Intermediate Responders Research Articles

Protein array screening reveals IgA autoantigenicity patterns predicting anti-TNFα therapy response in rheumatoid arthritis patients

BackgroundOne third of rheumatoid arthritis (RA) patients treated with biologicals targeting tumour necrosis factor α (TNFα) are therapy non-responders. The authors investigated the differences in seroreactivity of patients responding and...

Identification of responders/non-responders to 5-fluorouracil based on individual 50% inhibitory area under the concentration curve of 5-fluorouracil obtained with collagen gel droplet-embedded culture-drug sensitivity test in colorectal cancer

We previously reported the 5-fluorouracil (5-FU) sensitivity of cancer cells obtained from colorectal cancer (CRC) patients using the collagen gel droplet-embedded culture-drug sensitivity test (CD-DST). Multiple drug concentrations and contact durations, and the area under the concentration curve (AUC) and growth inhibition rate (IR) were combined, resulting in the AUC-IR curve, which was approximated to the logarithmic curve. Moreover, the individualized AUC(IR50), the AUC value which gives 50% growth inhibition, was calculated using the AUC-IR curve. This study aimed to identify responders/non-responders to 5-FU based on the individual AUC(IR50) obtained with CD-DST in order to establish individualized chemotherapy for CRC patients. The individual AUC(IR50) was calculated from each AUC-inhibition rate regression curve in all patients using the CD-DST. The cumulative distribution of the individual AUC(IR50) in CRC patients was evaluated. The cumulative distribution of the individual AUC(IR50) was regressed over the sigmoid curve (logarithmic scale). The approximate expression was almost exactly y=ab^exp(-cx) (a=0.9739, b=1.7096E-21, c=0.8990, the sum of square residuals, 0.0279). In the 80 cases examined, no notable change was observed in the regression curve when the number of patients increased. A standard curve was obtained describing responders to 5-FU among all CRC patients. From this standard curve, we ascertained that non-responders accounted for approximately 5% of all patients. Moreover, we were able to classify responders into good or intermediate responders to 5-FU. The standard curve describing response to 5-FU in CRC patients offers a useful tool in the establishment of individualized chemotherapy.

Prediction of Response to Preoperative Chemoradiotherapy in Rectal Cancer by Multiplex Kinase Activity Profiling

Tumor response of rectal cancer to preoperative chemoradiotherapy (CRT) varies considerably. In experimental tumor models and clinical radiotherapy, activity of particular subsets of kinase signaling pathways seems to predict radiation response. This study aimed to determine whether tumor kinase activity profiles might predict tumor response to preoperative CRT in locally advanced rectal cancer (LARC). Sixty-seven LARC patients were treated with a CRT regimen consisting of radiotherapy, fluorouracil, and, where possible, oxaliplatin. Pretreatment tumor biopsy specimens were analyzed using microarrays with kinase substrates, and the resulting substrate phosphorylation patterns were correlated with tumor response to preoperative treatment as assessed by histomorphologic tumor regression grade (TRG). A predictive model for TRG scores from phosphosubstrate signatures was obtained by partial-least-squares discriminant analysis. Prediction performance was evaluated by leave-one-out cross-validation and use of an independent test set. In the patient population, 73% and 15% were scored as good responders (TRG 1-2) or intermediate responders (TRG 3), whereas 12% were assessed as poor responders (TRG 4-5). In a subset of 7 poor responders and 12 good responders, treatment outcome was correctly predicted for 95%. Application of the prediction model on the remaining patient samples resulted in correct prediction for 85%. Phosphosubstrate signatures generated by poor-responding tumors indicated high kinase activity, which was inhibited by the kinase inhibitor sunitinib, and several discriminating phosphosubstrates represented proteins derived from signaling pathways implicated in radioresistance. Multiplex kinase activity profiling may identify functional biomarkers predictive of tumor response to preoperative CRT in LARC.

Abstract 1034: Combination therapy with gossypol reveals synergism against gemcitabine resistance in cancer cells with high Bcl-2 expression

