Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament.

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

STAT1 modulates tissue wasting or overgrowth downstream from PDGFRß.

Platelet-derived growth factor (PDGF) acts through two conserved receptor tyrosine kinases: PDGFRα and PDGFRß. Gain-of-function mutations in human PDGFRB have been linked recently to genetic diseases characterized by connective tissue wasting (Penttinen syndrome) or overgrowth (Kosaki overgrowth syndrome), but it is unclear whether PDGFRB mutations alone are responsible. Mice with constitutive PDGFRß signaling caused by a kinase domain mutation (D849V) develop lethal autoinflammation. Here we used a genetic approach to investigate the mechanism of autoinflammation in Pdgfrb+/D849V mice and test the hypothesis that signal transducer and activator of transcription 1 (STAT1) mediates this phenotype. We show that Pdgfrb+/D849V mice with Stat1 knockout (Stat1-/-Pdgfrb+/D849V ) are rescued from autoinflammation and have improved life span compared with Stat1+/-Pdgfrb+/D849V mice. Furthermore, PDGFRß-STAT1 signaling suppresses PDGFRß itself. Thus, Stat1-/-Pdgfrb+/D849V fibroblasts exhibit increased PDGFRß signaling, and mice develop progressive overgrowth, a distinct phenotype from the wasting seen in Stat1+/-Pdgfrb+/D849V mice. Deletion of interferon receptors (Ifnar1 or Ifngr1) does not rescue wasting in Pdgfrb+/D849V mice, indicating that interferons are not required for autoinflammation. These results provide functional evidence that elevated PDGFRß signaling causes tissue wasting or overgrowth reminiscent of human genetic syndromes and that the STAT1 pathway is a crucial modulator of this phenotypic spectrum.

Periodontal tissue stem cells and mesenchymal stem cells in the periodontal ligament.

Periodontal tissue stem cells, which play a crucial role in maintaining the homeostasis of periodontal tissues, are found in the periodontal ligament (PDL). These cells have long been referred to as mesenchymal stem/stromal cells (MSCs), and their clinical applications have been extensively studied. However, tissue stem cells in the PDL have not been thoroughly investigated, and they may be different from MSCs. Recent advances in stem cell biology, such as genetic lineage tracing, identification of label-retaining cells, and single-cell transcriptome analysis, have made it possible to analyze tissue stem cells in the PDL in vivo. In this review, we summarize recent findings on these stem cell populations in PDL and discuss future research directions toward developing periodontal regenerative therapy.

Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing.

Fibroblasts differentiate into myofibroblasts by acquiring new contractile function. This is important for tissue repair, but it also contributes to organ fibrosis. Platelet-derived growth factor (PDGF) promotes tissue repair and fibrosis, but the relationship between PDGF and myofibroblasts is unclear. Using mice with lineage tracing linked to PDGF receptor α (PDGFRα) gene mutations, we examine cell fates during skin wound healing. Elevated PDGFRα signaling increases proliferation but unexpectedly delays the fibroblast-to-myofibroblast transition, suggesting that PDGFRα must be downregulated for myofibroblast differentiation. In contrast, deletion of PDGFRα decreases proliferation and myofibroblast differentiation by reducing serum response factor (SRF) nuclear localization. Consequences of SRF deletion resemble PDGFRα deletion, but deletion of two SRF coactivators, MRTFA and MRTFB, specifically eliminates myofibroblasts. Our findings suggest a scenario where PDGFRα signaling initially supports proliferation of fibroblast progenitors to expand their number during early wound healing but, later, PDGFRα downregulation facilitates fibroblast differentiation into myofibroblasts.

Apparent diffusion coefficient as an MR imaging biomarker of low-risk ductal carcinoma in situ: a pilot study.

