Critical COVID-19 is associated with distinct leukocyte phenotypes and transcriptome patterns.

BACKGROUND: Prognostic markers for disease severity and identification of therapeutic targets in COVID-19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID-19 patients with different disease severity at admission and longitudinally during hospitalization. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS-CoV-2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS-CoV-2-negative bacterial sepsis (n = 5) and healthy controls (n = 10). RESULTS: COVID-19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA-DR) and exhaustion (PD-1) markers on T cells were increased compared with controls, but comparable between COVID-19 severity groups. Non-classical monocytes and monocytic HLA-DR expression decreased whereas monocytic PD-L1 and CD142 expression increased with COVID-19 severity. RNA sequencing exhibited increased plasma B-cell activity in critical COVID-19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease. CONCLUSION: Critical COVID-19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T-cell immune response. Plasma B-cell activity and calprotectin were higher in critical COVID-19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID-19 should be further explored.

Obesity and obesogenic growth are both highly heritable and modified by diet in a nonhuman primate model, the African green monkey (Chlorocebus aethiops sabaeus).

OBJECTIVE: In humans, the ontogeny of obesity throughout the life course and the genetics underlying it has been historically difficult to study. We compared, in a non-human primate model, the lifelong growth trajectories of obese and non-obese adults to assess the heritability of and map potential genomic regions implicated in growth and obesity. STUDY POPULATION: A total of 905 African green monkeys, or vervets (Chlorocebus aethiops sabaeus) (472 females, 433 males) from a pedigreed captive colony. METHODS: We measured fasted body weight (BW), crown-to-rump length (CRL), body-mass index (BMI) and waist circumference (WC) from 2000 to 2015. We used a longitudinal clustering algorithm to detect obesogenic growth, and logistic growth curves implemented in nonlinear mixed effects models to estimate three growth parameters. We used maximum likelihood variance decomposition methods to estimate the genetic contributions to obesity-related traits and growth parameters, including a test for the effects of a calorie-restricted dietary intervention. We used multipoint linkage analysis to map implicated genomic regions. RESULTS: All measurements were significantly influenced by sex, and with the exception of WC, also influenced by maternal and post-natal diet. Chronic obesity outcomes were significantly associated with a pattern of extended growth duration with slow growth rates for BW. After accounting for environmental influences, all measurements were found to have a significant genetic component to variability. Linkage analysis revealed several regions suggested to be linked to obesity-related traits that are also implicated in human obesity and metabolic disorders. CONCLUSIONS: As in humans, growth patterns in vervets have a significant impact on adult obesity and are largely under genetic control with some evidence for maternal and dietary programming. These results largely mirror findings from human research, but reflect shorter developmental periods, suggesting that the vervet offers a strong genetic model for elucidating the ontogeny of human obesity.

Fetal and maternal factors associated with infant mortality in vervet monkeys.

BACKGROUND: Causes of infant death remain unknown in significant proportions of human and non-human primate pregnancies. METHODS: A closed breeding colony with high rates of infant mortality had pregnancies assessed (n=153) by fetal measurements and maternal characteristics. Infant outcome was classified as neonatal death (stillborn or died <48 hours from birth), postnatal death (died 2-30 days) or surviving (alive after 30 days). RESULTS: Fetal size did not predict outcome. Poor maternal glycemic control and low social ranking increased odds for adverse outcome (OR=3.72, P=0.01 and 2.27, P=0.04, respectively). Male sex was over-represented in stillbirths (P=0.04), and many were macrosomic, but size did not associate with maternal glycemic control measured as glycated hemoglobin A1c. Postnatally dead infants were smaller (P<0.01), which associated with behavioral factors and glycemic control. CONCLUSIONS: Fetal growth estimates predicted gestational age but not fetal outcome. Maternal social status and metabolic health, particularly glycemic control, increased risks of adverse pregnancy outcome.

Simultaneous administration of vitamin A and DTP vaccine modulates the immune response in a murine cerebral malaria model.

