Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.     

Separate loci underlie resistance to root infection and leaf scorch during soybean sudden death syndrome

Soybean [Glycine max (L.) Merr.] cultivars show differences in their resistance to both the leaf scorch and root rot of sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Root susceptibility combined with reduced leaf scorch resistance has been associated with resistance to Heterodera glycines HG Type 1.3.6.7 (race 14) of the soybean cyst nematode (SCN). In contrast, the rhg1 locus underlying resistance to Hg Type 0 was found clustered with three loci for resistance to SDS leaf scorch and one for root infection. The aims of this study were to compare the inheritance of resistance to leaf scorch and root infection in a population that segregated for resistance to SCN and to identify the underlying quantitative trait loci (QTL). “Hartwig”, a cultivar partially resistant to SDS leaf scorch, F. virguliforme root infection and SCN HG Type 1.3.6.7 was crossed with the partially susceptible cultivar “Flyer”. Ninety-two F5-derived recombinant inbred lines and 144 markers were used for map development. Four QTL found in earlier studies were confirmed. One contributed resistance to leaf scorch on linkage group (LG) C2 (Satt277; P = 0.004, R ² = 15%). Two on LG G underlay root infection at R8 (Satt038; P = 0.0001 R ² = 28.1%; Satt115; P = 0.003, R ² = 12.9%). The marker Satt038 was linked to rhg1 underlying resistance to SCN Hg Type 0. The fourth QTL was on LG D2 underlying resistance to root infection at R6 (Satt574; P = 0.001, R ² = 10%). That QTL was in an interval previously associated with resistance to both SDS leaf scorch and SCN Hg Type 1.3.6.7. The QTL showed repulsion linkage with resistance to SCN that may explain the relative susceptibility to SDS of some SCN resistant cultivars. One additional QTL was discovered on LG G underlying resistance to SDS leaf scorch measured by disease index (Satt130; P = 0.003, R ² = 13%). The loci and markers will provide tagged alleles with which to improve the breeding of cultivars combining resistances to SDS leaf scorch, root infection and SCN HG Type 1.3.6.7. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.     

Computational and experimental determination of the alpha-helix unfolding reaction coordinate

We demonstrate a calculated alpha-helix peptide folding energy landscape which accurately simulates the first experimentally measured alpha-helix melting energy landscape. We examine a 21-amino acid, mainly polyalanine peptide and calculate the free energy along the Psi Ramachandran angle secondary folding coordinate. The experimental free energy landscape was determined using UV resonance Raman spectroscopy. The relative free energy values are very close as are the equilibrium peptide conformations. We find 2.3 kcal/mol activation barriers between the alpha-helix-like and PPII-like basins. We also find that the alpha-helix-like conformations are quite defective and the alpha-helix-like structure dynamically samples 310-helix and pi-bulges.     

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.     

Relatively normal human lymphopoiesis but rapid turnover of newly formed B cells in transplanted nonobese diabetic/SCID mice.

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2'-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of V(H) genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.     

The differential effects of fatigue on reflex response timing and amplitude in males and females.

We examined the effects of fatigue on patellar tendon reflex responses in males and females. A spring-loaded reflex hammer elicited a standardized tendon tap with the knee positioned in an isokinetic dynamometer and flexed to 85 degrees. We recorded vastus lateralis activity (SEMG) and knee extension force production at the distal tibia (force transducer). Reflex trials were performed before and after (immediate, 2, 4, and 6 min) an isokinetic fatigue protocol to 50% MVC (90 degrees /s). For each event, pre-motor time (PMT), electromechanical delay (EMD), and total motor time (TMT) were obtained, as well as EMG amplitude (EMG(amp)), time to peak EMG (EMG(tpk)), peak force amplitude (F(amp)), time to peak force (F(tpk)), EMG:force ratio (E:F), and rate of force production (F(rate)=N/ms). TMT increased significantly in females following fatigue, while males showed no change. The increased TMT was due to an increased EMD with fatigue, while PMT was unaffected. EMG(amp) and F(amp) were somewhat diminished in females yet significantly augmented in males following fatigue, likely accounting for the differential changes in EMD noted. Results suggest males and females may respond differently to isokinetic fatigue, with males having a greater capacity to compensate for contraction force failure when responding to mechanical perturbations.     

A chromosomal translocation causing multiple abnormalities including open eyelids at birth and glomerulonephritis

We have characterized the phenotype of a mouse with a t(2;13) reciprocal translocation induced by chlorambucil. It results in abnormal eyelid formation as well as a series of neurological, physiological, and immunological abnormalities. This mutant has been termed T(2;13)1Fla/+. T(2;13)1Fla/+ mice exhibit open eyelids at birth, a dilute coat color, hyperactivity, and occasional circling and stargazing activity. At 1-6 months, T(2;13)1Fla/+ mice show signs of immune complex-mediated glomerulonephritis and die prematurely. Additionally, double-stranded DNA autoantibodies have been found in sera of T(2;13)1Fla/+ mice. Cytogenetic analysis situated the translocation breakpoint at the proximal end of Chromosome (chr) 2 at band A2, and on Chr 13 at band A4. The mutant phenotype completely correlated with the presence of the translocation. Additional genetic studies have mapped the mutation and translocation breakpoint to Chr 13 between D13Mit16 and D13Mit64, and to Chr 2 proximal to D2Mit5. By fluorescent in situ hybridization (FISH), the position of this mutation/translocation on Chr 13 has been mapped to a region less than 1cM from D13Mit61.