Glucose-based optimization of CHO-cell perfusion cultures.

Perfusion cultures of CHO cells producing t-PA were performed using acoustic filter cell retention. A robust off-line glucose analysis and predictive control protocol was developed to maintain the process within approximately 0.5 mM of the glucose set point, without the need for a more fallible on-line sensor. Glucose usage (the difference between the inlet and reactor glucose concentrations) provided an easily measured indicator of overall medium utilization for mapping acceptable ranges of operation, including the edge of failure. Earlier onset of perfusion with a ramping glucose set point (1.5 mM/d) resulted in improved growth and consistency during the perfusion culture start-up. At steady state, the t-PA concentration variability increased gradually with increasing glucose usage up to approximately 22 mM, then up to 24 mM the variability increased threefold. Peak t-PA concentrations of over 90 mg/L were obtained by controlling at a glucose usage of approximately 24 mM, but these t-PA levels were not sustainable for more than 3 days. A consistent t-PA concentration of 40 mg/L was obtained at a glucose usage of 21.5 mM.

Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture.

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.

Characterization of long-term femoral-head-penetration rates. Association with and prediction of osteolysis.

BACKGROUND: We examined the relationship between long-term femoral-head-penetration patterns and osteolysis in a ten-year follow-up study of a well controlled patient population. The purposes of this study were to characterize the linearity of long-term head-penetration patterns over time, to describe the relationship between ten-year true wear rates and osteolysis, and to determine whether the occurrence of osteolysis at ten years could be predicted by penetration data obtained prior to five years. METHODS: Temporal femoral-head-penetration patterns were examined at a minimum of ten years after forty-eight primary total hip arthroplasties. The arthroplasties were performed with the use of an Arthropor acetabular cup (Joint Medical Products) and a thirty-two-millimeter-diameter cobalt-chromium femoral head (DePuy). Using a computer-assisted radiographic technique, we evaluated two-dimensional head penetration on serial annual radiographs. Linear regression analysis modeled penetration-versus-time data as a line for each patient. The slope of the regression line indicated the true wear rate for each patient. In a subgroup of thirty-four hips for which three annual radiographs had been made less than five years after the arthroplasty, we compared early head-penetration patterns with the later occurrence of osteolysis. RESULTS: For all forty-eight hips, the true wear rate averaged 0.18 millimeter per year (range, 0.01 to 0.44 millimeter per year) and temporal head-penetration patterns tended to be linear (mean r2 = 0.91 +/- 0.16). Osteolysis at ten years was strongly associated with increasing true wear rates (p < 0.001). Osteolysis did not develop in any of the nine hips with a true wear rate of less than 0.1 millimeter per year. However, osteolysis developed in nine (43 percent) of twenty-one hips with a rate between 0.1 and less than 0.2 millimeter per year, in eight of ten hips with a rate between 0.2 and 0.3 millimeter per year, and in all eight hips with a rate of greater than 0.3 millimeter per year. Evaluation of early true wear rates as a predictor of late osteolysis showed a similar relationship. CONCLUSIONS: This study demonstrates that true wear rates tend to be constant and that increased true wear is significantly associated with osteolysis at ten years after the operation. A similar relationship was also found at the early follow-up interval, indicating that early true wear rates (determined from serial radiographs) might enable orthopaedists to predict if patients are at risk for the development of osteolysis. CLINICAL RELEVANCE: On the basis of these findings, we use temporal femoral-head-penetration data in our practice to evaluate polyethylene inserts in asymptomatic patients, to estimate the time to component wear-through, and to adjust the frequency of follow-up evaluations for monitoring the development of osteolytic lesions in at-risk patients.

Predictive control of hollow-fiber bioreactors for the production of monoclonal antibodies.

