A first-in-human phase I study of a novel MDM2/p53 inhibitor alrizomadlin in advanced solid tumors.

BACKGROUND: The mouse double minute 2 homolog (MDM2) oncogene exerts oncogenic activities in many cancers and represents a potential therapeutic target. This trial evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of alrizomadlin (APG-115), a novel MDM2/p53 inhibitor, in patients with advanced solid tumors. PATIENTS AND METHODS: Patients with histologically confirmed advanced solid tumors who had progressed to standard treatment or lacked effective therapies were recruited. Alrizomadlin was administered once daily every other day for 21 days of a 28-day cycle until disease progression or intolerable toxicity. RESULTS: A total of 21 patients were enrolled and treated with alrizomadlin; 57.1% were male and the median age was 47 (25-60) years. The maximum tolerated dose of alrizomadlin was 150 mg and the recommended phase II dose was 100 mg. One patient in the 200-mg cohort experienced dose-limiting toxicity of thrombocytopenia and febrile neutropenia. The most common grade 3/4 treatment-related adverse events were thrombocytopenia (33.3%), lymphocytopenia (33.3%), neutropenia (23.8%), and anemia (23.8%). Alrizomadlin demonstrated approximately linear pharmacokinetics (dose range 100-200 mg) and was associated with increased plasma macrophage inhibitory cytokine-1, indicative of p53 pathway activation. Of the 20 assessable patients, 2 [10%, 95% confidence interval (CI) 1.2% to 31.7%] patients achieved partial response and 10 (50%, 95% CI 27.2% to 72.8%) showed stable disease. The median progression-free survival was 6.1 (95% CI 1.7-10.4) months, which was significantly longer in patients with wild-type versus mutant TP53 (7.9 versus 2.2 months, respectively; P < 0.001). Among patients with MDM2 amplification and wild-type TP53, the overall response rate was 25% (2/8) and the disease control rate was 100% (8/8). CONCLUSIONS: Alrizomadlin had an acceptable safety profile and demonstrated promising antitumor activity in MDM2-amplified and TP53 wild-type tumors. This study supports further exploration of alrizomadlin with recommended doses of 100 mg q.o.d. in 21 days on and 7 days off regimen.

[Automatic classification of immune-mediated glomerular diseases based on multi-modal multi-instance learning].

OBJECTIVE: To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy (OM), immunofluorescence microscopy (IM), and transmission electron microscopy (TEM). METHODS: We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi- instance model for classification of 3 immune-mediated glomerular diseases, namely immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), and lupus nephritis (LN). This model adopts an instance-level multi-instance learning (I-MIL) method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient. By comparing this model with unimodal and bimodal models, we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion. RESULTS: The multi-modal multi-instance model combining OM, IM, and TEM images had a disease classification accuracy of (88.34±2.12)%, superior to that of the optimal unimodal model [(87.08±4.25)%] and that of the optimal bimodal model [(87.92±3.06)%]. CONCLUSION: This multi- modal multi- instance model based on OM, IM, and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.

Activation of eIF4E-binding-protein-1 rescues mTORC1-induced sarcopenia by expanding lysosomal degradation capacity.

BACKGROUND: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. METHODS: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. RESULTS: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function. CONCLUSIONS: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia.

The changing landscape of phase II/III metastatic sarcoma clinical trials-analysis of ClinicalTrials.gov.

BACKGROUND: Well-designed clinical trials are of great importance in validating novel treatments and ensuring an evidence-based approach for sarcoma. This study aimed to provide a comprehensive landscape of the characteristics of metastatic or advanced sarcoma clinical trials using the substantial resource of the ClincialTrials.gov database. METHODS: We identified 260,755 trials registered with ClinicalTrials.gov in the last 20 years, and 277 of them were eligible for inclusion. The baseline characteristics were ascertained for each trial. The trials were systematically reviewed to validate their classification into 96 trials registered before 2008 and 181 trials registered between 2008 and 2017. RESULTS: We found that in the last decade, metastatic and advanced sarcoma trials were predominantly phase II-III studies (p = 0.048), were more likely to be ≥2 arms (17.7% vs 35.3%, respectively; p = 0.007), and were more likely to use randomized (13.5% vs 30.4%; p = 0.002) and double-blinded (2.1% vs 9.4%; p = 0.024) assignment than trials registered before 2008. Furthermore, in the last 10-year period, metastatic sarcoma trials were more likely to be conducted in Asia. Treatment involving target therapy and immunotherapy were more common (71.8% vs 37.5%; p < 0.001) than in previous years. CONCLUSIONS: Our data showed provocative changes in the sarcoma landscape and demonstrated that the incidence of clinical trials with target therapy and immunotherapy is increasing. These findings emphasize the desperate need for novel strategies, including target therapy and immunotherapy, to improve the outcomes for patients with advanced sarcoma.

