Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive.

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

The Future of Regulatory T Cell Therapy: Promises and Challenges of Implementing CAR Technology.

Cell therapy with polyclonal regulatory T cells (Tregs) has been translated into the clinic and is currently being tested in transplant recipients and patients suffering from autoimmune diseases. Moreover, building on animal models, it has been widely reported that antigen-specific Tregs are functionally superior to polyclonal Tregs. Among various options to confer target specificity to Tregs, genetic engineering is a particularly timely one as has been demonstrated in the treatment of hematological malignancies where it is in routine clinical use. Genetic engineering can be exploited to express chimeric antigen receptors (CAR) in Tregs, and this has been successfully demonstrated to be robust in preclinical studies across various animal disease models. However, there are several caveats and a number of strategies should be considered to further improve on targeting, efficacy and to understand the in vivo distribution and fate of CAR-Tregs. Here, we review the differing approaches to confer antigen specificity to Tregs with emphasis on CAR-Tregs. This includes an overview and discussion of the various approaches to improve CAR-Treg specificity and therapeutic efficacy as well as addressing potential safety concerns. We also discuss different imaging approaches to understand the in vivo biodistribution of administered Tregs. Preclinical research as well as suitability of methodologies for clinical translation are discussed.

Regulatory T cell-derived extracellular vesicles modify dendritic cell function.

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues.

Infant frontal EEG asymmetry in relation with postnatal maternal depression and parenting behavior.

Right frontal electroencephalogram (EEG) asymmetry associates with negative affect and depressed mood, which, among children, are predicted by maternal depression and poor parenting. This study examined associations of maternal depression and maternal sensitivity with infant frontal EEG asymmetry based on 111 mother-6-month-infant dyads. There were no significant effects of postnatal maternal depression or maternal sensitivity, or their interaction, on infant EEG frontal asymmetry. However, in a subsample for which the infant spent at least 50% of his/her day time hours with his/her mother, both lower maternal sensitivity and higher maternal depression predicted greater relative right frontal EEG asymmetry. Our study further showed that greater relative right frontal EEG asymmetry of 6-month-old infants predicted their greater negative emotionality at 12 months of age. Our study suggested that among infants with sufficient postnatal maternal exposure, both maternal sensitivity and mental health are important influences on early brain development.

Novel application of 3D printing in brachytherapy using MED610 3D printed insert for I-125 ROPES eye plaque.

The purpose of this study was to evaluate if MED610 3D printed material can be used as a surrogate for acrylic in the manufacturing of a replacement insert used in an eye plaque brachytherapy applicator. Measurement of the dose distributions from a standard acrylic insert were compared with dose obtained from MED610 3D printed replica using GafChromic® EBT3 films. The study used a 15 mm Radiation Oncology Physics and Engineering Services, Australia (ROPES) type eye plaque applicator loaded with I-125 (model 6711) seeds. GafChromic® EBT3 films were placed in a solid water phantom and dose distributions were measured three-dimensionally both along and perpendicular to a loaded ROPES eye plaque's central axis (CAX). Each measurement was performed with the stainless steel plaque backing attached to the eye plaque, to assess the variability of the dose distributions between the acrylic and MED 610 insert. Results of dose along the central axis were compared between acrylic and MED610 insert and the results found agreement within 1.5 %. Off-axis profiles were also compared between the acrylic insert and MED610 and were found to agree to within 7 % in the central 15 mm width centred on CAX at depths ranging from z = 2 mm to z = 8 mm in 2 mm increments. The aim of this investigation was to verify the consistency between doses profiles over a range of clinically relevant depths for a 15 mm loaded ROPES plaque using acrylic versus MED610 material. The results show an agreement between experimental measurements given the film uncertainty of 7 %.

Hg concentrations in fish from coastal waters of California and Western North America.