PURPOSE: To evaluate the potential of apparent diffusion coefficients (ADCs) obtained at quantitative diffusion-weighted magnetic resonance (MR) imaging of the breast as a biomarker of low-grade ductal carcinoma in situ (DCIS). MATERIALS AND METHODS: This retrospective study was approved by an institutional review board, and the requirement to obtain informed consent was waived. Twenty-two women (age range, 36-75 years; mean age, 56.4 years) with pure DCIS (seven with low-grade DCIS, five with intermediate-grade DCIS, and seven with high-grade DCIS) and three with microinvasion underwent breast MR imaging at 1.5 T between January 2008 and November 2010. MR examinations included contrast material-enhanced (gadoteridol) T1-weighted imaging and diffusion-weighted MR imaging with b values of 0 and 1000 sec/mm(2). ADC maps were generated. The distributions of the ADCs in regions of interest covering the lesions were compared among the three grades by using linear mixed-model analysis, and the discriminatory power of the lesion minimum ADC was determined with receiver operating characteristic analysis. RESULTS: The mean ADC was 1.42 × 10(-3) mm(2)/sec (95% confidence interval [CI]: 1.31 × 10(-3) mm(2)/sec, 1.54 × 10(-3) mm(2)/sec) for low-grade DCIS, 1.23 × 10(-3) mm(2)/sec (95% CI: 1.10 × 10(-3) mm(2)/sec, 1.36 × 10(-3) mm(2)/sec) for intermediate-grade DCIS, 1.19 × 10(-3) mm(2)/sec (95% CI: 1.08 × 10(-3) mm(2)/sec, 1.30 × 10(-3) mm(2)/sec) for high-grade DCIS, and 2.06 × 10(-3) mm(2)/sec (95% CI: 1.94 × 10(-3) mm(2)/sec, 2.18 × 10(-3) mm(2)/sec) for normal breast tissue. The mean ADCs for high- and intermediate-grade DCIS were significantly lower than that for low-grade DCIS (P < .01 and P = .03, respectively), and the mean ADC for low-grade DCIS was significantly lower than that for normal tissue (P < .001). The lesion minimum ADC for low-grade DCIS was also significantly higher than that for high- and intermediate-grade DCIS (P < .01). A threshold of 1.30 × 10(-3) mm(2)/sec for the minimum ADC in the diagnosis of low-grade DCIS had a specificity of 100% (12 of 12 patients; 95% CI: 73.5%, 100%) and a positive predictive value of 100% (four of four patients; 95% CI: 39.8%, 100%). CONCLUSION: These preliminary results suggest that quantitative diffusion-weighted MR imaging could be used to identify patients with low-grade DCIS with very high specificity. If the results of this study are confirmed, this approach could potentially spare those patients from invasive approaches such as mastectomy or axillary lymph node excision.

Pseudodysplastic regenerative mucosa associated with congenital ileal atresia.

A male neonate was clinically diagnosed with congenital intestinal atresia. Surgical operation was performed and the ileum including the atretic portion was resected. Grossly, there was a plaque-like elevation of mucosa at the proximal side of the ileal atresia. Microscopic examination of this lesion revealed proliferation of severely atypical glands. Although dysplasia was a serious diagnostic concern, we concluded that ischemia due to the intestinal atresia induced this benign pseudodysplastic regenerative mucosa, judging from the pattern of coexisting inflammation and the literature review.

Mice lacking PLAP-1/asporin counteracts high fat diet-induced metabolic disorder and alveolar bone loss by controlling adipose tissue expansion.

Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.

Mouse Model of Loeys-Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling.

Loeys-Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-ß receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-ß and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-ß compared with WT MEFs. To clarify the influence of TGF-ß signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-ß-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1 G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-ß signal dysfunction. This suggests that TGF-ß signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament.

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Mosaic Mutant Analysis Identifies PDGFRα/PDGFRß as Negative Regulators of Adipogenesis.