The World Health Organisation recommends vitamin A supplementation (VAS) to children aged 6 months to 5 years in low-income countries, and for logistic reasons, this has been linked to routine childhood immunizations. Observational studies suggest that VAS given with diphtheria-tetanus-pertussis (DTP) vaccine may increase mortality from non-targeted diseases. We investigated the non-targeted effect of pretreatment with VAS and DTP vaccine in a murine model of experimental cerebral malaria. Our a priori hypothesis was that VAS/DTP would aggravate the infection. We found that the effect of VAS and DTP depended on pathogenesis; VAS/DTP tended to increase parasitaemia and significantly depressed cytokine responses in mice, which developed cerebral malaria, but this was not seen in mice dying of anaemia. The divergent effect according to pathogenesis may help elucidate why VAS has divergent effects on different diseases in humans. Our results support the hypothesis that immunological effects of VAS/DTP may have detrimental implications for disease outcomes.

Clinicopathologic characterization of naturally occurring diabetes mellitus in vervet monkeys.

Diabetes mellitus (DM) is a group of chronic metabolic diseases characterized by persistent fasting hyperglycemia, and it can be of either polygenic or monogenic origin. Animal models have played an important role in elucidating the pathophysiology of the polygenic Type 1 and type 2 DM forms; however, useful animal models of the monogenic forms do not exist. The authors describe 4 cases of naturally occurring DM in vervet monkeys (Chlorocebus aethiops sabaeus), 1 of which has clinicopathologic findings consistent with type 2 DM, including persistent hyperglycemia, hypertriglyceridemia, islet amyloidosis, and reduced islet insulin immunostaining. In contrast, the 3 remaining animals have clinicopathologic similarities to a monogenic form of the disease, including a lack of islet amyloidosis and hypertriglyceridemia, as well as normal islet insulin immunostaining. In addition, pedigree analysis conducted on one of these animals is consistent with either an autosomal dominant or mitochondrial inheritance pattern, which supports a monogenic form of DM. The authors thus hypothesize that a naturally occurring monogenic form of diabetes may occur in vervet monkeys, making them a potential animal model for future studies.

Social impulsivity inversely associated with CSF 5-HIAA and fluoxetine exposure in vervet monkeys.

Animal and human research suggests that the central serotonin system is involved in the inhibition of impulsive behavior. Two studies were designed to assess this relationship in male vervet monkeys (Cercopithecus aethiops sabaeus) using a standardized test of impulsivity in a social context: the Intruder Challenge. In the first study, an index of impulsivity in response to an unfamiliar adult male intruder (including latency to approach and aggressive and assertive interactions) was inversely correlated with levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (r = -0.33, p <.01, n = 138). The approach, but not aggressive, component of the Impulsivity Index was the primary contributor to this relationship (partial r = -0.27, p <.01). The second experiment compared responses to the Intruder Challenge after 9 weeks of daily treatment with fluoxetine (2 mg/kg, i.m.) or vehicle. Fluoxetine-treated subjects (n = 6) had significantly lower Impulsivity Index scores than controls (n = 12). The results from these two investigations provide evidence for serotonergic influences on social impulsivity.

Physiological correlates of self-injurious behavior in captive, socially-reared rhesus monkeys.

This study examined the relationship between self-injurious behavior (SIB) in rhesus monkeys and several biological variables, including monoamine metabolites in cerebrospinal fluid (CSF) and circulating levels of ACTH, cortisol, and testosterone. Cisternal CSF and blood plasma samples were obtained from 23 individually housed male rhesus macaques, 14 of which had a veterinary record of self-inflicted wounding. CSF samples were analyzed for 5-hydroxyindole-3-acetic acid (5-HIAA), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) using isocratic high-performance liquid chromatography with electrochemical detection (HPLC-EC). Plasma samples were analyzed for ACTH, cortisol, and testosterone using commercially available radioimmunoassays (RIAs). Rates of self-directed biting were determined by systematic observation of all monkeys. Monkeys with SIB did not differ from controls in their basal monoamine or gonadal activity. However, the SIB group showed consistently lower mean plasma cortisol levels than the control group. Plasma cortisol was negatively correlated with rates of self-directed biting. These results suggest a persistent dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in monkeys with SIB. It is not yet clear whether this phenomenon of low cortisol represents chronically reduced adrenocortical secretion under basal conditions or a difference in response to the mild stress of capture and chemical restraint. The implications of these findings will be discussed with respect to SIB in humans as well as post-traumatic stress disorder (PTSD), a condition characterized by pituitary-adrenocortical hypoactivity.

