NCV covaries with facial size, but that the MS does not simply fill in the leftover space in the face. The role, if any, of the MSs in midfacial function and architecture remains unclear. Larger sample sizes, additional environmental variables, and assessment of MS and NC shape are necessary to resolve this issue.

Just how strapping was KNM-WT 15000?

For over twenty years, the young, male Homo erectus specimen KNM-WT 15000 has been the focus of studies on growth and development, locomotion, size, sexual dimorphism, skeletal morphology, and encephalization, often serving as the standard for his species. Prior research on KNM-WT 15000 operates under the assumption that H. erectus experienced a modern human life history, including an adolescent growth spurt. However, recent fossil discoveries, improvements in research methods, and new insights into modern human ontogeny suggest that this may not have been the case. In this study, we examine alternative life history trajectories in H. erectus to re-evaluate adult stature estimates for KNM-WT 15000. We constructed a series of hypothetical growth curves by modifying known human and chimpanzee curves, calculating intermediate growth velocities, and shifting the age of onset and completion of growth in stature. We recalculated adult stature for KNM-WT 15000 by increasing stature at death by the percentage of growth remaining in each curve. The curve that most closely matches the life history events experienced by KNM-WT 15000 prior to death indicates that growth in this specimen would have been completed by 12.3 years of age. These results suggest that KNM-WT 15000 would have experienced a growth spurt that had a lower peak velocity and shorter duration than the adolescent growth spurt in modern humans. As a result, it is likely that KNM-WT 15000 would have only attained an adult stature of 163 cm (∼ 5'4 ″), not 185 cm (∼ 6'1 ″) as previously reported. KNM-WT 15000's smaller stature has important implications for evolutionary scenarios involving early genus Homo.

Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay.

BACKGROUND: Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. METHODS: Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. RESULTS: These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100+/-150,900 islet equivalent number (IEQ) (4147+/-1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500+/-152,400 IEQ (2134+/-1524 IEQ/g pancreas). CONCLUSIONS: These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.