Incorporating blood-based liquid biopsy information into cancer staging: time for a TNMB system?

Tissue biopsy is the standard diagnostic procedure for cancer. Biopsy may also provide material for genotyping, which can assist in the diagnosis and selection of targeted therapies but may fall short in cases of inadequate sampling, particularly from highly heterogeneous tumors. Traditional tissue biopsy suffers greater limitations in its prognostic capability over the course of disease, most obviously as an invasive procedure with potential complications, but also with respect to probable tumor clonal evolution and metastasis over time from initial biopsy evaluation. Recent work highlights circulating tumor DNA (ctDNA) present in the blood as a supplemental, or perhaps an alternative, source of DNA to identify the clinically relevant cancer mutational landscape. Indeed, this noninvasive approach may facilitate repeated monitoring of disease progression and treatment response, serving as a means to guide targeted therapies based on detected actionable mutations in patients with advanced or metastatic solid tumors. Notably, ctDNA is heralding a revolution in the range of genomic profiling and molecular mechanisms to be utilized in the battle against cancer. This review will discuss the biology of ctDNA, current methods of detection and potential applications of this information in tumor diagnosis, treatment, and disease prognosis. Conventional classification of tumors to describe cancer stage follow the TNM notation system, heavily weighting local tumor extent (T), lymph node invasion (N), and detectable metastasis (M). With recent advancements in genomics and bioinformatics, it is conceivable that routine analysis of ctDNA from liquid biopsy (B) may make cancer diagnosis, treatment, and prognosis more accurate for individual patients. We put forward the futuristic concept of TNMB tumor classification, opening a new horizon for precision medicine with the hope of creating better outcomes for cancer patients.

Early-delayed, radiation-induced cognitive deficits in adult rats are heterogeneous and age-dependent.

Patients treated with whole-brain irradiation often develop cognitive deficits that are presumed to result from normal tissue injury. Age is a risk factor for these side effects. We compared the cognitive effects of fractionated whole-brain irradiation (300 kV X rays) in rats irradiated either as young adults or in middle age. A deficit in object memory was apparent at 3 months in rats irradiated as young adults, however, no comparable deficit was apparent in rats irradiated in middle age. In addition, the deficit in object memory in young adults was no longer apparent at 6 and 12 months after fractionated whole-brain irradiation and no radiation-induced deficit was detectable in a spatial memory task at any time, regardless of age at time of irradiation. Thus, clinically relevant fractionated whole-brain irradiation in adult rats resulted in early-delayed cognitive changes that were heterogeneous, transient and age-dependent. The results of the current and previous studies of radiation-induced cognitive changes support the continued investigation and validation of rodent models of radiation-induced brain injury, which are critical for developing and testing new therapies for treatment-induced cognitive dysfunction in cancer survivors.

Systemic effects of fractionated, whole-brain irradiation in young adult and aging rats.

Cranial irradiation is a critical and effective treatment for primary brain tumors and metastases. Unfortunately, most patients who are treated and survive for more than a few months develop neural and cognitive problems as the result of radiation-induced normal tissue injury. The neurobiological mechanisms underlying these cognitive deficits remain largely unknown and there are no validated treatments to prevent or ameliorate them; thus, there is a significant and continuing need for preclinical studies in animal models. Investigations from several laboratories have demonstrated neurobiological changes after cranial irradiation in rodents. To date, however, experimental studies in animal models have included little assessment of the systemic effects of cranial irradiation, despite evidence from the clinic that cranial irradiation results in changes throughout the body and recognition that systemic responses may influence the development of neural and cognitive deficits. This study evaluated systemic effects of clinically relevant, fractionated whole-brain irradiation in adult rats and demonstrates effects on the growth hormone/insulin-like growth factor-I axis, which may contribute to the development of neural changes. These and other systemic responses are important to consider in ongoing efforts to understand the mechanisms of radiation-induced normal tissue injury.

Gamma knife radiosurgery treatment planning for small animals using high-resolution 7T micro-magnetic resonance imaging.

