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PubMed2005

Stem cells and brain cancer.

One of the most devastating CNS pathologies is brain cancer. The undifferentiated character of brain tumor cells and recent reports of cancer stem cells prompt questions regarding the involvement of normal stem/progenitor cells in brain tumor biology, their potential contribution to the tumor itself, and whether they are the cause or the consequence of tumor initiation and progression. The cancer stem cell model proposes a clonally derived brain tumor arising from a cancer stem cell. This tumor cell-of-origin originates from a stem/progenitor or more differentiated cell via acquisition of oncogenic mutations that dysregulate or allow reacquisition of self-renewal mechanisms. The tumor cells differentiate unidirectionally from the cancer stem cell in a way parallel to normal development. However, several properties of brain tumors add complexity to this model. For example, the apparent lineage and differentiation status of tumor cells are significantly affected by signaling abnormalities that are causally related to formation of the tumor. In addition, these tumors recruit normal CNS stem and progenitor cells to the tumor mass leading to the possibility of a heterogeneous and polyclonal cell population. It is likely that a complete description of the role of stem cells in brain tumors will be more complex than our current models.

Animals↗

PubMed2004

Species differences in troxacitabine pharmacokinetics and pharmacodynamics: implications for clinical development.

PURPOSE: Troxacitabine is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Significant differences in troxacitabine toxicity between mice, rats, monkeys, and humans were observed during preclinical and clinical evaluations. To better understand the different toxicity and efficacy results observed between the human xenograft mouse tumor models used for preclinical assessment and the clinical study results, the pharmacodynamics and pharmacokinetics of troxacitabine were reassessed in murine and human models. EXPERIMENTAL DESIGN: Clonal and thymidine incorporation assays were used to investigate the in vitro antiproliferative activity of troxacitabine on a selected panel of mouse and human tumor cell lines and normal hemapoietic cells. Analysis of the intracellular metabolites of [14C]troxacitabine was determined in mouse and human T-lymphocytes obtained from peripheral blood. The antitumor efficacy of troxacitabine administered either as single or repeated high-dose bolus administrations or as low-dose continuous infusions was evaluated in the human colon HT-29 xenograft model. We also determined plasma concentrations of troxacitabine using the different administration schedules. RESULTS: Five to nine hundred-fold lower concentrations of troxacitabine were required to inhibit cell growth in human compared with murine tumor and normal hemapoietic cell lines. Furthermore, the sensitivity of cells of both species to troxacitabine was strongly time dependent, requiring >24 hours exposure for maximum activity. Analysis of the intracellular metabolites of [14C]troxacitabine in T-lymphocytes obtained from peripheral blood revealed subsequently higher levels of mono-, di-, and triphosphates in human compared with mouse. Antitumor efficacy studies revealed that prolonged exposure schedules (up to 6 days) showed equivalent efficacy to repeated high-dose bolus administrations. Five-day continuous infusion of 20 mg/mL troxacitabine via subcutaneous implanted mini-osmotic pump maintained systemic concentrations of 262 ng/mL (1.2 micromol/L) for the duration of administration, which are clinically achievable plasma concentrations, and led to significant antitumor activity [treated versus control (T/C) of 27% and tumor regression during treatment]. CONCLUSIONS: These studies support the hypothesis that troxacitabine infusions might be the administration regimen with the greatest likelihood of fully exploiting clinically the potent preclinical antitumor activity of troxacitabine.

Animals↗

PubMed2000

Longevity in vitro of human CD4+ T helper cell clones derived from young donors and elderly donors, or from progenitor cells: age-associated differences in cell surface molecule expression and cytokine secretion.