Abstract Gemcitabine is an anticancer agent used in breast, non-small cell lung, pancreatic and ovarian cancer. However, many cancers have been showed to be resistant to gemcitabine. Recent studies have demonstrated that gemcitabine resistance can be overcome when combined with gossypol. Gossypol is an inhibitor of Bcl-2 that induces apoptosis in various cancer cell lines. The aim of our study was to determine whether gossypol can overcome gemcitabine resistance via its interaction with the Bcl-2 protein. IC50 of gossypol and gemcitabine, as well as baseline expression of Bcl-2 were determined in 10 cell lines derived from nasopharyngeal (n=4), breast (n=3) and gastric (n=3)cancers. High Bcl-2 baseline expression that was observed in 3/10 cell lines, had higher IC50 to gemcitabine. Upregulation of Bcl-2 levels were also observed after treatment in cell lines that high Bcl-2 baseline expression. These findings suggest an interaction between Bcl-2 protein and gemcitabine resistance. Furthermore, in 7/10 cell lines that were sensitive to gemcitabine, five showed low Bcl-2 expression, suggesting that Bcl-2 levels plays a role in gemcitabine response in cancer. To determine whether gossypol can overcome gemcitabine resistance, combination treatments were carried out either by adding the drugs simultaneously or sequentially to the cell lines. Dual drug treatment was found to be synergistic to cell lines that have both high Bcl-2 level and were resistant to single agent, gemcitabine. Intrestingly cell lines that were sensitive to both drugs, and had high Bcl-2 level were also synergistic when treated with the combined drugs. In contrast, cell lines that were sensitive to both drugs and had low Bcl-2 level were antagonistic to combined drug treatments. The (-) enatiomer of gossypol (AT101) has been reported to be more potent than gossypol in inhibiting cell growth and inducing apoptosis in cancer cells. This suggests that AT101 is a more effective drug than gossypol in overcoming gemcitabine resistance. Cell lines treated with AT101 had lower IC50 compared to those treated with gossypol. Two cell lines (HK1 and AGS) that were intermediate responders to gossypol demonstrated higher sensitivity when treated with AT101, suggesting that AT101 is a more effective drug than gossypol. Futhermore, in vitro durg response studies comparing the 2 drugs in 35 gastric cell lines reveals an overall hypersensitive to AT101 compared to gossypol. Current studies are underway to investigate combination treatment of AT101 with gemcitabine, as well as its interactions with the Bcl-2 family proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1034.

Stage II/III rectal cancer with intermediate response to preoperative radiochemotherapy: Do we have indications for individual risk stratification?

BackgroundResponse to preoperative radiochemotherapy (RCT) in patients with locally advanced rectal cancer is very heterogeneous. Pathologic complete response (pCR) is accompanied by a favorable outcome. However, most patients show incomplete response. The aim of this investigation was to find indications for risk stratification in the group of intermediate responders to RCT.MethodsFrom a prospective database of 496 patients with rectal adenocarcinoma, 107 patients with stage II/III cancers and intermediate response to preoperative 5-FU based RCT (ypT2/3 and TRG 2/3), treated within the German Rectal Cancer Trials were studied. Surgical treatment comprised curative (R0) total mesorectal excision (TME) in all cases. In 95 patients available for statistical analyses, residual transmural infiltration of the mesorectal compartment, nodal involvement and histolologic tumor grading were investigated for their prognostic impact on disease-free (DFS) and overall survival (OS).ResultsResidual tumor transgression into the mesorectal compartment (ypT3) did not influence DFS and OS rates (p = 0.619, p = 0.602, respectively). Nodal involvement after preoperative RCT (ypN1/2) turned out to be a valid prognostic factor with decreased DFS and OS (p = 0.0463, p = 0.0236, respectively). Persistent tumor infiltration of the mesorectum (ypT3) and histologic tumor grading of residual tumor cell clusters were strongly correlated with lymph node metastases after neoadjuvant treatment (p < 0.001).ConclusionsAdvanced transmural tumor invasion after RCT does not affect prognosis when curative (R0) resection is achievable. Residual nodal status is the most important predictor of individual outcome in intermediate responders to preoperative RCT. Furthermore, ypT stage and tumor grading turn out to be additional auxiliary factors. Future clinical trials for risk-adapted adjuvant therapy should be based on a synopsis of clinicopathologic parameters.

Estimation of the Effective Doses of Nasal-Inactivated Influenza Vaccine in Humans from Mouse-Model Experiments

Mouse models of influenza play an important role in developing effective human influenza vaccines. We have demonstrated that intranasal immunization with inactivated subvirion (SV) vaccines, in conjunction with a cholera toxin B subunit adjuvant (CTB*), provides more effective cross-protection than parenteral immunization in BALB/c mice. In addition, the minimal effective dose of nasal vaccine for providing complete protection against a lethal influenza virus infection is 0.1 microg SV vaccine (containing about 30 ng hemagglutinin [HA]) (with 0.1 microg CTB*) in BALB/c mice (20 g body weight) immunized twice intranasally 4 weeks apart. The effective dose in humans can be estimated to be roughly 100 microg SV vaccine (containing approximately 30 microg HA) (with 100 microg CTB*)/20 kg body weight by using the dose/body weight ratio from actual vaccination trials. This estimation can be rationalized by the hypothesis that the distribution of strains (or individuals) in a mouse (or human) population, in relation to their HA antigen responsiveness, follows a normal distribution, and by the fact that BALB/c mice are intermediate responders for anti-HA antibody responses, and correspond to human intermediate responders, which form the largest population. We also discuss the use of innate immune responses, as well as antibody responses, in BALB/c mice to assess the efficacy of unknown adjuvants and the development of other adjuvants for nasal vaccines that should be clinically safer than CTB*.

