Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain: effect of exogenous IGF-1 and GDNF

Progenitor cells in the dentate gyrus of hippocampus (DG) and the subventricular zone of lateral ventricles (SVZ) generate new neurons throughout the life of mammals. Cerebral ischemia increases this basal progenitor cell proliferation. The present study evaluated the time frame of proliferation, length of survival and the phenotypes of the new cells formed after transient middle cerebral artery occlusion (MCAO) in adult spontaneously hypertensive rats. Compared to sham controls, ischemic rats showed a significantly higher number of newly proliferated cells (as defined by BrdU immunostaining) in both the DG (by fourfold, p < 0.05) and the SVZ (by twofold, p < 0.05). DG showed increased proliferation only in the first week of reperfusion and 49% of the cells formed in this period survived to the end of third week. Whereas, SVZ showed a continuous proliferation up to 3 weeks after MCAO, but the cells formed survived for less than a week. In both DG and SVZ, at the end of the first week of reperfusion, majority of the BrdU-positive (BrdU+) cells were immature neurons (DCX positive). In the DG, 28% of the cells formed in the first week after MCAO mature into neurons (NeuN positive). The ischemic cortex and striatum showed several BrdU+ cells which were ED-1 positive microglia/macrophages. At 1 week of reperfusion, MCAO-induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF-1 (by 127 +/- 14%, p < 0.05) and GDNF (by 91 +/- 5%, p < 0.05), compared to vehicle. In the growth factor treated rats subjected to transient MCAO, several BrdU+ cells formed in the first week survived up to the third week.     

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.     

Iso-antibodies to red cell antigens in pigs' sera 3. The effect of maternal red cell iso-antibodies on the red cells of piglets

The effect of the red cell antibody, anti-Ea, on the red cells of piglets in four litters was studied. Although the antibody was absorbed from the colostrum by all the piglets, it had little effect on their packed cell volume, haemoglobin and red cell counts, and no differences were noted between Ea-positive and Ea-negative piglets in the same litter, despite the fact that red cells of the former were positive for the direct Coomb's test for up to a week after birth.
Anti-Ea was not detected in the serum of Ea-positive piglets, all of it apparently being taken up by their red cells, unlike the Ea-negative ones, in whose serum anti-Ea could be detected for up to fifteen weeks of age.     

Postnatal expression of Doublecortin (Dcx) in the developing cerebellar cortex of mouse

We have investigated the expression of Doublecortin (Dcx) protein in the developing cerebellum of mouse from postnatal 2nd day to postnatal 22nd day and in young adults by immunohistochemistry. Strong expression of Dcx was present in the inner zone of the external granule cell layer, and remained strong while postmitotic granule cell precursors were present in this transitory layer. Descending granule cell precursors exhibited Dcx immunostaining not only while migrating but for a short time also after their settlement. Dcx-immunostained cells appeared in deep cerebellocortical territories and in the cerebellar white matter during the first postnatal week. These bipolar cells were arranged in the sagittal plane and built up transitory migratory streams during the second postnatal week and their number gradually decreased during the third postnatal week. Upward migration of bipolar cells was observed while leaving the migratory streams, penetrating the internal granule cell layer and the molecular layer. These cells were considered as precursors of late migrating molecular layer interneurons. However, a proportion of Dcx-immunostained cells underwent a bipolar-to-multipolar dendritic remodellation and - on the basis of strong morphological similarities - was taken for "multipotent progenitor cells", described recently in the neocortex of adult rat.     

Polyhormonal aspect of the endocrine cells of the human fetal pancreas

Histological studies were performed on 30 pancreases obtained from normal human fetuses aged between the 9th and 38th week. For immunocytochemistry, the avidin-biotin-peroxidase method was used to identify and colocalise insulin, glucagon, somatostatin, pancreatic polypeptide and proliferating cell nuclear antigen. In the 9th week, cells containing all investigated peptides were present. During the fetal period, two populations of endocrine cells have been distinguished, Langerhans islets and freely dispersed cells. The free cells were polyhormonal, containing insulin, glucagon, somatostatin and pancreatic polypeptide, and were localised in the walls of pancreatic ducts throughout the whole gland. During the development of the islets we have observed four stages: (1) the scattered polyhormonal cell stage (9th–10th week), (2) the immature polyhormonal islet stage (11th–15th week), (3) the insulin monohormonal core islet stage (16th–29th week), in which zonular and mantle islets are observed, and (4) the polymorphic islet stage (from the 30th week onwards), which is characterised by the presence of monohormonal cells expressing glucagon or somatostatin. Bigeminal and polar islets also appeared during this last stage. The islets consisted of an insulin core surrounded by a thick (in the part developing from the dorsal primordium) or thin rim (part of the pancreas concerned with the ventral primordium) of intermingled mono- or dihormonal glucagon-positive or somatostatin-positive cells. The most externally located polyhormonal cells exhibited a reaction for glucagon, somatostatin and pancreatic polypeptide. Apart from the above-mentioned types of islets, all arrangements observed in earlier stages were present. Proliferating cell nuclear antigen-positive cells (single in the large islets and more numerous in the smaller ones) were predominantly observed in the outermost layer. Taken together our data indicate that, during the human prenatal development of the islet, endocrine cells are able to synthesise several different hormones. Maturation of these cells involved or depended on a change from a polyhormonal to a monohormonal state and is concerned with decreasing proliferative capacity. This supports the concept of a common precursor stem cell for the hormone-producing cells of the fetal human pancreas. Accepted: 1 June 1999     