The State of California conducted an extensive and systematic survey of mercury (Hg) in fish from the California coast in 2009 and 2010. The California survey sampled 3483 fish representing 46 species at 68 locations, and demonstrated that methylHg in fish presents a widespread exposure risk to fish consumers. Most of the locations sampled (37 of 68) had a species with an average concentration above 0.3µg/gwet weight (ww), and 10 locations an average above 1.0µg/gww. The recent and robust dataset from California provided a basis for a broader examination of spatial and temporal patterns in fish Hg in coastal waters of Western North America. There is a striking lack of data in publicly accessible databases on Hg and other contaminants in coastal fish. An assessment of the raw data from these databases suggested the presence of relatively high concentrations along the California coast and in Puget Sound, and relatively low concentrations along the coasts of Alaska and Oregon, and the outer coast of Washington. The dataset suggests that Hg concentrations of public health concern can be observed at any location on the coast of Western North America where long-lived predator species are sampled. Output from a linear mixed-effects model resembled the spatial pattern observed for the raw data and suggested, based on the limited dataset, a lack of trend in fish Hg over the nearly 30-year period covered by the dataset. Expanded and continued monitoring, accompanied by rigorous data management procedures, would be of great value in characterizing methylHg exposure, and tracking changes in contamination of coastal fish in response to possible increases in atmospheric Hg emissions in Asia, climate change, and terrestrial Hg control efforts in coastal watersheds.

Maternal sensitivity, infant limbic structure volume and functional connectivity: a preliminary study.

Mechanisms underlying the profound parental effects on cognitive, emotional and social development in humans remain poorly understood. Studies with nonhuman models suggest variations in parental care affect the limbic system, influential to learning, autobiography and emotional regulation. In some research, nonoptimal care relates to decreases in neurogenesis, although other work suggests early-postnatal social adversity accelerates the maturation of limbic structures associated with emotional learning. We explored whether maternal sensitivity predicts human limbic system development and functional connectivity patterns in a small sample of human infants. When infants were 6 months of age, 20 mother-infant dyads attended a laboratory-based observational session and the infants underwent neuroimaging at the same age. After considering age at imaging, household income and postnatal maternal anxiety, regression analyses demonstrated significant indirect associations between maternal sensitivity and bilateral hippocampal volume at six months, with the majority of associations between sensitivity and the amygdala demonstrating similar indirect, but not significant results. Moreover, functional analyses revealed direct associations between maternal sensitivity and connectivity between the hippocampus and areas important for emotional regulation and socio-emotional functioning. Sensitivity additionally predicted indirect associations between limbic structures and regions related to autobiographical memory. Our volumetric results are consistent with research indicating accelerated limbic development in response to early social adversity, and in combination with our functional results, if replicated in a larger sample, may suggest that subtle, but important, variations in maternal care influence neuroanatomical trajectories important to future cognitive and emotional functioning.

MicroRNAs affect dendritic cell function and phenotype.

MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function.

Discovery of a novel long terminal repeat (LTR2i_SS) in Sus Scrofa.

Long terminal repeat (LTR) retrotransposons are transposable elements flanked by 5'/3' LTRs. They have a structure similar to endogenous retroviruses, but they lack the envelope (env) gene making them non-infectious. Long terminal repeats are motif-rich sequences and can act as bidirectional promoters or enhancers to regulate or inactivate genes by insertion. In this study, we identified a new chimeric LTR subfamily, LTR2i_SS, in the pig genome. This chimeric LTR family appears to be the ancestral form of the previously described LTR2_SS family. LTR2_SS appears to have deleted ~300 bp of un-annotated, ancestral sequence from LTR2i_SS. We identified no functional provirus sequences for either of these LTR types. LTR2i_SS sequences have been exapted into the untranslated regions of two protein-coding gene mRNAs. Both of these genes lie within previously mapped pig quantitative trait loci.

Tissue specific deletion of inhibitor of kappa B kinase 2 with OX40-Cre reveals the unanticipated expression from the OX40 locus in skin epidermis.

