Germinal Cell Population Research Articles

Autologous Peripheral Blood Mononuclear Cells can Lead to Pancreatic Beta Cell Regeneration

Introduction: Type-1 diabetes (T1D) is a disease that leads to pancreatic beta cells death, resulting in complete insulin loss. T1D occurs as a result of autoimmunity which takes insulin producing β cells as a target and is considered as a metabolic disorder causing hyperglycemia resulting from a serious loss of endogenous insulin roduction. Using cadaveric islets as a replacement therapy was done to reverse diabetes. Many various progenitor cell types have been found in the peripheral blood mononuclear cell (PBMC) fraction denoting hose PBMCs may have the ability to differentiate into many mature functional cell types in certain microenvironments. It is settled that circulating peripheral blood mononuclear cells (PBMC) contain germinal cell population which can share in the regeneration of tissues in different organs. Materials and Methods: 2 groups of type I diabetes patients had been monitored in a private clinic, number 40 each group, with 35 females and 45 males. It lied between 8-25 years old with 4-7 years of diabetes onset. The first group was on insulin therapy and they received peripheral blood mononuclear cells concentrate directly in the dorsal pancreatic artery. The second group was only on insulin injection. Results: The results show a significant increase in c peptide levels after direct injection of peripheral blood mononuclear cells in the dorsal pancreatic artery with p-value less than 0.0001. Conclusion: The peripheral blood mononuclear cells can induce beta cell regeneration and increase beta cell mass which is detected by an increase in c peptide levels.

Identification of EmSOX2, a member of the Sox family of transcription factors, as a potential regulator of Echinococcus multilocularis germinative cells

Larvae of the tapeworm Echinococcus multilocularis cause alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. The germinative cells, a population of stem cell-like cells, are considered to drive the continuous growth of the metacestodes within the host. The mechanisms and relative molecules controlling the behavior of germinative cells are poorly understood. Sox transcription factors play important roles in maintenance and regulation of stem/progenitor cells. We here describe the identification of a Sox family member in E. multilocularis, EmSOX2, as a potential regulator of germinative cells. Replacement of mouse Sox2 with EmSox2 could derive induced pluripotent stem cells (iPSCs) from somatic cells, suggesting that EmSOX2 is functionally related to mammalian SOX2. EmSOX2 is actively expressed in the proliferating germinative cells in E. multilocularis, and is significantly downregulated upon specific depletion of the germinative cell population by hydroxyurea treatment. These findings suggest that EmSOX2 may play a critical role in regulating E. multilocularis germinative cells.

Derivation of the human embryonic stem cell line RCe006-A (RC-2)

The human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Microsatellite DNA marker identity and HLA and blood group typing data are available.

Targeting Echinococcus multilocularis stem cells by inhibition of the Polo-like kinase EmPlk1.

BackgroundAlveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation.Methodology/Principal findingsWe herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation.Conclusions/SignificanceWe conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy.

Maize germinal cell initials accommodate hypoxia and precociously express meiotic genes

In flowering plants, anthers are the site of de novo germinal cell specification, male meiosis, and pollen development. Atypically, anthers lack a meristem. Instead, both germinal and somatic cell types differentiate from floral stem cells packed into anther lobes. To better understand anther cell fate specification and to provide a resource for the reproductive biology community, we isolated cohorts of germinal and somatic initials from maize anthers within 36 h of fate acquisition, identifying 815 specific and 1714 significantly enriched germinal transcripts, plus 2439 specific and 2112 significantly enriched somatic transcripts. To clarify transcripts involved in cell differentiation, we contrasted these profiles to anther primordia prior to fate specification and to msca1 anthers arrested in the first step of fate specification and hence lacking normal cell types. The refined cell-specific profiles demonstrated that both germinal and somatic cell populations differentiate quickly and express unique transcription factor sets; a subset of transcript localizations was validated by in situ hybridization. Surprisingly, germinal initials starting 5 days of mitotic divisions were enriched significantly in >100 transcripts classified in meiotic processes that included recombination and synapsis, along with gene sets involved in RNA metabolism, redox homeostasis, and cytoplasmic ATP generation. Enrichment of meiotic-specific genes in germinal initials challenges current dogma that the mitotic to meiotic transition occurs later in development during pre-meiotic S phase. Expression of cytoplasmic energy generation genes suggests that male germinal cells accommodate hypoxia by diverting carbon away from mitochondrial respiration into alternative pathways that avoid producing reactive oxygen species (ROS).