Adipocyte progenitors (APs) express platelet-derived growth factor receptors (PDGFRs), PDGFRα and PDGFRß. Elevated PDGFRα signaling inhibits adipogenesis and promotes fibrosis; however, the function of PDGFRs in APs remains unclear. We combined lineage tracing and functional analyses in a sequential dual-recombinase approach that creates mosaic Pdgfr mutant cells by Cre/lox recombination with a linked Flp/frt reporter to track individual cell fates. Using mosaic lineage labeling, we show that adipocytes are derived from the Pdgfra lineage during postnatal growth and adulthood. In contrast, adipocytes are only derived from the mosaic Pdgfrb lineage during postnatal growth. Functionally, postnatal mosaic deletion of PDGFRα enhances adipogenesis and adult deletion enhances ß3-adrenergic-receptor-induced beige adipocyte formation. Mosaic deletion of PDGFRß also enhances white, brown, and beige adipogenesis. These data show that both PDGFRs are cell-autonomous inhibitors of adipocyte differentiation and implicate downregulation of PDGF signaling as a critical event in the transition from AP to adipocyte.

Antidonor antibody in patients receiving ABO-identical and HLA-mismatched living donor liver transplants: effect on survival.

We retrospectively determined the correlation of results of lymphocyte crossmatch tests by direct complement-dependent cytotoxicity, to the outcomes of 585 consecutive ABO-identical and human leukocyte antigen (HLA)-mismatched living donor liver transplants (LDLTs) (male:female=276:309; median age, 18 years). Crossmatch test results were positive in 14 recipients (2.4%). Patient survival at eight years in the crossmatch-positive group was significantly lower than in the crossmatch-negative group (positive group, 56.3%; negative group, 77.6%; P=0.014). The survival at five years of the crossmatch-positive group was significantly lower than the negative group in both older recipients (>or=18 years of age: positive group, 41.7%; negative group, 76.4%; P=0.0065), and female recipients (positive group, 37.5%; negative group, 81.9%; P=3.3x10). We conclude that antidonor antibodies have adverse effects on the clinical outcome of LDLTs, and that being female and/or older aged (>or=18 years of age) are risk factors for LDLT.

Pivotal role of CXCR3 in melanoma cell metastasis to lymph nodes.

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to approximately 15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund's adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5-3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.

Gastric foveolar metaplasia with dysplastic changes in Brunner gland hyperplasia: possible precursor lesions for Brunner gland adenocarcinoma.

Cases of adenocarcinomas developed in Brunner gland hyperplasia (BGH) have been sporadically reported. Herein, we report the morphologic spectrum of hyperplastic changes culminating into dysplasia and carcinoma in 722 cases of BGH listed in our files. Fifteen of these cases showed dysplastic changes, with 8 graded as low-grade dysplasia, 5 as high-grade dysplasia, 11 as atypical hyperplasia, and 2 as invasive carcinoma, although each frequently coexisted in the same tumor. In two carcinomas, one had high-grade dysplasia in the mucosa, and another had only atypical hyperplasia. Interestingly, hyperplastic glands around dysplastic foci were associated with gastric foveolar metaplasia and papillary configuration in 13 cases, 11 of which showed a gradual increase in nuclear atypism in the transition from metaplastic to dysplastic glands. All of the metaplastic gastric glands showed diffuse and strong immunopositivity for gastric foveolar mucin (MUC5AC). Immunohistochemical profiles also supported the concept of a continuous spectrum in carcinogenesis from gastric foveolar hyperplasia through atypical hyperplasia or dysplasia and eventually to frank adenocarcinoma. The results of our study suggest, therefore, that dysplastic and/or carcinomatous change does occur in BGH, that they form the continuous morphologic spectrum, and that papillary foveolar metaplasia may be a precursor lesion in the process of carcinogenesis with a background of BGH.

Suppression of colonic polyposis by homeoprotein CDX2 through its nontranscriptional function that stabilizes p27Kip1.