NF-κB signalling plays an essential role in T cell activation and generation of regulatory and memory populations in vivo. In the present study, we aimed to investigate the role of NF-κB signalling in post-activation T cells using tissue specific ablation of inhibitor of kappa-B kinase 2 expression, an important component of the inhibitor of kappa-B kinase complex in canonical NF-κB signalling. The OX40 antigen is expressed on activated T cells. Therefore, we used previously described mouse strain expressing Cre recombinase from the endogenous OX40 locus. Ablation of IKK2 expression using OX40(Cre) mice resulted in the development of an inflammatory response in the skin epidermis causing wide spread skin lesions. The inflammatory response was characterised by extensive leukocytic infiltrate in skin tissue, hyperplasia of draining lymph nodes and widespread activation in the T cell compartment. Surprisingly, disease development did not depend on T cells but was rather associated with an unanticipated expression of Cre in skin epidermis, and activation of the T cell compartment did not require Ikbk2 deletion in T cells. Employment of Cre reporter strains revealed extensive Cre activity in skin epidermis. Therefore, development of skin lesions was rather more likely explained by deletion of Ikbk2 in skin keratinocytes in OX40(Cre) mice.

Comparing the use of cobalt chromium stents to stainless steel stents in primary percutaneous coronary intervention for acute myocardial infarction: a prospective registry.

OBJECTIVES: To determine clinical outcome and rates of target vessel revascularization (TVR) in patients undergoing primary percutaneous coronary intervention (PCI) for STEMI who were treated with cobalt-chromium stents compared to stainless steel bare metal stents (BMS). BACKGROUND: The newer generation cobalt chromium stents were reported to achieve lower rates of TVR compared with conventional BMS. METHODS: Consecutive STEMI cases admitted within 12 h of symptom onset and undergoing primary angioplasty and bare metal stent implantation 1 January 2002 and 31 December 2008 were identified. Primary outcomes were rates of clinically-driven TVR at six months as well as occurrence of major adverse cardiovascular events (MACE) either of all-cause death, repeat myocardial infarction or TVR at six months. RESULTS: 1030 cases with 1175 lesions (84% males) and median age of 58 years underwent primary PCI for STEMI in our registry. Overall procedural success rate was 98%. Stainless steel stents were inserted in 65% of the culprit lesions (stainless steel, n = 766 versus cobalt chromium, n = 264). Primary outcomes of TVR (3.5% in the stainless steel group and 3.4% in the cobalt chromium group, P = 0.93) and MACE (8.4% in the stainless steel group and 5.3% in the cobalt chromium group, P = 0.11) after six months were no different between the two groups. However, there were more deaths at 30 days in the stainless steel group compared to the cobalt chromium group (3.5% versus 0.4%, HR 4.04 (1.03-3.88), P = 0.04). CONCLUSION: Both cobalt-chromium and stainless steel coronary stents were associated with similar and low risk of clinically-driven TVR.

A prospective study to compare the diagnostic performance of breast elastography versus conventional breast ultrasound.

AIM: To compare the diagnostic performance of breast elastography versus conventional ultrasound in the assessment of breast lesions. MATERIALS AND METHODS: The study was approved by the hospital's institutional review board. A prospective study involving 99 consecutive women who gave informed consent were enrolled from September 2007 to March 2008. One hundred and ten breast lesions were evaluated separately by conventional ultrasound, elastography and combined conventional ultrasound with elastography. Ultrasound assessment was based on the BIRADS classification, whereas elastographic assessment was based on strain pattern and the elastographic size ratios. Histological diagnosis was used as the reference standard. The sensitivity, specificity, and accuracy of each technique were compared. RESULTS: The mean age of the patients was 46.7 years. Twenty-six lesions were malignant and 84 were benign. Sensitivity, specificity, and accuracy were 88.5, 42.9 and 53.6%, respectively, for conventional ultrasound, 100, 73.8, and 80%, respectively, for elastography, and 88.5, 78.6, and 80.9%, respectively, for combined imaging. The specificity and accuracy of elastography and combined imaging were significantly better than that of conventional ultrasound (p<0.0001), whereas there was no statistically significant difference in the sensitivity between all three groups. Two-thirds (66.7%) of sonographic false-positive lesions had benign elastogram findings, which might have been spared from biopsy. CONCLUSION: This initial experience with ultrasound breast elastography showed that it was more specific and more accurate than conventional ultrasound. Combining elastography with ultrasound improved specificity and accuracy of ultrasound and can potentially reduce unnecessary breast biopsies.

ATR-FTIR studies on ion interaction of lithium perchlorate in polyacrylate/poly(ethylene oxide) blends.