Increased clearance of cortisol by 5β-reductase in a subgroup of women with adrenal hyperandrogenism in polycystic ovary syndrome

Increased peripheral metabolism of cortisol may explain compensatory ACTH-dependent adrenal steroidogenesis and hence hyperandrogenism in polycystic ovary syndrome (PCOS). Previous studies have described an increased 5alpha-reduction of cortisol or impaired regeneration of cortisol by 11beta-HSD1 in PCOS. However, these observations may be confounded by obesity. Moreover, the relationship between alterations in cortisol metabolism and the extent of adrenal androgen hyper-secretion in response to ACTH has not been established. This study aimed to examine the association between cortisol metabolism and ACTH-dependent adrenal hyperandrogenism in PCOS, independently of obesity. We compared 90 PCOS women (age 18-45 yr) stratified by adrenal androgen responses to ACTH1-24 and 45 controls matched for age and body weight. PCOS women were stratified as normal responders (NR), intermediate responders (IR), and high responders (HR) to 250 microg ACTH1-24: NR (no.=27) had androstenedione and DHEA responses within 2 SD of the mean in controls; IR (no.=43) had DHEA responses >2 SD above controls; HR (no.=20) had both androstenedione and DHEA responses >2 SD above controls. All groups were similar for age, body weight, and body fat distribution. Basal testosterone, androstenedione, and 5alpha-dihydrotestosterone plasma levels were similarly elevated among the 3 groups of PCOS compared with controls, whereas basal DHEA-S was higher in HR (2.8+/-1.2 microg/ml) and IR (2.4+/-1.1 microg/ml) than in NR (1.8+/-0.8 microg/ml) and controls (1.7+/-0.6 microg/ml). The HR group had the lowest basal plasma cortisol levels (101+/-36 ng/ml vs IR 135+/-42 ng/ml, NR 144+/-48 ng/ml, and controls 165+/-48 ng/ml; all p<0.01), but the greatest cortisol response to ACTH1-24 (Delta(60-0)cortisol 173+/-60 ng/ml vs IR 136+/-51 ng/ml, NR 114+/-50 ng/ml, and controls 127+/-50 ng/ml; all p<0.01), and the highest urinary excretion of total and 5beta-reduced cortisol metabolites (eg 5beta-tetrahydrocortisol/ cortisol ratio 25.2+/-15.3 vs IR 18.8+/-10.7, NR 19.7+/-11.4, and controls 17.2+/-13.7; all p<0.05). There were no differences in urinary excretion of 5alpha-reduced cortisol metabolites or in 5alpha-dihydrotestosterone/testosterone ratio between groups. Adrenal androgen excess in PCOS is associated with increased inactivation of cortisol by 5beta-reductase that may lower cortisol blood levels and stimulate ACTH-dependent steroidogenesis.

α1-acid glycoprotein and α1-antitrypsin as early markers of treatment response in patients receiving the intensive phase of tuberculosis therapy

The identification of early markers that predict the response to anti-tuberculosis treatment would facilitate evaluation of new drugs and improve patient management. This study aimed to determine whether selected acute phase proteins and micronutrients measured at the time of diagnosis and during the first weeks of treatment could predict treatment responses during the 2-month standard intensive phase of therapy. For this purpose, alpha1-antitrypsin, alpha1-acid glycoprotein, alpha2-macroglobulin, C-reactive protein, C3, C4, zinc, copper and selenium concentrations were measured in Brazilian patients with smear-positive tuberculosis at the time of diagnosis and 1, 3, 5 and 8 weeks after initiation of therapy. Patients were classified into fast (n=29), intermediate (n=18) and slow responders (n=10) if they were smear-negative at 3, 5 or 8 weeks of treatment. alpha1-acid glycoprotein on enrolment and 1 week of treatment, alpha1-antitrypsin at week 1 and C-reactive protein and C3 after 3 weeks of therapy were higher in slow responders than in fast responders. alpha1-antitrypsin and alpha1-acid glycoprotein may be helpful in predicting treatment response at the time of initiation of therapy, and could be used as early markers to identify patients with an increased likelihood of treatment failure.

