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The 5-a-Day Power Plus program targeted multiethnic fourth- and fifth-grade students in 10 intervention and 10 control urban elementary schools in St. Paul, Minnesota, to increase fruit and vegetable consumption. The intervention included behavioral curricula in classrooms, parental involvement, school food service changes, and food industry support. Process evaluation was conducted by using surveys and classroom and lunchroom observations to assess the characteristics of teachers and food service staff, the degree the intervention was implemented as intended, and exteral factors that may have affected the program results. Results showed high levels of participation, dose, and fidelity for all of the intervention components, with the exception of parental involvement. The process evaluation findings help explain why the increase in fruit and vegetable consumption occurred mostly at school lunch and not at home. Future intervention research should focus on creating new and potent strategies for parental involvement and for increasing the appeal and availability of vegetables. [ABSTRACT FROM AUTHOR]

Involvement of the cerebellum in the pathophysiology of hypoxic-ischemic encephalopathy (HIE) in preterm infants is increasingly recognized. We aimed to assess the neuroprotective potential of intravenously administered multipotent adult progenitor cells (MAPCs) in the preterm cerebellum. Instrumented preterm ovine fetuses were subjected to transient global hypoxia-ischemia (HI) by 25 minutes of umbilical cord occlusion at 0.7 of gestation. After reperfusion, two doses of MAPCs were administered intravenously. MAPCs are a plastic adherent bone-marrow-derived population of adult progenitor cells with neuroprotective potency in experimental and clinical studies. Global HI caused marked cortical injury in the cerebellum, histologically indicated by disruption of cortical strata, impeded Purkinje cell development, and decreased dendritic arborization. Furthermore, global HI induced histopathological microgliosis, hypomyelination, and disruption of white matter organization. MAPC treatment significantly prevented cortical injury and region-specifically attenuated white matter injury in the cerebellum following global HI. Diffusion tensor imaging (DTI) detected HI-induced injury and MAPC neuroprotection in the preterm cerebellum. This study has demonstrated in a preclinical large animal model that early systemic MAPC therapy improved structural injury of the preterm cerebellum following global HI. Microstructural improvement was detectable with DTI. These findings support the potential of MAPC therapy for the treatment of HIE and the added clinical value of DTI for the detection of cerebellar injury and the evaluation of cell-based therapy., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy., Methods: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry., Results: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake., Discussion: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro ., (© 2019 The Author(s).)

Following spinal cord injury (SCI), inflammation amplifies damage beyond the initial insult, providing an opportunity for targeted treatments. An ideal protective therapy would reduce both edema within the lesion area and the activation/infiltration of detrimental immune cells. Previous investigations demonstrated the efficacy of intravenous injection of multipotent adult progenitor cells (MAPC ® ) to modulate immune response following SCI, leading to significant improvements in tissue sparing, locomotor and urological functions. Separate studies have demonstrated that tissue inhibitor of matrix metalloproteinase-3 (TIMP3) reduces blood-brain barrier permeability following traumatic brain injury in a mouse model, leading to improved functional recovery. This study examined whether TIMP3, delivered alone or in concert with MAPC cells, improves functional recovery from a contusion SCI in a rat model. The results suggest that intravenous delivery of MAPC cell therapy 1 day following acute SCI significantly improves tissue sparing and impacts functional recovery. TIMP3 treatment provided no significant benefit, and further, when co-administered with MAPC cells, it abrogated the therapeutic effects of MAPC cell therapy. Importantly, this study demonstrated for the first time that acute treatment of SCI with MAPC cells can significantly reduce the incidence of urinary tract infection (UTI) and the use of antibiotics for UTI treatment.

There is an urgent need for therapies that could reduce the disease burden of preterm hypoxic-ischemic encephalopathy. Here, we evaluate the long-term effects of multipotent adult progenitor cells (MAPC) on long-term behavioral outcomes in a preterm rat model of perinatal asphyxia. Rats of both sexes were treated with two doses of MAPCs within 24 h after the insult. Locomotor, cognitive and psychiatric impairments were evaluated starting at 1.5 (juvenile) and 6 months (adult). Hypoxia-ischemia affected locomotion, cognition, and anxiety in a sex-dependent manner, with higher vulnerability observed in males. The MAPC therapy partially attenuated deficits in object recognition memory in females of all tested ages, and in the adult males. The hypoxic insult caused delayed hyperactivity in adult males, which was corrected by MAPC therapy. These results suggest that MAPCs may have long-term benefits for neurodevelopmental outcome after preterm birth and global hypoxia-ischemia, which warrants further preclinical exploration., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Background: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals., Methods: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury., Results: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI., Conclusions: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.

Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive and trophic support functions. While MSCs from different sources frequently display a similar appearance in culture, they often show differences in their surface marker and gene expression profiles. Although bone marrow is considered the "gold standard" tissue to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from more easily accessible extra-embryonic tissues such as the umbilical cord. In this study, we defined the best way to isolate MSCs from the Wharton's jelly of the human umbilical cord (WJ-MSC) and assessed the mesenchymal and immunological phenotype of BM-MSC and WJ-MSC. Moreover, the gene expression profile of established WJ-MSC cultures was compared to two different bone marrow-derived stem cell populations (BM-MSC and multipotent adult progenitor cells or MAPC ® ). We observed that explant culturing of Wharton's jelly matrix is superior to collagenase tissue digestion for obtaining mesenchymal-like cells, with explant isolated cells displaying increased expansion potential. While being phenotypically similar to adult MSCs, WJ-MSC show a different gene expression profile. Gene ontology analysis revealed that genes associated with cell adhesion, proliferation, and immune system functioning are enriched in WJ-MSC. In vivo transplantation confirms their immune modulatory effect on T cells, similar to BM-MSC and MAPC. Furthermore, WJ-MSC intrinsically overexpress genes involved in neurotrophic support and their secretome induces neuronal maturation of SH-SY5Y neuroblastoma cells to a greater extent than BM-MSC. This signature makes WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated central nervous system disorders such as multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

Stem cell therapy modulates not only the local microenvironment of the brain but also the systemic immune responses. We explored the impact of human multipotent adult progenitor cells (MAPC) modulating splenic activation and peripheral immune responses after ischemic stroke. Hundred twenty-six Long-Evans adult male rats underwent middle cerebral artery occlusion. Twenty-four hours later, they received IV MAPC or saline treatment. At 3 days after infusion, RNA was isolated from the injured cortex and spleen for microarray analysis. Spleen mass, splenocyte phenotype, and releasing cytokines were measured. Serum cytokines, MAPC biodistribution, brain lesion sizes and neurofunctional deficits were compared in rats treated with MAPC or saline with and without spleens. Stroked animals treated with MAPC exhibited genes that more closely resembled animals with sham surgery. Gene categories downregulated by MAPC included leukocyte activation, antigen presentation, and immune effector processing, associated with the signaling pathways regulated by TNF-α, IL-1β, IL-6, and IFN-γ within the brain. MAPC treatment restored spleen mass reduction caused by stroke, elevated Treg cells within the spleen, increased IL-10 and decreased IL-1β released by splenocytes. MAPC reduced IL-6 and IL-1β and upregulated IL-10 serum levels. Compared with saline, MAPC enhance stroke recovery in rats with intact spleens but had no effects in rats without spleens. MAPC restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Immunomodulation of the splenic response by the intravenous administration of MAPC may create a more favorable environment for brain repair after stroke. Stem Cells 2017;35:1290-1302., (© 2017 AlphaMed Press.)

