In-vivo Response Research Articles

A Single-Chain H-2Db Molecule Presenting an Influenza Virus Nucleoprotein Epitope Shows Enhanced Ability at Stimulating CD8+ T Cell Responses In Vivo

We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.

Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C)

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8(+) T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8(+) T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8-16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp100(25-33) melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8(+) T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.

Coronin 1 Is Essential for IgM-Mediated Ca2+ Mobilization in B Cells but Dispensable for the Generation of Immune Responses In Vivo

Coronin 1 is a leukocyte specific regulator of Ca(2+)-dependent signaling and is essential for the survival of peripheral T lymphocytes, but its role in B cells is unknown. In this study, we show that coronin 1 is essential for intracellular Ca(2+) mobilization and proliferation upon triggering of the BCR. However, the presence of costimulatory signals rendered coronin 1 dispensable for B cell signaling, consistent with the generation of normal immune responses against a variety of Ags in coronin 1-deficient mice. We conclude that coronin 1, while being essential for T cell function and survival, is dispensable for B cell function in vivo.

Treatment with azathioprine and cyclic methylprednisolone has little or no effect on bioactivity in anti-interferon beta antibody-positive patients with multiple sclerosis

Background It is unknown whether immunosuppression of patients who have developed interferon-β (IFN-β) neutralizing antibodies (NAbs) hastens disappearance of NAbs in the blood. Objective We wanted to test whether immunosuppression with cyclic methylprednisolone (MP) in combination with azathioprine (AZA) for 6 months accelerates recovery of IFN-β bioactivity in patients with multiple sclerosis (MS) with abolished in-vivo myxovirus resistance protein A (MxA) mRNA response to IFN-β. Methods We included 13 patients with MS with NAbs and a low IFN-β bioavailability detected by the MxA-mRNA response in a descriptive, non-randomized trial. Another 14 NAb-positive patients with a low MxA-mRNA response served as controls. The primary outcome was the fraction of patients who regained an MxA-mRNA response to IFN-β. NAbs were measured by means of a clinically validated cytopathic effect assay and a new reporter gene assay. The in-vivo MxA-mRNA response was measured by real-time polymerase chain reaction. Results A total of 11 patients in the treatment group completed the trial. In all, two of these 11 patients regained an in-vivo MxA-mRNA response as compared to one of 14 patients in the control group. Conclusion Treatment with AZA and cyclic MP for 6 months has little or no effect on IFN-β bioactivity in NAb-positive patients with MS.

Upper extremity soft and rigid tissue mass prediction using segment anthropometric measures and DXA

Regression equations for predicting bone mineral content (BMC), fat mass (FM), lean mass (LM), and wobbling mass (WM) of living people from simple anthropometric measures (segment lengths, circumferences, breadths, and skin folds) have been reported in the literature for the lower extremities, but are lacking for the upper extremities. Multiple linear stepwise regression was used to generate such equations for the arm, forearm, and forearm and hand segments of healthy university aged people (38 males, 38 females). Actual tissue masses were obtained from full body Dual-energy X-ray Absorptiometry (DXA) scans and were used to validate the developed equations with an independent sample of 24 participants (12 male, 12 female). Prediction equations exhibited very high adjusted R 2 values (range from 0.854 to 0.968), with more explained variance for LM and WM than for BMC and FM. Scatter plots of actual versus predicted tissue masses revealed a close relationship ( R 2 range from 0.681 to 0.951). Relative errors between the predicted and actual tissue masses for the validation group ranged from −2.2% to 15.5%, and the root-mean-squared error (RMS error) ranged from 7.92 to 180.26 g, for BMC of the forearm and LM of the arm, respectively. These results suggest that accurate estimates of in-vivo tissue masses for the upper extremities can be predicted from simple anthropometric measurements in young adults. Access to tissue masses such as these will enable the development of more accurate models for predicting dynamic in-vivo response of the body to activities involving impact.

Identification of Genes Associated with Resistance and Response in Vivo to Therapy with Rituximab, Fludarabine and Cyclophosphamide in Patients with Chronic Lymphocytic Leukemia.