Time course of movement planning: selection of handgrips for object manipulation.

A goal of research on the cognitive control of movement is to determine how movements are chosen when many movements are possible. We addressed this issue by studying how subjects reached for a bar to be moved as quickly as possible from a home location to a target location. Ss generally grabbed the bar in a way that afforded a comfortable posture at the target location (the end-state comfort effect) and with the thumb toward the end of the bar that would be aligned with the target (the thumb-toward bias). The data suggested that subjects chose handgrips by retrieving instances of previous reaches, not by carrying out computations that treated candidate reaches as new behavioral events.

The effect of lumbar back support tension on trunk muscle activity.

OBJECTIVE: Assess the effect of different controlled lumbar back support tightness levels on trunk muscle activity. DESIGN: Two-way repeated measure design assessing lumbar back support tension and submaximal trunk extension moments on trunk muscle electromyographic activity. BACKGROUND: Biomechanical studies on lumbar back supports often use electromyography (EMG) to assess the affect on trunk muscle activity. However, the lumbar back support may alter the electromyographic signal by changing the electrode-muscle distance. METHODS: Subjects performed trunk extensions at three static submaximal extension moment levels (25%, 50% and 75% MVC) while stabilized at the hips and shoulders, with the back support tensioned to three different tightness levels (44.5, 66.7 and 89.0 N) as well as a no-back support condition. RESULTS: Statistical analysis failed to find a significant effect (P

Cross-sectional area of the lumbar back muscles as a function of torso flexion.

OBJECTIVE: Quantification of the maximum anatomical cross-sectional area of the lumbar back muscles as a function of torso flexion angle and development of prediction equations as a function of torso flexion and anthropometric measures. BACKGROUND: Cross-sectional areas of the lumbar back muscles used as inputs into biomechanical models have traditionally been derived from subjects lying in the neutral supine posture. However, it is known that the cross-sectional area of muscle is altered as the torso angle changes. DESIGN: Experimental design consisted of a two-factor multivariate analysis of variance on the cross-sectional area of the lumbar torso muscle across the lumbar levels, as a function of gender and torso angle. Hierarchical linear regression was utilized to assess the association between cross-sectional area and individual and torso posture characteristics. METHOD: Axial MRI scans, through and parallel to each of the lumbar intervertebral discs at four torso flexion positions were obtained from subjects in a lateral recumbent posture. Cross-sectional areas were quantified and converted into anatomical cross-sectional areas utilizing known fascicle orientations. RESULTS: The maximum anatomical cross-sectional area was located between the L(3)/L(4) and L(4)/L(5) level in the neutral posture. The anatomical cross-sectional areas at the L(4)/L(5) and L(5)/S(1) decreased during torso flexion, however, the percent change varied as a function of the individual level. The majority of the anatomical cross-sectional area variability was explained by gender and body mass. Lumbar curvature explained a larger proportion of the anatomical cross-sectional area variability at the lower lumbar levels than at the higher lumbar levels. CONCLUSIONS: The maximum anatomical cross-sectional area of the lumbar back muscles occur at the neutral torso posture and did not decrease as a function of torso flexion. When using maximum anatomical cross-sectional area or specific lumbar level anatomical cross-sectional areas, it appears necessary to account for gender and body mass. At the lower lumbar levels, knowledge of spinal curvature plays an increasing role in the estimation of the size of the lumbar torso muscle cross-sectional area. RELEVANCE: This research indicates the lower lumbar level trunk muscle anatomical cross-sectional area decrease as torso flexion increases, however, the maximum lumbar trunk muscle anatomical cross-sectional area does not vary as a function of torso flexion. Accounting for gender, body mass, torso characteristics and lumbar curvature may help increase accuracy of anatomical cross-sectional area prediction, as well as muscle force predictions from biomechanical models.