The effectiveness of the adaptive immune system relies upon extensive proliferation of an initially small number of antigen-specific T cells. At the end of a successful response, the majority die by apoptosis and a small minority joins the memory cell pool. Upon re-challenge with antigen, these memory cells must again undergo clonal expansion in order to mediate an effective response. Thus, T cells are subjected to marked proliferative stress which may result in clonal exhaustion due to replicative senescence. In other systems made up of rapidly proliferating cells (e.g. in the gut) individual clones are identical and are replaced at the end of their lifespan by differentiation from a stem cell reservoir. However, because of the unique clonal distribution of antigen receptors on T cells, mere replacement with other T cells is not sufficient to maintain the integrity of the system. Moreover, the very source of new T cells decreases with age (due to thymic involution). Therefore, the adaptive immune system may be uniquely susceptible to the deleterious effects of replicative senescence. Particularly in humans, in vivo studies of the behaviour of individual T-cell clones in the body is difficult. However, T-cell longevity, measured as proliferative capacity in terms of population doublings, can be usefully modelled at the clonal level in vitro. This paper discusses the surprisingly little that is known about the average longevity, variation between clones, and the maximal longevity of human T cells under clonal culture conditions in vitro. From our own studies, we show that average lifespan of human T cells is as little as 17 PD; however, established clones reach 35 PD on average, with maximum longevity generally in the region of 60-80 PD, regardless of the source of the cloned cells. Expression of surface molecules in general did not differ strikingly between young and old donors, but the frequency of clones secreting IL-10, and the amount secreted per clone was higher in the elderly than in the young. Conversely, the frequency of clones secreting IL-6 and the amount secreted per clone was higher in the young.

Adult↗

PubMed1993

Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells.

Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHI-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of beta 2-microglobulin and beta-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-mu was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗

PubMed1993

Aberrant induction of T cell tolerance in B cell suppressed mice.

Self-tolerance is the process by which the T cell repertoire develops without expressing self-reactive specificities. The mechanisms which functionally eliminate self-reactive T cells are clonal deletion and clonal inactivation, and both of these phenomena have been studied in T cell populations reactive to endogenous superantigens that are encoded by endogenous mouse mammary tumor proviruses (Mtv). The studies described here demonstrate that the kinetics of Etc-1 (encoded by the Mtv-9 open reading frame gene)-mediated deletion are much slower than that seen for Mls 1a (encoded by the Mtv-7 open reading frame), and that Etc-1-reactive T cells are present in the periphery up to 2 wk after birth. The deletion of peripheral Etc-1-reactive T cells late in ontogeny indicates an efficient mechanism of peripheral clonal deletion in these animals. The clonal deletion of Etc-1-reactive cells is abrogated in B cell-suppressed animals; however, clonal elimination of peripheral V beta 5+ or V beta 11+ (Etc-1 reactive) T cells can be induced when these mice are allowed to recover their B cell population after cessation of anti-mu treatment. Finally, we establish that peripheral Etc-1-reactive V beta 11+/CD4+ T cells remaining in B cell-suppressed and recovering animals are markedly less responsive to stimulation through the TCR than are control T cells. These data support the idea that peripheral self-reactive T cells can be rendered tolerant by two mechanisms which may be temporally related. This model suggests that clonal hyporesponsiveness may be followed by clonal deletion.

Animals↗

PubMed2026

Case Report: Immune-driven clonal selection underlying lineage switch from B-Precursor acute lymphoblastic leukemia to acute myeloid leukemia following inotuzumab ozogamicin.

Lineage switch (LS), defined as a change in leukemic lineage during the disease course, is a rare but clinically significant event in acute leukemia and is typically associated with poor prognosis. Although LS has been increasingly reported following targeted immunotherapies, the clonal mechanisms underlying this phenomenon remain incompletely understood, particularly in cases without KMT2A rearrangement. We report a case of LS from B-precursor acute lymphoblastic leukemia (BCP-ALL) to acute myeloid leukemia (AML) following treatment with the CD22-targeted antibody-drug conjugate inotuzumab ozogamicin. To elucidate the clonal architecture underlying LS, targeted next-generation sequencing was performed on bone marrow samples obtained at multiple time points throughout the disease course. Genomic analysis demonstrated that the lymphoid and myeloid disease phases shared ancestral genetic alterations but displayed distinct mutational profiles. At the time of LS, TP53 and SMC1A mutations newly emerged, whereas only a subset of mutations detected at ALL relapse was retained. These findings suggest that the AML phase most likely resulted from the selective expansion of a genetically distinct subclone derived from a common progenitor, rather than the direct transdifferentiation of the dominant ALL clone, consistent with immunotherapy-driven clonal selection. Longitudinal genomic profiling revealed stepwise clonal evolution during disease progression, supporting a model of immunotherapy-driven clonal selection leading to LS. This case provides molecular evidence suggesting that immune-targeted therapy can promote expansion of minor pre-existing subclones with alternative lineage potential within a common progenitor even in non-KMT2A-rearranged leukemia. Our findings highlight the importance of comprehensive genomic monitoring during immunotherapy to identify therapy-resistant subclones and better understand mechanisms of lineage plasticity in acute leukemia.