The selection of medium feed rates for perfusion bioreactors represents a challenge for process optimization, particularly in bioreactors that are sampled infrequently. When the present and immediate future of a bioprocess can be adequately described, predictive control can minimize deviations from set points in a manner that can maximize process consistency. Predictive control of perfusion hollow-fiber bioreactors was investigated in a series of hybridoma cell cultures that compared operator control to computer estimation of feed rates. Adaptive software routines were developed to estimate the current and predict the future glucose uptake and lactate production of the bioprocess at each sampling interval. The current and future glucose uptake rates were used to select the perfusion feed rate in a designed response to deviations from the set point values. The routines presented a graphical user interface through which the operator was able to view the up-to-date culture performance and assess the model description of the immediate future culture performance. In addition, fewer samples were taken in the computer-estimated cultures, reducing labor and analytical expense. The use of these predictive controller routines and the graphical user interface decreased the glucose and lactate concentration variances up to sevenfold, and antibody yields increased by 10% to 43%.

Failure of total hip arthroplasty with a precoated prosthesis. 4- to 11-year results.

A series of early femoral component failures prompted a detailed retrospective clinical and radiographic review of 176 hybrid cemented total hip arthroplasties using a polymethyl-methacrylate coated femoral prosthesis. All surgeries were performed using third generation cement techniques. Average length of followup was 6.3 years (range, 3-12 years). Twenty-one patients died, and one underwent revision surgery because of sepsis. Of the remaining 154 total hip arthroplasties, 23 (15%) of the femoral components failed (21 revised, two definitely loose). The average time to revision was 3.9 years. None of the acetabular components failed. Comparison between the failure and nonfailure groups revealed that poor cement mantles (Grades C or D) with distal cement mantle deficiencies were statistically significant predictors of femoral failure. The most common mechanism of failure was progressive, circumferential cement-bone interface osteolysis with relative preservation of the cement-metal interface. Debonding of the cement column from the prosthesis was a late finding and occurred in only 45% of failed cases. Incorporating the techniques of centralization and centrifugation significantly improved clinical results. Strengthening of the cement-prosthesis interface may magnify the deleterious effects of a poor cement mantle and predisposes the cement-bone interface to failure.

Functional activity of staphylococcal enterotoxin A requires interactions with both the alpha and beta chains of HLA-DR.

The staphylococcal enterotoxins, SEA and SEE, bind one zinc atom per molecule of protein. The presence of this metal atom enhances the binding of the toxins to MHC class II molecules, presumably through an interaction with histidine 81 of the beta chain. L cell transfectants expressing HLA-DR1 and HLA-DR7 molecules, with mutations in either the alpha1 or beta1 domains, were tested for their ability to bind SEA and present it to T cells. Cells expressing DR1 molecules with alanine at positions 77, 78, 80, 83, 84 and 85, or serine at position 79 could all bind SEA and present it to either polyclonal or monoclonal T cells. Most point mutations within the alpha-helical portion of the DR7 beta chain had no effect on binding and presentation. However, substitution of histidine 81 with alanine, glutamate, or aspartate, abrogated SEA binding as well as T cell stimulation by the superantigen. This effect was also observed when the non-polymorphic aspartate, at position 76 was changed to alanine. Mutation of the asparagine at position 82 had an intermediate effect. Point mutations of the DR alpha chain had little effect on binding of SEA as determined by a flow cytometric assay. However, mutation of lysine at position 39 of the alpha chain and, to a lesser extent methionine at position 36, markedly decreased the ability of SEA to stimulate toxin-responsive mouse T cell hybridomas. Finally, the monoclonal antibody, L243 binds to the alpha chain of HLA-DR, and was able to block T cell activation by SEA without blocking SEA binding. These data support the model whereby HLA-DR has two binding sites for SEA. A low affinity site, present on the alpha chain, is required for T cell stimulation by the superantigen, but is insufficient to mediate toxin binding. High affinity binding of HLA-DR to SEA occurs solely through residues on the beta chain, including both histidine 81 and aspartate 76.

Aseptic loosening in uncemented total hip arthroplasty in a canine model.