Efficacy and safety of morinidazole in pelvic inflammatory disease: results of a multicenter, double-blind, randomized trial.

This multicenter, double-blind, randomized, parallel-group, non-inferiority study compared the efficacy and safety of morinidazole with those of ornidazole in women with pelvic inflammatory disease. Women from 18 hospitals in China received a 14-day course of either intravenous morinidazole, 500 mg twice daily (n = 168), or intravenous ornidazole, 500 mg twice daily (n = 170). A total of 312 of 338 patients in the full analysis set (FAS) (92.3%) were included in the per protocol set (PPS) analyses, 61 (19.6%) of whom were included in the microbiologically valid (MBV) population. The clinical resolution rates in the PPS population at the test of cure (TOC, primary efficacy end point, 7-30 days post-therapy) visit were 96.86% (154/159) for morinidazole and 96.73% (148/153) for ornidazole (95% CI: -3.79% to 4.03%). The bacteriological success rates in the MBV population at the TOC visit were 100% (32/32) for morinidazole and 89.66% (26/29) for ornidazole (95% CI: -16.15% to 11.21%). Drug-related adverse events occurred less frequently with morinidazole (32.74%, 55/168) than with ornidazole (47.06%, 80/170) (p < 0.01). For women with pelvic inflammatory disease, twice-daily morinidazole for 14 days was clinically and bacteriologically as efficacious as twice-daily ornidazole for 14 days, while the former was associated with fewer drug-related adverse events than the latter.

[Normal values of otolith function tests and age-related changes].

Objective:To explore the normal values of otolith function tests and agerelated changes in health volunteers.Method: One hundred and seventyone health volunteers were distributed to seven age groups, all subjects accepted otolith function tests, including fundus photography, static subjective visual vertical(SSVV), cervical vestibular evoked myogenic potential(cVEMP)and ocular vestibular evoked myogenic potential(oVEMP), the fundus photographs was used to measure the discfovea angle(DFA). DFA, SSVV and VEMPs were analyzed and compared among groups. Result: For DFA and SSVV,there were no significant differences either between different ages or between the two eyes in one individual（P>0.05）. For cVEMP, the detection rate declined with age over 60 years old（P<0.01）; the cVEMP threshold increased with every 20 years old(P<0.05); and the cVEMP amplitude decreased with every 10 years old(P<0.05),however, there was an exception that no significant difference was found between 41-50 years old and 51-60 years old groups（P=0.93）;the cVEMP P1 latency prolonged with age over 70 years old（P<0.01）.For oVEMP, the detection rate also declined with age over 60 years old（P<0.01）; the oVEMP threshold was lowest at the age less than 30 years old and the largest threshold was found at the age over 70 years old（P<0.01）; consistently, the oVEMP amplitude was found largest at the age less than 20 years old and lowest at the age over 60 years old(P<0.05).The P1 and N1 latencies were found significantly longer in the group of over 70 years old than other groups（P<0.01）.No significant difference was found between both sides in one individual for oVEMP threshold,amplitude or latency (P>0.05). Conclusion: In health volunteers,there were no obvious aged related changes in DFA and SSVV. However,the detection rate, threshold, amplitude and latency of cVEMP and oVEMP greatly changed with age.

A phase I clinical trial utilizing autologous tumor-infiltrating lymphocytes in patients with primary hepatocellular carcinoma.