Female and male trunk geometry: size and prediction of the spine loading trunk muscles derived from MRI.

OBJECTIVE: Develop a gender specific database of trunk muscle cross-sectional areas across multiple levels of the thoracic and lumbar spine and develop prediction equations for the physiological cross-sectional area as a function of gender and anthropometry. DESIGN: This study quantified trunk muscle cross-sectional areas of male and female spine loading muscles. BACKGROUND: There is a lack of comprehensive data regarding the female spine loading muscle size. Although biomechanical models often assume females are the same as males, little is known regarding gender differences in terms of trunk muscle areas and no data exist regarding the prediction of trunk muscle physiological cross-sectional areas from commonly used external anthropometric measures. METHODS: Magnetic resonance imaging scans through the vertebral bodies from T(8) through S(1) were performed on 20 females and 10 males. Muscle fiber angle corrected cross-sectional areas were recorded at each vertebral level. Linear regression techniques taking into account anthropometric measures were utilized to develop prediction equations for the physiological cross-sectional area for each muscle of interest, as well as tests for differences in cross-sectional areas due to gender and side of the body. RESULTS: Significant gender differences were observed for the prediction of the erector spinae, internal and external obliques, psoas major and quadratus lumborum physiological cross-sectional areas. Anthropometric measures about the xyphoid process and combinations of height and weight resulted in better predictions of cross-sectional areas than when using traditional anthropometry. CONCLUSIONS: This study demonstrates that the trunk muscle geometry of females and males are different, and that these differences should be considered in the development of biomechanical models of the torso. Relevance. The prediction of physiological cross-sectional areas from external anthropometric measures provide gender specific equations to assist in estimation of forces of muscles which load the spine for biomechanical purposes.

MRI-derived moment-arms of the female and male spine loading muscles.

OBJECTIVE: Develop a comprehensive gender-specific database of trunk muscle moment-arms across multiple levels of the lower thoracic and lumbar spine, determine if gender differences exist across the different vertebral levels, and develop prediction equations for the moment-arms as a function of external anthropometric measures. DESIGN: This study quantified trunk muscle moment-arms relative to the spine from T(8) to S(1) of male and female spine loading muscles. BACKGROUND: Knowledge of trunk muscle geometry is important for biomechanical modeling of the low back and for understanding of spinal loading. However, there currently is a lack of comprehensive data regarding the moment-arms of the female spine loading muscles. Additionally, little is known regarding gender differences in moment-arms for the same muscles. METHODS: Magnetic resonance imaging scans through the vertebral bodies from T(8) through S(1) were performed on 20 females and 10 males. Moment-arms in the coronal and sagittal plane between the muscle centroid and vertebral body centroid were recorded at each vertebral level. Linear regression techniques taking into account anthropometric measures were utilized to develop prediction equations for the moment-arms for each muscle. RESULTS: Anthropometric measures were better predictors of coronal plane moment-arms than sagittal plane moment-arms for both genders. Measures consisting of height and weight were consistent predictors of female moment-arms. Measures about the xyphoid process and combinations of height and weight were consistent predictors of coronal plane moment-arms for males at several lower lumbar levels. Males exhibited larger moment-arms than for females, for most muscles at most levels. CONCLUSIONS: Trunk muscle moment-arms of females and males are different, and should be considered in the development of biomechanical models of the torso. Similar to other studies, external anthropometric measures were better predictors of coronal plane moment-arms than sagittal plane moment-arms.

Environmental stress alters genetic regulation of novelty seeking in vervet monkeys.