Mannose‐binding lectin gene polymorphism and resistance to therapy in women with recurrent vulvovaginal candidiasis

Women with recurrent vulvovaginal candidiasis (RVC) due to a polymorphism in codon 54 of the MBL2 gene respond better to fluconazole maintenance therapy than do women with other underlying causes. To explain differences in response rates to maintenance therapy with fluconazole in women suffering from RVC by evaluating associations with a polymorphism in the gene coding for mannose-binding lectin (MBL). Follow-up study, neted case-control group. Women attending vulvoginitis clinic for RVC. Women participating in a multicentric study in Belgium with a degressive dose of fluconazole for RVC (the ReCiDiF trial) were divided into good responders, intermediate responders and nonresponders according to the number of relapses they experienced during therapy. From 109 of these women with adequate follow-up data, vaginal lavage with 2 ml of saline were performed at the moment of a proven acute attack at inclusion in the study, before maintenance treatment was started. A buccal swab was obtained from 55 age-matched women without a history of Candida infections, serving as a control group. Extracted DNA from buccal or vaginal cells was tested for codon 54 MBL2 gene polymorphism by polymerase chain reaction and endonuclease digestion. Frequency of MBL2 condon 54 allele B in women with optimal or poor response to maintenance therapy in composition with controls. Results Women (n = 109) suffering from RVC were more likely to carry the variant MBL2 codon 54 allele B than control women (20 versus 6.6%, OR 3.4 [95% CI 1.3-8.2], P = 0.01). B alleles were present in 25% of the 36 women not suffering from any recurrence during the maintenance therapy with decreasing doses of fluconazole (OR 4.9 [95% CI 1.9-12.5], P = 0.0007 versus controls), in 20% of the 43 women with sporadic recurrences (OR 3.6 [95% CI 1.4-9.2], P = 0.007 versus controls) and in 15% of the 30 women who had to interrupt the treatment regimen due to frequent relapses (P = 0.097 versus controls). The MBL2 codon 54 gene polymorphism is more frequent in Belgian women suffering from RVC than in controls. The presence of the B allele is associated with a superior response to fluconazole maintenance therapy as compared with RVC patients without this polymorphism. We conclude that RVC due to deficient MBL production is more easily helped with antifungal medication than is RVC due to some other mechanism.

Selective Expansion of Influenza A Virus–Specific T Cells in Symptomatic Human Carotid Artery Atherosclerotic Plaques

Evidence is accumulating that infection with influenza A virus contributes to atherothrombotic disease. Vaccination against influenza decreases the risk of atherosclerotic syndromes, indicating that inflammatory mechanisms may be involved. We tested the hypothesis that influenza A virus-specific T cells contribute to atherosclerotic plaque inflammation, which mediates the onset of plaque rupture. T-cell cultures were generated from atherosclerotic segments and peripheral blood of 30 patients with symptomatic carotid artery disease. The response of plaque and peripheral blood T cells to influenza A virus was analyzed and expressed as a stimulation index (SI). Selective outgrowth of intraplaque influenza A-specific T cells was calculated by the ratio of plaque T cell SI and peripheral blood T cell SI for each patient. Accordingly, the patients were categorized as high- (SI ratio >or=5), intermediate- (5 <SI ratio <or=2), and non- (SI ratio <2) responders. The presence of influenza A virus in the vessel fragments was evaluated by reverse transcription-polymerase chain reaction. High proliferative responses of plaque-derived T cells to influenza A virus were frequently observed. Among the 30 patients, 5 were categorized as high responders, 10 were intermediate responders, and 15 were nonresponders. Live influenza A virus could not be detected in the atherosclerotic plaques by polymerase chain reaction. Selective outgrowth of influenza A virus-specific T cells occurs within the microenvironment of human atherosclerotic plaques. Influenza virus-derived antigens or alternatively, mimicry antigens, appear to be potential candidates for triggering or sustaining plaque inflammation, which eventually leads to symptomatic plaque complications.

The Use of the Point of Care in Evaluating the Anticoagulation Effect of Low Molecular Weight Heparin Nadroparin during Hemodialysis.