This report describes an ongoing Phase I clinical trial testing the safety of adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) in patients with primary hepatocellular carcinoma (HCC). Fifteen HCC patients were treated with their activated and expanded TILs following tumor resection. From a total of 17 patients with HCC, TIL were successfully expanded from 15 patients (88%), whereas two patients showed minimal or no expansion of TIL. Transient increase in the frequency of T cells was observed after adoptive transfer who was found only associated with grade I flu-like symptoms and malaise. After a median follow-up of 14 months, 15 patients (100%) were alive; and 12 patients (80%) showed no evidence of disease, 3 patients (patient 1,11,12) had tumor recurrence. The time to the diagnosis of tumor recurrence following therapy ranged from 105 to 261 days. These results indicate that immunotherapy with activated and expanded autologous TIL could be successfully performed with low toxicity, thus would serve as a novel treatment modality for patients with HCC.

Overexpression of WWP1 promotes tumorigenesis and predicts unfavorable prognosis in patients with hepatocellular carcinoma.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) has been speculated to play important roles in the development of several kinds of cancers. However, the role of WWP1 in hepatocellular carcinoma(HCC) is not clear. In the present study, we investigated the expression and prognostic role of WWP1 in primary hepatocellular carcinoma (HCC) using cell lines and 149 archived HCC samples. Correlation between the functions of WWP1 in HCC was also explored. We used human HCC cell lines (BEL-7402, SMMC-7721, Hep-G2, Hep-3B, SK-hep1 and Huh7) and a normal hepatocyte cell line (LO2) along with HCC samples from patients who had undergone resection for HCC previously at our hospital. A battery of methods (real-time quantitative polymerase chain reaction; western blotting; immunohistochemical analyses; cell proliferation and colony formation assays; cell migration and cell invasion assays) were employed to assess various aspects of WWP1. We found that WWP1 expression was upregulated aberrantly at mRNA and protein levels in human primary HCC tissues. Amplified expression of WWP1 was highly correlated with poor outcome. Silencing of WWP1 expression by siRNA inhibited the proliferation, colony formation, migration and invasion of HCC cells in vitro, and resulted in significant apoptosis and cycle arrest in HCC cells. Our findings suggest that WWP1 might have an oncogenic role in human primary HCC, and that it could be used as a prognostic marker as well as a potential molecular target for the treatment of HCC.

A novel pathogenic germline mutation in the adenomatous polyposis coli gene in a Chinese family with familial adenomatous coli.

Familial adenomatous polyposis (FAP) is an autosomal dominant disease manifesting as colorectal cancer in middle-aged patients. Mutations of the adenomatous polyposis coli (APC) gene contribute to both FAP and sporadic or familial colorectal carcinogenesis. Here we describe the identification of the causative APC gene defects associated with FAP in a Chinese pedigree. All patients with FAP were diagnosed by their combination of clinical features, family history, colonoscopy, and pathology examinations. Blood samples were collected and genomic DNA was extracted. Mutation analysis of APC was conducted by targeted next-generation sequencing, long-range PCR and Sanger sequencing. A novel mutation in exon 14-15(c.1936-2148 del) and intron 14 of the APC gene was demonstrated in all FAP patients and was absent in unaffected family members. This novel deletion causing FAP in Chinese kindred expands the germline mutation spectrum of the APC gene in the Chinese population.

Targeting the hsp70 gene delays mammary tumor initiation and inhibits tumor cell metastasis.

Elevated levels of the inducible heat-shock protein 70 (Hsp72) have been implicated in mammary tumorigenesis in histological investigations of human breast cancer. We therefore examined the role of Hsp72 in mice, using animals in which the hsp70 gene was inactivated. We used a spontaneous tumor system with mice expressing the polyomavirus middle T (PyMT) oncogene under control of the mouse mammary tumor virus (MMTV) long-terminal repeat (MMT mice). These mice developed spontaneous, metastatic mammary cancer. We then showed Hsp72 to be upregulated in a fraction of mammary cancer initiating cells (CIC) within the MMT tumor cell population. These cells were characterized by elevated surface levels of stem cell markers CD44 and Sca1 and by rapid metastasis. Inactivation of the hsp70 gene delayed the initiation of mammary tumors. This delay in tumor initiation imposed by loss of hsp70 was correlated with a decreased pool of CIC. Interestingly, hsp70 knockout significantly reduced invasion and metastasis by mammary tumor cells and implicated its product Hsp72 in cell migration and formation of secondary neoplasms. Impaired tumorigenesis and metastasis in hsp70-knockout MMT mice was associated with downregulation of the met gene and reduced activition of the oncogenic c-Met protein. These experiments therefore showed Hsp72 to be involved in the growth and progression of mammary carcinoma and highlighted this protein as a potential target for anticancer drug development.