Gamma Knife stereotactic radiosurgery is capable of providing small, high gradient dose distributions to a target with a high level of precision, which makes it an excellent choice for studies of focal irradiations with small animals. However, the Gamma Knife stereotactic radiosurgery process makes use of a human-sized fiducial marker system that requires a field of view of at least 200 mm(2) to relate computed tomography and magnetic resonance images to the Gamma Knife treatment planning software. Thus the Gamma Knife fiducial marker system is five to six times larger than a typical small animal subject. The required large field of view limits the spatial resolution and structural detail available in the animal treatment planning image set. In response to this challenge we have developed a custom-designed stereotactic jig and miniature fiducial marking system that allow small bore high-resolution micro-imaging techniques, such as 7T MR and micro-CT, to be used for treatment planning of Gamma Knife stereotactic radiosurgery focal irradiation of small animals.

Regulation of cytochrome oxidase activity in the rat forebrain throughout adulthood.

Measures of metabolic activity can provide useful indices of the effects of aging on neural function, since sustained changes in neural activity alter metabolic demand and the activity of metabolic enzymes. Previous reports of effects of aging on key enzymes for oxidative metabolism are mixed, however, with some reports that activity declines in the aging brain and others that activity remains stable or increases. We used high-resolution, quantitative histochemistry to test whether cytochrome oxidase (CO) activity changes in the forebrain during adulthood and senescence, measuring activity in each layer of the hippocampus and several cerebral cortical areas. In most forebrain regions, average cytochrome oxidase activity was slightly higher in middle-aged than in young adult rats but did not differ between middle-aged and old rats. Thus, there was no significant change in cytochrome oxidase activity with senescence. Additional analyses indicated that cytochrome oxidase activity is regulated regionally in the brain, as well as focally, and that differences in regional regulation may contribute to variation in CO activity among individuals, which was greater in young and old rats than in middle-aged animals.

Developmental changes in BDNF protein levels in the hamster retina and superior colliculus.

Quantitative studies of ontogenetic changes in the levels of brain-derived neurotrophic factor (BDNF) mRNA and its effector, BDNF protein, are not available for the retinal projection system. We used an electrochemiluminescence immunoassay to measure developmental changes in the tissue concentration of BDNF within the hamster retina and superior colliculus (SC). In the SC, we first detected BDNF (about 9 pg/mg tissue) on embryonic day 14 (E14). BDNF protein concentration in the SC rises about fourfold between (E14) and postnatal day 4 (P4), remains at a plateau through P15, then declines by about one-third to attain its adult level by P18. By contrast, BDNF protein concentration in the retina remains low (about 1 pg/mg tissue) through P12, then increases 4.5-fold to attain its adult level on P18. The developmental changes in retinal and collicular BDNF protein concentrations are temporally correlated with multiple events in the structural and functional maturation of the hamster retinal projection system. Our data suggest roles for BDNF in the cellular mechanisms underlying some of these events and are crucial to the design of experiments to examine those roles.

Intracerebroventricular infusion of insulin-like growth factor-I ameliorates the age-related decline in hippocampal neurogenesis.

The dentate gyrus of the hippocampus is one of few regions in the adult mammalian brain characterized by ongoing neurogenesis. Significantly, recent studies indicate that the rate of neurogenesis in the hippocampus declines with age, perhaps contributing to age-related cognitive changes. Although a variety of factors may influence the addition of new neurons in the adult dentate gyrus, the mechanisms responsible for the age-related reduction remain to be established. Insulin-like growth factor-I (IGF-I) is one promising candidate to regulate neurogenesis in the adult and aging brain since it influences neuronal production during development and since, like the rate of neurogenesis, it decreases with age. In the current study, we used bromodeoxyuridine labeling and multilabel immunofluorescence to assess age-related changes in neuronal production in the dentate gyrus of adult Brown Norway x Fischer 344 rats. In addition, we investigated the relationship between changes in neurogenesis and the age-dependent reduction in IGF-I by evaluating the effect of i.c.v. infusion of IGF-I on neurogenesis in the senescent dentate gyrus. The analyses revealed an age-dependent reduction in the number of newly generated cells in the adult dentate subgranular proliferative zone and, in addition, a 60% reduction in the differentiation of newborn cells into neurons. Restoration of IGF-I levels in senescent rats significantly restored neurogenesis through an approximately three-fold increase in neuronal production. The results of this study suggest that IGF-I may be an important regulator of neurogenesis in the adult and aging hippocampus and that an age-related decline in IGF-I-dependent neurogenesis could contribute to age-related cognitive changes.