Fludarabine and cyclophosphamide are the backbone of therapy for patients with CLL. The addition of rituximab leads to further improvement of response rates and progression free survival (results from the randomized CLL8 study of the GCLLSG). However, a considerable number of patients have insufficient or short responses to FC or RFC and there is a need to identify factors influencing response or resistance to therapy. The aims of this study areto identify gene expression associated with response or resistance before the start of therapy,to investigate changes in the expression of specific genes or pathways associated with response or resistance during the first cycle of FC and RFC,to provide a rationale for the additional use of novel drugs to improve remission and overcome resistance.We investigated peripheral blood samples from 20 patients receiving FC (n=10) or RFC (n=10) by gene expression profiling, flow cytometry, RT-PCR and western blotting before and during therapy. Sixteen patients received FC or RFC as first line (8 within the CLL8 study) and 4 as second line treatment. All patients were in stage Binet B or C. Gene expression was analyzed and correlated to good (CR or PR) or poor clinical response (SD or PD) at the end of therapy based on NCI-WG/IWCLL criteria. CD19+ cells were harvested by cell sorting before therapy, 24 hours after FC (FC arm), 24 hours after rituximab, and 24 hours after FC (RFC arm). Microarray analysis was performed using Affymetrix U133A gene chips. Genes with a consistent pattern of expression (high or low) in the majority of samples in the good or poor response group were further analyzed. Overall, 9 patients responded adequately to therapy (3 CR, 7 PR), while 11 did not (7 SD, 3PD). Unmutated IgVH status and poor risk cytogenetics were more frequent in poor responders. Gene expression signature before treatment showed that overexpression of 39 genes strongly correlated with response, while overexpression of 20 genes (including HSPA1B, IFI6, APP, CEACAM1, CD9, GAB1, INPP5F) was associated with resistance. Changes in expression after initiation of treatment was also analyzed. Seven genes (including CENTD1, HBA2, COL9A2 and APRIN) were significantly upregulated after rituximab in non-responders. Upregulation of 13 genes (including PMAIP1, SFRS11, CLK1, EFHC1, MRPL39, TUG1, TBRG1, CD49d, PTPRC) after R-FC and 7 genes (including ITPKB, LOC641298, CD44, TAF5) after FC was associated with poor response (resistance) to RFC and FC respectively. Many of these genes are involved in regulation of apoptosis, cell cycle, integrin and PI3-K signaling Therapeutic antibodies or inhibitors against some of these targets are already available. RT-PCR analysis demonstrated a significant downregulation of Akt1 mRNA 24 hours after rituximab infusion in RFC group but no significant changes were observed in patients receiving FC alone. In vitro exposure to rituximab confirmed its in vivo effect and resulted in a significant downregulation of Akt1 and PI3-K-p85 mRNA expression. FACS analysis demonstrated a decrease in the percentage and mean fluorescence intensity (MFI) of surface CD20 after rituximab infusion. This effect was associated with a significant change in total amount and phosphorylation state of CD20 in the RFC group. There was also a decrease in the MFI of CD44 and CD23 after rituximab in the majority of patients in the RFC group but this effect was not consistent in the FC group. In conclusion, we have identified a set of markers associated with good or poor response to FC or RFC before therapy and during the first cycle of treatment. The data provide a rationale for targeted drug combinations to overcome resistance and improve response to therapy in CLL.

The Absence of CCR7 on Donor T Cells Leads to Attenuated Graft-Versus- Host Disease (GVHD) with a Preserved Graft-Versus-Leukemia (GVL) Effect