Considerable attention has been paid to identifying genetic influences and gene-environment interactions that increase vulnerability to environmental stressors, with promising but inconsistent results. A nonhuman primate model is presented here that allows assessment of genetic influences in response to a stressful life event for a behavioural trait with relevance for psychopathology. Genetic and environmental influences on free-choice novelty seeking behaviour were assessed in a pedigreed colony of vervet monkeys before and after relocation from a low stress to a higher stress environment. Heritability of novelty seeking scores, and genetic correlations within and between environments were conducted using variance components analysis. The results showed that novelty seeking was markedly inhibited in the higher stress environment, with effects persisting across a 2-year period for adults but not for juveniles. There were significant genetic contributions to novelty seeking scores in each year (h(2) = 0.35-0.43), with high genetic correlations within each environment (rhoG > 0.80) and a lower genetic correlation (rhoG = 0.35, non-significant) between environments. There were also significant genetic contributions to individual change scores from before to after the move (h(2) = 0.48). These results indicate that genetic regulation of novelty seeking was modified by the level of environmental stress, and they support a role for gene-environment interactions in a behavioural trait with relevance for mental health.

Emotional support needs of gay males with AIDS.

Twenty-five gay and three bisexual males with AIDS (PWAs) rated their needs for four different kinds of emotional support (expressing love and concern, expressing encouragement and positive feedback, serving as a confidant, and providing a philosophical or spiritual perspective) from five different support providers (parents, partners, friends, HIV-positive friends and physicians). Findings support the importance of emotional support for this population. The results indicate that differences exist depending on type and provider of support. Participants expressed the greatest need for every category of emotional support from partners. Participants also rated their needs from physicians highly for each category of emotional support. Noticeably low was the need for providers to offer philosophical or spiritual perspectives.

Quantitative evaluation of pancreatic enhancement during dual-phase helical CT.

PURPOSE: To determine the improvement in pancreatic enhancement at helical computed tomography (CT) performed with an early delay after administration of contrast material compared with that performed with a standard delay. MATERIALS AND METHODS: Dual-phase helical CT of the abdomen was performed in 120 patients with a 150-mL bolus of contrast material infused at 5 mL/sec. Early and standard delayed scanning was performed beginning at 20 seconds and 49-71 seconds, respectively. Regions of interest were measured in the head, body, and tail of the pancreas in 92 patients. The difference in enhancement between early and standard delayed scanning was calculated. RESULTS: Mean pancreatic enhancement was 82 HU +/- 3 (standard error) with an early delay, whereas enhancement on standard delay scans was 62 HU +/- 2 (P < .001). An improvement in enhancement greater than 10 HU was attained in 66 of 92 cases (72%). CONCLUSION: Pancreatic enhancement at helical CT with an early delay after contrast material administration is often significantly greater than the enhancement seen with a standard delay when a monophasic, rapidly infused bolus of contrast material is used.

Effect of foot movement and an elastic lumbar back support on spinal loading during free-dynamic symmetric and asymmetric lifting exertions.

The aim of this study was to assess the effect of an elastic lumbar back support on spinal loading and trunk, hip and knee kinematics while allowing subjects to move their feet during lifting exertions. Predicted spinal forces and moments about the L5/S1 intervertebral disc from a three-dimensional EMG-assisted biomechanical model, trunk position, velocities and accelerations, and hip and knee angles were evaluated as a function of wearing an elastic lumbar back support, while lifting two different box weights (13.6 and 22.7 kg) from two different heights (knee and 10 cm above knee height), and from two different asymmetries at the start of the lift (sagittally symmetric and 60 degrees asymmetry). Subjects were allowed to lift using any lifting style they preferred, and were allowed to move their feet during the lifting exertion. Wearing a lumbar back support resulted in no significant differences for any measure of spinal loading as compared with the no-back support condition. However, wearing a lumbar back support resulted in a modest but significant decrease in the maximum sagittal flexion angle (36.5 to 32.7 degrees), as well as reduction in the sagittal trunk extension velocity (47.2 to 40.2 degrees s(-1)). Thus, the use of the elastic lumbar back support provided no protective effect regarding spinal loading when individuals were allowed to move their feet during a lifting exertion.

An investigation of perceived exertion via whole body exertion and direct muscle force indicators during the determination of the maximum acceptable weight of lift.