Background: Multipotent adult progenitor cells are a bone marrow-derived, allogeneic, cell therapy product that modulates the immune system, and represents a promising therapy for acute stroke. We aimed to identify the highest, well-tolerated, and safest single dose of multipotent adult progenitor cells, and if they were efficacious as a treatment for stroke recovery., Methods: We did a phase 2, randomised, double-blind, placebo-controlled, dose-escalation trial of intravenous multipotent adult progenitor cells in 33 centres in the UK and the USA. We used a computer-generated randomisation sequence and interactive voice and web response system to assign patients aged 18-83 years with moderately severe acute ischaemic stroke and a National Institutes of Health Stroke Scale (NIHSS) score of 8-20 to treatment with intravenous multipotent adult progenitor cells (400 million or 1200 million cells) or placebo between 24 h and 48 h after symptom onset. Patients were ineligible if there was a change in NIHSS of four or more points during at least a 6 h period between screening and randomisation, had brainstem or lacunar infarct, a substantial comorbid disease, an inability to undergo an MRI scan, or had a history of splenectomy. In group 1, patients were enrolled and randomly assigned in a 3:1 ratio to receive 400 million cells or placebo and assessed for safety through 7 days. In group 2, patients were randomly assigned in a 3:1 ratio to receive 1200 million cells or placebo and assessed for safety through the first 7 days. In group 3, patients were enrolled, randomly assigned, and stratified by baseline NIHSS score to receive 1200 million cells or placebo in a 1:1 ratio within 24-48 h. Patients, investigators, and clinicians were masked to treatment assignment. The primary safety outcome was dose-limiting toxicity effects. The primary efficacy endpoint was global stroke recovery, which combines dichotomised results from the modified Rankin scale, change in NIHSS score from baseline, and Barthel index at day 90. Analysis was by intention to treat (ITT) including all patients in groups 2 and 3 who received the investigational agent or placebo. This study is registered with ClinicalTrials.gov, number NCT01436487., Findings: The study was done between Oct 24, 2011, and Dec 7, 2015. After safety assessments in eight patients in group 1, 129 patients were randomly assigned (67 to receive multipotent adult progenitor cells and 62 to receive placebo) in groups 2 and 3 (1200 million cells). The ITT populations consisted of 65 patients who received multipotent adult progenitor cells and 61 patients who received placebo. There were no dose-limiting toxicity events in either group. There were no infusional or allergic reactions and no difference in treatment-emergent adverse events between the groups (64 [99%] of 65 patients in the multipotent adult progenitor cell group vs 59 [97%] of 61 in the placebo group). There was no difference between the multipotent adult progenitor cell group and placebo groups in global stroke recovery at day 90 (odds ratio 1·08 [95% CI 0·55-2·09], p=0·83)., Interpretation: Administration of multipotent adult progenitor cells was safe and well tolerated in patients with acute ischaemic stroke. Although no significant improvement was observed at 90 days in neurological outcomes with multipotent adult progenitor cells treatment, further clinical trials evaluating the efficacy of the intervention in an earlier time window after stroke (<36 h) are planned., Funding: Athersys Inc., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Macrophages and microglia are key effector cells in immune-mediated neuroinflammatory disorders. Driving myeloid cells towards an anti-inflammatory, tissue repair-promoting phenotype is considered a promising strategy to halt neuroinflammation and promote central nervous system (CNS) repair. In this study, we defined the impact of multipotent adult progenitor cells (MAPC), a stem cell population sharing common mesodermal origin with mesenchymal stem cells (MSCs), on the phenotype of macrophages and the reciprocal interactions between these two cell types. We show that MAPC suppress the secretion of tumor necrosis factor alpha (TNF- α ) by inflammatory macrophages partially through a cyclooxygenase 2- (COX-2-) dependent mechanism. In turn, we demonstrate that inflammatory macrophages trigger the immunomodulatory properties of MAPC, including an increased expression of immunomodulatory mediators (e.g., inducible nitric oxide synthase (iNOS) and COX-2), chemokines, and chemokine receptors. Macrophage-primed MAPC secrete soluble factors that suppress TNF- α release by macrophages. Moreover, the MAPC secretome suppresses the antigen-specific proliferation of autoreactive T cells and the T cell stimulatory capacity of macrophages. Finally, MAPC increase their motility towards secreted factors of activated macrophages. Collectively, these in vitro findings reveal intimate reciprocal interactions between MAPC and inflammatory macrophages, which are of importance in the design of MAPC-based therapeutic strategies for neuroinflammatory disorders in which myeloid cells play a crucial role.