Caudal-related homeoprotein CDX2 is expressed in intestinal epithelial cells, in which it is essential for their development and differentiation. A tumor suppressor function is suggested by evidence that CDX2 levels are decreased in human colon cancer specimens and that an inactivating mutation of Cdx2 in Apc(Δ716) mice markedly increases the incidence of colonic polyps. In this study, we investigated roles for transcriptional and nontranscriptional functions of CDX2 in suppression of colonic tumorigenesis. Mutagenic analysis of CDX2 revealed that loss of function stabilizes CDK inhibitor p27Kip1 by a nontranscriptional but homeodomain-dependent mechanism that inhibits cyclin E-CDK2 activity and blocks G0/G1-S progression in colon cancer cells. p27Kip1 stabilization was mediated by an inhibition of ubiquitylation-dependent proteolysis associated with decreased phosphorylation of Thr187 in p27Kip1. siRNA-mediated knockdown of p27Kip1 relieved the decrease in cyclin E-CDK2 activity and S-phase cell fraction elicited by CDX2 expression. Together, these results implicate a nontranscriptional function of CDX2 in tumor suppression mediated by p27Kip1 stabilization. Up to approximately 75% of low-CDX2 human colon cancer lesions show reduced levels of p27Kip1, whereas approximately 68% of high-CDX2 lesions retain expression of p27Kip1. These results show that low levels of CDX2 accelerate colon tumorigenesis by reducing p27Kip1 levels.

Acute Epstein-Barr virus infection presenting as severe gastroenteritis without infectious mononucleosis-like manifestations.

Primary Epstein-Barr virus (EBV) infection is usually a self-limiting disease. Although it is sometimes accompanied by severe complications such as thrombocytopenia, hemolytic anemia, and splenic rupture, predominantly gastrointestinal complications are rarely reported. We studied an unusual case of primary EBV infection associated with severe hemorrhagic gastroenteritis. EBV infection was confirmed in the biopsy specimen by demonstrating the presence of EBV DNA by polymerase chain reaction, and of EBV-encoded small RNA (EBER)-positive cells by in-situ hybridization. Our patient was suspected of having primary EBV infection from the serological findings-EBV-viral capsid antigen IgM (+) and EBV nuclear antigen (-)-but he did not show typical clinical features of infectious mononucleosis such as lymph node swelling, pharyngitis, liver dysfunction, and splenomegaly. A definite diagnosis of primary EBV infection was made using biopsy specimens by demonstrating the presence of EBV DNA and EBER-positive cells.

Requirement of protocol biopsy before and after complete cessation of immunosuppression after liver transplantation.

BACKGROUND: Operational tolerance is defined as long-term acceptance of a transplanted organ after complete cessation of immunosuppression (IS), but may not always protect against antigen-dependent changes in graft morphology. METHOD: IS free patients after living-donor liver transplantation (LDLT) underwent protocol biopsy (tolerance group [Gr-Tol]) and were evaluated for rejection and fibrosis. The degree of fibrosis was compared with those in the patients on maintenance IS group (Gr-IS) and the base line normal liver group (Gr-BS). When bridging fibrosis or progression of fibrosis was observed, IS was reintroduced or increased in Gr-Tol or in the patients in the weaning process. RESULTS: Neither acute nor chronic rejection was observed. The degree of fibrosis, however, was significantly greater in Gr-Tol than those in Gr-IS and Gr-BS. In Gr-Tol, the number of graft infiltrating FOXP3 cells was significantly increased, the interval between LDLT and biopsy plus the donor age was significantly longer, and recipient age at LDLT was significantly younger, compared with those in Gr-IS. However, none of these three parameters correlated with the degree of fibrosis. In 7 of 11 patients in whom IS was reintroduced or increased, the improvement of fibrosis was observed by the subsequent biopsy. CONCLUSION: Grafts of operationally tolerant patients after LDLT did not exhibit acute or chronic rejection, but they exhibited fibrosis. It remains elusive whether fibrosis observed in tolerant grafts is antigen dependent. The finding that after [corrected] the reintroduction or the increase of IS fibrosis was improved supported the possibility that fibrosis in operationally tolerant patients was antigen dependent.

Significance of C4d staining in ABO-identical/compatible liver transplantation.