"In vitro" assay of rat spermatogonial chalone in prepuberal seminiferous tubules.

The spermatogonial chalone activity is analyzed in correlation to the germinal cell population present in the seminiferous tubules of prepuberal rats. The assay is done in an "in vitro" system and aims at identifying the cell type that produces the chalone. It was found that the spermatogonial chalone is produced by the germinal cells, its production increases with the age of the animals and attains its maximum in 40 days old rats. There is a direct relationship between the amount of chalone produced and the number of spermatocytes present in the seminiferous tubules of prepuberal rats. The response of the germ cells to the chalone is maximal by 25 days of age. The apparent decrease of response in 40 days old rats may be related to the establishment of a complete germ cell line.

Identification of various testicular cell populations in pubertal and adult cockerels

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/ Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia

Genomic imprinting marks in the male germ line are already established in the adult germinal stem cell population. We studied the methylation patterns of H19 and MEST imprinted genes in sperm of control and oligozoospermic patients, by bisulphite genomic sequencing. We here report that 7 out of 15 (46.7%) patients with a sperm count below 10 x 10(6)/ml display defective methylation of H19 and/or MEST imprinted genes. In these cases, hypomethylation was observed in 5.54% (1.2-8.3%) and complete unmethylation in 2.95% (0-5.9%) of H19 clones. Similarly, for the CTCF-binding site 6, hypomethylation occurred in 4.8% (1.2-8.9%) and complete unmethylation in 3.7% (0-6.9%) of the clones. Conversely, hypermethylation occurred in 8.3% (3.8-12.2%) and complete methylation in 6.1% (3.8-7.6%) of MEST clones. Of the seven patients presenting imprinting errors, two had both H19 hypomethylation and MEST hypermethylation, whereas five displayed only one imprinted gene affected. The frequency of patients with MEST hypermethylation was highest in the severe oligozoospermia group (2/5 patients), whereas H19 hypomethylation was more frequent in the moderate oligozoospermia (2/5 patients). In all cases, global sperm genome methylation analysis (LINE1 transposon) suggested that defects were specific for imprinted genes. These findings could contribute to an explanation of the cause of Silver-Russell syndrome in children born with H19 hypomethylation after assisted reproductive technologies (ART). Additionally, unmethylation of the CTCF-binding site could lead to inactivation of the paternal IGF2 gene, and be linked to decreased embryo quality and birth weight, often associated with ART.

Spermatogenesis in the turkey ( Meleagris gallopavo): Quantitative approach in immature and adult males subjected to various photoperiods

The objectives of this study were to identify and quantitate the germ cell populations of the testes in sexually mature male turkeys (Trial 1), determine the duration of meiosis based on BrdU labeling and stereological analyses (Trial 2), and examine the impact of various photoperiods on germinal and somatic cell populations in immature and adult males (Trial 3). In Trial 1, both testes within a male had similar stereological components ( P > 0.05) for all parameters analyzed. In Trial 2, the duration of Type-1 spermatocytes and round spermatids in turkeys lasted 4.5 ± 0.5 and 2.0 ± 0.5 days, respectively. In Trial 3, the short photoperiod (7L:17D) delayed testicular growth (in the stereological parameters analyzed). In contrast, the effect of a moderately short photoperiod (10.5L:13.5D) was comparable to the effect of a long (14L:10D) or increasing photoperiod (7L:17D to 14L:10D) on the stereological parameters examined. With the exception of the short photoperiod, all other photoperiods used in this study induced comparable early testicular maturation, with maximum testis weight at 29–35 weeks of age. As the males got older, there was a progressive, linear decline in testis weight through 60 weeks, at which time there were no significant differences among photoperiods. In conclusion, the duration of meiosis in the turkey was similar to that observed in the fowl and guinea-fowl. The existence of a threshold of photosensitivity to gonad stimulation in male turkeys is suggested to be between 7.0 and 10.5 h of light.

