Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

El Alimento y la muerte en la festividad de Día de Muertos en México

En la mayor parte de México se celebra los días 1 y 2 de noviembre el Día de Muertos, celebración reconocida por la UNESCO como Obra Maestra del Patrimonio Oral e Intangible de la Humanidad. La fiesta, que ha sido objeto de estudio de numerosos trabajos antropológicos, ha sido relegada en el ámbito de la estética, tanto así, que antropólogos como Stanley Brandes subrayan la carencia de un tratamiento procesual de la dimensión artística de la festividad.

Proliferation is a prerequisite for bacterial superantigen-induced T cell apoptosis in vivo.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex class II molecules and selectively interacts with T cells that bear certain T cell receptor (TCR) V beta domains. Administration of SEB in adult mice results in initial proliferation of V beta 8+ T cells followed by a state of unresponsiveness resulting from a combination of clonal deletion and clonal anergy in the SEB-reactive population. At this time, it is unclear what relationship exists between the T cells that have proliferated and those that have been deleted or have become anergic. Here we show that only a fraction of the potentially reactive V beta 8+ T cells proliferate in response to SEB in vivo, and that all the cells that have proliferated eventually undergo apoptosis. Virtually no apoptosis can be detected in the nonproliferating V beta 8+ T cells. These data demonstrate a causal relationship between proliferation and apoptosis in response to SEB in vivo, and they further indicate that T cells bearing the same TCR V beta segment can respond differently to the same superantigen. The implications of this differential responsiveness in terms of activation and tolerance are discussed.

In vivo mechanisms leading to transplantation tolerance induced by regulatory T cells

Purpose: The mechanisms by which CD4+CD25+Foxp3+ T cells (Tregs) regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer and skin transplantation model, we analyzed the in vivo expansion, effector function and trafficking of effector T cells and donor-specific Tregs, in response to an allograft. Methods and materials: Antigen-specific Tregs were generated and expanded in vitro by culturing freshly isolated Tregs from BALB/c mice (H2d) with syngeneic dendritic cells pulsed with an allopeptide (here the Kb peptide derived from the MHC class I molecule of allogeneic H2b mice). Fluorescent-labelled CD4+CD25- naive T cells and donor-antigen-specific Tregs were transferred alone or coinjected into syngeneic BALB/c-Nude recipients transplanted with allogeneic C57BL/6xBALB/c donor skin. Results: As opposed to their in vitro hyporesponsiveness, Tregs divided in vivo, migrated and accumulated in the allograft draining lymph nodes (drLN) and within the graft. The co-transfer of Tregs did not modify the early proliferation and homing of CD4+CD25- T cells to secondary lymphoid organs. But, in the presence of Tregs, effector T cells produced significantly less IFN- and IL-2 effector cytokines, while higher amounts of IL-10 were detected in the spleen and drLN of these mice. Furthermore, time-course studies showed that Tregs were recruited into the allograft at a very early stage posttransplantation and prevented infiltration by effector T cells. Conclusion: Overall, our results suggest that suppression of graft rejection involves the early recruitment of donor-specific Tregs at the sites of antigenic challenge and that Tregs mainly regulate the effector arm of T cell alloresponses.

Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring.

The authors developed a standardized approach for immune monitoring of antigen-specific CD8+ T cells within peripheral blood lymphocytes (PBLs) that combines direct ex vivo analysis of Melan-A/MART-1 and influenza-specific CD8+ T cells with HLA-A2/peptide multimers and interferon-gamma ELISPOT assays. Here the authors assessed the quality of results obtained with 180 PBLs from healthy donors and melanoma patients. Reproducibility of the multimer assay was good (average of 15% variation). In the absence of in vivo antigen-specific T-cell responses, physiologic fluctuations of multimer-positive T cells was low, with variation coefficients of 20% for Melan-A and 28% for influenza-specific T cells. In contrast, patients with vaccination-induced T-cell responses had significantly increased T-cell frequencies clearly exceeding physiologic fluctuations. Comparable results were obtained with ELISPOT assays. In conclusion, this approach is well suited to assess T-cell responses as biologic endpoints in clinical vaccine studies.

Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.

Secretory immunoglobulin (Ig) A (SIgA) is essential in protecting mucosal surfaces. It is composed of at least two monomeric IgA molecules, covalently linked through the J chain, and secretory component (SC). We show here that a dimeric/polymeric IgA (IgA(d/p)) is more efficient when bound to SC in protecting mice against bacterial infection of the respiratory tract. We demonstrate that SC ensures, through its carbohydrate residues, the appropriate tissue localization of SIgA by anchoring the antibody to mucus lining the epithelial surface. This in turn impacts the localization and the subsequent clearance of bacteria. Thus, SC is directly involved in the SIgA function in vivo. Therefore, binding of IgA(d/p) to SC during the course of SIgA-mediated mucosal response constitutes a crucial step in achieving efficient protection of the epithelial barrier by immune exclusion.

Feasibility of in vivo 15N MRS detection of hyperpolarized 15N labeled choline in rats.

