Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Efficacy and safety of immunization with phosphorylated tau against neurofibrillary tangles in mice.

As an abnormally folded and aggregated protein, tau composed of neurofibrillary tangles (NFTs) in Alzheimer's disease and other tauopathies seems to be a candidate for immunotherapy. Yet, the encephalitogenicity of full-length tau protein, recently reported by us in immunized mice, demands to carefully and selectively target pathological tau and address both efficacy (anti-NFT effect) and safety (free of encephalitis). We immunized NFT mice with NFT-related phosphorylated (phos) tau peptides, using an immunization protocol aimed to predispose a proinflammatory milieu in CNS as a set up to detect biohazard, an approach we used when the neurotoxicity of full-length tau was detected [use of complete Freund adjuvant (CFA) with pertussis toxin (PT)]. A decrease of about 40% in NFT burden in CNS was demonstrated and was accompanied with an increase in microglial burden. Anti-phos-tau antibodies were detected in serum and blood vessels in the CNS, while no encephalitogenicity (free of clinical neurological deficits, of adverse effects on brain inflammatory cells and of axonal damage) was recorded. The level of the lysosomal proteases, cathepsins D and L, was affected in the immunized mice suggesting the possible involvement of the lysosomal system in the decrease of NFTs. The robust anti-NFT effect and the lack of encephalitogenicity in NFT mice immunized with phos-tau peptides, even though CFA with PT was included in vaccine, point to their anti-NFT therapeutic potential.

Conversion to sirolimus immunosuppression in liver transplantation recipients with hepatocellular carcinoma: Report of an initial experience.

AIM: To report a retrospective analysis of preliminary results of 36 patients who received sirolimus (SRL, Rapamune, rapamycin) in a consecutive cohort of 248 liver allograft recipients. METHODS: Thirty-six liver transplant patients with hepatocellular carcinoma (HCC) who were switched to SRL-based immunosuppression therapy from tacrolimus were enrolled in this study. The patients who were diagnosed as advanced HCC before orthotopic liver transplantation (OLT) were divided into group A (n = 11), those who were found to have HCC recurrence and/or metastasis after OLT were assigned to group B (n = 18), and those who developed renal insufficiency caused by calcineurin inhibitor (CNI) were assigned to group C (n = 7) after OLT. RESULTS: The patients were followed up for a median of 10.4 mo (range, 3.8-19.1 mo) after conversion to SRL therapy and 12.3 mo (range, 5.1-34.4 mo) after OLT. Three patients developed mild acute cellular rejection 2 wk after initiating SRL therapy, which was fully reversed after prednisolone pulse therapy. In group A, only 1 patient was found to have HCC recurrence and metastasis 12 mo after OLT. In group B, 66.7% (12/18) patients (2 with progressive tumor, 7 with stable tumor and 3 without tumor) were still alive due to conversing to SRL and/or resection for HCC recurrence at the end of a median follow-up of 6.8 mo post conversion and 10.7 mo posttransplant. In group C, no HCC recurrence was demonstrated in 7 patients, and renal function became normal after SRL therapy. Thrombocytopenia (n = 2), anemia (n = 8), and oral aphthous ulcers (n = 7) found in our cohort were easily manageable. CONCLUSION: The conversion to SRL-based immunosuppression may inhibit the recurrence and metastasis of HCC and improve CNI-induced renal insufficiency in OLT patients with HCC.

Effect of enamel matrix derivative on periodontal ligament cells in vitro is diminished by Porphyromonas gingivalis.

BACKGROUND: Enamel matrix derivative (EMD) has been shown to possess a mitogenic effect to induce effective periodontal regeneration, however, it is unclear whether periodontal pathogens can modulate the effect of EMD. The present study examined the influence of Porphyromonas gingivalis on EMD-stimulated periodontal ligament (PDL) cells. METHODS: P. gingivalis ATCC33277 and its mutants deficient in fimbriae (delta fimA) or gingipains (delta rgpA delta rgpB, delta kgp, and delta rgpA delta rgpB delta kgp) were employed. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis wild strain or a mutant. Cell migration and proliferation were then evaluated with an in vitro wound healing assay. The expression of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor I (IGF-I) mRNA by PDL cells was examined. Further, the degradation and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) as well as paxillin in infected PDL cells were estimated using Western blot analysis. RESULTS: P. gingivalis ATCC33277 inhibited the migration and proliferation of PDL cells on EMD-coated dishes, and the mutants delta fimA, delta rgpA delta rgpB, and delta kgp showed the same effects. Further, each of these organisms diminished the expression of TGF-beta1 and IGF-I mRNA, as well as the phosphorylation of ERK1/2 from EMD-stimulated PDL cells. In addition, total paxillin protein was markedly degraded by both the wild-type strain and each of the mutants except for delta rgpA delta rgpB delta kgp, which showed a negligible effect in all of the assays with EMD-stimulated PDL cells. CONCLUSION: These results suggest that P. gingivalis diminishes the effect of EMD on PDL cells in vitro through a cooperative action of gingipains.

The inhibitory effects of manganese on steroidogenesis in rat primary Leydig cells by disrupting steroidogenic acute regulatory (StAR) protein expression.

