Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays.

Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of "misleading" positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of "misleading" positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by "misleading positive" genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.

Cellular Therapies Clinical Research Roadmap: lessons learned on how to move a cellular therapy into a clinical trial.

BACKGROUND AIMS: A clinical research roadmap has been developed as a resource for researchers to identify critical areas and potential pitfalls when transitioning a cellular therapy product from the research laboratory, by means of an Investigational New Drug (IND) application, into early-phase clinical trials. The roadmap describes four key areas: basic and preclinical research, resource development, translational research and Good Manufacturing Practice (GMP) and IND assembly and submission. METHODS: Basic and preclinical research identifies a new therapeutic concept and demonstrates its potential value with the use of a model of the relevant disease. During resource development, the appropriate specialists and the required expertise to bring this product into the clinic are identified (eg, researchers, regulatory specialists, GMP manufacturing staff, clinicians and clinical trials staff, etc). Additionally, the funds required to achieve this goal (or a plan to procure them) are identified. In the next phase, the plan to translate the research product into a clinical-grade therapeutic is developed. Finally regulatory approval to start the trial must be obtained. In the United States, this is done by filing an IND application with the Food and Drug Administration. RESULTS: The National Heart, Lung and Blood Institute-funded Production Assistance for Cellular Therapies program has facilitated the transition of a variety of cellular therapy products from the laboratory into Phase1/2 trials. CONCLUSIONS: The five Production Assistance for Cellular Therapies facilities have assisted investigators by performing translational studies and GMP manufacturing to ensure that cellular products met release specifications and were manufactured safely, reproducibly and at the appropriate scale. The roadmap resulting from this experience is the focus of this article.

Shipping of therapeutic somatic cell products.

BACKGROUND AIMS: Shipment of therapeutic somatic cells between a current good manufacturing practice (cGMP) facility and a clinic or between different cGMP facilities requires validated standard operating procedures (SOP). Under National Heart Lung & Blood Institute (NHLBI) sponsorship, the Production Assistance for Cellular Therapies (PACT) group conducted a validation study for the shipping SOP it has created, including shipments of cryopreserved somatic cells, fresh peripheral blood specimens and apheresis products. METHODS: Comparisons of pre- and post-shipped cells and cell products at the three participating facilities included measurements of viability, phenotypic profiles and cellular functions. The data were analyzed at the University of Pittsburgh Biostatistics Facility. RESULTS: No consistent shipping effects on cell viability, phenotype or functions were detected for cryopreserved and shipped peripheral blood mononuclear cells (PBMC), monocytes, immature dendritic cells (iDC), NK-92 or cytotoxic T cells (CTL). Cryopreserved mesenchymal stromal cells (MSC) had a significantly decreased viability after shipment, but this effect was in part because of inter-laboratory variability in the viable cell counts. Shipments of fresh peripheral blood and apheresis products for the generation of CTL and dendritic cells (DC), respectively, had no significant effects on cell product quality. MSC were successfully generated from fresh bone marrow samples shipped overnight. CONCLUSIONS: This validation study provides a useful set of data for guiding shipments of therapeutic somatic cells in multi-institutional clinical trials.

Developments in clinical cell therapy.

Immunotherapy has become an important part of hematopoietic stem cell (HSC) transplantation and cancer therapy. Regenerative and reparative properties of somatic cell-based therapies hold tremendous promise for repairing injured tissue, preventing and reversing damage to organs, and restoring balance to compromised immune systems. The principles and practices of the diverse aspects of immune therapy for cancer, HSC transplantation and regenerative medicine have many commonalities. This meeting report summarizes a workshop sponsored by the National Heart, Lung and Blood Institute (NHLBI) and Production Assistance for Cellular Therapies (PACT), held on 23-24 April 2009 at the National Institutes of Health (NIH, USA). A series of scientific sessions and speakers highlighted key aspects of the latest scientific, clinical and technologic developments in cell therapy, involving a unique set of cell products with a special emphasis on converging concepts in these fields.

CD34(+) cell selection using small-volume marrow aspirates: a platform for novel cell therapies and regenerative medicine.

BACKGROUND AIMS: This study was initiated to determine whether CD34(+) cell selection of small-volume bone marrow (BM) samples could be performed effectively on the Isolex(R) 300i Magnetic Cell Selection System device and whether the results obtained from these samples were comparable with results from large standard-volume samples. The impact on CD34(+) recovery using a full versus half vial of Isolex(R) CD34 reagent and the effects of shipping a post-selection product were evaluated. METHODS: A protocol to evaluate CD34(+) cell selection with two ranges of smaller volume BM samples (c. 50 mL and c. 100 mL) was developed and instituted at three Production Assistance for Cellular Therapies (PACT) facilities. The study was performed in two phases. RESULTS: In phase I, the mean post-selection CD34(+) recoveries from the two sizes of samples were 104.1% and 103.3% (smallest and largest volumes, respectively), and mean CD34(+) recoveries were 115.6% and 88.7%, with full and half vials of reagent, respectively. Mean CD34(+) recoveries for post-shipment smaller volume samples were 106.8% and for larger volume samples 116.4%; mean CD34(+) recoveries were 99.9% and 127.4% for post-shipment samples processed with full and half vials of reagent, respectively. In phase II, mean CD34(+) recovery was 76.8% for post-selection samples and 74.0% for post-shipment samples. CONCLUSIONS: The results suggest that smaller volume BM sample processing on the Isolex(R) system is as efficient or more efficient compared with standard-volume sample processing. Post-processing mean CD34(+) recovery results obtained using a full or half vial of CD34 reagent were not significantly different.