Effect of selenium-induced oxidative stress on the cell kinetics in testis and reproductive ability of male mice

Objective The present study evaluated the role of experimental oxidative stress (induced by feeding diets with different concentrations of selenium [Se], a trace nutrient and potent antioxidant) on male reproductive activity in mice. Methods To create different levels of oxidative stress in male mice, three diets with different levels of Se were fed to different groups for 8 wk. Mice in group 1 were fed a yeast-based diet, which is considered a Se-deficient diet (0.02 ppm). Mice in groups 2 and 3 were fed with an Se-deficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively. Results After completion of the feeding schedule, a significant decrease in Se levels were observed in Se-deficient mice (group 1), whereas Se levels greatly increased in the Se-excess mice (group 3). Glutathione peroxidase activity was greatly decreased in the liver and testis in group 1, whereas glutathione-S-transferase activity was significantly increased in the testis. No significant change was found in activities of glutathione peroxidase and glutathione-S-transferase in group 3 compared with group 2. Cell kinetics showed a significant decrease in the number of pachytene spermatocytes and young and mature spermatids in group 1 compared with group 2. No appreciable change was observed in the germinal cell population in group 3. A significant decrease in sperm number was observed in group 1 compared with group 2. No change in these parameters was observed in group 3. The fertility status of mice in terms of percent fertility and litter size also exhibited a significant decrease in the reproductive ability of group 1. No change in these parameters was observed in group 3 compared with group 2. Conclusion The present results clearly demonstrate the effect of oxidative stress generated by feeding different concentrations of Se on cell kinetics in the testis and, hence, its effect on the reproductive ability of male mice.

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone.

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.

Hair follicle stem cells.

The workshop on Hair Follicle Stem Cells brought together investigators who have used a variety of approaches to try to understand the biology of follicular epithelial stem cells, and the role that these cells play in regulating the hair cycle. One of the main concepts to emerge from this workshop is that follicular epithelial stem cells are multipotent, capable of giving rise not only to all the cell types of the hair, but also to the epidermis and the sebaceous gland. Furthermore, such multipotent stem cells may represent the ultimate epidermal stem cell. Another example of epithelial stem cell and transit amplifying cell plasticity, was the demonstration that adult corneal epithelium, under the influence of embryonic skin dermis could form an epidermis as well as hair follicles. With regards to the location of follicular epithelial stem cells, immunohistochemical and ultrastructural data was presented, indicating that cells with stem cell attributes were localized to the prominent bulge region of developing human fetal hair follicles. Finally, a new notion was put forth concerning the roles that the bulge-located stem cells and the hair germ cells played with respect to the hair cycle.

Morphological alterations in mouse testis by a single dose of malathion

Malathion((R)) is a widely used organophosphorate agropesticide. In spite of its low toxicity for mammalian cells, it provokes cytogenetic and genotoxic damage both in vivo and in vitro. The effect of Malathion was analyzed in CF-1 young adult male mice. Commercial Malathion (96.6% purity) was injected intraperitoneally in a single dose (250 mg/kg body weight corresponding to 1/12 LD50). Four, 14, 18, and 26 days after injection animals were sacrificed to study epididymal sperm (count and morphology), testicular histology (percentage of depleted seminiferous tubules), and ultrastructural alterations in the germinal epithelium. The effect of Malathion on different germinal cell populations was studied. Teratozoospermia was induced by Malathion at all times studied. Spermatozoa midpiece and flagella were the most affected and at day 18 we observed less alterations of the head. The sperm count at different time intervals was significatively increased compared to controls and there was a parallel increase in depletion of the seminiferous tubules. In conclusion, all germinal cell populations studied were affected by Malathion. Malathion has a teratogenic effect on mice spermatid differentiation, which compromises mostly the flagella, perhaps due to an alkylating effect that disturbs the normal assembling of tail structural protein components. Apparently, the pachytene spermatocyte stage may be relatively more resistant to the pesticide. The Sertoli cells were affected by the insecticide and their damage at an ultrastructural level is highly significant. Cytoplasmatic vacuolization probably revealed metabolic alteration of these cells.

Morphological alterations in mouse testis by a single dose of malathion.

Malathion((R)) is a widely used organophosphorate agropesticide. In spite of its low toxicity for mammalian cells, it provokes cytogenetic and genotoxic damage both in vivo and in vitro. The effect of Malathion was analyzed in CF-1 young adult male mice. Commercial Malathion (96.6% purity) was injected intraperitoneally in a single dose (250 mg/kg body weight corresponding to 1/12 LD50). Four, 14, 18, and 26 days after injection animals were sacrificed to study epididymal sperm (count and morphology), testicular histology (percentage of depleted seminiferous tubules), and ultrastructural alterations in the germinal epithelium. The effect of Malathion on different germinal cell populations was studied. Teratozoospermia was induced by Malathion at all times studied. Spermatozoa midpiece and flagella were the most affected and at day 18 we observed less alterations of the head. The sperm count at different time intervals was significatively increased compared to controls and there was a parallel increase in depletion of the seminiferous tubules. In conclusion, all germinal cell populations studied were affected by Malathion. Malathion has a teratogenic effect on mice spermatid differentiation, which compromises mostly the flagella, perhaps due to an alkylating effect that disturbs the normal assembling of tail structural protein components. Apparently, the pachytene spermatocyte stage may be relatively more resistant to the pesticide. The Sertoli cells were affected by the insecticide and their damage at an ultrastructural level is highly significant. Cytoplasmatic vacuolization probably revealed metabolic alteration of these cells.

