Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor alpha chain (IL-3Ralphahi). The CD34+IL-3Ralphahi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3Ralphahi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.     

Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.     

Neurotransmission and the ontogeny of human brain

The early appearance of neurotransmitters in brain tissue refers to their regulative functions on the neuronal circuits. Many neurotransmitters have direct effects on neuronal outgrowth and differentiation during brain development, which precede their role in synaptic information coding. Both the neurotrophic and neurotoxic properties of excitatory amino acids (EAAs) have focused special interest on glutamatergic neurotransmission during brain development. Therefore, this work intends to review and discuss developmental alterations of the EAA neurotransmitter system in the human brain, their relation to human brain maturation and implications for pathological processes during early human brain development.     

The capacity of central and peripheral catecholaminergic neurons to innervate the pineal organ and cerebral cortex of the rat: in vitro immunohistochemical observations

The locus coeruleus (LC) or superior cervical ganglion (SCG) of neonatal rats were co-cultured either with the pineal organ or cerebral cortex (CX) to investigate the innervating capacity of central and peripheral catecholamine neurons under these experimental conditions. After 2 weeks of co-culturing, cultures were fixed for tyrosine hydroxylase (TH) immunohistochemistry to examine the distribution of catecholamine neurons and their fibers. Glial fibrillary acidic protein and fibronectin immunohistochemistry was performed to determine the cell types proliferating around the explants. In LC/CX co-cultures, numerous astrocytes spread between the two explants, and TH-immunoreactive neurites were generally seen to invade CX explants. In contrast, neurite extension from LC to pineal explants occurred only when a glial cell sheet grew between the two explants, and when the pineal explants were not surrounded by a tight fibronectin-positive cell layer. Neurites of the SCG usually invaded both CX and pineal explants, regardless of the existence of glial or non-glial cell layer. These results indicate that central and peripheral catecholamine neurites have the potential of invading both the cortex and pineal, although they are distributed only in particular regions of the intact brain. The distribution of LC neurites, however, seems to be profoundly affected by the cell types spreading around the explants; glial cells appear to support LC neurite extension, whereas non-glial cells appear to inhibit it.     

Two receptor tyrosine phosphatases of the LAR family are expressed in the developing leech by specific central neurons as well as select peripheral neurons, muscles, and other cells

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. We have cloned and sequenced two Hirudo medicinalis rPTPs that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. In addition, HmLAR2 is expressed on the cell body, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, we hypothesize that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. Furthermore, we hypothesize that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. Lastly, we propose that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2.     

Expression of the brain creatine kinase gene in rat RT4 peripheral neurotumor cell lines and its modulation by cell confluence

Creatine kinases (CK) catalyze the reversible transfer of a high energy phosphate group between creatine phosphate and ADP to regenerate ATP in cell types where the requirements for ATP are extensive and/or sudden. Previously, we have shown in primary rat brain cell cultures that brain CK (CKB) mRNA levels are highest in astrocytes and oligodendrocytes and much lower in neuronal cells. However, little is known of the factors which regulate CKB expression in the central nervous system and peripheral nervous system. To begin to investigate these factors, we asked in this report (1) if this pattern of CKB expression was also characteristic of some established glial and neuronal cell lines derived from the PNS; (2) whether CKB expression could be rapidly modulated by culture conditions, and (3) if CKB is expressed in cells with characteristics of glial cell progenitors. In subconfluent cells, CKB mRNA and enzyme activity were found to be high in both the rat RT4 peripheral neurotumor stem cell RT4-AC36A and its glial cell derivative RT4-D6. Conversely, CKB mRNA and activity were 5- and 8-fold lower, respectively, in the neuronal derivative RT4-E5 and, more dramatically, CKB was undetectable in neuronal RT4-B8 cells. Maintaining RT4-D6 glial cells at confluence rapidly increased CKB enzyme activity by 7-fold, such that D6 cells contained about 25% of the CKB level in lysates prepared from either whole adult rat brain or primary cultures of rat brain astrocytes. The levels of CKB mRNA and immunoreactive protein were also correspondingly increased in confluent D6 cells. These confluence-mediated increases in CKB appeared to be due to cell-cell contact and not the depletion of serum growth factors or an increase in intracellular cAMP. This study indicates that CKB expression is highest in cells displaying glial properties and can be rapidly modulated by appropriate culture conditions. The results are discussed in relation to the factors which may regulate CKB expression in vivo.     