Anticoagulation in hemodialysis is targeted to prevent the activation of coagulation during the procedure. Although unfractionated heparin (UFH) is the anticoagulant commonly used, recent studies suggested the safety and efficacy of low-molecular-weight heparin (LMWH) and possibly the most cost-effective anticoagulation for hemodialysis. The activated clotting time is usually used to monitor UFH while the chromogenic anti-Xa is the standard monitoring test for LMWH. The anti-Xa is not readily available; a bedside test may be helpful to ensure optimal anticoagulation. The objective of this study was to evaluate the utility of a whole blood (WB) point of care (POC) test, HEMONOX™ (HEMOCHRON Signature+) in measuring the anticoagulation effect of Nadroparin (FRAXIPARINE®, Sanofi, France) in normal blood samples spiked with different concentrations of Nadroparin and in clinical samples from patients during hemodialysis. Following IRB approval, Nadroparin was administered to the patients (n=11) at 56 IU/Kg 3–4 min before dialysis as a single bolus dose injected into the arterial needle pre-dialysis. Blood samples were obtained at baseline, 15 min and 2 hrs post bolus and at the end of the hemodialysis for performing the following POC tests: HEMONOX, low range activated clotting time (ACT-LR) and WB-activated partial thromboplastin time (WB-aPTT). Plasma samples were separated from each blood draw for the determination of the laboratory plasma based aPTT test (Lab-aPTT, Biomerieux France) and the anti-Xa assay (STAGO, France). In vitro analyses were performed by adding Nadroparin at increasing concentrations to fresh WB from 4 healthy donors. These samples were tested for clotting time using WB-aPTT, ACT-LR and HEMONOX. All WB-POC tests showed a linear dose response to Nadroparin in concentrations of 0–2.5 IU/ml and the overall clotting time results suggest variable levels of sensitivity to Nadroparin. The HEMONOX assay showed more sensitivity to the Nadroparin concentration with increases of 2–6 fold from baseline at LMWH levels of 1 and 1.5 IU/ml blood, compared to 1.2–1.5 and 1.5–3.0 fold for the ACT-LR and the WB-aPTT respectively. The clinical data confirmed these results and also correlated well to the anti-Xa activity levels. HEMONOX values obtained at baseline time (72–109 seconds) corresponded to undetectable anti-Xa. HEMONOX peak values obtained at 15 min post bolus, corresponded to therapeutic anti-Xa level but patients showed variable response to Nadroparin. HEMONOX clotting time (CT) at peak response suggested 3 patient subgroups with different levels of sensitivity to Nadroparin: low (range: CT 92–98 seconds and anti-Xa 0.56–1.0 U/ml), intermediate (range: CT 123–160 seconds and anti-Xa 0.90–1.37 U/ml) and high responders (range: CT 319–870 seconds and anti-Xa 1.2–1.30 U/ml). The HEMONOX test was the most sensitive indicator of Nadroparin anticoagulation when compared to POCT ACT-LR and WB-aPTT coagulation tests. The HEMONOX method showed a good correlation to the Lab-aPTT (r= 0.86, n= 36) and both methods yielded comparable correlation coefficient to anti-Xa (r= 0.71, n= 27). The results from this pilot study suggest that the WB-POC test HEMONOX is sensitive to the anticoagulant effect of Nadroparin during hemodialysis. More studies are needed to confirm the significance of these findings.

Therapeutic resistance to angiotensin converting enzyme (ACE) inhibition is related to pharmacodynamic and -kinetic factors in 5/6 nephrectomized rats

Proteinuria plays a pathogenic role in the development of end stage renal disease. Angiotensin converting enzyme (ACE) inhibitors lower proteinuria and are renoprotective. However, large inter-individual variation in antiproteinuric response to ACE inhibitors exists. In this study, we explored the mechanism of therapeutic resistance to an ACE inhibitor in the rat 5/6 nephrectomy model. At week 6 after 5/6 nephrectomy, treatment with lisinopril was initiated for 6 weeks. Proteinuria and blood pressure were evaluated weekly. At the end of the experiment, rats were divided into tertiles according to their antiproteinuric response: (1) responders ( n = 9), (2) intermediate responders ( n = 8) and (3) non-responders to ACE inhibitor therapy ( n = 9). At the start of treatment, proteinuria had progressively increased to 154 (95% confidence interval [CI]: 123–185) mg/24 h in the entire cohort, with comparable proteinuria and blood pressure in all groups. Following treatment with ACE inhibitor, proteinuria was significantly lower in the responders (68, CI: 46–89 mg/24 h) compared to the non-responders (251, CI: 83–420) mg/24 h). Similarly, blood pressure was reduced in the responders, but unaffected in the non-responders. At autopsy, renal ACE activity and renal ACE expression were significantly lower in the responders compared to the non-responders. Although lisinopril intake was comparable in all animals, urinary drug excretion was increased in the non-responders, demonstrating increased drug clearance. Average urinary lisinopril excretion was correlated with antiproteinuric response ( R 2 = 0.32, P = 0.003). In conclusion, both pharmacodynamic and -kinetic factors account for the non-response to lisinopril. Whether these can be overcome simply by increasing drug dosage in non-responders should be investigated.

Aberrant Repertoire and Deficient Proliferation of TCR γδ T Cells in Myelodysplastic Syndromes (MDS).