Rat fetuin: distribution of protein and mRNA in embryonic and neonatal rat tissues.

Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E 10Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E16-E18. Fetuin expression was present in germinal cell populations, e.g., in the basal layer in the skin, in the germinal cell populations in the brain anlage and the gonads, and it was heavily expressed in the fetal hemopoietic liver. Furthermore, fetuin was expressed in the gastrointestinal epithelium prior to the development of glands and crypts. Fetuin was widely distributed in mesenchymal derived tissues, e.g., bone and muscle. In the developing kidney fetuin was heavily expressed is both mesenchymal condensations and glomerular anlages. Thus, fetuin was located in cells or structures undergoing differentiation and transformation. As fetuin has been shown previously to interfere with hormone signaling of transforming growth factor-beta, insulin and hepatocyte-growth factor, fetuin might be involved in cell differentiation and tissue transformation during the initial histogenesis, i.e., the time period in which cellular phenotypic characteristics are established.

Ki-67 Immunostaining of Normal Human Epidermis: Comparison with 3H-Thymidine Labelling and PCNA Immunostaining

Background: The size of the germinative growth fraction (i.e. the number of actively proliferating germinative cells) of normal human epidermis is still a subject of debate. Ki-67 antigen and PCNA, an auxiliary protein of d-polymerase, are considered as markers of the growth fraction when used under optimal conditions. Method: In the present work, we have compared Ki-67 expression (detected with MIB1 antibody) with PCNA expression (detected with PC10 antibody) in biopsies of normal human epidermis fixed in neutral formalin and using antigen retrieval by microwave processing. To obtain additional information, such as the percentage of cells in S phase, biopsies were also incubated in ³H-thymidine before immunostaining. Results: Before microwave treatment, 8% of the basal cells were positive for MIB1 antibody and 7.8% were positive for PC10 antibody. The ³H-thymidine labelling index was 2.8%. The proportion of MIB1-positive cells rose to 19% after antigen retrieval by microwave processing. In the same way, the ³H labelling index rose to 9%. In contrast, PC10 became positive in all epidermal nuclei. Conclusion: These results suggest that the growth fraction of the germinative cell population of normal human epidermis is not larger than 20% and is composed of cells with a short cell cycle time.

The growth fraction of normal human epidermis.

In human epidermis, germinative cell production is determined by three parameters: the cell cycle time, the loss of cycling cells by programmed cell death and the proportion of keratinocytes actively engaged in the cell cycle or growth fraction. The last parameter is still a subject of debate as the identification of a proliferative cell with certainty is impossible. Two indirect methods (grain count dilution of labelled cells and continuous labelling) suggest that non-proliferative cells may exist in the germinative compartment of human epidermis. Results obtained in vitro demonstrate that keratinocytes can be blocked in certain conditions in a state of quiescence similar to a G0 phase. Increasing evidence suggests that, as in mouse epidermis, the germinative compartment of human epidermis is and is divided into a small proportion of stem cells (10%) with a high proliferative potential, a larger proportion of transit-amplifying cells with a limited proliferative potential (50%) and postmitotic cells committed to undergo terminal differentiation (40%). In this hierarchical arrangement, most of the nonproliferative cells belong to the postmitotic compartment. If this model is correct, the germinative cell population in a quiescent or G0 state is a small proportion of the stem cell compartment and has therefore little influence on the kinetics of normal human epidermis.