Background: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties., Methods: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology., Results: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain., Conclusions: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation, and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

Introduction: Stem cell-based therapies are currently widely explored as a tool to treat neuroimmune diseases. Multipotent adult progenitor cells (MAPC) have been suggested to have strong immunomodulatory and neuroprotective properties in several experimental models. In this study, we investigate whether MAPC are of therapeutic interest for neuroinflammatory disorders such as multiple sclerosis by evaluating their capacities to modulate crucial pathological features and gain insights into the molecular pathways involved., Methods: Rat MAPC were treated with combinations of pro-inflammatory cytokines that are closely associated with neuroinflammatory conditions, a process called licensing. mRNA expression of immunomodulatory molecules, chemokines and chemokine receptors was investigated. The migratory potential of licensed rat MAPC towards a broad spectrum of chemokines was tested in a Transwell assay. Furthermore, the effect of licensing on the ability of rat MAPC to attract and suppress the proliferation of encephalitogenic T cells was assessed. Finally, neuroprotective properties of rat MAPC were determined in the context of protection from oxidative stress of oligodendrocytes. Therefore, rat MAPC were incubated with conditioned medium of OLN93 cells subjected to sublethal doses of hydrogen peroxide and the gene expression of neurotrophic factors was assessed., Results: After licensing, a wide variety of immunomodulatory molecules and chemokines, including inducible nitric oxide synthase and fractalkine, were upregulated by rat MAPC. The migratory properties of rat MAPC towards various chemokines were also altered. In addition, rat MAPC were found to inhibit antigen-specific T-cell proliferation and this suppressive effect was further enhanced after pro-inflammatory treatment. This phenomenon was partially mediated through inducible nitric oxide synthase or cyclooxygenase-2. Activated rat MAPC secreted factors that led to attraction of myelin-specific T cells. Finally, exposure of rat MAPC to an in vitro simulated neurodegenerative environment induced the upregulation of mRNA levels of vascular endothelial growth factor and ciliary neurotrophic factor. Factors secreted by rat MAPC in response to this environment partially protected OLN93 cells from hydrogen peroxide-induced cell death., Conclusions: Rat MAPC possess immune modulatory and neuroprotective properties which are enhanced in response to neuroinflammatory signals. These findings thereby warrant further research to evaluate MAPC transplantation as a therapeutic approach in diseases with an immunological and neurodegenerative component such as multiple sclerosis.

Background: There is growing interest in neurorestorative and reparative therapies after acute stroke. MultiStem is an allogeneic cell therapy treatment comprising a population of multipotent adherent bone marrow cells that has shown safety in clinical trials of myocardial infarction and graft vs. host disease, as well as preclinical evidence of activity in stroke and other neurological damage models. MultiStem is now being evaluated in a clinical trial in patients that have suffered an ischemic stroke, in which the product is administered intravenously 24-36 h after the ischemic event., Methods: The Phase 2 randomized, double-blind, placebo-controlled, multicenter dose-escalation trial will consist of three treatment cohorts, including a placebo group, and two treatment groups involving dose tiers of either 400 million or 1200 million cells per patient. Patients will be treated at 24-36 h after stroke. The two primary objectives are to determine the highest well-tolerated and safe single dose of MultiStem up to a maximum of 1200 million total cells in subjects with ischemic stroke and to determine the efficacy of MultiStem on functional outcome in subjects with stroke as measured by the modified Rankin Scale at 90 days. Patients will also be evaluated using the National Institutes of Health Stroke Scale and Barthel Index. The study will explore other aspects including, uniquely, the measurement of spleen size after stroke by magnetic resonance imaging or computed tomography imaging., Conclusions and Future Direction: If MultiStem is safe and there is a signal of efficacy, a late stage phase IIb-III trial is planned., (© 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization.)

We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation.

Introduction: We have demonstrated previously that the intravenous delivery of multipotent adult progenitor cells (MAPC) after traumatic brain injury affords neuroprotection via interaction with splenocytes, leading to an increase in systemic anti-inflammatory cytokines. We hypothesize that the observed modulation of the systemic inflammatory milieu is related to T regulatory cells and a subsequent increase in the locoregional neuroprotective M2 macrophage population., Methods: C57B6 mice were injected with intravenous MAPC 2 and 24 hours after controlled cortical impact injury. Animals were euthanized 24, 48, 72, and 120 hours after injury. In vivo, the proportion of CD4(+)/CD25(+)/FOXP3(+) T-regulatory cells were measured in the splenocyte population and plasma. In addition, the brain CD86(+) M1 and CD206(+) M2 macrophage populations were quantified. A series of in vitro co-cultures were completed to investigate the need for direct MAPC:splenocyte contact as well as the effect of MAPC therapy on M1 and M2 macrophage subtype apoptosis and proliferation., Results: Significant increases in the splenocyte and plasma T regulatory cell populations were observed with MAPC therapy at 24 and 48 hours, respectively. In addition, MAPC therapy was associated with an increase in the brain M2/M1 macrophage ratio at 24, 48 and 120 hours after cortical injury. In vitro cultures of activated microglia with supernatant derived from MAPC:splenocyte co-cultures also demonstrated an increase in the M2/M1 ratio. The observed changes were secondary to an increase in M1 macrophage apoptosis., Conclusions: The data show that the intravenous delivery of MAPC after cortical injury results in increases in T regulatory cells in splenocytes and plasma with a concordant increase in the locoregional M2/M1 macrophage ratio. Direct contact between the MAPC and splenocytes is required to modulate activated microglia, adding further evidence to the central role of the spleen in MAPC-mediated neuroprotection.