The development of GVHD depends on the trafficking of donor effector T cells (Teffs) into recipient secondary lymphoid tissue early after hematopoietic stem cell transplantation (HSCT). Chemokine receptor 7 (CCR7) has been shown to be critical for the movement of naïve T cells into lymph nodes and for the organization of T cell and B cell regions in the spleen. Using a murine transplant model, we set out to study the contribution of CCR7 to both GVHD induction and the immunomodulatory properties of regulatory T cells (Tregs) in the HSCT setting.Methods: C57BL/6 (H-2b; termed B6) mice served as bone marrow (BM) donors, and B6xDBA/2 F1 (H-2bxd; termed B6D2) mice functioned as recipients. Teffs and Tregs were obtained from wild-type (WT) B6 or CCR7 knockout (CCR7−/−) mice extensively backcrossed on a B6 background. For Teff studies, recipient animals were lethally irradiated to 950 rads on day –1 and administered 3 ×106 T-cell depleted (TCD) BM cells +/− 4×106 splenic Teffs from WT or CCR7−/− donors on day 0. For GVL studies, 25,000 P815 murine mastocytoma cells were coadministered with the BM/Teff inoculum on day 0. For Treg studies, mice received TCD BM +/− 1×106 WT or CCR7−/− Tregs on transplant day 0, with 4×106 WT Teffs dosed on transplant day +2.Results: WT and CCR7−/− Teffs generated GVHD responses that were nearly identical during the first 7–10 post transplant. Thereafter, those mice receiving CCR7−/− Teffs demonstrated a significant attenuation of their GVHD, with 83% surviving long term. In contrast, those animals receiving WT Teffs exhibited relentlessly progressive disease and a 92% mortality rate by day +70 (see figure; P=0.0006 for comparison between WT and CCR7−/− Teff groups by the log-rank test). In spite of their relatively impaired capacity to generate lethal GVHD, CCR7−/− Teffs were found to produce complete donor reconstitution of the CD4+ and CD8+ T cell compartments by transplant day +40, albeit at a slower rate than that observed with WT Teffs. CCR7−/− Teffs also demonstrated significant anti-tumor activity in-vivo. B6D2 mice challenged with P815 tumor cells and TCD BM all died with massive splenomegaly and diffuse tumor infiltration of the liver by transplant day +20. Animals receiving WT Teffs at the time of P815 challenge all died of GVHD, with no tumor noted at autopsy. In contrast, 75% of the mice administered P815 cells and CCR7−/− Teffs survived long term with no signs of malignancy and only mimimal evidence of GVHD clinically. These findings did not appear to be the result of an impaired ability of CCR7−/− Teffs to respond to alloantigens in-vitro, as proliferative responses of sort-purified CD25- CD4+ and CD8+ T cells from WT and CCR7−/− mice were similar in one-way mixed lymphocyte reactions. Surprisingly, CCR7−/− Tregs appeared to be capable of protecting against lethal GVHD when administered two days prior to WT Teffs, with 100% of recipients surviving to day +70. Their GVHD scores, however, were somewhat higher than in those mice receiving WT Tregs.Conclusions: CCR7 is not required for the initial phase of GVHD induction, but appears important for the perpetuation and augmentation of disease 10–14 days post transplantation. The attenuated in-vivo allo-immune responses observed with CCR7−/− Teffs appear to be independent of any intrinsic deficiency in the proliferative capacity of the T cells themselves. CCR7−/− Teffs are capable of generating a potent GVL effect in our P815 tumor model. CCR7 does not appear to be required for Tregs to protect against lethal GVHD when they are administered in advance of WT Teffs. At the time of this writing we have successfully generated CCR7−/− eGFP+ mice, and in-vivo trafficking studies are currently planned to elucidate a mechanism for the above findings. [Display omitted]

The in Vivo Response of Patients Suffering from Severe Congenital Neutropenia (CN) to G-CSF Is Due to “Emergency” Granulopoiesis Induced by C/EBPβ