Background: We recently showed that MDS natural killer cells display altered cytolytic function and proliferation in response to IL-2 (Leukemia , 2006; 20:463–70). TCR γδ T cells are other important components of innate immunity, recently shown to be involved in the immune response against various tumors. Furthermore, a TCR γδ agonist synthetic phosphoantigen, the BrHPP molecule, is able to expand and activate in vitro Vδ2 cells (the peripheral blood circulating subset of γδ T cells). We studied γδ T cell population in MDS patients.Methods: PBMCs from 31 MDS patients (WHO: 3 pure RA, 2 pure RAS, 1 RCMD-RS, 6 RCMD, 2 del5q, 8 RAEB1, 4 RAEB2, 5 MDS-U; IPSS: 9 low, 12 Int-1, 6 Int-2, 4 High) and 15 controls were stimulated with BrHPP and IL-2 during 8 to 15 days. Immunophenotyping of αβ and γδ T cells was determined by multi-color flow cytometry. Cytolytic capacity of activated Vδ2 T cells was assessed using 51Cr release and CD107a degranulation assays. IFNγ was measured by ELISA after TCR γδ triggering of expanded γδ T cells.Results: % and absolute numbers of γδ T cells from MDS were comparable to those of controls, but MDS Vδ2 T cell repertoire was significantly skewed: 10/19 MDS Vδ2 T cells were characterized by a “naive” predominant population, 5/19 had a majority of “effectors” cells, and only 4 of 19 had a predominant “central memory population”, which was always the main Vδ2 compartment in healthy donors. Stimulation with BrHPP induced a significant increase of MDS Vδ2 T cells subset, indicating their specific activation. However, MDS Vδ2 T cells poorly proliferated in 2 week cultures in response to IL-2, in contrast with the high proliferation index of controls derived cells. On day 8 of culture, only 6 of the 29 MDS samples studied clearly responded to BrHPP+IL-2 (i.e. >60% of Vδ2 cells in culture) compared to 13/15 in controls, while 12/29 were intermediate responders (20 to 60% of Vδ2), and 11/29 non-responders. We found no correlation between response to BrHPP and WHO, cytogenetics or IPSS category. However, coexistence of immunologic abnormalities (rheumatoid arthritis in 5 patients, temporal arteritis in 1, antiphospholipid syndrome in 1, Raynaud's phenomenon in 2, and thyroiditis in 2) was more frequent in non-responders. The 3 IL-2 receptor (IL-2-R) chains were normally expressed in MDS Vδ2 T cells and IL-2-Rα expression was normally induced in response to IL-2. We are currently investigating downstream signaling molecules of the IL-2-R pathway. Functional activity of MDS γδ T cells (cytotoxicity, degranulation, and IFNg secretion) was similar to that of healthy donor cells. In particular, those cells were cytotoxic against the MDS-derived P39 cell line.Conclusion: in MDS, γδ T cell repertoire is profoundly skewed, and those cells do not proliferate in response to potent agonists, although their numbers and function seem normal. Those alterations did not correlate with MDS characteristics, but were more frequent in patients with associated immunological abnormalities. Our results further support the existence of important alterations of innate immunity effectors in MDS that could play a role in disease pathophysiology or progression.

In vivo murine CD23 destabilization enhances CD23 shedding and IgE synthesis

To investigate the effects of in vivo CD23 destabilization on CD23 shedding and IgE production, an anti-CD23 stalk monoclonal (19G5), previously shown to enhance proteolysis of CD23 in vitro, was utilized. Compared to isotype control-treated mice, BALB/cJ mice injected with 19G5 displayed significantly enhanced serum soluble CD23 and IgE. Soluble CD23 and IgE levels were also increased in 19G5-treated C57BL/6J mice (intermediate IgE responders); however, the kinetics of the responses differed between the high (BALB/cJ) and intermediate responder mice, suggesting a potential role for CD23 in regulating IgE responder status. The 19G5-induced IgE response was dependent on IL-4 and independent of CD21 as demonstrated through use of IL-4Rα and CD21/35-deficient mice, respectively. Overall, the data provide a direct demonstration for CD23’s role in regulating IgE production in vivo and suggest that therapies aimed at stabilizing cell surface CD23 would be beneficial in controlling allergic disease.

Up-regulation of asparagine synthetase expression is not linked to the clinical response l-asparaginase in pediatric acute lymphoblastic leukemia

Abstractl-asparaginase (l-Asp) is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Asparagine synthetase (AS) opposes the action of l-Asp by resynthesis of asparagine. In vitro, resistance to l-Asp has been associated with up-regulation of AS mRNA expression. We monitored AS mRNA levels in leukemic cells before and during 5 days after intravenous administration of 1000 IU/m2 pegylated l-asparaginase (PEG-Asp) in a therapeutic window in children with ALL at initial diagnosis. Within 24 hours, AS mRNA levels increased by 3.5-fold and remained stable in the following 4 days. Baseline and l-Asp–induced expression levels of AS did not differ between clinically good, intermediate, and poor responders to PEG-Asp. No significant difference of AS mRNA up-regulation was found between precursor B- and T-ALL or between hyperdiploids, TEL/AML1 rearranged ALL or absence of genetic abnormalities. In 3 of 12 patients with T-ALL even a slight down-regulation of AS mRNA expression upon l-Asp exposure was found. In conclusion, although l-Asp exposure induces the expression of AS mRNA, the up-regulated gene expression does not correlate with an early clinical poor response to this drug in children with ALL.