Mucopolysaccharidosis type I (MPS-I; Hurler syndrome) is an inborn error of metabolism caused by lack of the functional lysosomal glycosaminoglycan (GAG)-degrading enzyme α-L-iduronidase (IDUA). Without treatment, the resulting GAG accumulation causes multisystem dysfunction and death within the first decade. Current treatments include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy. HSCT ameliorates clinical features and extends life but is not available to all patients, and inadequately corrects the most devastating features of the disease including mental retardation and skeletal deformities. Recent developments suggest that stem cells can be used to deliver needed enzymes to the central nervous system. To test this concept, we transplanted bone marrow-derived normal adult human MultiStem® cells into the cerebral lateral ventricles of immunodeficient MPS-I neonatal mice. Transplanted cells and human-specific DNA were detected in the hippocampal formation, striatum, and other areas of the central nervous system. Brain tissue assays revealed significant long-term decrease in GAG levels in the hippocampus and striatum. Sensorimotor testing 6 months after transplantation demonstrated significantly improved rotarod performance of transplanted mice in comparison to nontransplanted and sham-transplanted control animals. These results suggest that a single injection of MultiStem cells into the cerebral ventricles of neonatal MPS-I mice induces sustained reduction in GAG accumulation within the brain, and modest long-term improvement in sensorimotor function.

Introduction: Due to the publicity about stem cell transplantation for the treatment of cerebral palsy, many families seek information on treatment, and many travel overseas for cell transplantation. Even so, there is little scientific confirmation of benefit, and therefore existing knowledge in the field must be summarized., Areas Covered: This paper addresses the clinical protocols examining the problem, types of stem cells available for transplant, experimental models used to test the benefit of the cells, possible mechanisms of action, potential complications of cell treatment and what is needed in the field to help accelerate cell-based therapies., Expert Opinion: While stem cells may be beneficial in acute injuries of the CNS the biology of stem cells is not well enough understood in chronic injuries or disorders such as cerebral palsy. More work is required at the basic level of stem cell biology, in the development of animal models, and finally in well-conceived clinical trials.

Macrophage-mediated axonal dieback presents an additional challenge to regenerating axons after spinal cord injury. Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to ameliorate this detrimental inflammation-related process has not been investigated. Using an in vitro model of axonal dieback as well as an adult rat dorsal column crush model of spinal cord injury, we found that multipotent adult progenitor cells (MAPCs) can affect both macrophages and dystrophic neurons simultaneously. MAPCs significantly decrease MMP-9 (matrix metalloproteinase-9) release from macrophages, effectively preventing induction of axonal dieback. MAPCs also induce a shift in macrophages from an M1, or "classically activated" proinflammatory state, to an M2, or "alternatively activated" antiinflammatory state. In addition to these effects on macrophages, MAPCs promote sensory neurite outgrowth, induce sprouting, and further enable axons to overcome the negative effects of macrophages as well as inhibitory proteoglycans in their environment by increasing their intrinsic growth capacity. Our results demonstrate that MAPCs have therapeutic benefits after spinal cord injury and provide specific evidence that adult stem cells exert positive immunomodulatory and neurotrophic influences.