Complement degradation product C4d has become an important marker of humoral or antibody-mediated rejection in renal and heart allograft biopsies. Although there have been several reports on the detection of C4d in liver allografts, the significance of C4d in liver transplantation and its relationship with humoral rejection are still not clear. We investigated the frequency and pattern of C4d staining in liver allograft biopsies with reference to preoperative lymphocyte crossmatch tests, which detect donor-reactive lymphocyte antibody. Survival rates at 5 years were 77% for crossmatch-negative patients and 53% for crossmatch-positive patients (P=0.009). In crossmatch-negative patients, reproducible positive staining was obtained in 28 of 86 (33%) biopsies taken within 90 days after transplantation and 33 of 96 (34%) biopsies 90 days or after transplantation. Most C4d staining was observed in the portal areas, and no clear correlation was observed between C4d positivity and histological diagnosis. In crossmatch-positive patients, 9 of 11 (82%) biopsies showed positivity for C4d. C4d stained perivenular areas as well as portal areas. Histology of crossmatch-positive patients included acute rejection and cholangitis, but did not include periportal changes that were seen in humoral rejection in ABO-incompatible liver transplantation. In summary, focal C4d deposition was seen in various types of liver allograft injury and had little clinical impact on crossmatch-negative patients, but extensive C4d staining in crossmatch-positive patients may be associated with humoral rejection and poor graft survival.

FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts.

Fas-Fas ligand (FasL) interaction and apoptosis are important in the mechanism of allograft rejection. However, the interaction between donor and recipient cells, specifically focusing on antigen-presenting cells (APCs), under various conditions is poorly understood in human liver allografts. FasL expression on APCs, its association with apoptosis, and the origin of apoptotic lymphocytes in human liver allografts were assessed by immunohistochemistry and in situ hybridization. We found increased expression of FasL on Kupffer cells (KCs) and endothelium in acute cellular rejection (n = 20) and to lesser extent in chronic rejection (n = 6) and septic cholangitis (n = 5) compared with stable grafts and normal controls. In addition, the graft specificity of infiltrating T cells was confirmed by polymerase chain reaction examination of T-cell receptor-gamma loci. T-cell apoptosis occurred at a higher rate in acute cellular rejection than in chronic rejection or septic cholangitis. The number of apoptotic bodies derived from recipient lymphocytes correlated with the severity of rejection and was reversed by treatment. FasL(+) KCs phagocytosed CD4(+) interferon-gamma(+) T cells, rather than CD4(+) interleukin-4(+) T cells, suggesting a role of KCs in regulating CD4(+) T-cell subset differentiation. In conclusion, our data suggest that FasL expression on APCs and phagocytosis of apoptotic T cells by FasL(+) KCs are indicators of rejection activity in human liver allografts.

Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor 1 alpha and its significance.

BACKGROUND/AIMS: We analyzed the expressions of hexokinase II (HK II), a key enzyme in glycolysis, and VEGF in hepatocellular carcinoma (HCC) and metastatic liver cancer in relation to tumor vascularity, and the participation of hypoxia-inducible factor-1 (HIF-1) was studied. METHODS: A real-time quantitative reverse transcription-polymerase chain reaction was performed to examine the HK II and VEGF mRNA expression. Expression of HIF-1 alpha and HK II protein, and microvessel density (MVD) were examined immunohistochemically. RESULTS: MVD was significantly higher in HCCs than in metastatic liver cancers, and VEGF mRNA expression was positively correlated only with MVD of HCCs. HK II mRNA expression was significantly higher in metastatic liver cancers, however, some cases of HCC pretreated with transcatheter arterial embolization (TAE) showed marked HK II mRNA expression. Both HIF-1 alpha and HK II protein expressions were co-localized in the cancer cells near necrosis, and the intensity of HIF-1 alpha protein expression was significantly correlated with HK II mRNA expression in both tumors. CONCLUSIONS: These results suggest that, in metastatic liver cancers, glycolysis induced by HIF-1 is the predominant energy source under the hypoxic environment and, at least in some TAE-pretreated HCC cases, cancer cells obtain energy for growth by switching the metabolic profile to glycolysis through HIF-1.