Using quality improvement to address pain management practices in nursing homes.

The objective of this study was to test whether a quality improvement intervention can improve pain management in nursing homes. Experts in quality improvement and clinical pain management provided nursing home staff leaders with feedback on pain quality indicator data, education in pain management, and technical assistance to apply the Plan-Do-Study-Act model of quality improvement. Trained abstractors completed structured chart audits at baseline and five months to capture quality indicator data related to pain assessment and treatment. Residents in pain who underwent pain assessments increased from 8% to 29% (P < 0.001). Residents receiving non-pharmacological pain treatments increased from 31% to 42% (P = 0.010), but pain medication use did not change. Among residents with daily moderate or excruciating pain, complete pain assessment was associated with increased probability of pain medication use. Quality improvement is a promising method to improve pain management in nursing homes.

Violent traumatic brain injury: occurrence, patient characteristics, and risk factors from the Traumatic Brain Injury Model Systems project.

OBJECTIVES: To examine the occurrence of and characteristics associated with violent traumatic brain injury (TBI) in the Traumatic Brain Injury Model Systems (TBIMS) project for 4 of the 5 original Model Systems centers and to determine the patient characteristics of this group, as well as the risk factors for sustaining such an injury. DESIGN: Prospective evaluation of individuals with violent TBI over a 10-year period. SETTING: Four TBIMS centers. PARTICIPANTS: A total of 1,229 individuals who received acute hospitalization and inpatient rehabilitation care for TBI. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE: The occurrence of a violent TBI. RESULTS: Twenty-six percent of the participants in the TBIMS project sustained a violent TBI. This type of injury was more common in African-American men who were single and slightly older than the average TBI patient, were unemployed before injury, and had had a previous TBI. A higher injury rate was noted in the earlier part of the evaluation period. Those who sustained a violent TBI had higher levels of caregiver burden and disability, as well as decreased productivity and community reintegration at rehabilitation discharge and at 1 and 2 years postinjury. CONCLUSIONS: The occurrence of violent TBI in the TBIMS project is consistent with national trends of decreasing incidence of violent injuries in the 1990s. These results present a profile of those who have been injured through violence. The relative risks for sustaining such an injury appear to be well defined when considering demographic and temporal factors.

Spinal epidural abscess: study of early outcome.

OBJECTIVE: To evaluate the course, complications, and outcomes of individuals with spinal epidural abscess (SEA) and to compare these factors in individuals who had sustained a traumatic spinal cord injury (TSCI). METHOD: This is a retrospective study evaluating risk factors, functional change, and neuromedical complications. Thirty-two adults with SEA, treated on a rehabilitation unit at an urban university medical center, were compared with 32 individuals with TSCI. Groups were matched by lesion level and American Spinal Injury Association classification. RESULTS: Both groups made significant functional improvement as measured by the functional independence measure (FIM), although the SEA group only averaged a 15-point increase, whereas the TSCI group averaged approximately 30 points. When compared with the TSCI group, the SEA group had a higher frequency of pressure ulcers (P < 0.04), and exhibited greater intravenous drug use (P < 0.008). There were no differences between the groups with respect to discharge placement or neuromedical risk factors. CONCLUSION: A number of predisposing factors and neuromedical complications that have significant medical implications were noted in the SEA group. In general, predisposing factors and outcomes were similar between those with SEA and those with TSCI, except for drug use and rate of pressure ulcers. These factors do not appear to relate to differential outcome in discharge placement, however. Although it is important to be aware of factors that place an individual at risk for SEA and appreciate the risks for complications, significant functional improvement can be achieved in this population.

Blunt versus penetrating violent traumatic brain injury: frequency and factors associated with secondary conditions and complications.

OBJECTIVE: To compare types and frequency of medical complications and comorbidities associated with violence-related penetrating traumatic brain injury (TBI) as compared to violence-related blunt TBI. METHOD: Data were collected prospectively at four medical centers participating in the TBI Model Systems (TBIMS) of Care project. A total of 317 individuals met the inclusion criteria for the TBIMS (i.e., showed evidence of a TBI, were age 16 or older, presented to the TBIMS emergency department within 24 hours of injury, and received acute and rehabilitation services within the model system). MAIN OUTCOME MEASURES: Frequency of medical complications and comorbid diseases. RESULTS: Patients with penetrating injuries suffered significantly higher rates of respiratory failure (P =.004), pneumonitis/pneumonia, (P =.002), skull fracture (P =.001), cerebrospinal fluid leak (P =.0005), and hypotonia (P =.001) than did patients with blunt injuries. Prediction of complications and comorbidities via multiple regression revealed that a penetrating violent injury and the severity of injury were independent predictors of a higher rate of medical complications, whereas age and gender did not account for unique variance in the equation. CONCLUSIONS: Penetrating injuries are associated with higher rates of certain medical complications, especially to the pulmonary and central nervous systems. Acute care physicians and physiatrists must be prepared to treat these complications more often in patients with penetrating injuries.