Recent investigation has shown an interaction between transplanted progenitor cells and resident splenocytes leading to the modulation of the immunologic response in neurological injury. We hypothesize that the intravenous injection of multipotent adult progenitor cells (MAPC) confers neurovascular protection after traumatic brain injury through an interaction with resident splenocytes, subsequently leading to preservation of the blood brain barrier. Four groups of rats underwent controlled cortical impact injury (3 groups) or sham injury (1 group). MAPC were injected via the tail vein at two doses (2*10(6) MAPC/kg or 10*10(6) MAPC/kg) 2 and 24h after injury. Blood brain barrier permeability was assessed by measuring Evans blue dye extravasation (n=6/group). Additionally, splenic mass was measured (n=12/group) followed by splenocyte characterization (n=9/group) including: cell cycle analysis (n=6/group), apoptosis index (n=6/group), cell proliferation (n=6/group), and inflammatory cytokine measurements (n=6/group). Vascular architecture was determined by immunohistochemistry (n=3/group). Traumatic brain injury results in a decrease in splenic mass and increased blood brain barrier permeability. Intravenous infusion of MAPC preserved splenic mass and returned blood brain barrier permeability towards control sham injured levels. Splenocyte characterization indicated an increase in the number and proliferative rate of CD4+ T cells as well as an increase in IL-4 and IL-10 production in stimulated splenocytes isolated from the MAPC treatment groups. Immunohistochemistry demonstrated stabilization of the vascular architecture in the peri-lesion area. Traumatic brain injury causes a reduction in splenic mass that correlates with an increase in circulating immune cells leading to increased blood brain barrier permeability. The intravenous injection of MAPC preserves splenic mass and the integrity of the blood brain barrier. Furthermore, the co-localization of transplanted MAPC and resident CD4+ splenocytes is associated with a global increase in IL-4 and IL-10 production and stabilization of the cerebral microvasculature tight junction proteins., (Copyright © 2010 Elsevier Inc. All rights reserved.)

CRTh2 (DP(2)) is a prostaglandin D(2) receptor implicated in the recruitment of eosinophils and basophils within the asthmatic lung. Here we report the discovery of a novel series of 3-indolyl sultam antagonists with low nM affinity for CRTh2. These compounds proved to be selective over the other primary prostaglandin D(2) receptor (DP1) as well as the thromboxane A(2) receptor (TP)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Once hypoxic-ischemic (HI) injury ensues in the human neonate at birth, the resulting brain damage lasts throughout the individual's lifetime, as no ameliorative treatments are currently available. We have recently shown that intracerebral transplantation of multipotent adult progenitor cells (MAPCs) results in behavioral improvement and reduction in ischemic cell loss in neonatal rat HI-injury model. In an attempt to advance this cellular therapy to the clinic, we explored the more practical and less invasive intravenous administration of MAPCs. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, then 7 days later received intracerebral or intravenous injections of allogeneic rat MAPCs. On post-transplantation days 7 and 14, the animals that received MAPCs via the intracerebral or intravenous route exhibited improved motor and neurologic scores compared with those that received vehicle infusion alone. Immunohistochemical evaluations at day 14 after transplantation revealed that both intracerebrally and intravenously transplanted MAPCs were detected in the ischemic hippocampal area. The degree of hippocampal cell preservation was almost the same in the two treatment groups and greater than that in the vehicle group. These results show that intravenous delivery of MAPCs is a feasible and efficacious cell therapy with potential for clinical use.

The use of adult stem cells as therapeutic agents to treat disease has become increasingly prevalent. During the last decade, isolated and expanded stem and progenitor cells have demonstrated the capacity to differentiate into multiple cell types. Early optimism that in vitro differentiation capacity would translate into in vivo tissue regeneration has lessened and identifying the mechanisms that underlie the benefit of stem cell repair is an emerging area of investigation. This review considers several of the pathways and mechanisms required for adult stem cell repair. These mechanisms include the mobilization and the homing of stem cells to sites of injury, immunomodulatory effect of stem cells, and the association of stem cells with increased vascularization of injured tissue. These data suggest that the unique properties of adult stem cells can be utilized to treat a wide variety of diseases that cannot be treated with existing pharmacological agents, and prompt new paradigms for the bio-pharmacokinetics of biological expressed by efficacious stem cells.

