Bridge to recovery in children on ventricular assist devices-protocol, predictors of recovery, and long-term follow-up.

BACKGROUND: The majority of children supported with ventricular assist devices (VADs) are bridged to heart transplantation. Although bridge to recovery has been reported, low recovery patient numbers has precluded systematic analysis. The aim of this study was to delineate recovery rates and predictors of recovery and to report on long-term follow-up after VAD explantation in children. METHODS: Children bridged to recovery at our institution from January 1990 to May 2016 were compared with a non-recovery cohort. Clinical and echocardiographic data before and at pump stoppages and after VAD explantation were analyzed. Kaplan‒Meier estimates of event-free survival, defined as freedom from death or transplantation after VAD removal, were determined. RESULTS: One hundred forty-nine children (median age 5.8 years) were identified. Of these, 65.2% had cardiomyopathy, 9.4% had myocarditis, and 24.8% had congenital heart disease. The overall recovery rate was 14.2%, and was 7.1% in patients with dilated cardiomyopathy. Predictors of recovery were age <2 years (recovery rate 27.8%, odds ratio [OR] 5.64, 95% confidence interval [CI] 2.0 to 16.6) and diagnosis of myocarditis (rate 57.1%; OR 17.56, 95% CI 4.6 to 67.4). After a median follow-up of 10.8 years, 15 patients (83.3%) were in Functional Class I and 3 (16.7%) in were in Class II. Mean left ventricular ejection fraction was 53% (range 28% to 64%). Ten- and 15-year event-free survival rates were both 84.1 ± 8.4%. CONCLUSIONS: Children <2 years of age and those diagnosed with myocarditis have the highest probability of recovery. Long-term survival after weaning from the VAD was better than after heart transplantation, as demonstrated in the excellent long-term stability of ejection fraction and functional class.

A natural infection by the red sea bream iridovirus-type Megalocytivirus in the golden mandarin fish Siniperca scherzeri.

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)-subgroup I. Additionally, this train clustered with the ehime-1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV-type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 102 to 7.95 × 106  copies/mg of tissue individually, suggesting that the infected fish were in the disease-transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV-type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.

Novel strategy for a bispecific antibody: induction of dual target internalization and degradation.

Activation of the extensive cross-talk among the receptor tyrosine kinases (RTKs), particularly ErbB family-Met cross-talk, has emerged as a likely source of drug resistance. Notwithstanding brilliant successes were attained while using small-molecule inhibitors or antibody therapeutics against specific RTKs in multiple cancers over recent decades, a high recurrence rate remains unsolved in patients treated with these targeted inhibitors. It is well aligned with multifaceted properties of cancer and cross-talk and convergence of signaling pathways of RTKs. Thereby many therapeutic interventions have been actively developed to overcome inherent or acquired resistance. To date, no bispecific antibody (BsAb) showed complete depletion of dual RTKs from the plasma membrane and efficient dual degradation. In this manuscript, we report the first findings of a target-specific dual internalization and degradation of membrane RTKs induced by designed BsAbs based on the internalizing monoclonal antibodies and the therapeutic values of these BsAbs. Leveraging the anti-Met mAb able to internalize and degrade by a unique mechanism, we generated the BsAbs for Met/epidermal growth factor receptor (EGFR) and Met/HER2 to induce an efficient EGFR or HER2 internalization and degradation in the presence of Met that is frequently overexpressed in the invasive tumors and involved in the resistance against EGFR- or HER2-targeted therapies. We found that Met/EGFR BsAb ME22S induces dissociation of the Met-EGFR complex from Hsp90, followed by significant degradation of Met and EGFR. By employing patient-derived tumor models we demonstrate therapeutic potential of the BsAb-mediated dual degradation in various cancers.

Dosimetric advantages of generalised equivalent uniform dose-based optimisation on dose-volume objectives in intensity-modulated radiotherapy planning for bilateral breast cancer.