The interaction behaviours between components of polyacrylate (PAc)/poly(ethylene oxide) (PEO) and lithium perchlorate (LiClO(4)) were investigated in detail by Attenuated Total Reflectance (ATR)-Fourier Transformed Infrared (FTIR) spectroscopy. Solution cast films of the PAc/PEO and PAc/PEO/LiClO(4) were examined. No obvious shifting of the characteristic ether and ester group stretching modes of PEO and PAc was observed, indicating incompatibility of the binary PAc/PEO blend. The spectroscopic studies on the PAc/PEO/LiClO(4) blends reveal that Li(+) ions coordinate individually to the polymer components at the ether oxygen of PEO and the C-O of the ester group of PAc. Frequency changes observed on the nu(C-O-C) and omega(CH(2)) of PEO confirm the coordination between PEO and Li(+) ions resulting in crystallinity suppression of PEO. The absence of experimental evidence on the formation of PEO-Li(+)-PAc complexes suggests that LiClO(4) does not enhance the compatibility of PAc/PEO blend.

Outpatient-based therapy of oral fludarabine and subcutaneous alemtuzumab for asian patients with relapsed/refractory chronic lymphocytic leukemia.

Background. Intravenous alemtuzumab and fludarabine are effective in combination for the treatment of chronic lymphocytic leukemia (CLL), but require hospital visits for intravenous injection. We performed a pilot study to assess the safety and efficacy of outpatient-based oral fludarabine with subcutaneous alemtuzumab (OFSA) for the treatment of relapsed/refractory CLL. Results. Depending on their response, patients were given two to six 28-day cycles of subcutaneous alemtuzumab 30 mg on days 1,3, and 5 and oral fludarabine 40 mg/m(2)/day for 5 days. Median patient age was 74. The lymphocyte counts of all five patients fell after the 1st cycle of treatment and reached normal/low levels on completion of 2 to 6 cycles of therapy. Platelet counts and hemoglobin were unaffected. All five patients achieved complete hematological remission, while two attained minimal residual disease negativity on 4-color flow cytometry. Conclusions. Our OFSA regimen was effective in elderly Asian patients with relapsed/refractory CLL, and it should be investigated further.

Enhanced IFNgamma production in adenosine-treated CHO cells: a mechanistic study.

Adenosine causes growth arrest in recombinant mammalian cell cultures, which results in enhanced productivity of the recombinant protein. Adenosine is also known to increase intracellular ATP level when added to mammalian cells. As a cell's energy level affects its protein expression capacity, we investigated the factors that contribute to the increase in recombinant protein productivity. Chinese hamster ovary (CHO) cells expressing human interferon-gamma (IFNgamma) were treated with 1 mM adenosine on Day 2 of culture. The growth arrest resulted in 60% reduction in integral viable cell density when compared with control. However, IFNgamma titer improved 1.4-fold alongside a 2.5-fold increase in average specific productivity. The adenosine-treated cells also experienced a two-fold increase in ATP level that sustained for 3 days. Western blot studies revealed a relatively short-lived but strong activation of the energy sensor AMP-activated protein kinase (AMPK) in adenosine-treated cells. Activation of AMPK was probably due to adenosine being temporarily converted to AMP. Activated AMPK should have down-regulated protein translation by preventing mammalian target of rapamycin (mTOR) from phosphorylating and inactivating 4E-binding protein 1 (4E-BP1), a key repressor of protein translation initiation. However, Western blots showed increased phosphorylation of 4E-BP1 on Day 2 that lasted 3 days. This implied that a high concentration of ATP could keep 4E-BP1 inhibited, probably by directly modulating mTOR. This corroborated with an earlier in vitro observation (Dennis et al., Science. 2001;294:1102-1105). Inhibition of translation initiation repression is thus likely to contribute in part to the improvement in IFNgamma-specific productivity and titer.

Upright stereotactic vacuum-assisted needle biopsy of suspicious breast microcalcifications.

Stereotactic core needle biopsy is a useful technique for evaluation of suspicious breast microcalcifications. The development of the 11-G vacuum-assisted biopsy system offers another method of minimally invasive biopsy carried out on a conventional mammography unit. We evaluate its usefulness, efficacy and safety in Asian women. Vacuum-assisted biopsy was carried out through the lateral approach using an add-on stereotactic device attached to a mammography unit. One hundred and five lesions were sampled in 97 patients. Excisional biopsy was subsequently Carried out for diagnosis of atypical ductal hyperplasia or carcinoma in high-risk patients. Patients with benign diagnosis underwent mammographic follow up. The technical success rate was 97%. An average of 13.5 tissue cores were retrieved for each lesion. The histopathological result obtained from mammotome was benign in 84.8% and malignant in 15.2%. The benign microcalcifications were predominantly fibrocystic change (n = 42) whereas the malignant microcalcifications included ductal carcinoma in situ (n = 15) and invasive carcinoma (n = 1). Twenty-two patients underwent subsequent open surgical biopsy but no underestimation of disease was seen. Only two patients had vasovagal syncope and three others felt unwell during the biopsy. Nine patients had small haematomas, which resolved spontaneously. Vacuum-assisted biopsy carried out on an upright stereotactic mammography unit is a safe and effective method for evaluation of suspicious microcalcifications.

Comparison of performance of computer display monitors for radiological diagnosis; "diagnostic" high brightness monochrome LCD, 3MP vs "clinical review" colour LCD, 2MP.

A study to compare performance of the following display monitors for application as PACS CR diagnostic workstations is described. 1. Diagnostic quality, 3 Mega Pixel, 21 inch monochrome LCD monitors--Planar C3i. 2. Clinical review quality, 2 Mega Pixel, 21 inch colour LCD monitors--Planar PX212. Two sets of seventy radiological studies were presented to four senior radiologists on two occasions, using different displays on each occasion. The clinical condition used for this investigation was to query for the presence of a solitary pulmonary nodule. Receiver Operating Characteristic (ROC) curves were constructed for diagnostic performance for each presentation. Areas under the ROC curves (AUC) for diagnosis using different monitors were compared and the following results obtained: Monochrome AUC = 0.813 +/- 0.02, Colour AUC = 0.801 +/- 0.021. These results indicate that there is no statistically significant difference in the performance of these monitor types at a 95% confidence level.

Numerical modelling of biopotential field for detection of breast tumour.

Breast cancer is a disease characterised by the uncontrolled growth of abnormal cells. These cancer cells can travel through the body by way of blood or lymph nodes. Previous studies have indicated that, changes in the electrical properties of abnormal breast are more significant compared to the breast normal tissues. In the present study, a simple 2D models of breast (close to realistic), with and without artificially inserted malignant cancer were simulated, based upon electrical activity within the breast. We developed an inhomogeneous female breast model, closer to the actual, by considering a breast as a hemisphere with various layers of unequal thickness in supine condition. In order to determine the potential distribution developed due to a dipole source, isotropic homogeneous conductivity was assigned to each of these compartments and the volume conductor problem was solved using finite element method. Significant changes in the potential distribution were recoded in the malignant and normal breast regions. The surface potential decreases about 0.5%, for the small malignant region of surface area 13 mm(2) (spherical diameter=2mm). And it (surface potential) decreases about 16.4% for large malignant surface area of 615 mm(2) (spherical diameter=14 mm). Hence, the results show that, the sizes of tumours result in the reduction of surface potential and follows a fourth order polynomial equation. Thus, biofield analysis yields promising results in the detection of the breast cancer of various sizes.

Testing by Sysmex UF-100 flow cytometer and with bacterial culture in a diagnostic laboratory: a comparison.

A large proportion of the samples tested in routine diagnostic microbiology laboratory are urine samples. The gold standard is bacterial culture, but a high proportion of samples cultured are negative. Unnecessary testing can be reduced and an improved service provided by an effective screening test. The Sysmex UF-100 flow cytometer has been developed to count cells and casts accurately in urine samples. Its performance in a screening test was compared with bacterial culture by using 1005 consecutive urine samples, and cut-off criteria were established. Cut-off values of 3000 bacteria/microl and 111 WBC/microl provided the best discrimination. Of 1005 samples, 606 (60%) would be cultured. Sixteen samples that were not selected according to these criteria were culture positive. This was considered acceptable for our routine use. The use of a testing algorithm incorporating the Sysmex UF-100 flow cytometer has improved the quality and efficiency of urine testing within the routine microbiology laboratory.