Ontogeny of the mouse motor cortex. The polymorph layer or layer VI. A Golgi and electronmicroscopical study

Summary The development of neurons and their synapses of the mouse motor cortex has been studied from the first postnatal day up to an age of three weeks both electronmicroscopically and with the Golgi method. Special attention has been paid to the maturation of the different cell types in the sixth cortical layer and their dendritic organization within this layer.The polymorph layer is subdivided into two zones: an internal (VIb) and an external one (VIa). In these zones six different cell types can be identified both electronmicroscopically and with the Golgi method: large, small and inverted pyramidal cells in VIa; horizontal cells, star cells and small pyramidal cells in VIb.Spines of apical dendrites of large pyramidal cells in sublayer VIa can be detected as early as the 6th postnatal day. About the ninth day the basal dendrites as well show emerging spines. Somatic spines are found only on the large pyramidal cells and disappear slowly towards the end of the 3rd postnatal week.The small pyramidal cells show developing spines on their apical dendrite in the first half of the second postnatal week. The final density and distribution of spines is reached by the stem dendrites towards the end of the second week, by the basal dendrites during the third week. The maturation process of the improperly orientated neurons occurs in time in between the large and the small pyramidal cells.The axo-somatic synapses appear in general at a later date than the axo-dendritic ones. In the horizontal cells axo-somatic synapses are visible already at the seventh postnatal day.At the end of the first week especially in layer VIb many immature neurons with an ovoid or round nucleus are present having little if any endoplasmic reticulum organised as ergastoplasm.Towards the end of the second week however most neurons in the polymorph layer have a well developed endoplasmic reticulum.Electronmicroscopical pictures reveal in outgrowing dendrites many enlargements filled with vesicles, these correspond to the varicosities seen in Golgi pictures. At nine days postnatally the first myelinated fibres appear.Aided by grant (R-209-67) from the United Cerebral Palsy Research and Educational Foundation, New York.     

Effect of Maraviroc Intensification on HIV-1-Specific T Cell Immunity in Recently HIV-1-Infected Individuals

Background

The effect of maraviroc on the maintenance and the function of HIV-1-specific T cell responses remains unknown.

Methods

Subjects recently infected with HIV-1 were randomized to receive anti-retroviral treatment with or without maraviroc intensification for 48 weeks, and were monitored up to week 60. PBMC and in vitro-expanded T cells were tested for responses to the entire HIV proteome by ELISpot analyses. Intracellular cytokine staining assays were conducted to monitor the (poly)-functionality of HIV-1-specific T cells. Analyses were performed at baseline and week 24 after treatment start, and at week 60 (3 months after maraviroc discontinuation).

Results

Maraviroc intensification was associated with a slower decay of virus-specific T cell responses over time compared to the non-intensified regimen in both direct ex-vivo as well as in in-vitro expanded cells. The effector function profiles of virus-specific CD8⁺ T cells were indistinguishable between the two arms and did not change over time between the groups.

Conclusions

Maraviroc did not negatively impact any of the measured parameters, but was rather associated with a prolonged maintenance of HIV-1-specific T cell responses. Maraviroc, in addition to its original effect as viral entry inhibitor, may provide an additional benefit on the maintenance of virus-specific T cells which may be especially important for future viral eradication strategies.     