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.

Oral administration of a low dose of midazolam (75 microg) as an in vivo probe for CYP3A activity.

OBJECTIVE: We investigated whether the oral administration of a low dose (75 micro g) of midazolam, a CYP3A probe, can be used to measure the in vivo CYP3A activity. METHODS: Plasma concentrations of midazolam, 1'OH-midazolam and 4'OH-midazolam were measured after the oral administration of 7.5 mg and 75 micro g midazolam in 13 healthy subjects without medication, in four subjects pretreated for 2 days with ketoconazole (200 mg b.i.d.), a CYP3A inhibitor, and in four subjects pretreated for 4 days with rifampicin (450 mg q.d.), a CYP3A inducer. RESULTS: After oral administration of 75 micro g midazolam, the 30-min total (unconjugated + conjugated) 1'OH-midazolam/midazolam ratios measured in the groups without co-medication, with ketoconazole and with rifampicin were (mean+/-SD): 6.23+/-2.61, 0.79+/-0.39 and 56.1+/-12.4, respectively. No side effects were reported by the subjects taking this low dose of midazolam. Good correlations were observed between the 30-min total 1'OH-midazolam/midazolam ratio and midazolam clearance in the group without co-medication (r(2)=0.64, P<0.001) and in the three groups taken together (r(2)=0.91, P<0.0001). Good correlations were also observed between midazolam plasma levels and midazolam clearance, measured between 1.5 h and 4 h. CONCLUSION: A low oral dose of midazolam can be used to phenotype CYP3A, either by the determination of total 1'OH-midazolam/midazolam ratios at 30 min or by the determination of midazolam plasma levels between 1.5 h and 4 h after its administration.

Thalamic connectivity in patients with essential tremor treated with MR imaging-guided focused ultrasound: in vivo fiber tracking by using diffusion-tensor MR imaging.

PURPOSE: To use diffusion-tensor (DT) magnetic resonance (MR) imaging in patients with essential tremor who were treated with transcranial MR imaging-guided focused ultrasound lesion inducement to identify the structural connectivity of the ventralis intermedius nucleus of the thalamus and determine how DT imaging changes correlated with tremor changes after lesion inducement. MATERIALS AND METHODS: With institutional review board approval, and with prospective informed consent, 15 patients with medication-refractory essential tremor were enrolled in a HIPAA-compliant pilot study and were treated with transcranial MR imaging-guided focused ultrasound surgery targeting the ventralis intermedius nucleus of the thalamus contralateral to their dominant hand. Fourteen patients were ultimately included. DT MR imaging studies at 3.0 T were performed preoperatively and 24 hours, 1 week, 1 month, and 3 months after the procedure. Fractional anisotropy (FA) maps were calculated from the DT imaging data sets for all time points in all patients. Voxels where FA consistently decreased over time were identified, and FA change in these voxels was correlated with clinical changes in tremor over the same period by using Pearson correlation. RESULTS: Ipsilateral brain structures that showed prespecified negative correlation values of FA over time of -0.5 or less included the pre- and postcentral subcortical white matter in the hand knob area; the region of the corticospinal tract in the centrum semiovale, in the posterior limb of the internal capsule, and in the cerebral peduncle; the thalamus; the region of the red nucleus; the location of the central tegmental tract; and the region of the inferior olive. The contralateral middle cerebellar peduncle and bilateral portions of the superior vermis also showed persistent decrease in FA over time. There was strong correlation between decrease in FA and clinical improvement in hand tremor 3 months after lesion inducement (P < .001). CONCLUSION: DT MR imaging after MR imaging-guided focused ultrasound thalamotomy depicts changes in specific brain structures. The magnitude of the DT imaging changes after thalamic lesion inducement correlates with the degree of clinical improvement in essential tremor.

Increase of [(18)F]FLT Tumor Uptake In Vivo Mediated by FdUrd: Toward Improving Cell Proliferation Positron Emission Tomography.

PURPOSE: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D: -glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. METHODS: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. RESULTS: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. CONCLUSION: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration.

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated "memory" cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

Pharmacokinetic and pharmacodynamic analysis of efavirenz dose reduction using an in vitro-in vivo extrapolation model.

The pharmacokinetics (PK) of efavirenz (EFV) is characterized by marked interpatient variability that correlates with its pharmacodynamics (PD). In vitro-in vivo extrapolation (IVIVE) is a "bottom-up" approach that combines drug data with system information to predict PK and PD. The aim of this study was to simulate EFV PK and PD after dose reductions. At the standard dose, the simulated probability was 80% for viral suppression and 28% for central nervous system (CNS) toxicity. After a dose reduction to 400 mg, the probabilities of viral suppression were reduced to 69, 75, and 82%, and those of CNS toxicity were 21, 24, and 29% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. With reduction of the dose to 200 mg, the probabilities of viral suppression decreased to 54, 62, and 72% and those of CNS toxicity decreased to 13, 18, and 20% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. These findings indicate how dose reductions might be applied in patients with favorable genetic characteristics.