Long-term allograft tolerance is characterized by the accumulation of B cells exhibiting an inhibited profile.

Numerous reports have highlighted the central role of regulatory T cells in long-term allograft tolerance, but few studies have investigated the B-cell aspect. We analyzed the B-cell response in a rat model of long-term cardiac allograft tolerance induced by a short-term immunosuppression. We observed that tolerated allografts are infiltrated by numerous B cells organized in germinal centers that are strongly regulated in their IgG alloantibody response. Moreover, alloantibodies from tolerant recipients exhibit a deviation toward a Th2 isotype and do not activate in vitro donor-type endothelial cells in a pro-inflammatory way but maintained expression of cytoprotective molecules. Interestingly, this inhibition of the B-cell response is characterized by the progressive accumulation in the graft and in the blood of B cells blocked at the IgM to IgG switch recombination process and overexpressing BANK-1 and the inhibitory receptor Fcgr2b. Importantly, B cells from tolerant recipients are able to transfer allograft tolerance. Taken together, these results demonstrate a strong regulation of the alloantibody response in tolerant recipients and the accumulation of B cells exhibiting an inhibited and regulatory profile. These mechanisms of regulation of the B-cell response could be instrumental to develop new strategies to promote tolerance.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Indirect CD4+ TH1 response, antidonor antibodies and diffuse C4d graft deposits in long-term recipients conditioned by donor antigens priming.

Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.D(u) molecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4+ indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.

Metastatic hypervascular lymph nodes in malignant glomus vagale tumor: angiography findings.

Malignant vagal paraganglioma is very uncommon and the diagnosis of malignancy is made on the basis of presence of distant metastasis rather than the histological findings. We report angiography findings of metastatic cervical lymph nodes in a case of malignant vagal paraganglioma.

Sonographic and MRI findings in prepubertal adnexal hemorrhagic cyst with torsion.

Adnexal torsion is rare before menarche. We report the case of a 10-year-old girl with persistent left lower quadrant pain proven by surgery to be caused by adnexal torsion due to a hemorrhagic cyst. Sonography showed a well-defined, complex, predominantly solid mass with some sound through-transmission and a small amount of fluid. The left ovary could not be distinguished from the mass; the right ovary appeared normal. Doppler sonography demonstrated no blood flow within the mass. MRI revealed a circumferential region of high signal intensity in the periphery of the mass and multiple hyperintense foci in the left ovary.

Mammographic and sonographic findings in the diagnosis of idiopathic granulomatous mastitis.

The aim of this study was to describe the mammographic and sonographic findings of idiopathic granulomatous mastitis which is a rare inflammatory disease of the breast of unknown etiology. The clinical, radiologic, and pathologic features of 12 cases with idiopathic granulomatous mastitis were retrospectively reviewed. Mammography was performed in all cases, 8 of which showed a focal asymmetric density, 3 had a mass with irregular margins, and 1 had no abnormality. Sonography was performed in 10 cases, and a focal area with inhomogeneous and hypoechoic pattern was depicted in 6 cases, 4 of which were associated with internal tubular hypoechoic structures. One case revealed a hypoechoic mass consistent with malignancy. In 1 case sonography showed an edematous pattern involving nearly the entire breast. Two patients had normal sonograms. If a focal asymmetric density is seen in mammography and inhomogeneous hypoechogenity with internal hypoechoic tubular structures accompany ultrasonographically, these findings should suggest the possibility of idiopathic granulomatous mastitis; however, very often idiopathic granulomatous mastitis mimics a breast carcinoma clinically and the final diagnosis should be reached histopathologically due to high false-positive and false-negative mammographic appearances.

Transient neonatal hypoglycemia: cranial US and MRI findings.

A case of transient neonatal hypoglycemia with patchy hyperechogenic white matter abnormalities in the frontal and parietooccipital lobes on cranial US is presented. An MRI examination revealed T1 and T2 shortening of the lesions in the occipital and frontal white matter. Follow-up cranial US demonstrated recovery of white matter changes in the patient with normal neurological outcome.