C/EBP transcription factors are crucial for the regulation of granulopoiesis in vitro and in vivo. C/EBPa is considered to be the master regulator of “steady state” granulopoiesis via upregulation of several myeloid genes (e.g. ELA2, CSFR3, etc.). The absence of C/ EBPa results in a complete loss of neutrophils. We were able to show that in patients with severe congenital neutropenia (CN) harbouring HAX-1 or ELA2 mutations C/EBPa is severely abrogated secondary to defective expression of LEF-1 (Skokowa J, et al. Nat Med. 12, 1191–7 ( 2006)). Therefore, we were interested, whether other transcription factors are capable of substituting C/EBPa, since these patients respond to G-CSF with slight increase in neutrophils from less than 200/ul to above 1500/ul depending on the dose of G-CSF. C/EBPβ has recently been shown to be required for cytokine induced “emergency” granulopoiesis (Hirai H, et al. Nat Immunol. 7, 732-9 ( 2006)). Therefore, we investigated the expression pattern of C/EBPβ during G-CSF treatment of CN patients. Indeed, C/EBPβ mRNA was upregulated 2.8-fold in CD33+ myeloid cells from CN patients by G-CSF treatment, as compared to healthy individuals. It was associated with upregulation of G-CSFR mRNA and –protein expression as well as ligand binding to G-CSFR in myeloid cells, and elevated levels of biologically active G-CSF in serum from CN patients. To confirm C/EBPβ-dependent activation of G-CSFR and G-CSF gene expression, we performed reporter gene assays in CD34+ bone marrow hematopoietic progenitor cells from two CN patients co-transfected with C/EBPβ and reporter constructs containing upstream regulatory regions of G-CSF or G-CSFR genes, −1470bp and −670bp, respectively. Indeed, C/EBPβ activated G-CSFR promoter 3.2-fold and G-CSF promoter 5.4-fold. These data demonstrate that in CN patients, G-CSF induces C/EBPa independent granulopoiesis and that C/EBPβ is required for the response to G-CSF treatment in these patients. C/EBPβ leads in response to G-CSF to induction of differentiation of neutrophil precursors to mature neutrophils in vivo. Our hypothesis is that in CN the steady state granulopoiesis is abrogated whereas the emergency granulopoiesis still leads to sufficient numbers of neutrophils.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity >98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p<0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p<0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change >2, FDR <0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

Short-Term Culture and In-Vivo Modeling of Primary HNSCC

Problem Head and neck squamous cell carcinoma (HNSCC) accounts for 4% of annual U.S. cancer deaths. In-vivo models exist using established HNSCC lines, but currently there is no such model that allows consistent growth of HNSCC from primary tumors. Methods Primary HNSCC tissue was obtained from 103 patients at biopsy/resection, disaggregated and seeded onto collagen-coated plates in keratinocyte growth media with 10% FBS, additives and antibiotics. After short-term growth in culture, cells were transferred to denuded rat tracheas and implanted subcutaneously in nude mice. Indirect immunofluorescent staining using antibodies specific to cytokeratin, vimentin and nuclear Ku was performed to determine cell lineage and origin. Results Cultured cells exhibited morphology consistent with epithelial or stromal derivation. 80% of cultures had viable cells present at 10 days and 24% were maintained 30 days or longer. 5 cultures (5%) proliferated after multiple passages and thrived on uncoated plates in the absence of mesenchymal cells. The xenograft model was able to successfully establish tumors in vivo from 59% of primary tumors. Immunostaining for nuclear Ku and cytokeratin confirmed human origin and epithelial cell lineage, respectively. Conclusion The high success rate indicates that selective growth and survival pressures for short-term culture of primary HNSCC may be considerably less than for establishment of cell lines. Additionally, the techniques permit tumor-derived epithelial and mesenchymal cells to be cultured simultaneously. Preliminary data for the in-vivo trachea xenograft model is promising. A luciferase lentiviral system has been created to allow monitoring of tumor growth in vivo with serial live animal imaging. Significance These short-term culture techniques may more accurately characterize both the biological diversity of HNSCC and tumor-stromal cell interactions. Once optimized, the trachea xenograft model can be used to determine the in-vivo response of a heterogeneous group of HNSCC to standard and novel therapies. Support Funds provided by an endowment for the Barry Baker Laboratory for Head and Neck Oncology, the Vanderbilt Ingram Cancer Center Endowed Professorship Fund, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.