Cholinergic, serotonergic and catecholaminergic neurons are not affected in Ts65Dn mice

Ts65DN mice were developed as a model of Down syndrome (DS); they are trisomic for the distal segment of chromosome 16 (MMU16), which contains genes syntenic with some of the genes located on the critical region of human chromosome 21 (HSA21). Since behavioral and neurochemical disturbances have been observed in this animal model, it seemed interesting to perform an immunohistochemical characterization of the main cholinergic, catecholaminergic and serotonergic nuclei. However, when the brains of Ts65Dn mice were compared with those of control littermates, no differences were found either in the morphology of the neurons of the three systems or in the number of immunoreactive cells. The results indicate that these systems are not affected by the triplication of some of the genes present on chromosome 16.     

Cell death in the Schwann cell lineage and its regulation by neuregulin

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.     

Microsomal protein changes in the brain of hens in ontogeny

Studies have been made on the peripheral (extracted by 1 M KCl, pH 9.0) and integral (successively extracted by a 1% solution of Triton X-100 and 0.3% solution of sodium dodecyl sulfate) microsomal proteins from the brain of chick embryos beginning from the 8th day of incubation, chicks and adult hens up to 1 year 4 months. Investigation was made of the quantity, fractional composition (by means of polyacrylamide gel electrophoresis) and the rate of incorporation of 14C-lysine. It was found that the content of these membrane proteins and their composition depend on the age of animals. Integral proteins as compared with peripheral ones exhibit higher rate of incorporation of the labeled amino acid, this rate increasing with the age of animals.     

Selective neural cell adhesion molecule signaling by Src family tyrosine kinases and tyrosine phosphatases

Nerve growth cone guidance is a highly complex feat, involving coordination of cell adhesion molecules, trophic factor gradients, and extracellular matrix proteins. While navigating through the developing nervous system, the growth cone must integrate diverse environmental signals into a singular response. The repertoire of growth cone responses to these extracellular cues includes axonal growth, fasciculation, and synaptic stabilization, which are achieved through dynamic changes in the cytoskeleton and modulation of gene expression. It has become evident that interactions between cell adhesion molecules can activate intracellular signaling pathways in neurons. Such signaling pathways are just beginning to be defined for the axonal growth promoting molecules L1 and NCAM which are members of the immunoglobulin (Ig) superfamily. Recent findings have revealed that L1 and NCAM induce neurite outgrowth by activating intracellular signaling pathways in the growth cone mediated by two different members of the src family of nonreceptor protein tyrosine kinases (PTKs), pp60(c-src) and p59(fyn5,6). Growth cones display diverse morphologies and variable motility on these different cell adhesion molecules, which are likely to be generated by src kinases. In this review we will address novel features of nonreceptor PTKs of the src family which dictate their distinctive molecular interactions with cell adhesion molecules and signaling components.     

Understanding sleep ontogeny to assess brain dysfunction in neonates and infants

The ontogenetic framework onto which a child's sleep is constructed undergoes significant developmental alterations during early life. Sleep state behaviors, in large part, reflect continuities from fetal through neonatal time periods. Major changes in sleep organization subsequently occur throughout infancy. Maturational expressions of sleep behaviors must be understood by the pediatric neurologist before specific physiologic phenomena can be assessed as transient sleep disturbances or clinically relevant sleep disorders. The first part of this two-part review article focuses on the major aspects of developmental sleep physiology in the first few months of life. Recognition of age-specific electroencephalographic/polysomnographic patterns will facilitate the child neurologist's evaluation of the newborn with suspected seizures and interictal encephalopathies, as well as the prediction of neurologic sequelae.     

Differential distribution of tyrosine hydroxylase fibers on small and large neurons in layer II of anterior cingulate cortex of schizophrenic brain