An in vivo canine model was developed to investigate the histologic and biochemical parameters associated with aseptic loosening. Thirty-eight canines had cementless total hip arthroplasty. Experimental groups were designed specifically to investigate the relative contributions of implant motion and particulate debris (cobalt chrome alloy, titanium aluminum vanadium, and polyethylene) on the resultant periprosthetic tissues. Tissues from a stable, well-ingrown prosthesis provided a control. Importantly, the histologic and biochemical characteristics of the experimentally induced membranes consistently correlated with previous in vitro reports of tissues retrieved at revision surgery for aseptic loosening. Implant motion and all 3 particulate debris groups resulted in increased numbers of macrophages in the periprosthetic membranes. The histologic findings paralleled the increase in levels of biochemical mediators of bone resorption as measured by collagenase, gelatinase, prostaglandin E2, and interleukin-1 activity. The most striking results were seen in the histology and biochemistry of the particle groups with highly cellular membranes showing increased biochemical activity when compared with controls. The clinical relevance of this work lies in the description of an in vivo model of aseptic loosening that can be used to investigate the effects of numerous variables implicated in aseptic loosening. Ultimately, the model may serve as a basis for developing therapeutic interventions.

Nonsteroidal antiinflammatory drug-induced gastroduodenal injury in children.

OBJECTIVE: To determine the incidence of abdominal pain and gastroduodenal injury in children with arthritis taking nonsteroidal antiinflammatory drugs (NSAIDs). METHODS: A retrospective review of the records of all children (570 patients) receiving followup care in an academic rheumatology clinic between 1991 and 1993 was performed. RESULTS: There were 344 patients who used NSAIDs during the study period. Abdominal pain was recorded in 27.9% of patients taking NSAIDs and 14.6% of patients not taking NSAIDs. Abdominal pain in 47 patients (49%) taking NSAIDs and 14 patients (42%) not taking NSAIDs was evaluated radiographically and/or endoscopically. Among those patients evaluated, gastric or duodenal injury was found in 16 (34.0%) who were taking NSAIDs and 1 (7.1%) who were not. This represented a relative risk for gastroduodenal injury of 4.8 for patients taking NSAIDs (P = 0.09). The incidence of injury did not change when analyses were controlled for prednisone or slow-acting antirheumatic drug use. None of the children were hospitalized or died as a result of gastroduodenal injury during the 3-year period. CONCLUSION: We conclude that NSAID use in children with arthritis frequently leads to gastroduodenal injury, with an estimated incidence and relative risk that are comparable to the rates found in adults with arthritis taking NSAIDs, but that hospitalization or death as a result of this injury is uncommon.

Inhibition of antigen-specific T cell activation by staphylococcal enterotoxins.

The staphylococcal enterotoxins SEA, SEB, SEC2, and TSST-1 bind to MHC class II molecules and stimulate polyclonal T cell populations on the basis of the expression of responsive TCR V beta domains. CL-1 is a human T cell clone that is specific for a peptide derived from influenza hemagglutinin (HA 307-319) presented in the context of HLA-DR1. CL-1 expresses the TCR V beta 13.1 domain, and does not respond to SEA, SEB, or TSST-1. This T cell was used to test the effect of nonstimulatory staphylococcal enterotoxins on a response to antigenic peptide. These toxins inhibit peptide-specific activation of CL-1 in a concentration-dependent manner. These toxins also inhibit the response of an HLA-DR1-specific alloreactive T cell clone. This inhibition seems to be a result of impaired access of TCR to the MHC/peptide complex rather than negative signaling by toxin via class II interaction or induction of T cell anergy. SEA, but neither SEB nor TSST-1 impedes avidin access to a biotin group attached to the amino terminus of HA 307-319. SEA partially impairs access of avidin to HA peptide biotinylated at residue 313, but is unable to inhibit avidin access to biotin at residue 318. This demonstrates that SEA binds to HLA-DR molecules that have also bound the antigenic peptide and suggests a topology for the interaction of SEA with class II, whereby the toxin interferes with peptide/MHC-TCR contact.

Time-varying covariates in models of human mortality and aging: multidimensional generalizations of the Gompertz.