Effects of early visual experience and diurnal rhythms on BDNF mRNA and protein levels in the visual system, hippocampus, and cerebellum.

The expression of brain-derived neurotrophic factor (BDNF) mRNA and the secretion of BDNF protein are tightly regulated by neuronal activity. Thus, BDNF has been proposed as a mediator of activity-dependent neural plasticity. Previous studies showed that dark rearing (DR) reduces BDNF mRNA levels in the primary visual cortex (V1), but the effects of visual experience on BDNF protein levels are unknown. We report that rearing in constant light or DR alters BDNF mRNA and protein levels in the retina, superior colliculus (SC), V1, hippocampus (HIPP), and cerebellum (CBL), although the changes in mRNA and protein are not always correlated. Most notably, DR increases BDNF protein levels in V1 although BDNF mRNA is decreased. BDNF protein levels also undergo diurnal changes. In the retina, V1, and SC, BDNF protein levels are higher during the light phase of the circadian cycle than during the dark phase. By contrast, in HIPP and CBL, the tissue concentration of BDNF protein is higher during the dark phase. The discrepancies between the experience-dependent changes in BDNF mRNA and protein suggest that via its effects on neuronal activity, early sensory experience alters the trafficking, as well as the synthesis, of BDNF protein. The circadian changes in BDNF protein suggest that BDNF could cause the diurnal modulation of synaptic efficacy in some neural circuits. The fluctuations in BDNF levels in nonvisual structures suggest a potential role of BDNF in mediating plasticity induced by hormones or motor activity.

Overexpression of brain-derived neurotrophic factor enhances sensory innervation and selectively increases neuron number.

Target-derived neurotrophin growth factors have significant effects on the development and maintenance of the mammalian somatosensory system. Studies of transgenic mice that overexpress neurotrophins NGF and neurotrophin 3 (NT-3) at high levels in skin have shown increased sensory neuron number and enhanced innervation of specific sensory ending types. The effects of two other members of this family, BDNF and NT-4, on sensory neuron development are less clear. This study examined the role of brain-derived neurotrophic factor (BDNF) using transgenic mice that overexpress BDNF in epithelial target tissues of sensory neurons. BDNF transgenic mice had an increase in peripheral innervation density and showed selective effects on neuron survival. Neuron number in trigeminal ganglia, DRG, and SCG were unchanged, although a 38% increase in neurons comprising the placode-derived nodose-petrosal complex occurred. BDNF transgenic skin showed notable enhancement of innervation to hair follicles as detected by PGP9.5 immunolabeling. In nonhairy plantar skin, Meissner corpuscle sensory endings were larger, and the number of Merkel cells with associated innervation was increased. In trigeminal ganglia, neurons expressing trkB receptor were increased threefold, whereas trkA-positive neurons doubled. Analysis of trkB by Northern, reverse transcription-PCR, and Western assays indicated a modest increase in the expression of the T1 truncated receptor and preferential distribution to the periphery. These data indicate that skin-derived BDNF does not enhance survival of cutaneous sensory neurons, although it does promote neurite innervation of specific sites and sensory end organs of the skin.

BDNF injected into the superior colliculus reduces developmental retinal ganglion cell death.