乌毛蕨配子体发育的研究

采用混合土培养乌毛蕨（Blechnum orientale）孢子,显微镜下观察记录其孢子萌发及配子体发育过程。结果表明：孢子黑褐色,赤道而豆形,极而观椭圆形,单裂缝。播种1周左右孢子萌发,萌发类型为书带蕨型,配子体发育为叉蕨型。丝状体5—10细胞时开始发育为片状体。播种2周后发育形成幼原叶体,成熟原叶体呈心脏形。原叶体边缘及表面均可产生毛状体,数量丰富,为单细胞。播种后1个月左右开始有颈卵器出现,成熟颈卵器颈部由4列细胞组成,3—5层细胞高。精子器产,扛时间较颈卵器早10d左右,精子器近圆球形,由3细胞组成。精卵受精后2周左右即可观察到从原叶体上生成的幼胚。     

稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定

实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此,通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系,研究CD19 CAR-T细胞对其的杀伤,能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞,证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实,连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比,CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后,通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明,成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系,且该细胞系能够被CD19 CAR-T细胞特异性杀伤。     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.     

The gastric mucosa of the human fetus. I. The surface epithelium

The ontogenesis of the surface epithelium in the gastric mucosa was studied by means of light and electron microscopy in 41 human foetuses ranging from 7th to 12th week of gestational age. The results obtained can be summarized as follows: 1) At the 7th week the gastric mucosa shows a simple pseudostratified epithelium; the epithelial cells are undifferentiated and filled, with glycogen clusters. 2) From the 8th week the epithelial surface shows small depressions that become deeper in the mesenchyme making the first bud of the gastric foveolae. 3) At the 9-10th week the gastric foveolae are more developed. The cells of the gastric epithelium can be therefore separated in two populations: a) the cells of the foveolae; b) the cell of the mucosal surfaces. 4) At the 12th week the cells of the mucosal surface become, on the basis of their histochemical and ultrastructural characteristics, surface mucous cells. The morphological differentiation is testified mainly by the transposition of the nuclei in the basal parts of the cells and by the gradual substitution of the cytoplasmic glycogen by mucous granules.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

Cell lines from the soft tick <Emphasis Type="Italic">Ornithodoros moubata</Emphasis>

Primary cell cultures (n = 16) were initiated from tissues of embryonic and neonatal larval Ornithodoros moubata following methods developed for hard ticks. After maintenance for 20–25 months in vitro, cell multiplication commenced in surviving cultures, leading to the establishment of six cell lines designated OME/CTVM21, 22, 24, 25, 26 and 27. All lines are maintained at 28°C, with subculture at 2–8 week intervals. The cultures comprise heterogeneous populations of large cells of 15–100 μm in diameter, often with finger-like protrusions and/or intracellular crystals, rarely attached, predominantly floating and forming clumps or hollow multicellular vesicles up to 1 mm in diameter. Attempts to cryopreserve the cells are described. Tick-borne encephalitis virus has been serially passaged ten times in OME/CTVM21 cells without significant decrease in virus production and with no change in its biological properties as shown by the size and morphology of plaques produced in porcine kidney cells.     

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus. immunohistochemistry; cell cycle; proliferation; renal stem cells; proximal tubule; renal epithelial cells     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Implantation of Tumoral XC Cells Induces Chronic, Endothelin-Dependent, Thermal Hyperalgesia in Mice

1. We describe here the alterations in the nociceptive sensitivity of Swiss CD1 mice receiving an intraplantar (i.pl.) administration of XC Rous sarcoma-virus-transformed rat fibroblasts (XC cells). 2. Histological studies reveal that XC cells remain at the injection site 2-3 weeks after implantation, a time at which an inflammatory reaction is also detected. No tumoral growth was found and 5 weeks after inoculation neither XC cells nor inflammatory reaction were observed. 3. Measures to different types of noxious stimuli were performed. At week 1 after XC cell inoculation, hyperalgesia to thermal, but not mechanical, stimuli as well as to capsaicin injection is present in the implanted paw. At week 5 after XC cell implantation, only thermal hyperalgesia is present, and this enhanced reactivity persisted for even 25 weeks after the disappearance of XC tumoral cells. 4. Pharmacological studies on thermal hyperalgesia were conducted at two different stages, week 1 and week 5 after XC cell inoculation. The systemic administration of morphine (1-10 mg/kg i.p. (intraperitoneal); 30 min before testing) prevents this thermal hyperalgesic reaction both at week 1 and week 5. The endothelin type A (ETA) receptor antagonist BQ-123 (10 nmol; i.pl.; 90 min before testing) abolishes both the early (week 1) and the late (week 5) thermal hyperalgesia. In contrast, the selective endothelin type B (ETB) receptor antagonist, BQ-788 (10 nmol; i.pl.; 90 min before) abolishes thermal hyperalgesia only at week 1, but not at week 5 after XC cell inoculation. 5. It might be concluded that endothelins are probably involved in this type of long-term thermal hyperalgesia produced by the transitory presence of the XC tumoral cell line.     

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.