Over the past 5 years, adult pluripotent stem cell lines have been isolated from multiple organs and tissues in laboratories worldwide. Adult pluripotent stem cells are capable of regenerating tissues of all three primitive germ layers and express pluripotency markers, such as Oct4 or telomerase, which are associated with the primitive stem cell properties of embryonic stem cells. As our collective understanding of the biology of these unique cells has improved, so has our ability to isolate, expand and subsequently evaluate them as therapeutics in preclinical models of acute injury and disease. Pluripotent adult stem cells, as opposed to tissue-restricted adult stem cells, such as mesenchymal stromal cells; have extensive replicative capacity enabling large-scale clinical expansion. This is essential to achieving consistent clinical response data and enabling the cost-effective production necessary for commercial adoption. In addition, investigators have reported effective use of allogeneic adult pluripotent stem cells in acute ischemic injury models in the heart and brain, supporting the 'off the shelf' product concept for this cellular therapy. In this article, the authors review preclinical animal data demonstrating the benefit of pluripotent adult progenitor cells in the treatment of ischemic injuries of the heart, vascular system and CNS.

Children born with hypoxic-ischemic (HI) brain injury account for a significant number of live births wherein no clinical treatment is available. Limited clinical trials of stem cell therapy have been initiated in a number-of neurological disorders, but the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. One major postulated mechanism underlying therapeutic benefits of stem cell therapy involves stimulation of endogenous neurogenesis via transplantation of exogenous stem cells. To this end, transplantation has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents as highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. Here, we investigated the efficacy of intrahippocampal transplantation of multipotent progenitor cells (MPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, which involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI injury, animals received stereotaxic hippocampal injections of vehicle or cryopre-served MPCs (thawed just prior to transplantation) derived either from Sprague-Dawley rats (syngeneic) or Fisher rats (allogeneic). All animals were treated with daily immunosuppression throughout the survival period. Behavioral tests were conducted on posttransplantation days 7 and 14 using the elevated body swing test and the rotarod to reveal general and coordinated motor functions. MPC transplanted animals exhibited reduced motor asymmetry and longer time spent on the rotarod than those that received the vehicle infusion. Both syngeneic and allogeneic MPC transplanted injured animals did not significantly differ in their behavioral improvements at both test periods. Immunohistochemical evaluations of graft survival after behavioral testing at day 14 posttransplantation revealed that syngeneic and allogeneic transplanted MPCs survived in the hippocampal region. These results demonstrate for the first time that transplantation of MPCs ameliorated motor deficits associated with HI injury. In view of comparable behavioral recovery produced by syngeneic and allogeneic MPC grafts, allogeneic transplantation poses as a feasible and efficacious cell replacement strategy with direct clinical application. An equally major finding is the observation lending support to the hippocampus as an excellent target brain region for stem cell therapy in treating HI injury.

There is currently no treatment for neonatal hypoxic-ischemic (HI) injury. Although limited clinical trials of stem cell therapy have been initiated in a number of neurological disorders, the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. Stem cell therapy, via stimulation of endogenous stem cells or transplantation of exogenous stem cells, has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has also been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents a highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. In the present study, we investigated the efficacy of intrahippocampal transplantation of multipotent adult progenitor cells (MAPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day old Sprague-Dawley rats were initially subjected to unilateral HI injury, that involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI

The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of beta-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative Galpha(q)-(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

The 5-a-Day Power Plus program targeted multiethnic fourth- and fifth-grade students in 10 intervention and 10 control urban elementary schools in St. Paul, Minnesota, to increase fruit and vegetable consumption. The intervention included behavioral curricula in classrooms, parental involvement, school food service changes, and food industry support. Process evaluation was conducted by using surveys and classroom and lunchroom observations to assess the characteristics of teachers and food service staff, the degree the intervention was implemented as intended, and exteral factors that may have affected the program results. Results showed high levels of participation, dose, and fidelity for all of the intervention components, with the exception of parental involvement. The process evaluation findings help explain why the increase in fruit and vegetable consumption occurred mostly at school lunch and not at home. Future intervention research should focus on creating new and potent strategies for parental involvement and for increasing the appeal and availability of vegetables.