Osseointegration of one-piece zirconia implants compared with a titanium implant of identical design: a histomorphometric study in the dog.

OBJECTIVE: The aim of this study was to evaluate osseointegration of one-piece zirconia vs. titanium implants depending on their insertion depth by histomorphometry. MATERIAL AND METHODS: Four one-piece implants of identical geometry were inserted on each side of six mongrel dogs: (1) an uncoated zirconia implant, (2) a zirconia implant coated with a calcium-liberating titanium oxide coating, (3) a titanium implant and (4) an experimental implant made of a synthetic material (polyetheretherketone). In a split-mouth manner they were inserted in submerged and non-submerged gingival healing modes. After 4 months, dissected blocks were stained with toluidine blue in order to histologically assess the bone-to-implant contact (BIC) rates and the bone levels (BL) of the implants. RESULTS: All 48 implants were osseointegrated clinically and histologically. Histomorphometrically, BL in the crestal implant part did not differ significantly with regard to material type or healing modality. The submerged coated zirconia implants tended to offer the most stable crestal BL. The histometric results reflected the different healing modes by establishing different BL. The median BIC of the apical implant part of the zirconia and titanium group amounted to 59.2% for uncoated zirconia, 58.3% for coated zirconia, 26.8% for the synthetic material and 41.2% for titanium implants. CONCLUSIONS: Within the limits of this animal study, it is concluded that zirconia implants are capable of establishing close BIC rates similar to what is known from the osseointegration behaviour of titanium implants with the same surface modification and roughness.

Angiozyme: a novel angiogenesis inhibitor.

Several inhibitors of angiogenesis are being developed for the treatment of cancer. One dominant strategy involves disruption of the vascular endothelial growth factor (VEGF) pathway by inhibition of the receptors for VEGF. Inhibition of the VEGF receptor activity can be accomplished using catalytic RNA molecules known as ribozymes, which downregulate VEGF receptor function by specifically cleaving the mRNAs for the primary VEGF receptors, Flt-1 and KDR. Significant inhibition of angiogenesis using ribozymes against both receptors has been demonstrated. In animal tumor models, antitumor effects are most pronounced with the anti-Flt-1 ribozyme known as Angiozyme (Ribozyme Pharmaceuticals, Boulder, CO). Extensive preclinical studies have demonstrated no significant toxicities. Clinical trials of Angiozyme are currently in progress for patients with advanced malignancy. Preliminary results demonstrate Angiozyme to be well tolerated, without significant side effects. Several phase II trials are underway for patients with advanced malignancy to test therapeutic efficacy.

Helper-independent, L-selectinlow CD8+ T cells with broad anti-tumor efficacy are naturally sensitized during tumor progression.

We recently reported that the CD4(+) T cell subset with low L-selectin expression (CD62L(low)) in tumor-draining lymph nodes (TDLN) can be culture activated and adoptively transferred to eradicate established pulmonary and intracranial tumors in syngeneic mice, even without coadministration of IL-2. We have extended these studies to characterize the small subset of L-selectin(low) CD8(+) T cells naturally present in TDLN of mice bearing weakly immunogenic tumors. Isolated L-selectin(low) CD8(+) T cells displayed the functional phenotype of helper-independent T cells, and when adoptively transferred could consistently eradicate, like L-selectin(low) CD4(+) T cells, both established pulmonary and intracranial tumors without coadministration of exogenous IL-2. Whereas adoptively transferred L-selectin(low) CD4(+) T cells were more potent on a cell number basis for eradicating 3-day intracranial and s.c. tumors, L-selectin(low) CD8(+) T cells were more potent against advanced (10-day) pulmonary metastases. Although the presence of CD4(+) T cells enhanced generation of L-selectin(low) CD8(+) effector T cells, the latter could also be obtained from CD4 knockout mice or normal mice in vivo depleted of CD4(+) T cells before tumor sensitization. Culture-activated L-selectin(low) CD8(+) T cells did not lyse relevant tumor targets in vitro, but secreted IFN-gamma and GM-CSF when specifically stimulated with relevant tumor preparations. These data indicate that even without specific vaccine maneuvers, progressive tumor growth leads to independent sensitization of both CD4(+) and CD8(+) anti-tumor T cells in TDLN, phenotypically L-selectin(low) at the time of harvest, each of which requires only culture activation to unmask highly potent stand-alone effector function.