Marked Impairment of Protease-Activated Receptor Type 1-Mediated Vasodilation and Fibrinolysis in Cigarette Smokers: Smoking, Thrombin, and Vascular Responses In Vivo

We sought to test the hypothesis that cigarette smoking adversely alters protease-activated receptor type 1 (PAR-1)-mediated vascular effects in vivo in humans. Distinct from its role in the coagulation cascade, thrombin exerts its major cellular and cardiovascular actions via PAR-1. The activation of PAR-1 causes endothelium-dependent arterial vasodilation and the release of endogenous fibrinolytic factors. Forearm blood flow was measured with venous occlusion plethysmography in 12 cigarette smokers and 12 age- and gender-matched nonsmokers during intrabrachial infusions of PAR-1-activating-peptide (SFLLRN; 5 to 50 nmol/min), bradykinin (100 to 1,000 pmol/min), and sodium nitroprusside (2 to 8 mug/min). Plasma tissue plasminogen activator (t-PA) and plasminogen-activator inhibitor 1 antigen and activity concentrations were measured throughout the experiment. All agonists caused dose-dependent increases in forearm blood flow (p < 0.0001 for all). Although bradykinin and sodium nitroprusside caused similar vasodilation, SFLLRN-induced vasodilation was attenuated in smokers (p = 0.04). Smokers had modest reductions in bradykinin-induced active t-PA release (reduced by 37%, p = 0.03) and had a marked impairment of SFLLRN-induced t-PA antigen (p = 0.02) and activity (p = 0.006) release, with a 96% reduction in overall net t-PA antigen release. The use of SFLLRN also caused similar (p = NS) increases in inactive plasminogen-activator inhibitor 1 in both smokers and nonsmokers (p <or= 0.002 for both). Cigarette smoking causes marked impairment of PAR-1-mediated endothelial vasomotor and fibrinolytic function. Relative arterial stasis and near abolition of t-PA release will strongly promote clot propagation and vessel occlusion. These findings suggest a major contribution of impaired endothelial PAR-1 action to the increased atherothrombotic risk of cigarette smokers.

In Vivo Responses to Urological Biomaterials as Utilized for Urological Reconstruction

In Vivo Responses to Urological Biomaterials as Utilized for Urological Reconstruction

Genetic basis of variation in tenofovir drug susceptibility in HIV-1

To develop an improved model for the genetic basis of reduced susceptibility to tenofovir in vitro. A dataset of 532 HIV-1 subtype B reverse transcriptase genotypes for which matched phenotypic susceptibility data were available was assembled, both as a continuous (transformed) dataset and a categorical dataset generated by imposing a cut-off on the basis of earlier studies of in-vivo response of 1.4-fold. Models were generated using stepwise regression, decision tree and random forest approaches on both the continuous and categorical data. Models were compared by mean squared error (continuous models), or by misclassification rates by nested crossvalidation. From the continuous dataset, stepwise linear regression, regression tree and regression forest methods yielded models with MSE of 0.46, 0.48 and 0.42 respectively. Amino acids 215, 65, 41, 67, 184 and 151 in HIV-1 reverse transcriptase were identified in all three models and amino acid 210 in two. The categorical data yielded logistic regression, classification tree and forest models with misclassification rates of 26, 24 and 23%, respectively. Amino acids 215, 65 and 67 appeared in all; 41, 184, 210 and 151 were also included in the classification forest model. The random forests approach has yielded a substantial improvement in the available models to describe the genetic basis of reduced susceptibility to tenofovir in vitro. The most important sites in these models are amino acid sites 215, 65, 41, 67, 184, 151 and 210 in HIV-1 reverse transcriptase.

Femoral stem wear in cemented total hip replacement

The great success of cemented total hip replacement to treat patients with endstage osteoarthritis and osteonecrosis has been well documented. However, its long-term survivorship has been compromised by progressive development of aseptic loosening, and few hip prostheses could survive beyond 25 years. Aseptic loosening is mainly attributed to bone resorption which is activated by an in-vivo macrophage response to particulate debris generated by wear of the hip prosthesis. Theoretically, wear can occur not only at the articulating head-cup interface but also at other load-bearing surfaces, such as the stem-cement interface. Recently, great progress has been made in reducing wear at the head-cup interface through the introduction of new materials and improved manufacture; consequently femoral stem wear is considered to be playing an increasingly significant role in the overall wear of cemented total hip replacement. In this review article, the clinical incidences of femoral stem wear are comprehensively introduced, and its significance is highlighted as a source of generation of wear debris and corrosion products. Additionally, the relationship between femoral stem surface finish and femoral stem wear is discussed and the primary attempts to reproduce femoral stem wear through in-vitro wear testing are summarized. Furthermore, the initiation and propagation processes of femoral stem wear are also proposed and a better understanding of the issue is considered to be essential to reduce femoral stem wear and to improve the functionality of cemented total hip replacement.