The objective of this study was to identify the perceived exertion mechanisms (direct muscle force and whole body exertion) associated with the decision to change the weight of lift during the determination of the maximum acceptable weight of lift (MAWL). Fifteen males lifted a box of unknown weight at a rate of 4.3 lifts/min, and adjusted the weight until their MAWL was reached. Variables such as the predicted muscle forces and heart rate were measured during the lifting exertion, as well as the predicted spinal loading in three dimensions using an EMG-assisted biomechanical model. Multiple logistic regression techniques were used to identify variables that were associated with the decision to change the weights up and down prior to a subsequent lift. Results indicated that the force in the left erector spinae, right internal oblique, and left latissimus dorsi muscles as well as heart rate were associated with decreases in the weight prior to the next lift. It appears that a combination of local factors (muscle force) and whole body exertion factors (heart rate) provide the feedback for the perceived exertion when decreasing the weight. The up-change model indicated that the forces of the right erector spinae, left internal oblique, and the right latissimus dorsi muscles were associated with the decision to increase the weight prior to the next lift. Thus, local factors provide feedback during the decision to increase the weight when starting from light weights. Collectively, these findings indicate that psychophysically determined weight limits may be more sensitive to muscular strain rather than spinal loading.

Detection and follow-up of important extra-arterial lesions with helical CT angiography.

AIM: To determine the prevalence and significance of extra-arterial findings detected prospectively on helical computed tomographic angiography (CTA). SUBJECTS AND METHODS: The official reports of 802 consecutive CTAs performed over a 4.5-year period on 624 patients and the reports of all radiographic follow-up studies were reviewed for identification of important extra-arterial findings. Medical records and imaging studies of all patients with previously unknown extra-arterial findings on CTA were reviewed to assess follow-up. In cases where follow-up was not indicated in the medical record, referring physicians were contacted directly. RESULTS: Important, previously unknown, extra-arterial findings were detected on 35 CTAs (4.4% of all CTAs, 5.6% of all patients), with 33 of 35 detected prospectively. Six lesions were consistent with and/or proven to be malignant. Important non-tumoural lesions were discovered on nine CTAs. Of 13 lesions with imaging features that were suspicious for malignancy. Five of these lesions proved to be benign, but radiographic and/or clinical follow-up was not obtained or could not be documented in eight patients. CONCLUSION: With the increasing use of CTA as a replacement for conventional angiography, careful attention should be paid to the visualized extra-arterial structures. Extra-vascular findings that are believed to be significant, may not be adequately followed-up by referring vascular specialists.

Techniques for collecting saliva from awake, unrestrained, adult monkeys for cortisol assay.

Cortisol levels serve as an index of pituitary-adrenal activity in nonhuman primates. In adult monkeys, cortisol is normally measured in blood (typically requiring restraint or sedation) or urine (reflecting a state rather than point estimate). In contrast, saliva collection is less invasive than drawing blood and allows for repeated sampling within a short period of time. Although protocols exist for collecting saliva from young monkeys, these procedures are inadequate for awake, unrestrained adult animals. Our laboratory has developed two methods for collecting saliva from adult rhesus monkeys: a "screen" method, which involves licking screen-covered gauze, and a "pole" method, which involves sucking and chewing on an attached rope. Twenty-three adult male rhesus monkeys were used to evaluate these two methods. After a period of adaptation, saliva samples were collected from 21 of 23 subjects. Saliva collection was faster with the pole than with the screen method (P < 0.01), but the pole method was not suitable for some animals because of their tendency to bite off the attached rope. An analysis of 19 saliva samples revealed a mean cortisol concentration of 0.84 microg/dl (range 0.27-1.77 microg/dl). There was no statistically significant difference in cortisol value between methods used (P > 0.22). The influence of the flavoring on the cortisol assay was tested, and was found to have no significant effect (P > 0.28). Our results indicate that either technique can be used to safely collect saliva from unrestrained adult monkeys. Choice of technique will depend on the proclivities of individual monkeys.

Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events.

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.