Models of mortality and aging depend on assumptions about physiological change even if they are not made explicit. Standard models, like the Gompertz, often fail to describe mortality at extreme ages, suggesting a need for biologically more detailed and flexible models. One solution is to model the interaction of time-varying covariates with mortality to better describe the age dependence of mortality, test hypotheses about the relation of physiological change and mortality, and use longitudinal data to generalize assumptions about physiological change. This model is applied to (a) a 34-year follow-up of risk factors and mortality and (b) a 9.5-year follow-up of function and mortality from longitudinal surveys of the U.S. elderly population.

Sjögren's syndrome presenting as hypokalemic periodic paralysis.

We describe a 21-year-old Hispanic woman who presented with hypokalemic paralysis as the initial manifestation of Sjögren's syndrome (SS). Our review of the English literature revealed 12 previously reported cases of SS and renal tubular acidosis (RTA). Paralysis often preceded the sicca complex in those patients. Renal function in the patients with hypokalemic paralysis was reduced compared with that in patients who had primary SS and RTA but no history of hypokalemic paralysis (P < 0.002). Hypokalemic periodic paralysis is a rare manifestation of SS. It is seen more often in patients with primary SS, may precede the classic sicca complex, and may serve as a clinical marker for more severe renal disease in patients who have primary SS and RTA.

An overview of relevant data sources in the former USSR for studies in demographic trends, aging and noncommunicable disease problems.

This article provides an overview of health data available in the former USSR. It is not all-inclusive in terms of chronic diseases covered or in details of data collection activities carried out. However, several broad conclusions can be drawn: There is a system of population and mortality data collection which covers the former USSR and which can be disaggregated to smaller administrative areas. The system is being exploited by population specialists, demographers, medical demographers and epidemiologists, both nationally and internationally, both for analytical purposes and as part of health monitoring systems. A national-level data-collection system for morbidity and disability, based on delivery of health services, is in place and is exploited by both health researchers and health planners. The shortcomings of such a health service-based statistical system are well recognized. Further standardization or calibration of measures of total and cause-specific morbidity and disability measures should be examined. A potential calibration tool is the 1988-1993 health examination and interview survey covering a representative (but highly clustered) sample of the former USSR population. The possibilities of greater standardization of measurement procedures used in this survey should also be investigated. In certain disease areas, e.g. cardiovascular diseases, cancer, rheumatic diseases and gerontology, clinical and epidemiological studies involving international collaboration have been carried out. This has resulted in the use of internationally accepted disease definitions, diagnostic procedures, and of clinical and laboratory standardization of demographic, social and biological measurements. Participation in multilateral or bilateral studies should be encouraged in research in disease areas where these types of programmes have not yet been instituted.(ABSTRACT TRUNCATED AT 250 WORDS)

Projections of disability consequences in Indonesia.

With the populations in many developing countries growing older due to declining fertility and infant mortality, there is concern that chronic disease and disability will rapidly increase in prevalence. Such an increase could create a conflict of priorities in developing countries with scarce public health resources between, the continuing need for infectious disease and infant mortality programs, and an emerging need to deal with the health, and health related social problems of adult and elderly populations. To assess the potential magnitude of the emerging problems of chronic disease and disability - and to assess when those needs are likely to emerge - we present projections of chronic disability and its sequelae using data from a large Indonesian survey focusing on the prevalence of impairments and handicaps as envisaged in the WHO (1980) ICIDH classification.

Forecasting chronic disease risks in developing countries.

Declining fertility and infant mortality has caused the population in many developing countries to age. Population ageing can produce a rapid shift in the predominant public health problems from infant mortality and infectious diseases to chronic disease mortality at later ages. Designing public health strategies to deal with the health consequences of population ageing in developing countries is difficult both because of a remaining burden of infectious diseases and because of changes in life style associated with economic development that may raise chronic disease risks. Because there are few longitudinal studies of chronic disease risks in developing countries, we investigate the use of a planning and forecasting model, which combines data from multiple sources, in six developing countries.