Manganese is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, the effects of manganese on the biosynthesis of testosterone by primary rat Leydig cells were examined. Primary Leydig cells were exposed to various concentrations of manganese chloride for different periods of time. Dose and time-dependent reductions of human chorionic gonadotropin (hCG)-stimulated testosterone level were observed in the culture medium. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were also detected. The expression of StAR protein stimulated by hCG was suppressed by manganese chloride at all concentrations (0.01, 0.1, 1.0 mM) and time points (2, 4, 24, 48 h) tested. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced after treated by manganese chloride for 24 or 48 h, respectively. The manganese exposure effect on cell viability was significant at 1.0 and 1.5 mM at 24 h, while at 48 h it was significant at every concentration tested. The decreasing effect of manganese on mitochondrial membrane potential was significant at every concentration measured and every time point tested. These data suggest that manganese exposure for 2 and 4 h inhibited rat primary Leydig cell steroidogenesis by decreasing StAR protein expression while 24 and 48 h exposure of manganese chloride caused adverse effects on both StAR protein and P450scc and 3beta-HSD enzyme activity to reduce steroidogenesis. Manganese may also disrupt StAR expression and/or function secondary to mitochondrial dysfunction.

Ativaçäo osteoblástica e mecanismos de migraçäo de neutrófilos induzidos pela dentina / Osteoblastic activation and mechanisms of neurophil migration induced by dentin

Após a mineralizaçäo, as moléculas de dentina permanecem imobilizadas pela fase mineral da matriz, sendo expostas ou liberadas como conseqüência de danos ao ligamento periodontal e à polpa. Uma vez liberadas, estas moléculas poderiam participar na migraçäo e ativaçäo de células ósseas e inflamatórias influenciando o curso dos processos associados à dissoluçäo da matriz dentinária. Com o objetivo de avaliar a capacidade da dentina em induzir eventos inflamatórios, verificamos o efeito de extratos brutos na liberaçäo in vitro de mediadores inflamatórios por macrófagos e osteoblastos. Avaliamos ainda, a capacidade de extratos brutos e das Sialoproteína (DSP) e Fosfoproteína (DPP) dentinárias em induzir quimiotaxia de neutrófilos in vivo e caracterizamos os mediadores envolvidos neste processo. Os extratos dentinários induziram osteoblastos a produzir IL-1 , TNF- , IL-6, CINC-1 e IL-10, sem interferir com a morfologia e a diferenciaçäo destas células. Os extratos dentinários, a DSP e a DPP induziram macrófagos a produzir IL-1 , TNF- e as quimiocinas MIP-1 , KC e MIP-2, bem como estimularam a migraçäo de leucócitos de maneira dose-dependente. A investigaçäo dos mecanismos envolvidos revelou a participaçäo de IL-1 , TNF- , KC e MIP-2 e excluiu a participaçäo de prostaglandinas, leucotrienos e MIP-1 na migraçäo leucocitária. Observamos ainda que macrófagos e mastócitos respectivamente estimulam e inibem o recrutamento de neutrófilos induzido por DSP e DPP e que macrófagos estimulados in vitro produzem fator(es) quimiotático(s) para neutrófilos. A partir destes resultados, podemos concluir que a dentina é capaz de estimular a produçäo de mediadores inflamatórios, com reconhecida atividade sobre osteoclastos, por células osteoblásticas e macrófagos in vitro. Os mecanismos pelos quais as proteínas dentinárias induzem migraçäo de neutrófilos in vivo säo indiretos e dependentes de IL-1 , TNF- e das quimiocinas KC e MIP-2, sendo modulados por macrófagos e mastócitos

Characterization of a potent uveitopathogenic site derived from rat phosducin.

Phosducin is a retinal and pineal phosphoprotein assumed to play an important role in visual phototransduction. Phosducin is also a uveitopathogenic retinal antigen, but its potency has been reported to be mild. During the course of studies aimed at identifying uveitopathogenic sites in phosducin, we found that rat phosducin possessed a potent uveitopathogenic site. In this study, we characterize the potent uveitopathogenic site by using synthetic peptides. Several synthetic peptides from this region plus adjuvants were injected into Lewis rats, and the uveitopathogenic core sequence was defined. We also determined the pivotal amino acid residues by using synthetic peptides with single residue substitution. Immunization with PDC(R)65-96 (amino acid residues 65 through 96 derived from rat phosducin) at doses of 0.83 nmol or more induced severe experimental autoimmune uveitis (EAU) in all rats within 12 days. Experimental autoimmune pinealitis (EAP) was also observed in all rats after immunization with 0.83 nmol or higher doses of the peptide. The lowest dose of the peptide to induce EAU and EAP was 0.24 nmol. The smallest peptide that induced EAU as severe as PDC(R)65-96 was PDC(R)77-87, which consisted of 11 amino acid residues (YELIHQDKEDE). The core sequence within the uveitopathogenic site was a pentapeptide (LIHQD), amino acid residues from 79 to 83. To determine the role of individual residues within PDC(R)77-87, we tested the uveitopathogenicity of analogues of PDC(R)75-85 and PDC(R)77-89, respectively, in which each of the residues from 77 to 87 was replaced by alanine (A). Analogous peptides bearing a single residue substitution at 80 (I-->A) and 82 (Q-->A), respectively, were not uveitopathogenic. Our findings demonstrated the presence of a potent uveitopathogenic site in PDC(R)65-96 whose potency in Lewis rats was comparable to that of S-antigen. The pivotal amino acid residues for uveitopathogenicity were the residues at 80 (I) and 82 (Q). The clinical and histological features of this EAU closely resembled those of the EAU induced by S-antigen and recoverin.