The use of a HEMOCHRON® JR. HEMONOX™ point of care test in monitoring the anticoagulant effects of enoxaparin during interventional coronary procedures

Enoxaparin is increasingly used for the anticoagulation of patients undergoing percutaneous coronary intervention (PCI). Several reports have suggested the utility of using point of care tests in monitoring the anticoagulation levels of enoxaparin in patients undergoing PCI. The objective of this study was to evaluate a new point-of-care test (POCT) HEMONOX in monitoring the anticoagulant effect of enoxaparin in non citrated fresh whole blood samples from patients undergoing elective PCI procedure. Following IRB approval, blood samples were obtained from fifty-four patients who received two sequential intravenous doses of enoxaparin; 0.1 mg/kg followed 5 min later by 0.4 mg/kg for a total of 0.5 mg/kg. Blood was drawn at baseline and at 5, 10, 30 and 60 min post first bolus for evaluation in the clot-based POCT HEMONOX, ACT and aPTT and the chromogenic anti-Xa activity assay. HEMONOX clotting time (CT) at baseline was 62.6 +/- 6.2 secs, (n = 32) in healthy donors and statistically higher in PCI patients (71.6 +/- 9.1 secs, p = 0.0001). The peak HEMONOX response that was always achieved at 10 min post bolus was >100 secs in all 54 patients, of these 83% yielded CT >150 secs (range: 150-466). There was no detectable anti-Xa activity level at baseline while peak HEMONOX CT corresponded to therapeutic levels (0.85 +/- 0.14 U/ml; range: 0.61-1.34). Both HEMONOX CT and anti-Xa level significantly decreased at the time of sheath removal. HEMONOX CT at peak response suggested 3 patient subgroups with different levels of sensitivity to enoxaparin: low, intermediate and high responders. The correlation between anti-Xa activity level and HEMONOX CT was >or=0.85 in each patient subgroup when data from the 3 critical time points; baseline (absence of drug), peak response (10 min post bolus) and sheath removal (60 min post bolus) were analyzed. The correlation diminished to >or=0.83 when the analyses included data from all 5 time points [baseline, 5, 10, 30, and 60 min post bolus]. The HEMONOX test was the most sensitive POCT to measure the anticoagulant effects of enoxaparin. All patients completed PCI successfully. The HEMONOX test may be able to guide anticoagulation with enoxaparin during PCI. The HEMONOX assay is a one step whole blood coagulation test performed on the HEMOCHRON Jr. Signature + POC system. The method was evaluated to monitor the anticoagulant level of enoxaparin in blood samples from patients undergoing PCI after receiving an intravenous dose of 0.5 mg/kg. The results suggest a clear distinction of HEMONOX CT between the baseline value of untreated patients and patients achieving therapeutic enoxaparin levels.

Ocular Hypertensive Response to Topical Dexamethasone Ointment in Children

PurposeTo investigate the rate and the degree of the ocular hypertensive response to dexamethasone ointment in children undergoing eyelid surgery.MethodsDexamethasone ointment (Dexcosil®) was applied three times a day for the first week and twice a day for the second to third week postoperatively to children undergoing epiblepharon surgery. Intraocular pressure (IOP) was measured on the day before surgery, postoperative day 1, 7, 14, 21, 28 and 2 weeks thereafter until the IOP reached preoperative levels. Peak IOP, IOP net increase and time to reach a peak IOP were analyzed. Dexamethasone ointment was discontinued if the IOP was 25 mmHg or more.ResultsA total of 96 children (mean age, 6.5±2.7 years) were included. Preoperative mean IOP was 13.6±2.9 mmHg (range 7-19). After dexamethasone ointment treatment, the children showed a significant rise in IOP as compared with the preoperative values. The peak IOP was 20.6±4.9 mmHg (range 11-39) and the time to reach peak IOP was 8.5±5.9 days. The low responders (delta IOP ≤5 mmHg) of our group comprised 35.4% (34/96) of patients, intermediate responders (delta IOP 6-15 mmHg) comprised 56.3% (54/96) of patients and high responders (delta IOP≥16) comprised 8.3% (8/96) of patients. A net increase in IOP was significantly higher in children 5 years old or less as compared with those older than 5 years (age ≤5, 9.4±7.5 mmHg vs age >5, 6.3±4.4 mmHg; p=0.015, unpaired t-test).ConclusionsOcular hypertensive response after dexamethasone ointment to the eyelids occurred frequently in children, especially those 5 years old or younger.