Humans↗

PubMed1989

Two-stage models of tumor incidence for historical control animals in the National Toxicology Program's carcinogenicity experiments.

The tumor incidence rate is modeled for various tumors in a database of control animals [Fischer 344 rats and (C57BL/6 x C3H)F1 mice] originally developed by the national Toxicology Program and recently augmented to include additional sacrifice data by Portier et al. (1986). These rates are assumed to follow a clonal two-stage model of carcinogenesis where cells in the various tissues are allowed to be in one of three states arbitrarily defined as a "normal" state, an "initiated" state, and a "tumor present" state. The parameters of this clonal two-stage model have a direct interpretation with regard to the mechanism of action and in some cases may suggest a common mechanism for various tumors in the different sex/species groups. Also, the risk of dying (from all causes) in tumor-bearing animals is compared to the risk of dying in non-tumor-bearing animals via estimates of relative risk. In general, it was found that the risk of dying was elevated for most tumors. The purpose of this analysis is to provide estimates of baseline tumor rates and relative risks, which are useful in the design and analysis of future carcinogenicity experiments.

Animals↗

PubMed2003

NOD/SCID mice transplanted with marrow from patients with myelodysplastic syndrome (MDS) show long-term propagation of normal but not clonal human precursors.

Sublethally irradiated NOD/SCID mice were transplanted with hematopoietic progenitor cells obtained from the marrow of patients with myelodysplastic syndromes (MDS). Engraftment of MDS cells, as determined by flow cytometry, was delayed compared to marrow from normal donors. Human CD38(+)CD34(-) cells were prominent in marrows and spleens of MDS chimeras. CD34(+)CD38(-), CD34(+)CD38(+) and T cells were also easily detected. Human myeloid cells (CD33(+); CD15(+)) were present in low proportions. No clonal precursors were identified by fluorescent in situ hybridization (FISH) or by molecular analysis of polymorphic X-linked markers in mice with documented engraftment of human cells more than 2 months after transplantation. These data indicate that human cells present in murine MDS chimeras, at the levels of sensitivity of our assays, were derived from residual normal cells in human MDS marrow, and suggest that the NOD/SCID environment was not conducive to the expansion of clonal MDS precursors. This model may allow identification of factors relevant for sustaining or expanding clonal precursors.

Adult↗

PubMed1996

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse.

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2Kb-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10(-7) M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells.

Adipocytes↗

PubMed2005

Immunophenotypic differences between diagnosis and relapse in childhood AML: Implications for MRD monitoring.

BACKGROUND: Determination of antigen expression patterns is, in addition to morphologic analysis, essential to the diagnosis of acute myeloid leukemia (AML). The present study was performed to determine (a) the degree of changes in immunophenotype and their consequences on the monitoring of minimal residual disease (MRD) in childhood AML and (b) whether certain clusters of changes in antigen expression patterns exist between diagnosis and relapse. METHODS: Bone marrow specimens of 48 children enrolled in the German AML-BFM-93/98 (Acute Myeloid Leukemia-Berlin-Frankfurt-Munster) studies were analyzed immunologically, morphologically, and genetically at diagnosis and at first relapse. RESULTS: The immunophenotypes by flow cytometry differed by at least one antigen between samples at presentation and relapse in 42 of 48 children (88%). More children displayed an immature phenotype at relapse (43 of 47, 91.5%, vs. 37 of 48, 77%; P = 0.05) with expression of CD34 and/or CD117. This was reflected by a gain of markers that are associated with lineage immaturity in 18 of 25 (72%) of cases, whereas the loss of such antigens was observed in 6 of 25 (24%) patients. We did not observe significant changes for lineage specific markers, with comparable occurrences of loss or gain of myeloid and lymphoid antigens in the sample pairs. Only minimal changes were seen for morphologic and genetic features. CONCLUSION: An antigenic shift was observed in 88% of cases in this study. The antibody panels used for MRD monitoring in childhood AML should therefore not be restricted to the immunophenotype detected at presentation but should include in particular markers of lineage immaturity. The clinical observation of a shift toward a more immature phenotype of the myeloblasts is consistent with the model of a clonal evolution of a leukemic stem cell.