OBJECTIVE: We compared and evaluated the differences between two models for treating bilateral breast cancer (BBC): (i) dose-volume-based intensity-modulated radiation treatment (DV plan), and (ii) dose-volume-based intensity-modulated radiotherapy with generalised equivalent uniform dose-based optimisation (DV-gEUD plan). METHODS: The quality and performance of the DV plan and DV-gEUD plan using the Pinnacle(3) system (Philips, Fitchburg, WI) were evaluated and compared in 10 patients with stage T2-T4 BBC. The plans were delivered on a Varian 21EX linear accelerator (Varian Medical Systems, Milpitas, CA) equipped with a Millennium 120 leaf multileaf collimator (Varian Medical Systems). The parameters analysed included the conformity index, homogeneity index, tumour control probability of the planning target volume (PTV), the volumes V(20 Gy) and V(30 Gy) of the organs at risk (OAR, including the heart and lungs), mean dose and the normal tissue complication probability. RESULTS: Both plans met the requirements for the coverage of PTV with similar conformity and homogeneity indices. However, the DV-gEUD plan had the advantage of dose sparing for OAR: the mean doses of the heart and lungs, lung V(20) (Gy), and heart V(30) (Gy) in the DV-gEUD plan were lower than those in the DV plan (p<0.05). CONCLUSIONS: A better result can be obtained by starting with a DV-generated plan and then improving it by adding gEUD-based improvements to reduce the number of iterations and to improve the optimum dose distribution. Advances to knowledge The DV-gEUD plan provided superior dosimetric results for treating BBC in terms of PTV coverage and OAR sparing than the DV plan, without sacrificing the homogeneity of dose distribution in the PTV.

Topical calcitriol restores the impairment of epidermal permeability and antimicrobial barriers induced by corticosteroids.

BACKGROUND: The active form of vitamin D(3) , calcitriol, is widely used for the treatment of psoriasis, with or without topical corticosteroids. Topical corticosteroids are known to disrupt permeability and antimicrobial barriers, even with short-term use. Yet, the effect of topical calcitriol on epidermal permeability and antimicrobial barriers disrupted by topical corticosteroids has not been determined. OBJECTIVES: To examine the effect of calcitriol on epidermal permeability and antimicrobial barrier function that has been impaired by corticosteroids, as well as to elucidate the mechanism of improvement. MATERIAL AND METHODS: Topical calcitriol or the control vehicle was applied to each flank of hairless mice 20 min after treatment with topical clobetasol propionate and repeated every 12 h for 3·5 days. Barrier function assessment, Nile red staining, electron microscopy, immunohistochemistry, Western blotting, and real-time reverse transcriptase-polymerase chain reaction studies were performed 24 h after the last application. RESULTS: Epidermis co-treated with topical calcitriol showed an improvement of stratum corneum integrity and barrier recovery, more intense fluorescence staining with Nile red, and an increase in lamellar body (LB) maturation and density, as well as upregulation of major epidermal lipid synthesis-related enzymes (3-hydroxy-3-methylglutaryl-CoA, serine-palmitoyl transferase and fatty acid synthase), mouse beta-defensin 3, cathelin-related antimicrobial peptide and vitamin D receptor. CONCLUSIONS: We found that topical calcitriol restored both the epidermal permeability and antimicrobial barrier that had been impaired by corticosteroids. This restoration was mediated by both an activation of the cutaneous vitamin D pathway and an increase of epidermal lipids and antimicrobial peptides, promoted by the formation of the LB and the activity of epidermal lipid synthesis-related enzymes.

T-lymphocyte subsets in patients with AJCC stage III gastric cancer during postoperative adjuvant chemotherapy. American Joint Committee on Cancer.