Altered Self-Erythrocyte Recognition and Destruction in an Inbred Line of Tilapia (Oreochromis aureus)

Carboxyfluorescein diacetate (cFDA)-stained autologous and syngeneic tilapia (Oreochromis aureus) erythrocytes are recognized by effector peripheral blood leukocytes and lysed after a short culture period of 4 h. The hemolysis level was evaluated by measuring the fluorescence of the released cFDA. The degree of lysis of stained target erythrocytes of 60 individuals revealed a trimodal distribution statistically stratified into three groups of low (LR), intermediate (IR), and high (HR) responders. Depletion of the majority of phagocytes from leukocytes lowered the lysis level of HR to that of LR. A highly significant increase of LR cytotoxicity was obtained after the addition of conditioned medium from HR but only in the presence of phagocytes. Genetic analysis of offspring from four crosses (IR x HR, IR x LR, HR x LR, and LR x LR) revealed a quantitative trait locus (QTL) segregating for the level of response linked to markers UNH207 and UNH231 on linkage group 6 of tilapia. Based on segregation analysis of 58 gynogenetic BIU-1 offspring, the distances from the centromere were estimated as 21.5, 11.5, and 9.0 cM for UNH207, UNH231, and the QTL, respectively. It is suggested that 1) self-target recognition and destruction requires both cFDA-altered self-erythrocyte membrane and membrane structures normally present in autologous, syngeneic, and xenogeneic targets; 2) natural cytotoxic cells and/or macrophages are involved in erythrocyte lysis; and 3) the lysis level is codominantly inherited by a QTL segregating on tilapia linkage group 6.

Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene.

Sugar-induced anthocyanin accumulation has been observed in many plant species. We observed that sucrose (Suc) is the most effective inducer of anthocyanin biosynthesis in Arabidopsis (Arabidopsis thaliana) seedlings. Other sugars and osmotic controls are either less effective or ineffective. Analysis of Suc-induced anthocyanin accumulation in 43 Arabidopsis accessions shows that considerable natural variation exists for this trait. The Cape Verde Islands (Cvi) accession essentially does not respond to Suc, whereas Landsberg erecta is an intermediate responder. The existing Landsberg erecta/Cvi recombinant inbred line population was used in a quantitative trait loci analysis for Suc-induced anthocyanin accumulation (SIAA). A total of four quantitative trait loci for SIAA were identified in this way. The locus with the largest contribution to the trait, SIAA1, was fine mapped and using a candidate gene approach, it was shown that the MYB75/PAP1 gene encodes SIAA1. Genetic complementation studies and analysis of a laboratory-generated knockout mutation in this gene confirmed this conclusion. Suc, in a concentration-dependent way, induces MYB75/PAP1 mRNA accumulation. Moreover, MYB75/PAP1 is essential for the Suc-mediated expression of the dihydroflavonol reductase gene. The SIAA1 locus in Cvi probably is a weak or loss-of-function MYB75/PAP1 allele. The C24 accession similarly shows a very weak response to Suc-induced anthocyanin accumulation encoded by the same locus. Sequence analysis showed that the Cvi and C24 accessions harbor mutations both inside and downstream of the DNA-binding domain of the MYB75/PAP1 protein, which most likely result in loss of activity.

The Association Between the DRD2 TaqI A Polymorphism and Smoking Cessation in Response to Acupuncture in Koreans

The response to acupuncture is presumed to differ with the individual; this variation might be due to genetic differences. Therefore, we examined whether the DRD2 TaqI A polymorphism is associated with the response to acupuncture in the treatment of smoking cessation in a Korean population. A total of 231 healthy Korean male smokers participated in this study. The subjects underwent three manual auricular acupuncture treatments in one week. After 1 week, high and low responders to the acupuncture treatment were selected according to their cigarette consumption and the desire to smoke. A DRD2 TaqI A restriction enzyme fragment length polymorphism (RFLP) study examined genomic DNA extracted from blood samples. The proportions of high, intermediate, and low responders were 28.6, 55.4, and 16.0%, respectively. The RFLP study showed that the distribution of the DRD2*A genotype and allele frequencies differed significantly between the high and low responders (chi(2) test, p = 0.031 and 0.016, respectively), and that high responders had a higher DRD2*A2 allele frequency than low responders. Our findings suggest that DRD2 TaqI A polymorphism is related to acupuncture response in smoking cessation treatment.