In Vivo Response to Methotrexate Forecasts Outcome of Acute Lymphoblastic Leukemia and Has a Distinct Gene Expression Profile

BackgroundChildhood acute lymphoblastic leukemia (ALL) is the most common cancer in children, and can now be cured in approximately 80% of patients. Nevertheless, drug resistance is the major cause of treatment failure in children with ALL. The drug methotrexate (MTX), which is widely used to treat many human cancers, is used in essentially all treatment protocols worldwide for newly diagnosed ALL. Although MTX has been extensively studied for many years, relatively little is known about mechanisms of de novo resistance in primary cancer cells, including leukemia cells. This lack of knowledge is due in part to the fact that existing in vitro methods are not sufficiently reliable to permit assessment of MTX resistance in primary ALL cells. Therefore, we measured the in vivo antileukemic effects of MTX and identified genes whose expression differed significantly in patients with a good versus poor response to MTX.Methods and FindingsWe utilized measures of decreased circulating leukemia cells of 293 newly diagnosed children after initial “up-front” in vivo MTX treatment (1 g/m2) to elucidate interpatient differences in the antileukemic effects of MTX. To identify genomic determinants of these effects, we performed a genome-wide assessment of gene expression in primary ALL cells from 161 of these newly diagnosed children (1–18 y). We identified 48 genes and two cDNA clones whose expression was significantly related to the reduction of circulating leukemia cells after initial in vivo treatment with MTX. This finding was validated in an independent cohort of children with ALL. Furthermore, this measure of initial MTX in vivo response and the associated gene expression pattern were predictive of long-term disease-free survival (p < 0.001, p = 0.02).ConclusionsTogether, these data provide new insights into the genomic basis of MTX resistance and interpatient differences in MTX response, pointing to new strategies to overcome MTX resistance in childhood ALL.Trial registrations: Total XV, Therapy for Newly Diagnosed Patients With Acute Lymphoblastic Leukemia, http://www.ClinicalTrials.gov (NCT00137111); Total XIIIBH, Phase III Randomized Study of Antimetabolite-Based Induction plus High-Dose MTX Consolidation for Newly Diagnosed Pediatric Acute Lymphocytic Leukemia at Intermediate or High Risk of Treatment Failure (NCI-T93-0101D); Total XIIIBL, Phase III Randomized Study of Antimetabolite-Based Induction plus High-Dose MTX Consolidation for Newly Diagnosed Pediatric Acute Lymphocytic Leukemia at Lower Risk of Treatment Failure (NCI-T93-0103D).

Covalent Binding of the Nitroso Metabolite of Sulfamethoxazole Is Important in Induction of Drug-Specific T-Cell Responses in Vivo

Immune-mediated drug hypersensitivity reactions (IDHRs) represent a significant problem due to their unpredictable and severe nature, as well as the lack of understanding of the pathogenesis. Sulfamethoxazole (SMX), a widely used antibiotic, has been used as a model compound to investigate the underlying mechanism of IDHRs because it has been associated with a relatively high incidence of hypersensitivity. Previous studies by others showed that administration of 4-(nitroso)-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide (SMX-NO), the reactive metabolite of SMX, to rats resulted in the generation of SMX-specific antibodies and ex vivo splenocyte proliferative responses, as well as haptenation of skin keratinocytes, circulating peripheral blood mononuclear cells, and splenocytes. The objective of the present study was to further investigate SMX-NO-protein binding in relationship to its immunogenicity. In female DBA/1 mice treated with SMX-NO, varying degrees of SMX-NO-dependent T-cell responses and SMX-NO-protein adduct formation were observed in the spleen and in inguinal, brachial, and axillary lymph nodes. The data suggested a tissue-specific threshold of SMX-NO dosage that triggers the detection of adducts and immune response. Furthermore, serum albumin and immunoglobulin were identified as protein targets for SMX-NO modification. It seemed that these adducts were formed in the blood, circulated to lymphoid tissues, and initiated SMX-NO-dependent immune responses. Collectively, these data revealed a causal link between the deposition of SMX-NO-protein adducts in a lymphoid tissue and the induction of immune response in that tissue. Our findings also suggest that the immunogenicity of SMX-NO is determined by the immunogenic nature of the hapten, rather than special characteristics of the adducted protein.