Comparison of bioabsorbable and non-resorbable membranes in the treatment of dehiscence-type defects. A histomorphometric study in dogs.

BACKGROUND: The goal of this investigation was to compare, histologically and histometrically, the healing process of dehiscence-type defects treated by guided tissue regeneration (GTR) with bioabsorbable polylactic acid (PLA) membranes and non-resorbable expanded polytetrafluoroethylene (ePTF) membranes. METHODS: Six mongrel dogs were used. Buccal osseous dehiscences were surgically created on the distal roots of the mandibular third and fourth premolars. The defects were exposed to plaque accumulation for 3 months. After this period, the defects were randomly assigned to one of the treatments: GTR with bioabsorbable membrane (PLA), GTR with non-resorbable membrane (ePTFE), open flap debridement (OFD), and non-treated control (NTC). After 3 months of healing, the dogs were sacrificed and the blocks were processed. The histometric parameters evaluated included: gingival recession, epithelial length, connective tissue adaptation, new cementum, and new bone area. RESULTS: A superior length of new cementum was observed in the sites treated by GTR, regardless of the type of barrier used, in comparison with OFD (P <0.05). No statistically significant differences were found between PLA and ePTFE in any of the parameters with the exception of bone area. PLA presented a greater bone area when compared to ePTFE, OFD, and NTC (P <0.05). CONCLUSIONS: Within the limits of this study, it can be concluded that both barriers are equally effective for new cementum formation. The bioabsorbable membrane may provide a greater bone area than the non-resorbable membrane.

Detection of circulating tumor cells and micrometastases in stage II, III, and IV breast cancer patients utilizing cytology and immunocytochemistry.

Evaluation for circulating tumor cells and bone marrow micrometastases has generated considerable interest due to a potential association with disease recurrence and poor prognosis. In this study, we examined bone marrow and apheresis samples from Stage II, III, and IV patients (n 120) enrolled in various clinical breast cancer trials at the National Institutes of Health/National Cancer Institute. For each patient sample, two Diff-Quik-stained cytospins were reviewed for morphology, and approximately 1 x 10(6) cells were analyzed for the expression of cytokeratins using an avidin-biotin immunoperoxidase method. Keratin-positive malignant cells appearing as single cells or in small clusters were detected in bone marrow samples from Stage IV patients only (9/68, 13%) and detected in apheresis samples from both Stage III and IV patients (13/245, 5%). These findings indicate that the combination of cytomorphology with immunocytochemistry can be utilized for the investigation of circulating tumor cells and bone marrow micrometastases, and that positive results appear to correlate with high tumor stage/burden.

CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection.

Tumor-specific CD4+ effector T cells often play a decisive role in immunologic tumor rejection, in some cases without evident co-participation of CD8+ T cells. During such CD4+ T-cell-mediated rejection there is often no detectable direct contact between T cells and tumor cells. Optimally prepared, adoptively transferred CD4+ T cells can reject established tumors with great efficiency even when targeted tumor cells express no MHC Class II molecules, implying that recognition of tumor antigen (Ag) occurs via MHC Class II-expressing host antigen-presenting cells (APC) within the tumor. Because consequent rejection also excludes Ag-specific contact between CD4+ T cells and MHC Class IIneg tumor cells, the most critical CD4+ T-cell-mediated event is likely cytokine release, resulting in an accumulation and activation of accessory cells such as tumoricidal macrophages and lymphokine-activated killer cells. Although such an indirect rejection mechanism may appear antithetical to popular strategies centered on CD8+ cytotoxic T cell (CTL), current evidence suggest that even CD8+ T-cell-mediated recognition/rejection often bypasses direct tumor cell contact and is largely cytokine mediated. While CTL are likely to participate prominently in many models of tumor rejection, indirect mechanisms of recognition/rejection have the theoretical advantage of remaining operative even when individual tumor cells evade direct contact by down-regulating MHC and/or Ag expression.

Contribution of the periosteum to bone formation in guided bone regeneration. A study in monkeys.

The periosteum has been referred to as a protective barrier in the regeneration of bone defects. The objective of this study was to determine the contribution of periosteum as a natural barrier to bone formation in guided bone regeneration. Mucoperiosteal flaps were elevated bilaterally on the buccal aspect of the mandibular angle in 5 cynomolgus monkeys. Bleeding was induced by perforating the cortical bone. A hemispherical titanium mesh was fixed over the areas thus creating a void 5 mm in height between the mesh and the bone surface. One one side the mesh was covered with an ePTFE membrane (test side). The contralateral side did not receive further treatment (control side). After 4 month healing, histomorphometric analyses were used to determine the percentage of new bone in the void underneath the mesh, and the ratio between mineralized tissue and marrow spaces in new and old bone. The mean percentage of new bone tissue was 77.2 +/- 7.5% for the test sides and 68.6 +/- 8.4% for the control sides (P = 0.018, t-test). This new bone contained 80.0 +/- 3.6% mineralized tissue in the test group and 82.5 +/- 5.0% in the control group (P > 0.05, t-test). In both groups the newly formed bone exhibited significantly less mineralized tissue than the old bone (P < 0.05, t-test). It is concluded from this study that new bone formation was enhanced by the additional use of an ePTFE membrane under a periosteum-lined mucoperiosteal flap when space maintenance was excluded as a critical factor.

Calcium ionophore-treated myeloid cells acquire many dendritic cell characteristics independent of prior differentiation state, transformation status, or sensitivity to biologic agents.

We previously reported that treatment of human peripheral blood monocytes or dendritic cells (DC) with calcium ionophore (CI) led to the rapid (18 hour) acquisition of many characteristics of mature DC, including CD83 expression. We therefore investigated whether less-mature myeloid cells were similarly susceptible to rapid CI activation. Although the promyelocytic leukemia line HL-60 was refractory to cytokine differentiation, CI treatment induced near-uniform overnight expression of CD83, CD80 (B7.1), and CD86 (B7. 2), as well as additional characteristics of mature DC. Several cytokines that alone had restricted impact on HL-60 could enhance CI-induced differentiation and resultant T-cell sensitizing capacity. In parallel studies, CD34(pos) cells cultured from normal donor bone marrow developed marked DC-like morphology after overnight treatment with either rhCD40L or CI, but only CI simultaneously induced upregulation of CD83, CD80, and CD86. This contrasted to peripheral blood monocytes, in which such upregulation could be induced with either CI or rhCD40L treatment. We conclude that normal and transformed myeloid cells at many stages of ontogeny possess the capacity to rapidly acquire many properties of mature DC in response to CI treatment. This apparent ability to respond to calcium mobilization, even when putative signal-transducing agents are inoperative, suggests strategies for implementing host antileukemic immune responses.

Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways.

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.

Functional and esthetic outcome enhancement of periodontal surgery by application of plastic surgery principles.

The closure of surgical wounds in a layer-by-layer fashion, a common principle of plastic surgery, is applied in this article to the field of periodontal surgery with the introduction of a new flap design. The suggested technique is indicated with all periodontal procedures that aim for hard and soft tissue augmentation (guided bone regeneration, mucogingival surgery, or plastic periodontal surgery) where passive, tension-free wound closure is fundamental for wound healing and a successful functional and esthetic outcome. By means of a series of incisions, buccal and lingual flaps are split several times; this results in a double-partial thickness flap and a coronally positioned palatal sliding flap, respectively. Thus, several tissue layers are obtained and the passive advancement of flaps becomes possible for the coverage of augmented areas. Wound closure with microsurgical suture material is accomplished in a multilayer approach, which ensures adaptation and closure of the outer tissue layers without any tension. Two case reports demonstrate the new plastic periodontal approach.