Acute Disease↗

PubMed1991

2,4,6-trinitrophenyl (TNP) responsiveness of anti-TNP (Sp6) transgenic mice.

Transgenic mouse models have demonstrated clonal deletion as well as clonal anergy of monospecific, high-avidity autoreactive B cell. The function and fate of naturally activated B cells, many of them displaying degenerate specificity including autoreactivity, are still a matter of debate. The question was pursued in Sp6-transgenic mice. Sp6, a monoclonal anti-2,4,6-trinitrophenyl (TNP) IgM has been shown to react with a variety of self antigens. Responsiveness of antibody-secreting B cells was followed throughout postnatal development of Sp6-transgenic mice and was related to the availability of antigen- and idiotype-specific help. Thymus as well as spleen cells of transgenic mice contained a significantly higher number of TNP-specific B cell than non-transgenic controls. In contrast to control mice, the number of TNP-specific B cells remained unchanged or decreased in thymus and spleen of transgenic mice after antigenic stimulation with TNP in T-dependent (TD) and T-independent (TI) form. Since the relative frequency of transgenic B cells was in particular diminished after repeated stimulation with TD antigen, it was examined whether limited responsiveness was linked to the available repertoire of helper T cells. Early after birth of transgenic individuals, thymic as well as splenic T cells which proliferated in response to TNP and Sp6 and provided help for B cells were found to be significantly augmented. Their number decreased rapidly during postnatal maturation and Th cells did not expand after antigenic stimulation. There was no indication that in the naive host transgenic B cells would suppress proliferation of TNP- and Sp6-specific T cells, but they did so after antigenic stimulation. Furthermore, and in contrast to B cells of non-transgenic mice, transgenic B cells were unable to present nominal antigen in a stimulatory way. The decrease in the number of B cells after antigenic stimulation indicated that autoreactive transgenic B cells may be subject to (functional) deletion under selected circumstances. In addition, idiotype- and antigen-specific help was impaired in Sp6-transgenic mice and this clearly was due to interactions with B cells expressing the immunoglobulin transgene.

Age Factors↗

PubMed1994

Ceramide mediates the apoptotic response of WEHI 231 cells to anti-immunoglobulin, corticosteroids and irradiation.

We demonstrate for the first time how immature B cells kill themselves. Ceramide is identified as the mediator of apoptosis in the murine B lymphoma line WEHI 231 commonly used as a model to study clonal deletion in B lymphocytes. We show that exogenous ceramide induces apoptosis in WEHI 231 cells. To maintain self tolerance, immature lymphocytes readily undergo apoptotic death in response to the cross-linking of their antigen-specific receptors. We demonstrate that endogenously produced ceramide accumulates in WEHI 231 cells exposed to anti-IgM, an antigen surrogate before the onset of apoptosis. We also show that two other inducers of apoptosis, irradiation and dexamethasone, cause intracellular accumulation of ceramide.

Animals↗

PubMed2001

Involvement of polyamines in B cell receptor-mediated apoptosis: spermine functions as a negative modulator.

The B cell lymphoma WEHI231 has been used as a model for studying clonal deletion of B cells on the basis of its ability to undergo growth arrest and apoptosis by B cell antigen receptor (BCR) cross-linking. To comprehensively analyze the genes involved in BCR-mediated apoptosis, we applied the technique of serial analysis of gene expression (SAGE) to WEHI231. Comparison of expression patterns revealed that BCR cross-linking caused coordinate changes in the expression of genes involved in polyamine metabolism. Polyamines are ubiquitous compounds required for cell proliferation and homeostasis. The coordinate expression of the polyamine-related genes was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction analysis. During apoptosis, the genes involved in polyamine biosynthesis were downregulated, whereas those involved in polyamine catabolism were upregulated, suggesting that intracellular polyamines play a role in BCR-mediated apoptosis. Levels of intracellular putrescine, spermidine, and spermine were reduced after BCR cross-linking. These effects were prevented by concurrent CD40 stimulation, which blocked BCR-mediated apoptosis. Furthermore, addition of spermine could repress the BCR-mediated apoptosis by attenuating the mitochondrial membrane potential (Deltapsim) loss and activation of caspase-7 induced by BCR signaling. These findings strongly suggest that polyamine regulation is involved in apoptosis during B cell clonal deletion.