Exponential Enhancement of Oncolytic Vesicular Stomatitis Virus Potency by Vector-mediated Suppression of Inflammatory Responses In Vivo

Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

Comparison of PD0348292, a selective factor Xa inhibitor, to antiplatelet agents for the inhibition of arterial thrombosis

The objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n = 6-10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of (111)In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x10(6)/cm(2)) at 4.3(549 +/- 1,066), 0.9 (399 +/- 162) and 0.4 mg/kg (531 +/- 470) compared to vehicle (2,242 +/- 1,443). Aspirin (992 +/- 973), clopidogrel (537 +/- 483), clopidogrel plus aspirin (228 +/- 66) or PD0348292 plus aspirin (558 +/- 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.

Preparation of Artificial Kidney Stones of Reproducible Size, Shape, and Mass by Precision Injection Molding

To prepare artificial kidney stones of defined shape, size, mass, and material composition via precision injection molding of Ultracal 30 cement slurries into an inexpensive biodegradable mold. A calcium alginate and silica-based mold was used to prepare casts of varying shapes in a reproducible manner. Ultracal 30 cement slurries mixed 1:1 with water were injected into these casts and allowed to harden. The artificial stones were recovered and their physical properties determined. Ex-vivo and in-vivo responses to holmium laser lithotripsy were examined. Spheres, half spheres, cylinders, cubes, tapered conical structures, and flat angulated structures were prepared with high precision without post-molding manipulations. Large spheres of average mass 0.661 g (+/- 0.037), small spheres of average mass 0.046 g (+/- 0.0026), and hexagons of average mass 0.752 g (+/- 0.0180) were found to have densities (1610-1687 kg/m(3)) within the expected range for Ultracal 30 cement stones. Ex-vivo holmium laser lithotripsy of small spheres in saline showed uniformly reproducible efficiencies of comminution. Implantation of a tapered conical stone into the ureter of a porcine model demonstrated stone comminution in vivo consistent with that seen in the ex-vivo models. We present an environmentally safe, technically simple procedure for the formation of artificial kidney stones of predetermined size and shape. The technique does not require the use of hazardous solvents or postprocedural processing of the stones. These stones are intended for use in standardized experiments of lithotripsy efficiency in which the shape of the stone as well as the mass can be predetermined and precisely controlled.

A New Role of CTLA-4 on B Cells in Thymus-Dependent Immune Responses In Vivo

The expression of CTLA-4 (CD152) on the cell surface of B cells and its consequences for the humoral immune response in vivo are unknown. We investigated the expression of CTLA-4 mRNA and protein in B cells in T cell-independent or -dependent ways. B cells in the presence of Ag-stimulated Th2 cells expressed mRNA of CTLA-4 and up-regulated intracellular CTLA-4 protein. Using a liposome-enhanced staining technique, we show for the first time, that surface CTLA-4 protein is expressed by 11-15% of B cells in a T cell-dependent culture system. To dissect the role of CTLA-4 on B cells in vivo, we used bone marrow chimeric mice in which only B cells were CTLA-4 deficient. These mice showed that early B cell development and homeostasis is not influenced by CTLA-4 deficiency of B cells. Ag-specific responses after immunization of the chimeric mice revealed elevated levels of IgM Abs in mice deficient for B cell CTLA-4. We propose that CTLA-4 signals on B cells determine the early fate of B cells in thymus-dependent immune responses.