Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.     

IgE recognition patterns of profilin, PR-10, and tropomyosin panallergens tested in 3,113 allergic patients by allergen microarray-based technology

Background

IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed.

Methods

We evaluated the IgE recognition profile of profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2), PR-10 proteins (Aln g 1, Api g 1, Bet v 1.0101, Bet v 1.0401, Cor a 1, Dau c 1 and Mal d 1.0108) and tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7) using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion.

Results

3,113 individuals (49.9% female) were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgE⁺), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE⁺) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE⁺). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis.

Conclusions

Unless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients'' immunological phenotypes.     

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.     

Chiral arylpyrrolidinols: preparation and biological profile

The preparation and biological evaluation of a new class of arylpyrrolidinols is reported. The antinociceptive activity was evaluated in vivo with the hot plate test (HPT) and formalin test (FT), excluding any involvement on motor coordination with the rota-rod test (RRT). The nociceptive behavior in the late phase of FT (representative of chronic pain) suggests an involvement of the antiinflammatory process and it is clearly influenced by the stereochemical features, being the eutomer of phenylpyrrolidinols, the (2R,3S) enantiomer. Despite this, a specific mechanism of action is not yet clarified.     

A Statistical Evaluation of Sediment Quality Triad Benthic Community Measurements in PAH-Impacted Sediments at a Manufactured Gas Plant Site along the Hudson River

The sediment quality triad (SQT) assumes that three measurements (sediment chemistry, laboratory bioassay, and benthic macroinvertebrate counts) comprise an independent assessment of impact, which when integrated using a weight-of-evidence approach provides a comprehensive assessment of risk. An SQT assessment was conducted on 41 sediment samples collected adjacent to a manufactured gas plant site on the freshwater reach of the Hudson River in New York State. The assessment shows that the benthic macroinvertebrate data did not correlate with either sediment or pore water polycyclic aromatic hydrocarbon (PAH) concentrations, nor did these data show consistent relationships to the results of laboratory bioassay testing (Hyalella azteca 28-day survival or biomass). The benthic community across the site and reference areas was comprised of few taxa, all of which were pollution-tolerant organisms with tolerance values greater than or equal to five. Only in significantly impacted sediment samples with PAH concentrations in the thousands of milligrams per kilogram, pore water concentrations above 100 toxic units, and visible non-aqueous phase liquid present in the sample did the benthic macroinvertebrate data show a response. In contrast, sediment and pore water PAH measurements and H. azteca toxicity testing provided consistent interpretation of impact. These results illustrate that benthic macroinvertebrate data may contain less information value and be a more challenging line of evidence to interpret in triad studies conducted in certain ecological settings; in this case, a large-order river with a relatively depauperate benthic community dominated by species tolerant of PAHs.     

Epigenetics Beyond Fetal Growth Restriction: A Comprehensive Overview

Molecular Diagnosis & Therapy - Fetal growth restriction is a pathological condition occurring when the fetus does not reach the genetically determined growth potential. The etiology of fetal...     

Assessment of CD4<Superscript>+</Superscript> T cells specific for the tumor antigen SSX-1 in cancer-free individuals

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.     

20S proteasome mediated degradation of DHFR: implications in neurodegenerative disorders

The 20S proteasome is responsible for the degradation of protein substrates implicated in the onset and progression of neurodegenerative disorders, such as alpha-synuclein and tau protein. Here we show that the 20S proteasome isolated from bovine brain directly hydrolyzes, in vitro, the dihydrofolate reductase (DHFR), demonstrated to be involved in the pathogenesis of neurodegenerative diseases. Furthermore, the DHFR susceptibility to proteolysis is enhanced by oxidative conditions induced by peroxynitrite, mimicking the oxidative environment typical of these disorders. The results obtained suggest that the folate metabolism may be impaired by an increased degradation of DHFR, mediated by the 20S proteasome.     

Influence of Water Activity and System Mobility on Peroxidase Activity in Maltodextrin Solutions

Maltodextrins influenced the enzymatic activity in aqueous solutions by affecting the water activity (a_w) and mobility as described by viscosity and T’g. In diluted solutions, viscosity being equal, (1) maltodextrin with dextrose equivalents (DE) of 33 was more effective than glucose in limiting horseradish peroxidase (HRP) activity; (2) an increase in the maltodextrin chain length from DE 33 to DE 8.7 did not further limited enzymatic activity; (3) the maltodextrin with the highest chain length (DE 2.5) determined the highest enzyme inhibition. In general, the increase of molecular weight negatively affected the HRP activity by increasing the viscosity and T’g (decreasing molecular mobility) but it positively affected the a_w and, in some cases, this compensated the HRP activity inhibition. In concentrated solutions (apparent viscosity ≈ 40 mPa s) the HRP activity decreased with the increase of the maltodextrin molecular weight, and it showed a dependence on T’g which could be described by a William, Landel and Ferry (WLF)-type equation. On the contrary, in the solution added with the maltodextrin with the highest chain length (DE 2.5) the HRP activity was much higher than that predicted by the WLF-type equation. The maltodextrin with DE 2.5 contains intact starch fragments and in water forms a suspension. In such a discontinuous system, the viscosity in the vicinity of the protein is lower than the bulk viscosity, and thus, the enzyme activity is higher than expected. Moreover, since T’g is a property of the soluble phase, it does not explain the mobility in discontinuous systems.