Animals↗

PubMed1995

Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane.

The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in vitro mouse neuroblastoma-rat glioma hybrid NG108-15 clonal cell line model. Following 0.3 mM SM exposure, cell viability remained high (> 80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover, there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ([3H]AA) from cellular membranes. Increase in [3H]AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in [3H]AA release was inhibited by quinacrine (20 microM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in [3H]AA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca(2+)-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

Animals↗

PubMed1995

Identification of epidemiologic markers for Neisseria meningitidis using difference analysis.

The feasibility of identifying epidemiologic markers based solely on the identification of DNA fragments present in outbreak-associated isolates was investigated using Neisseria meningitidis (Nm) as a model system. The clonal structure of Nm has been well characterized using multilocus electrophoresis. In Canada, electrophoretic types ET1, ET5, ET9 and ET21 are being displaced from the natural population by type ET15, and the latter type is associated with an increased prevalence of serogroup C meningococcal disease. Difference analysis, which uses subtractive hybridization and polymerase chain reaction (PCR) amplification, was employed to identify amplifiable DNA fragments (amplicons) that differ between the ET15 and the ET1, ET5, ET9 and ET21 genomes. 14 amplicons were cloned which were further characterized by Southern blot analysis to identify six amplicons that represent fragments either unique to or highly polymorphic in the ET15 genome. Oligodeoxyribonucleotide primer pairs were designed for each of the six amplicons, and PCR amplification was used to determine their prevalence across a panel of 167 Nm isolates representative of other serogroups and ETs. Among group C isolates only two of the six amplicons, designated as A and G, were effective in discriminating ET15 from non-ET15 isolates. Amplicon A detects a deletion in the dhps gene which effectively differentiates sulfonamide-sensitive and -resistant serogroup C isolates. The frequency of amplicon A and G detection in the other serogroups and ETs was too great to facilitate their direct use as diagnostic markers for the differentiation of virulent Nm isolates.

Bacterial Outer Membrane Proteins↗

PubMed2005

Spindle cells and their role in Kaposi's sarcoma.

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.

Cell Lineage↗

PubMed2005

Molecular cloning and characterization of two types of CD8alpha from ginbuna crucian carp, Carassius auratus langsdorfii.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

Amino Acid Sequence↗

PubMed2026

Postzygotic biallelic inactivation of FDFT1 underlies solitary lesion formation in porokeratosis of Mibelli.

BACKGROUND: Porokeratosis reflects clonal expansion of keratinocytes with biallelic inactivation of mevalonate-cholesterol biosynthesis pathway genes. In disseminated porokeratosis (DP), lesions arise through independent somatic second hits in carriers of heterozygous germline pathogenic variants, whereas porokeratosis of Mibelli (PM) is usually solitary, and its molecular basis remains incompletely defined. OBJECTIVE: To elucidate the molecular basis of solitary PM. METHODS: We analyzed blood and lesional epidermis from seven patients with solitary PM within a 156-patient porokeratosis cohort using deep sequencing, copy-number/SNP profiling, and methylation analysis. RESULTS: Solitary PM plaques were larger and more irregular than the annular DP lesions. No pathogenic germline variants were detected in MVK, PMVK, MVD, FDPS, or FDFT1. Three patients had somatic biallelic genetic inactivation of FDFT1 through putative deleterious variants and/or focal microdeletions. The remaining four showed FDFT1 promoter hypermethylation with loss of heterozygosity (LOH) at the FDFT1 locus due to copy-neutral LOH or a monoallelic 8p deletion, consistent with early monoallelic epigenetic silencing, followed by genetic loss of the remaining active allele. In one patient, part of the plaque expanded centrifugally over 7.5 years. CONCLUSION: Solitary PM can be driven by postzygotic, lesion-restricted, biallelic inactivation of FDFT1 through genetic or epigenetic mechanisms within a single epidermal clone, promoting clonal expansion. This model may explain the tendency toward solitary PM lesions. The low probability of acquiring postzygotic biallelic inactivation without germline predisposition may underlie solitary PM and suggest a low recurrence risk for offspring, unlike DP driven by germline heterozygosity.

General dermatology↗