BACKGROUND AND AIMS: Advanced neoplastic diseases alter the immune response in cancer patients. The aim of this study was to evaluate the changes of T-lymphocyte subsets during postoperative adjuvant chemotherapy, and the relationship between T-lymphocyte subsets and tumor recurrence in AJCC stage III gastric cancers. MATERIAL AND METHODS: Analysis of T-lymphocyte subsets was performed in 39 patients with stage III gastric adenocarcinoma who had undergone a curative gastric resection and postoperative chemotherapy. CirculatingT-lymphocyte subsets were measured on venous blood by using flow cytometry and monoclonal antibodies on preoperative day 1, and postoperative months 1, 3, and 6. RESULTS: The 5-year disease-free survival rates of patients with stage 3a and 3b gastric cancer were 57.1% and 33.3%, respectively (p = 0.06). Values of CD3+ and CD4+ T-cells, and CD4+/CD8+ ratios were consistently lower in the recurrence group throughout the observation period. CD4+ T-cell counts were significantly lower in the recurrence group on preoperative day 1, and postoperative months 1 and 6. However, most values of the T-lymphocyte subsets showed no statistically significant difference when comparing the stage 3a and 3b disease patient groups. CONCLUSIONS: The results of this study suggest that immunosuppression associated with CD3+ and CD4+ T-cell depression is a risk factor for postoperative recurrence in patients with stage III gastric cancer.

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae.

To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellular defense reactions.

Chemically induced rat mammary tumor treated with tamoxifen showed decreased expression of cyclin D1, cyclin E, and p21(Cip1).

We studied the effects of tamoxifen (TAM) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor and the expression of cyclin D1, cyclin E, p21(Cip1), and estrogen receptors (ER) by performing immunohistochemistry and Western blot analysis. When tumor size reached between 10 and 15mm in the largest dimension, the rats were divided into a DMBA-control group and a DMBA-TAM group. The administration of TAM markedly decreased the tumor development and showed decreased expression of bromodeoxyuridine, cyclin D1, cyclin E, and p21(Cip1) when compared with those of the DMBA-control group; however, a few tumors showed progressive growth in spite of TAM treatment. These tumors had decreased expression of ER. This study suggests that TAM suppresses tumor development through the down-expression of cyclin D1 and cyclin E.

Mucinous cystadenoma coexisting with stromal tumor with minor sex-cord elements of the ovary: a case report.

Mucinous neoplasms occur rarely in association with cystic teratoma, Sertoli-Leydig cell tumor, granulosa cell tumor or carcinoid tumor. Several cases of an ovarian stromal tumor with minor sex-cord elements have been reported in the literatures. However, there has been no report about an ovarian mucinous neoplasm coexisting with a stromal tumor with sex-cord elements yet. We report a case of an ovarian neoplasm composed of both mucinous cystadenoma and stromal tumor with minor sex-cord elements in a 58-yr-old female. The ovary including the mass measured 5 cm in size. On section, it revealed an unilocular cyst (4.5 cm in diameter) filled with mucinous fluid. There was a round, yellow, solid nodule, 1.5 cm in diameter within the wall. Microscopically, the cyst was lined by a single layer of endocervical mucinous epithelium and the nodule was composed of spindle cells showing an intersecting and whorled arrangement. There were cell nests showing polygonal shape with abundant cytoplasm among the spindle cells. They showed immunoreactivity for inhibin and did not have any connection with the adjacent mucinous epithelium. Therefore, we interpret the mucinous cystadenoma as having arisen de novo.

Comparison of the efficacy and safety of different formulations of omeprazole-based triple therapies in the treatment of Helicobacter pylori-positive peptic ulcer.