beta-2 Adrenergic receptors (B2ARs) are endocytosed by clathrin-coated pits. This process serves specialized functions in signal transduction and receptor regulation, raising the question of whether B2ARs are associated with biochemically specialized membrane vesicles during their endocytic trafficking. Here we show that B2ARs are endocytosed by a distinct subpopulation of clathrin-coated pits, which represent a limited subset of coated pits present in the plasma membrane, even in cells overexpressing both B2ARs and beta-arrestin. Coated pits mediating agonist-induced endocytosis of B2ARs differ from other coated pits mediating constitutive endocytosis of transferrin receptors in their temperature dependence for fission from the plasma membrane and in the association of their membrane coats with beta-arrestin. Endocytosis of these coated pits generates endocytic vesicles selectively enriched in B2ARs, which fuse within approximately 10 min after their formation with a common population of endosomes containing both B2ARs and transferrin receptors. These observations demonstrate, for the first time, the existence of a functionally and biochemically distinct subpopulation of clathrin-coated pits that mediate the agonist-regulated endocytosis of G-protein-coupled receptors, and they suggest a new model for the formation of compositionally specialized membrane vesicles at the earliest stage of the endocytic pathway.

Objectives: A randomized school based trial sought to increase fruit and vegetable consumption among children using a multicomponent approach., Methods: The intervention, conducted in 20 elementary schools in St. Paul, targeted a multiethnic group of children who were in the fourth grade in spring 1995 and the fifth grade in fall 1995. The intervention consisted of behavioral curricula in classrooms, parental involvement, school food service changes, and industry support and involvement. Lunchroom observations and 24-hour food recalls measured food consumption. Parent telephone surveys and a health behavior questionnaire measured psychosocial factors., Results: The intervention increased lunchtime fruit consumption and combined fruit and vegetable consumption, lunchtime vegetable consumption among girls, and daily fruit consumption as well as the proportion of total daily calories attributable to fruits and vegetables., Conclusions: Multicomponent school-based programs can increase fruit and vegetable consumption among children. Greater involvement of parents and more attention to increasing vegetable consumption, especially among boys, remain challenges in future intervention research.

We have combined long synthetic arrays of alpha satellite DNA with telomeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6-10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-generation system for the construction of human artificial chromosomes should be suitable for dissecting the sequence requirements of human centromeres, as well as developing constructs useful for therapeutic applications.

We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clone (II/G); Na/K-ATPase is selectively retained in the basal-lateral membrane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is sorted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells. A glycosyl-phosphatidylinositol (GPI)-anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, GlcCer) are preferentially transported to the apical membrane in clone II/G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equally to both apical and basal-lateral membranes, similar to Na/K-ATPase. To examine this apparent inter-relationship between sorting of GlcCer, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B1 (FB1). In the presence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and basal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membranes, respectively. These results show that the generation of complete cell surface polarity of Na/K-ATPase involves a hierarchy of sorting mechanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclusion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane proteins.

Development and maintenance of cell-surface polarity in epithelial cells requires specialized localization of proteins to functionally and structurally distinct plasma membrane domains. The organization of these domains is dependent upon targeted delivery of transport vesicles between different membrane compartments, and upon protein sorting in the membranes of the Golgi complex and cell surface. Increasing evidence has been gathered in recent years that cytoskeletal components facilitate these processes.

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Restriction of sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase) to either the apical or basal-lateral membrane domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted membrane distribution of Na+,K(+)-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na+,K(+)-ATPase may be generated in normal polarized epithelial and in disease states.

We have compared lysosomal enzyme distributions on density gradients and rates of transport of endocytic markers for actively-growing and confluent cells. While it has been previously established that mammalian cells accumulate lysosomal enzymes during quiescence, we show that this accumulation is predominantly in residual bodies (p greater than 1.12 g/ml) rather than in dense lysosomes (p = 1.08-1.10 g/ml) and does not represent a change in the endosomal and lysosomal enzyme content. The accumulation is not caused by a change in the rate of production of dense lysosomes, since the rate of transfer of epidermal growth factor (EGF) from light to dense compartments is the same between confluent and subconfluent cells. Confluent cultures have a higher rate of initial pinocytosis, and a higher rate of retroendocytosis and/or recycling, causing a net lower rate of accumulation of fluid-phase material. The accumulation of residual bodies in confluent cultures may be caused by a lower rate of exocytosis of their contents and/or a lack of dilution by cell division. The data indicate that the impact of culture confluence must be carefully assessed in experiments designed to analyze endocytic pathways.