Poly(ADPribosyl)ation System in Rat Germinal Cells at Different Stages of Differentiation

In order to study the possible functional relationship between poly(ADP-ribosyl)ation and spermatogenesis, the three main germinal cell types have been isolated and characterized as haploid spermatids and diploid and tetraploid spermatocytes. Purified germinal cell populations and rats of different age were used for activity-, immuno-, and Northern blot experiments, to determine at which level poly(ADPR)polymerase (PARP) is regulated at various stages of spermatogenesis. Poly(ADPR)glycohydrolase (PARG) activity was also determined, as was the subcellular distribution of both PARP and PARG enzymes. The results show that the maximum of both PARP amount and PARP activity can be detected on tetraploid spermatocytes which undergo meiotic division, whereas PARG activity does not differ in germinal cells; the cytoplasmic form of this enzyme is prevalent in testis. Moreover, a difference in timing was observed in maximal level between PARP expression, determined on testis from 60-day-old rats, and PARP activity, detected on testis from 30-day-old animals. It seems that different mechanisms modulate the poly(ADPribosyl)ation system during spermatogenesis. Regulation of the poly(ADPribose) turnover, variations of PARP amount, as well as changes of PARP transcription level, seem to accompany germinal cell differentiation, possibly being implicated in DNA replication, repair, and transcription.

Germinal‐center cell proliferation in response to T‐independent antigens: A stathmokinetic, morphometric and immunohistochemical study in vivo

Although the nature of the germinal center reaction during responses to T-dependent antigens has been well documented, much less is known regarding the relationship between germinal centers and T-independent antigens. In this study, germinal-center cell proliferation was determined at specific time points in spleens of C3H/HeN mice following immunization with either the type-1, T-independent antigen dinitrophenol-lipopolysaccharide (DNP-LPS), or the type-2, T-independent antigen DNP-Ficoll. A stathmokinetic technique was employed to assess proliferation in terms of germinal center cell birth rate and morphometry was used to measure actual growth and regression of the germinal center cell population. An estimate of the absolute rate of germinal-center (GC) cell proliferation was derived from these two values. In addition, immunohistochemistry was performed to correlate changes in GC cell proliferation with the presence or absence of antigen within GC. Following immunization with both antigens, there was an initial reduction of proliferation within pre-existing germinal centers which manifested as either GC dissociation (DNP-LPS) or a suppression of birth rates (DNP-Ficoll). This was followed by a period of increased GC cell proliferation in animals immunized with DNP-LPS, but not in those exposed to DNP-Ficoll. GC cell proliferation was then measured in mice treated with cyclosporin A from 1 day before to 2 days after immunization with DNP-LPS. In these animals, the expected increase in GC cell birth rates did not take place. Immunohistochemistry showed that DNP-Ficoll and DNP-LPS were present in GC from 1 day after immunization until the end of the experiment on day 7. Treatment with cyclosporin A did not affect the deposition of DNP-LPS in GC. These results show that only some T-independent antigens are able to stimulate GC cell proliferation, and we propose that this is related to their ability to recruit precursors of GC B cells into the GC reaction. In addition, the results indicate that GC proliferation seen in response to a so-called T-independent antigen is at least partly driven by T cell-derived cytokines.

Radiation‐Induced Apoptosis and Developmental Disturbance of the Brain*

AbstractThe developing mammalian brain is highly susceptible to ionizing radiation. A significant increase in small head size and mental retardation has been noted in prenatally exposed survivors of the atomic bombing, with the highest risk in those exposed during 8–15 weeks after fertilization. This stage corresponds to day 13 of pregnancy for mice and day 15 for rats in terms of brain development. The initial damage produced by radiation at this stage is cell death in the ventricular zone (VZ) of the brain mantle, the radiosensitive germinal cell population. During histogenesis of the cerebellum, on the other hand, the external granular layer (EGL) is also radiosensitive. Although extensive cell death results in microcephaly and histological abnormality, both VZ and EGL have an ability to recover from a considerable cell loss and form the normal structure of the central nervous system. The number of cell deaths to induce tissue abnormalities in adult brain rises in the range of 15–25% of the germinal cell population; and the threshold doses are about 0.3 Gy for cerebral defects and 1 Gy for cerebellar anomalies in both mice and rats. A similar threshold level is suggested in human cases in induction of mental retardation.Radiation‐induced cell death in the VZ and EGL has been revealed as apoptosis, by the nuclear and cytoplasmic condensation, transglutaminase activation, required macromolecular synthesis, and internucleosomal DNA cleavage. Apoptosis of the germinal cell is assumed to eliminate acquired genetic damage. Once an abnormality in DNA has been induced and fixed in a germinal cell, it would be greatly amplified during future proliferation, bringing with it the risk of producing malformations and tumors. These cells would commit suicide when injured for replacement by healthy cells, rather than undertake DNA repair which is rarely complete. In fact they show very slow repair of cellular damage. Thus the high sensitivity of undifferentiated neural cells to the lethal effect of radiation may constitute a biological defense mechanism.