The role of neurotrophins as survival factors for developing CNS neurons, including retinal ganglion cells (RGCs), is uncertain. Null mutations for brain-derived neurotrophic factor (BDNF) or neurotrophin 4 (NT4), individually or together, are without apparent effect on the number of RGCs that survive beyond the period of normal, developmental RGC death. This contrasts with the BDNF dependence of RGCs in vitro and the effectiveness of BDNF in reducing RGC loss after axotomy. To investigate the effect of target-derived neurotrophins on the survival of developing RGCs, we injected BDNF into the superior colliculus (SC) of neonatal hamsters. At the age when the rate of developmental RGC death is greatest, BDNF produces, 20 hr after injection, a 13-15-fold reduction in the rate of RGC pyknosis compared with the rates in vehicle-injected and untreated hamsters. There is no effect 8 hr after injection. Electrochemiluminescence immunoassay measurements of BDNF protein in the retinae and SC of normal and BDNF-treated hamsters demonstrate that the time course of BDNF transport to RGCs supports a role for target-derived BDNF in promoting RGC survival. The effectiveness of pharmacological doses of BDNF in reducing developmental RGC death may be useful in further studies of the mechanisms of stabilization and elimination of immature central neurons.

Polyoma middle T antigen in HL-60 cells accelerates hematopoietic myeloid and monocytic cell differentiation.

Expression of the polyoma virus middle T antigen in HL-60 cells accelerates their differentiation in response to both monocytic and granulocytic differentiation-inducing agents. Middle T-expressing cells treated with the granulocytic inducer retinoic acid or the monocytic inducer 1,25-dihydroxy vitamin D3 differentiated 24 h earlier than parental, mock-electroporated, or vector control cell lines. The rapid onset of differentiation correlated with an increase in the cellular level of the middle T protein as well as two known retinoic-acid-inducible markers in HL-60 cells: the paxillin and transglutaminase gene products. The accelerated functional differentiation response and expression of retinoic-acid-inducible markers indicate that middle T played a causal role in differentiation. Thus, expression of the polyoma middle T antigen in HL-60 cells enhanced a variety of molecular changes associated with cellular differentiation.

K-252a promotes survival and choline acetyltransferase activity in striatal and basal forebrain neuronal cultures.

The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.

K-252a induces tyrosine phosphorylation of the focal adhesion kinase and neurite outgrowth in human neuroblastoma SH-SY5Y cells.

The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.

Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells.

Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.

C-FMS dependent HL-60 cell differentiation and regulation of RB gene expression.

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of G0 arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driven by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions.

K-252a and staurosporine promote choline acetyltransferase activity in rat spinal cord cultures.

The protein kinase inhibitor K-252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose-dependent manner (EC50 of approximately 100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K-252a completely prevented the marked decline in ChAT activity occurring over a 5-day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor staurosporine also increased ChAT activity (EC50 of approximately 0.5 nM). Effective concentrations of K-252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin-like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose-dependent increases in ChAT activity in spinal cord cultures. The combination of K-252a with insulin-like growth factor-I or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K-252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K-252a and staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.

Temporal restriction of neural crest development in vitro: changes in the adrenergic differentiation of neural crest clusters in the presence and absence of an overlay of reconstituted basement membrane-like matrix.

Neural crest clusters were isolated after 18 or 42 h of growth in suspension and plated on a fibronectin substrate in the presence or absence of an overlay of a reconstituted basement membrane-like (RBM) matrix. After 1 week in vitro, cultures of 18-h clusters grown with the RBM gel overlay exhibited a 60-fold increase in the number of catecholamine-positive cells, while the number of melanocytes was decreased to one-quarter the control value. In contrast, clusters isolated after 42 h in suspension developed few adrenergic cells in the presence or absence of the RBM gel overlay. Melanocyte cell number was not altered in 42-h clusters grown in the presence of the RBM gel overlay. Total cell number after 1 week in vitro was similar under all conditions for both 18- and 42-h clusters. The distribution of tyrosine hydroxylase-immunoreactive cells among the experimental conditions was similar to that of catecholamine-positive cells. The presence of the neural tube was not necessary to generate the different developmental properties of clusters isolated at 18 h versus 42 h. These results show that the RBM gel can enhance adrenergic cell development and inhibit melanogenesis in young neural crest cluster cultures. Also, they demonstrate that the capacity of these neural crest cluster cell populations to differentiate and to respond to environmental cues changes as a function of time. These results are compatible either with a model in which the survival of adrenergic precursors within the clusters declines with time or one in which the adrenergic precursors switch their fate to another phenotype.