A series of recent postmortem investigations of the anterior cingulate cortex in schizophrenic brain have suggested that there may be a loss and/or impairment of inhibitory interneurons in layer II. It has been postulated that changes of this type could secondarily result in a relative increase of dopaminergic inputs to GABAergic interneurons. To test this hypothesis, an immunoperoxidase technique was developed to extensively and reliably visualize tyrosine hydroxylase-immunoreactive (TH-IR) varicose fibers in human postmortem cortex. This method has been applied to the anterior cingulate (ACCx; Brodmann area 24) and prefrontal (PFCx: Brodmann area 10) cortices from a cohort of 15 normal control and 10 schizophrenic cases. The number of TH-IR varicosities in contact with large neurons (LN), small neurons (SN), and neuropil (NPL) was blindly analyzed using a computer-assisted microscopic technique. There was no significant difference in density of TH-IR varicosities in apposition with either LN or SN cell bodies observed in either ACCx or PFCx of schizophrenics when compared to normal controls. The density of varicosities was significantly reduced in NPL of layers V and VI in ACCx, but 2 neuroleptic-free cases did not show this change, suggesting that these decreases of TH-IR varicosities may be related to antipsychotic effects on corticostriatal projection cells in this region. When the density of TH-IR varicosities on SNs was compared to that observed on LNs, both groups showed a higher density on SNs. In ACCx, this pattern was much more pronounced for the schizophrenic group, particularly in layer II where the density on SNs was three times higher than that for LNs (P = 0.01). Unlike the changes in layer V, this latter change in layer II showed no relationship to neuroleptic exposure. There was a positive correlation between age and the density of TH-IR varicosities on SNs of layer II in ACCx; however, the patients were younger than the controls and would have been expected to show a lower density, rather than a higher one, if age considerations had accounted for the group differences. Overall, the results reported here suggest that there are no gross differences in the distribution of TH-IR varicosities in various laminae of the dorsolateral prefrontal cortex. In the anterior cingulate region, however, there may be a significant shift in the distribution of TH-IR varicosities from large neurons to small neurons that occurs selectively in layer II of schizophrenic subjects. Using size criteria, the majority of small neurons are likely nonpyramidal, while the majority of large neurons are predominantly pyramidal in nature. Taken together with other accumulating evidence of preferential abnormalities in this lamina of the cingulate region, the findings reported here are consistent with a model of schizophrenia in which a subtle "miswiring" of ventral tegmental inputs may result in a relative, though not absolute, hyperdopaminergic state with respect to an impaired population of GABAergic interneurons.     

Excitation of central and peripheral terminals of primary afferent neurons by capsaicin in vivo

In three groups of rats discharge activity was recorded (i) from the peripheral stump of the cut saphenous nerve (saphenous-receptor preparation); (ii) from the central stump of the cut L4 or L5 dorsal root (dorsal root preparation); or (iii) from the peripheral stump of the saphenous nerve segment cut at both ends (axon preparation) during slow intraarterial infusion of capsaicin (30-300 micrograms/kg/min for 5 min) into the carotid artery. Capsaicin produced excitation, i.e. an increase in frequency of action potentials in the same dose range (100-300 micrograms/kg/min) in both the saphenous-receptor and dorsal root preparations, while the axon preparations remained unresponsive. In the cat, close arterial injection of capsaicin (up to 20 micrograms) into a collateral branch of the saphenous artery failed to evoke discharges in the saphenous axon preparation, although similar injection of 4-aminopyridine (60 micrograms), a K+ channel blocking agent was readily effective. These results indicate that after systemic application of capsaicin the peripheral and central endings of primary afferent neurons are equally important sites for activation and are much more sensitive to capsaicin than the axons of the nerve trunk.     

Estrogen-dependent ontogeny of sex differences in somatostatin neurons of the hypothalamic periventricular nucleus

The sexually dimorphic profile of GH secretion is thought to be engendered by gonadal steroids acting in part on hypothalamic periventricular somatostatin (SOM) neurons. The present study set out to examine and characterize the development of sex differences in these SOM neurons. In the first series of experiments, we used in situ hybridization to examine SOM messenger RNA (mRNA) expression within the periventricular nucleus (PeN) of male and female rats on postnatal day 1 (P1), P5, and P10. Cellular SOM mRNA content was found to increase from P1 to P10 in both sexes (P < 0.01), but was 24% (P < 0.05) and 38% (P < 0.01) higher in males on P5 and P10, respectively. A second series of experiments examined the SOM peptide content of the PeN in developing rats and found increasing levels from P1 to P10, with a 44% higher SOM content in males compared with females on P10 (P < 0.05). The third series of experiments questioned the role of gonadal steroids in engendering sex differences in SOM mRNA expression by determining the effects of neonatal gonadectomy (GDX) and replacement of dihydrotestosterone or estradiol benzoate. The SOM mRNA content of PeN neurons in P5 males gonadectomized on the day of birth was the same as that in P5 females and was significantly reduced compared with that in sham-operated P5 males (P < 0.05). Male rats GDX on P1 and treated with estradiol benzoate from P1 to P5 had cellular SOM mRNA levels similar to those in intact males on P5, whereas dihydrotestosterone treatment had no effect. Treatment of intact males with an androgen receptor antagonist, cyproterone acetate, on P1 had no effect on cellular SOM mRNA on P5, whereas male rats given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione from P1 to P5 had lower (P < 0.05) SOM mRNA levels than controls. In the final set of experiments, dual labeling immunocytochemistry showed that SOM neurons in the PeN of P5 rats did not contain estrogen receptor-alpha, but expressed androgen receptors in a sexually dimorphic manner. These results demonstrate that a sex difference in SOM biosynthesis, which persists into adulthood, develops between P1 and P5 in PeN neurons. Despite the absence of estrogen receptor-alpha in these neurons, the organizational influence of testosterone only occurs after its aromatization to estrogen.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.     