Primary prevention of type 2 (non-insulin-dependent) diabetes mellitus.

Type 2 (non-insulin-dependent) diabetes mellitus is the major form of the disease in all societies. Its public health impact appears to be increasing and the greatest genetic predisposition to the disease is encountered in developing communities. The reduction or elimination of disease in whole populations is a fundamental goal in public health. Whilst several factors are associated with the development of Type 2 diabetes, it is not clear how they cause the disease, if indeed they do, nor whether they act in the same way in all populations. Risk factors may be true determinants of a disease but alternatively they may be associated with its occurrence only by virtue of an innocent relationship with the true causes. Furthermore, known risk factors usually explain only a small proportion of any chronic disease. The role of risk factors in disease causation is therefore of fundamental importance in considering disease prevention. Two alternative strategies for prevention of disease in populations have been proposed. The population strategy seeks to remove the causes of disease in communities as a whole, whilst the high-risk strategy aims to identify subjects at increased risk, and to intervene selectively. The population approach should be tried and carefully evaluated in selected communities at above-average risk of several noncommunicable diseases. However, certain epidemiological features of Type 2 diabetes, including the distributional characteristics of glycaemia and the complications of hyperglycaemia, the clustering of cardiovascular risk factors in the diabetic subpopulation, as well as uncertainties over the causal nature of known risk factors, suggest that a high-risk approach to prevention is also appropriate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of interventions on community awareness and treatment of hypertension: results of a WHO study.

A WHO-coordinated study of the community control of hypertension in six countries (Cuba, Finland, France, Italy, Mongolia and Portugal) has shown that a comprehensive approach clearly improves the care of hypertensives in various populations. Hypertension control programmes were individually designed in each country and were implemented in defined communities. The intervention strategies varied between countries, the major components being: establishment of hypertension clinics and hypertension registers, involvement of health care personnel, and health education of the entire community. As a result of this programme, the blood pressure in the age group 30-59 years decreased on average by 3/2 mmHg among men and by 6/3.5 mmHg among women; the mean blood pressure level decreased twice as much in hypertensive subjects as in the entire population in the intervention areas of the study.

The dynamics of blood pressure in populations and hypertensive cohorts.

Two sets of data, derived from the WHO Cooperative Hypertension Community Control Project and concerned with spontaneous changes of blood pressure over a period of five years, are described. The first deals with the community as a whole, studied through the examination of two independent random samples of the same population made five years apart. The second pertains to cohorts of hypertensive subjects included in the hypertension registers from various centres participating in the WHO programme and followed up for four years.The population blood pressure distribution showed a clear shift towards lower levels for both systolic and diastolic values. The mean changes, however, were smaller than 5 mmHg. In the subjects initially labelled as "hypertensive", the effects of "regression to the mean" were apparent both in the short-term evaluation (five months) and, more importantly, after four years of follow-up. In the latter case, the decreases were more remarkable in the first year but continued to show until the third year.These findings suggest that a "controlled" design is necessary not only in prospective clinical trials but also in community projects where the effects of an intervention on blood pressure are to be evaluated.

Conceptual and measurement issues in assessing disability cross-nationally: Analysis of a WHO-sponsored survey of the disablement process in Indonesia.

The World Health Organization, in collaboration with the Indonesian Institute of Health Research and Development, conducted a survey of disability in 14 of 24 Indonesian provinces. This survey was designed to assess the validity of the disablement process as described in WHO's (1980) classification of impairments, disabilities, and handicaps. We analyzed these data with a multivariate procedure which simultaneously identifies subgroups in the surveyed population and the typical attributes of those subgroups. The purpose of the analysis was to (a) determine the association of basic patterns of physical and psychological impairments with disabilities (limitations of the ability to perform certain functions) and handicaps (limitations in the ability to fulfill social roles); (b) assess how those disabilities and handicaps were expressed in urban and rural contexts in a developing country, and (c) determine how the relation of impairments with disabilities and handicaps varied with age.