Little is known about the efficacy and safety of different formulations of omeprazole-based triple therapy regimens for the treatment of Helicobacter pylori-positive peptic ulcer. We compared the efficacy and safety of two formulations of omeprazole used in triple therapies in patients with H. pylori-positive active peptic ulcer. Seventy-four patients with endoscopically proven H. pylori-positive active peptic ulcer were randomized to two groups, each with 37 patients, to receive either OAC-I (6 weeks of "A" formulation of omeprazole [20 mg twice daily] plus 2 weeks of amoxicillin [1.0 g twice daily] and clarithromycin [500 mg twice daily] or OAC-II (6 weeks of "B" formulation of omeprazole [20 mg twice daily] plus 2 weeks of the same antibiotics. The H. pylori and ulcer healing status were assessed at the baseline and at the 6-week endpoint of therapy. Gastrointestinal symptoms, documentation of adverse events, and standard laboratory examinations were assessed at each visit. Eradication of H. pylori (intention to treat [n = 74]/per protocol [n = 66]) and healing of the ulcer were successful in 83.8%/96.9% and 93.8%, respectively, of the OAC-I group patients, and in 91.9%/100% and 97.1%, respectively, of the OAC-II group patients (P = 0.477; P = 0.608). The OAC-I group experienced rapid resolution of symptoms, but no significant differences were found between the two groups for number of days taken for resolution of gastrointestinal symptoms, adverse events, and laboratory findings. The two different formulations of omeprazole used in triple therapy regimens produced similar efficacy and safety results after 6 weeks of treatment in patients with H. pylori-positive active peptic ulcer.

The fate of small-diameter homografts in the pulmonary position.

BACKGROUND: Limited durability is expected for small homograft valves that are used to correct congenital cardiac disease. METHODS: All 76 homograft valves with an internal annulus diameter ranging from 8 to 13 mm that were implanted from 1987 through 2000 in the pulmonary position were retrospectively analyzed. In each case, homograft size was normalized to the patient's body surface area: z-value. For 93% (14 of 15) of the 8 to 9 mm grafts, z was less than 2. For 56% (5 of 9) of the 10 mm grafts and 98% (51 of 52) of the 11 to 13 mm allografts, z was greater than 2. Survival and freedom from complications were estimated by the Kaplan-Meier method. Homograft failure was defined as homograft replacement or late death; significant dysfunction, as homograft obstruction with an echo-Doppler gradient greater than 50 mm Hg or grade III or IV valvular insufficiency. The log-rank test was used to compare outcomes. RESULTS: Seven patients died early after operation; three, late. Survival was 86.5% +/- 3.8% at 1 year and remained stable during the succeeding years. Freedom from failure for all homografts was 90.6% +/- 3.7%, 71.8% +/- 6.9%, and 61.8% +/- 9.0% at 1, 5, and 10 years, respectively. Corresponding freedom from significant dysfunction was 87.6% +/- 4.1%, 51.2% +/- 7.4%, and 10.1% +/- 8.3%. The smaller homografts (z less than 2) failed and deteriorated faster (p < 0.0001): only 32.1% +/- 13.0% were still functioning at 24 months. The larger grafts (z at least 2) retained function for the first 4 years, and 73.7% +/- 10.4% had not yet failed at 10 years. CONCLUSIONS: Smaller (z less than 2) homografts (the great majority of 8 to 9 mm grafts) have to be replaced early, usually within 2 years of implantation. Larger (z at least 2) grafts (nearly all 11 to 13 mm grafts) show remarkable durability and are suitable valved conduits for establishing right ventricle to pulmonary artery continuity in neonates and young infants.

Regulation of leptin gene expression by insulin and growth hormone in mouse adipocytes.

