Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge

Based on the fact that the 24 amino acid extracellular domain of M2 protein (M2e) is nearly invariant in all influenza A strains, several different M2e vaccine constructs and vaccination modalities have been developed by others and us. Although most of these vaccines could induce efficient and broad-spectrum immunity inhibiting influenza A virus infection in mice model, information of the refined protective epitope on M2e was scarce. In this paper, two M2e specific monoclonal antibodies (mAbs) conferring protective immunity in vivo were reported, which in passive administration could protect 75% mice from five LD(50) (50% lethal dose) challenge of influenza virus A/PR/8/34. In addition, higher M2e specific antibody titer (over 1:1600) could be detected after 12h of intraperitoneal passive administration in mice sera. Peptide mapping assay indicated that both mAbs strongly interacted with N-terminus and middle part peptides of M2e (NM2, aa2-12; MM2, aa8-18), but not with the C-terminus peptide (CM2, aa13-24). More importantly, M2e specific mAbs could recognize EVETPIRN (aa6-13) peptide, which were the overlapping region of NM2 and MM2 peptide and the neighboring amino acid residues. In contrast, M2e domain that was deleted EVETPIR sequence could not be recognized by either mAb in immunoblotting assay. All these results indicated that the epitope EVETPIRN (aa6-13) on M2e could be responsible for the induction of the protective immunity.     

Composite articular cartilage engineered on a chondrocyte-seeded aliphatic polyurethane sponge

To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.     

Expression and intracellular localization of FKHRL1 in mammary gland neoplasms

FKHRL1 (FOXO3a), a member of the Forkhead family of genes, has been considered to be involved in the development of breast tumors; however, the in vivo expression and activation status of FKHRL1 in breast tumors still remains unclear. We immunohistochemically demonstrated the expression and intracellular localization of FKHRL1 in human breast tumors by the novel anti-FKHRL1 antibody which is available for formalin-fixed paraffin-embedded specimens. In a total of 51 cases of benign tumors, FKHRL1 was diffusely expressed in all cases, and its intracellular localization was revealed to be cytoplasmic (inactive form) in 94% of cases of intraductal papillomas (16/17) and 91% cases of fibroadenomas (31/34), with a similar pattern to normal glandular epithelium. In invasive ductal carcinomas, 83% of the cases (93/112) diffusely expressed FKHRL1; however, unlike benign tumors, 71% of the cases (66/93) showed the nuclear-targeted, active form of FKHRL1. Moreover, activated FKHRL1 was predominantly observed in scirrhous (29/36, 81% of the cases) and papillotubular (30/38, 79% of the cases) subtypes, compared to the solid-tubular subtype (7/19, 37% of the cases). Furthermore, the cases with nuclear-targeted FKHRL1 showed a tendency to have lymph nodal metastasis with statistical significance (P < 0.0001). Thus, the activation of FKHRL1 seems to be recognized as one of the specific features of invasive ductal carcinoma of the breast.     

Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.     

Molecular mechanism for loss of visual cortical responsiveness following brief monocular deprivation

A dramatic form of experience-dependent synaptic plasticity is revealed in visual cortex when one eye is temporarily deprived of vision during early postnatal life. Monocular deprivation (MD) alters synaptic transmission such that cortical neurons cease to respond to stimulation of the deprived eye, but how this occurs is poorly understood. Here we show in rat visual cortex that brief MD sets in motion the same molecular and functional changes as the experimental model of homosynaptic long-term depression (LTD), and that prior synaptic depression by MD occludes subsequent induction of LTD. The mechanisms of LTD, about which there is now a detailed understanding, therefore contribute to visual cortical plasticity.     

Induction of heme oxygenase-1 expression inhibits platelet-dependent thrombosis

Heme oxygenase-1 (HO-1) plays a key role in protecting tissue from oxidative stress. Although some studies implicate HO-1 in modulating thrombosis after vascular injury, the impact of HO-1 on the rate of clot formation in vivo is poorly defined. This study examined the potential function of HO-1 in regulating platelet-dependent arterial thrombosis. Platelet-rich thrombi were induced in C57BL/6J mice by applying 10% ferric chloride to the exposed carotid artery. Mean occlusion time of wild-type mice (n = 10) was 14.6 +/- 1.0 min versus 12.9 +/- 0.6 min for HO-1-/- mice (n = 11, p = 0.17). However, after challenge with hemin, mean occlusion time was significantly longer in wild-type mice (16.3 +/- 1.2 min, n = 15) than HO-1-/- mice (12.0 +/- 1.0 min, n = 9; p = 0.021). Hemin administration induced an approximately twofold increase in oxidative stress, measured as plasma thiobarbituric acid reactive substances. Immunohistochemical analysis revealed that hemin induced a robust increase in HO-1 expression within the carotid arterial wall. Ex vivo blood clotting within a collagen-coated perfusion chamber was studied to determine whether the accelerated thrombosis observed in HO-1-/- mice was contributed to by effects on the blood itself. Under basal conditions, mean clot formation during perfusion of blood over collagen did not differ between wild-type mice and HO-1-/- mice. However, after hemin challenge, mean clot formation was significantly increased in HO-1-/- mice compared with wild-type controls. These results suggest that, under basal conditions, HO-1 does not exert a significant effect on platelet-dependent clot formation in vivo. However, under conditions that stimulate HO-1 production, platelet-dependent thrombus formation is inhibited by HO-1. Enhanced HO-1 expression in response to oxidative stress may represent an adaptive response mechanism to down-regulate platelet activation under prothrombotic conditions.     

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Utilization of the 2017 diagnostic criteria for hEDS by the Toronto GoodHope Ehlers–Danlos syndrome clinic: A retrospective review

The new 2017 diagnostic criteria for hypermobile Ehlers–Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy.