gebhart/patrepeval huggingface dataset:
Perfluorocarbons as Blood Substitutes: The Early Years: Experience with Fluosol DA-20% in the 1980s (1994)

Clinical testing of perfluorocarbons (PFC) as blood substitutes began in the early 1980's in the form of Fluosol DA-20% (FDA), a mixture of perfluorodecalin and perfluorotripropylamine emulsified with Pluronic F68. We have treated 55 patients (Treatment (T) = 40; Control (C) = 15) with intravenous infusions of 30 cc/kg of FDA as part of either a randomized, clinical trial or a humanitarian protocol. All patients were Jehovah's Witnesses who refused blood transfusion and were severely anemic (mean hemoglobin = 4.6 g/d). FDA successfully increased dissolved or plasma oxygen content (P1O2 in ml/dl), but not overall oxygen content (T group: P1O2 baseline = 1.01 +/- .27, P1O2 12hrs = 1.58 +/- .47 [p = < .0001, t-test]; P1O2 12 hrs: T = 1.58 +/- .47, C = 1.00 +/- .31, p = < .0002, t-test). This effect persisted for only 12 hours post infusion, and had no apparent effect on survival. FDA is an ineffective blood substitute because of low concentration and short half-life. Improved emulsion design may resolve these problems, thereby producing a more effective agent. Our discussion will include a review of our data plus a summary of other reports of FDA efficacy as a blood substitute.
Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits. (1991)

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.
Specificity and detection of insulin-reactive CD4+T cells in type 1 diabetes in the nonobese diabetic (NOD) mouse (2011)

In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4(+) T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA(g7). This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4(+) T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA(g7) tetramers that bind to most insulin-specific T-cell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4(+) T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA(g7) in register 3 may explain the escape of insulin-specific CD4(+) T cells from the mechanisms that usually eliminate self-reactive T cells.
Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells (2004)

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-alpha and TCR-beta genes. We demonstrate that Th1 cells transduced with the TCR-alpha and -beta genes from the I-A(d)-restricted ovalbumin (OVA)(323-339)-specific T-cell clone DO11.10 produce IFN-gamma but not interleukin-4 in response to stimulation with OVA(323-339) peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.
Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis (2017)

Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world’s working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31+ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5–3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.
Functional characterization of green fluorescent protein–profilin fusion proteins (2000)

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.
Characterization of cell phenotype by a novel cDNA library subtraction system: expression of CD8 alpha in a mast cell-derived interleukin-4- dependent cell line (1994)

We have established a unique variant cell line, MC/9.IL-4, which continuously proliferates in the presence of interleukin-4 (IL-4), from a murine interleukin-3 (IL-3)-dependent mast cell line, MC/9 (referred to as MC/9.IL-3). Compared with MC/9.IL-3 cells, MC/9.IL-4 cells are smaller, lack cytoplasmic granules and metachromasia, carry a very small amount of histamine, and express fewer high-affinity IgE receptors (IgERs) and IL-3 receptors. To further characterize MC/9.IL-4, we developed a novel method to enrich cell type-specific cDNAs by cDNA library subtraction and applied it for MC/9.IL-3 versus MC/9.IL-4. Sequence analysis of cDNA clones isolated by this technique showed that MC/9.IL-4 cells specifically express CD8 alpha and expression of mast cell-specific proteases and major histocompatibility complex class II (MHCII) is considerably decreased. It was also noted that responsiveness to the IL-3-agonistic antibody F9 and expression of the transcription factor GATA-2 is diminished in MC/9.IL-4, indicating that MC/9.IL-4 have lost major characteristics of the bone marrow-derived cultured mast cells. Because other T-cell marker antigens, CD8 beta, CD4, Thy-1, were not detected on MC/9.IL-4 cells, MC/9.IL-4 cells may represent an unknown class of hematopoietic cells that express CD8 alpha. This cell line will be useful in studies of IL-4-mediated signal transduction, as well as transcriptional regulation of mast cell characteristic genes. This study also demonstrates the effective use of the cDNA library subtraction strategy to characterize unknown types of hematopoietic cells at the molecular level.
Improved Pressure–Frequency Sensing Subxiphoid Pericardial Access System: Performance Characteristics During In Vivo Testing (2010)

We have designed, synthesized, and tested an improved version of our original subxiphoid access system intended to facilitate epicardial electrophysiology. The new version of the system incorporates a precision fiber-optic pressure sensor and a novel signal analysis algorithm for identifying pressure-frequency signatures which, in the clinical setting, may allow for safer access to the pericardial space. Following in vivo studies on ten adult canine models, we analyzed 215 pressure-frequency measurements made at the distal tip of the access needle, of which 98 were from nonpericardial, 112 were from pericardial, and five were from ventricular locations. The needle locations as identified by the algorithm were significantly different from each other (p &lt; 0.01), and the algorithm had improved performance when compared to a standard fast Fourier transform (FFT) analysis of the same data. Moreover, the structure of the algorithm can potentially overcome the time lags intrinsic to FFT analysis such that the needle's location can be determined in near-real time. Hydrodynamic pressure-frequency measurements made during traversal of the pericardial membrane revealed a distinct change in signal structure between the pericardial and nonpericardial anatomy. We present and discuss the design principles, details of construction, and performance characteristics of this system.
Precision of Seismic Wave Propagation Methods in Construction Applications (2000)

Seismic wave propagation methods provide in situ wave velocity profiles from which various elastic moduli can be determined. The tests are applicable to a wide range of civil engineering materials, including soil and portland cement concrete (PCC). Wave velocities obtained from seismic testing can also be used to calculate other salient design properties such as Poisson's ratio. Although the accuracy of seismic surveying is well researched and accepted by the engineering community, little research has been reported on uncertainty or precision of parameters obtained from the tests. In this study, a large sample of replicate field data was collected from multiple borehole and surface tests. Measurements of vertical (SV) shear waves were obtained at two independent soil sites. Seismic surveying was conducted at one PCC site to obtain measurements of compression (P) and Rayleigh waves. From this data, variation in seismic wave velocities collected from typical seismic testing was determined. In addition, variation in seismic wave velocities was used with error propagation techniques to evaluate uncertainty of Poisson's ratio. Based upon data presented within this study, coefficients of variation of SV, P, and Rayleigh wave velocities indicate a high degree of precision for these velocities. Also, the coefficient of variation for Poisson's ratio attests to the high precision of determining this property with seismic surveying techniques.
Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase (2002)

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.
Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis. (1982)

New details of NADPH binding to Lactobacillus casei dihydrofolate reductase have become visible as a result of crystallographic refinement to an R factor of 0.152 at 1.7 A resolution. Conformational torsion angles for bound NADPH have been extensively revised and specific interatomic contacts responsible for cofactor binding have been identified. In addition, several structurally conserved water molecules are seen to mediate the protein-ligand interaction. In the nicotinamide binding site three oxygen atoms of the enzyme lie in the plane of the pyridine ring and close to ring carbons 2, 4, and 6. The placement of these polar groups suggests that the enzyme stabilizes a C4-carbonium electronic isomer of oxidized nicotinamide in the transition state. Pyramidalization of ring nitrogen N1 in the transition state might be promoted by a fixed water molecule positioned to donate a hydrogen bond to the N1 lone pair orbital. Pyramidalization could also relieve an unfavorable steric contact due to the observed rotation of the nicotinamide's carboxamide group by 180 degrees from its most stable conformation.
Avian-Induced Pluripotent Stem Cells Derived Using Human Reprogramming Factors (2011)

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.
A Transformed Cell Population Derived from Cultured Mesenchymal Stem Cells has no Functional Effect after Transplantation into the Injured Heart (2009)

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells characterized by their self-renewal and differentiation potential. Accumulating clinical and preclinical evidence indicate MSCs are a promising cell source for regenerative medical therapies. However, undesirable immortalization, spontaneous transformation, and tumorigenic potential from long-term cultured MSCs have been reported in human and mouse. We report rat MSCs isolated from young donors could undergo transformation in early passage culture. We aimed to characterize the transformed population and determine their therapeutic effects after intracardiac transplantation in the infarcted myocardium. MSCs were isolated from bone marrow of Lewis rats according to standard protocols and cultured under standard conditions. Phenotype of growing cells was assessed by flow cytometry. Following acute myocardial infarction in rats, cells were delivered by intracardiac injection. Cardiac functions were assessed by pressure-volume loops. Infarction size and pathologic effects were evaluated after 6 weeks. The abnormal colonies were detected in culture as early at passage 3. They were noted to appear as distinctly different morphology from typical MSCs, which changed from a normal elongated spindle shape to a compact abnormal morphology. They exhibited rapid cell proliferation. Some subclones lost contact inhibition of cell division and formed multilayer aggregates. Chromosomal instability was detected. They were devoid of surface markers CD29, CD44, CD90, and CD117. Furthermore, there was no significant improvement on infarction size and cardiac function 6 weeks after cell transplantation. Our study highlights the need for establishment of biosafety criteria in regulating culture- expanded MSCs to achieve the full clinical therapeutic benefits.
Changes in serum levels of TNF-alpha, IL-6, OPG, RANKL and their correlation with radiographic and clinical assessment in fragility fractures and high energy fractures. (2013)

Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-alpha, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-alpha concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-alpha and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-alpha, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair.
Properties of purified T cell subsets. I. In vitro responses to class I vs. class II H-2 alloantigens. (1985)

In light of the widely accepted view that Ia-restricted L3T4+ T helper cells play a decisive role in controlling the differentiation of Lyt-2+ cells, experiments were designed to examine whether Lyt-2+ cells can respond to antigen in the absence of L3T4+ cells. The results showed that highly purified Lyt-2+ cells gave high primary mixed lymphocyte reactions (MLR) to various class I differences, including both mutant and allelic differences; responses to class II (Ia) differences were generally undetectable with Lyt-2+ cells. The intensity of MLR to class I differences was not affected by addition of anti-L3T4 monoclonal antibodies (mAb) to the cultures or by removing T cells from the stimulator populations. Negative selection experiments showed that Lyt-2+ cells could respond to class I differences across Ia barriers. MLR of purified Lyt-2+ cells peaked on days 3-4 and then fell sharply; background responses with syngeneic stimulators (auto-MLR) were virtually absent. Parallel experiments with purified L3T4+ cells showed that this subset responded in MLR only to class II (Ia) and not class I differences, reached peak responses only on day 6 rather than days 3-4, and often gave high auto-MLR. Within the first 3-4 d of culture, MLR were generally higher with Lyt-2+ cells than L3T4+ cells. Although no evidence could be found that Ia-restricted L3T4+ cells were required for the response of Lyt-2+ cells, presentation of antigen by Ia+ cells appeared to be essential. Thus, responses were ablated by pretreating stimulator cells with anti-Ia mAb plus C'. Significantly the failure of Lyt-2+ cells to respond to anti-Ia plus C'-treated stimulators could not be restored by adding syngeneic spleen cells; addition of IL-2 led to only a minor (15%) restoration of the response. It is suggested that Ia+ cells provide an obligatory second signal required by Lyt-2+ cells.