Early radiation effects in highly apoptotic murine lymphoma xenografts monitored by 31P magnetic resonance spectroscopy

PURPOSE: To assess the efficacy and toxicity of combined modality therapy with short intensive primary chemotherapy in the treatment of primary CNS lymphoma (PCL). METHODS AND MATERIALS: Prospective study of 31 nonimmunodeficient patients with PCL treated with initial chemotherapy (13 shortened MACOP-B; and 18 modified MACOP with high dose methotrexate) followed by radiotherapy (whole brain and a boost). Patients were aged 18-72 years (median 51 years). Eight patients had positive CSF cytology of which one had spinal meningeal disease; one patient had vitreous involvement. RESULTS: The overall complete response (CR) rate after chemotherapy and radiotherapy was 69% (95% Confidence Interval: 49-84%). At a median follow-up of 24 months (4 months to 10 years) median survival was 23 months and 5-year survival 34%. Age, sex, performance status, number of lesions, CSF cytology, and extent of surgery were not of prognostic significance for survival on univariate analysis. Eleven patients developed mucositis (Grade 3+) and 21 hematological toxicity (Grade 3+) with 22 septicemic episodes in 15 patients. Three patients developed dementia, one assumed to be treatment related, and two due to recurrent disease. CONCLUSION: The survival results of short intensive primary chemotherapy followed by radiotherapy are similar to the results of chemotherapy in Stage IV aggressive systemic non-Hodgkin's lymphoma, although the treatment was associated with high morbidity. The apparently favorable results when compared to radiotherapy alone may at least in part be due to selection of patients with good prognostic factors. To confirm the benefit of combined chemotherapy and radiotherapy over either of the two modalities alone requires evaluation in large prospective and ideally randomized studies.     

Perinatal asphyxia-induced changes in rat brain tyrosine hydroxylase-immunoreactive cell body number: effects of nicotine treatment

We previously reported that the amyloid-beta protein (Abeta) reduces the synthesis of acetylcholine (ACh) in a mouse septal cell line, SN56, without causing death of the cells. Here, we report that the ACh-reducing effect of either Abeta 1-28 or Abeta 1-42 (100 nM; 48 h) in SN56 cells can be prevented by a co-treatment with the tyrosine kinase inhibitors, genistein (75 microM) and tyrphostin A25 (50 microM). Treatment of the cells with either of these inhibitors alone increased ACh levels. An enhancement of the cellular ACh content was also obtained with aphidicolin, a compound which inhibits DNA synthesis. However, co-treatment of the cells for 48 h with aphidicolin (500 nM) and Abeta 1-42 (100 nM) did not prevent the reduction in ACh levels caused by the peptide. Furthermore, this effect could not prevented by a pre-treatment with vitamin E (50 microg/ml). These results suggest that the ACh-reducing effect of Abeta in SN56 cells is dependent on tyrosine phosphorylation, but is not dependent on DNA synthesis and may not be mediated by free radicals.     

Decrease of medullary catecholaminergic neurons in multiple system atrophy and Parkinson's disease and their preservation in amyotrophic lateral sclerosis

We investigated the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the C1 and A2 regions of the medulla, the sites of the baroreflex arc, in 7 patients with multiple system atrophy (MSA), 8 with Parkinson's disease (PD), 9 with amyotrophic lateral sclerosis (ALS), and 12 age-matched normal subjects to analyze the relationship between cardiovascular dysfunction and medullary catecholaminergic neurons. Orthostatic hypotension (OH) was marked in all the MSA patients and moderate in three PD patients. Three of the five ALS patients who had been on respirators showed lability of blood pressure; paroxysmal hypertension and nocturnal hypotension without compensatory tachycardia. All the MSA patients showed extremely marked decrease of TH-immunoreactive neurons in both the C1 and A2 regions. In the patients with Parkinson's disease, numerous TH-immunoreactive neurons contained Lewy bodies that were immunostained by antibody to TH. TH-immunoreactive neurons were decreased very markedly in the A2 regions of two patients with OH, and three patients without OH showed fairly marked decreases in the C1 or A2 region. In contrast, the number of TH-immunoreactive neurons in ALS was the same as in normal subjects. In MSA and some PD patients, orthostatic hypotension may partly be due to the involvement of the medullary catecholaminergic neurons. The lability of blood pressure in ALS probably is not related to the medullary catecholaminergic neurons.