Morphologic and radiologic anatomy of the occipital bone.

Several diseases may cause craniovertebral instability warranting occiput-cervical fusion. As occipital screw and rod constructs are becoming more popular, requiring that screws be placed either medially or laterally in the occipital bone, the need for clearer anatomical and computed tomography (CT)-confirmed data regarding the relative thickness of the occiput in its various localities has become more critical. In 18 cadaveric specimens, the occipital bone was divided into 35 measurable segments. Transversely, the occipital bone was divided into five lines starting at the level of the inion; horizontal lines then proceeded inferiorly in 1-cm segments, 1, 2, 3, and 4 cm below the level of inion. In a comparable fashion, the occipital bone was divided vertically, starting at the midline, and proceeding laterally also in 1-, 2-, and 3-cm segments. Anatomical measurements of thickness were directly performed using a Vernier caliper. Results were directly correlated with axial CT measurements of bony thickness. Anatomical and CT measurements closely correlated within the same specimen, but there was significant interspecimen variability. The marked differences in the occipital bone anatomy noted between specimens indicates that patients undergoing occipital screw placement for cranial-cervical instability would benefit from preoperative occipital CT evaluations.

Identification of immunodominant donor MHC peptides following rejection and donor strain transfusion-induced tolerance of heart allografts in adult rats.

Pre-graft priming of heart allograft recipients with donor strain blood induces tolerance in 100% of adult rats in the congenic LEW.1W to LEW.1A combination. This tolerant state is specific for donor MHC antigens as third-party blood transfusions fail to induce tolerance, and third-party skin grafts are promptly rejected by tolerant graft recipients. In this study we have characterized the immunodominant donor (RT1u) class I and II allogenic peptides which elicit an in vitro proliferative response to splenocytes from recipients (RT1a) undergoing acute rejection or tolerant to a LEW.1A cardiac allograft. Paradoxically, splenocytes from tolerant animals responded more vigorously to a broader set of donor peptides than splenocytes from rejecting animals. In addition, several of these peptides were observed to be stimulatory only for tolerant splenocytes. These findings suggest that regulatory cells may be involved in tolerance induction or maintenance and are selected by specific motifs, which could be utilized for manipulating the immune system of graft recipients.

Anti-TCR-specific DNA vaccination demonstrates a role for a CD8+ T cell clone in the induction of allograft tolerance by donor-specific blood transfusion.

Donor-specific allograft tolerance can be induced in the adult rat by pregraft donor-specific blood transfusion (DST). This tolerance appeared to be mediated by regulatory cells and to the production of the suppressive cytokine TGF-beta1. A potential immunoregulatory CD8+ clone bearing a Vbeta18-Dbeta1-Jbeta2.7 TCR gene rearrangement was previously identified in DST-treated recipients. To assess the functional role of this T cell clone in the induction of tolerance by DST, we have vaccinated DST-treated recipients with a plasmid construct encoding for the Vbeta18-Dbeta1-Jbeta2.7 TCR beta-chain. DST-induced allograft tolerance was abolished by anti-TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination in six of seven recipients, whereas vaccination with the vector alone, or with the construct encoding a TCR Vbeta13 beta-chain, had no effect. However, the transcript number of the Vbeta18-Dbeta1-Jbeta2.7 chain was unchanged in allografts from vaccinated DST-treated rats, suggesting that this clone was not depleted by vaccination, but rather was altered in its function. Moreover, TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination restored the anti-donor alloantibody production, partially restore the capacity of spleen cells from tolerized recipients to proliferate in vitro against donor cells, and decreased the inhibitory effect of TGF-beta1, seen in DST-treated recipients, in spleen cells from vaccinated DST-treated ones. This study strongly suggests that this CD8+ TCR Vbeta18-Dbeta1-Jbeta2.7 T cell clone has an effective immunoregulatory function in allograft tolerance induced by DST.

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression.

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.