The role of leptin in the control of obesity, insulin resistance and type II diabetes has been reported, however, the regulatory mechanism of leptin in animals affected by hormones is not clearly understood. In this study, the effects of insulin, epinephrine, growth hormone or dexamethasone on the expression of leptin was examined in mouse primary adipocytes. The leptin expression was also studied in the adipose tissue of the mouse treated with insulin or growth hormone (0.3 or 0.6 units/animal). Insulin (100 nM) or dexamethasone (100 nM) stimulated leptin mRNA transcription while epinephrine (100 nM) alleviated its transcription in mouse primary adipocytes. The level of leptin protein in cultured media of adipocytes treated with insulin or dexamethasone was higher than that of the control group but growth hormone or epinephrine treatment had no effect on them. Insulin administration (0.6 units/mouse) enhanced leptin mRNA as well as leptin protein in mouse adipose tissue but growth hormone administration (0.3 or 0.6 units/mouse) had no effect on them. Leptin protein level in sera of mice injected with insulin or growth hormone was not significantly different from that of control group. These results indicate that both insulin and dexamethasone stimulate leptin gene expression and secretion of its product, whereas, growth hormone has no effect on the expression of leptin gene in mouse adipocytes.

Endoscopic mucosal resection with a ligation device for early gastric cancer and precancerous lesions: comparison of its therapeutic efficacy with surgical resection.

Endoscopic mucosal resection with a ligation device (EMR-L) has become important in the curative treatment of precancerous lesions and early gastric cancers (EGCs), but little is known of the long-term efficacy and survival rates of EMR-L compared with surgical resection. We analyzed the therapeutic efficacy and safety of EMR-L in cases of EGC and precancerous lesions and compared the results of EMR-L with those of gastrectomy in patients with EGC over the same periods. EMR-L was performed on 20 EGCs and 54 precancerous lesions including tubular adenomas with or without severe dysplasias in 74 patients. Macroscopic type, size and location of the lesion were determined by endoscope, and the depth of invasion in EGCs was determined by endoscopic ultrasonography and confirmed by pathologic examination of the resected specimens. All the EGC cases were endoscopically followed up for at least 18 months (range, 18-66 months). Patients were selected that underwent subtotal gastrectomy and the survival rates were compared with those that underwent EMR-L. Complete resection was made in a single EMR-L treatment session in 61 cases (82.4%; 91.5%, were precancerous lesions and 65% were EGCs). After a repeat trial of EMR-L, the total rate of complete resection of precancerous lesions and EGCs was 92.6% and 85.0%, respectively. The survival rate of EGCs showed that complete resection by EMR-L resulted in 2 and 5 year survival rates of 100% and 95%, which are comparable to those of surgery (100% and 100%). This study suggests that EMR-L is a technically simple, minimally invasive and highly safe and effective treatment modality for selective EGCs, and offers an alternative to surgical treatment.

A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.

Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-kDa protein was cleaved to a 35-kDa protein. RNA blot hybridization revealed that expression of the 45-kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge.

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine.

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and beta-1, 3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and beta-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.

Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae.

Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine. The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.

Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae.

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the biochemical process of encapsulation is still obscure. To isolate and characterize the early-stage encapsulation-relating protein (ERP), we used the coleopteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted pieces of surgical suture were recovered 10 min after injection or insertion, and were analyzed on SDS/PAGE under reducing conditions. Four different proteins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymph. Among them, we purified 56-kDa and 48-kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity-purified antibodies of the 56-kDa and 48-kDa ERPs cross-reacted with the 48-kDa and 56-kDa ERPs, respectively. Analysis of the cDNA of 56-kDa ERP consisted of 579 amino acid residues and showed a novel glutamine-rich protein. Positive clones of the 48-kDa ERP showed the same DNA sequence as 56-kDa ERP. Interestingly, the chemically determined N-terminal amino acid sequence and the three partial amino acid sequences of the 48-kDa protein were found in the 56-kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101-Gly102 of the 56-kDa ERP by a limited proteolysis. Western blotting analysis showed that these ERPs were detected exclusively on membrane fractions of hemocytes. Also, when the early-stage encapsulated beads were coated with both the 56-kDa and 48-kDa ERP antibodies and re-injected into larvae, no further encapsulation reaction was observed. However, when the early-stage encapsulated beads were incubated with 56-kDa ERP antibody, 48-kDa ERP antibody or nonimmunized rabbit IgG and re-injected into larvae, further encapsulation did occur.