Coupled down-regulation of the RB retinoblastoma and c-myc genes antecedes cell differentiation: possible role of RB as a "status quo" gene.

The ability of the well known morphogen, retinoic acid (RA), as well as 1,25-dihydroxy-vitamin D3 (VD), whose receptor complex binds a DNA consensus sequence related to that of the retinoic acid receptor, to regulate expression of the retinoblastoma (RB) tumor suppressor gene in a context of induced cell differentiation was characterized. HL-60 human promyelocytic leukemia cells were induced to undergo myeloid or monocytic terminal cell differentiation by these agents. To investigate the potential coupling between down-regulation of RB and c-myc oncogene expression with cell differentiation, dose response relationships for the induced down-regulation of RB and c-myc expression were compared with each other and with induced cell differentiation. The total amount of RB protein per cell increased as cells advanced through the cell cycle, but the amount of RB protein relative to the total cell mass remained approximately constant. Treated with RA or VD, an early progressive decrease in cellular content of the RB protein occurred in all cell cycle phases well before any cell cycle modulation or phenotypic differentiation. For a differentiation-defective variant HL-60 cell line, failure to differentiate was preceded by a failure to down-regulate cellular levels of the RB protein. In dose response experiments, progressively increasing RA or VD concentrations caused progressively greater reductions in RB as well as c-myc expression with an increasing fraction of cells terminally differentiating. For both RA and VD, the dose response relationships for reductions in RB and c-myc expression were similar suggesting that their down-regulation may be coupled. These observations are consistent with a model whereby RB expression acts as a cellular brake to sustain a developmentally ordained state of differentiation (i.e., preserve the "status quo"); and the down-regulation of heterogeneously distributed RB protein per cell below a threshold is part of the metabolic cascade culminating in terminal cell differentiation. Thus, RB may have a role in this developmental context.

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations.

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-(1+)-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1+ cells compared to the remaining HNK-1+ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1+ subpopulations were grown together with HNK-1- cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1+ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1- cells. In the second part of this study, we investigate the specificity of differentiation of HNK-(1+)- and HNK-(1-)-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-(1+)-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-(1-)-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-(1+)- and the HNK-(1-)-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.

Studies related to reaction of supporting soft tissue to denture wear: the histological response of vervet monkey oral epithelium to a -80 mmHg vacuum.

The histological response of vervet monkey oral epithelium to a negative force similar to that experienced under dentures was investigated. Using impression trays with adherent impressions linked to a vacuum pump, the epithelia of the hard palate, attached gingiva and alveolar mucosa of 16 monkeys were subjected to a continuous vacuum of -80 mmHg, and then fixed by perfusion and immersion while in situ. After processing for light microscopy, sections were measured to obtain the rete peg length, supra-papillary width, epithelial width and basal, spinous and superficial cell density 700 microns-2. Mean values and standard deviations were calculated for each measurement and, using Student's t-test, these data were compared with results obtained for normal tissue from nine additional monkeys. The vacuum caused an increase in epithelial width in the palate and attached gingiva, and a decrease in epithelial width in the alveolar mucosa. The cell density 700 microns-2 decreased significantly in all layers of the palate, but increased in the basal layer of the attached gingiva and the basal and superficial layers of the alveolar mucosa. The alveolar mucosa within 0.5 mm of the mucogingival junction showed a variable response. This study demonstrates that a vacuum of -80 mmHg modifies the structure of the oral epithelium, and this response is directly related to the functional demands of the tissue.