Median Lysis Research Articles

Point-of-care viscoelastic coagulation monitor parameters in Amazon parrots (Amazona spp.).

To evaluate the feasibility of the point-of-care viscoelastic coagulation monitor (VCM) in Amazon parrots (Amazona spp.) and describe the parameters with fresh whole-blood samples in healthy Amazon parrots. A total of 18 Amazon parrots were enrolled. Physical examinations, a CBC, and a biochemistry profile, including bile acids, were performed on all parrots. VCM tracings were obtained at the time of venipuncture for baseline laboratory work. The median clot time was 2102seconds (range: 38.6-3599s), median clot formation time was 929seconds (range: 21.4-1711s), median alpha angle was 20 (range: 6-67), and the median maximum clot formation was 8.5 (range: 0-36). The median lysis index at 30minutes (LI30) was 100 (range: 98-100), and the median lysis index at 45minutes (LI45) was 100 (range: 90-100). Of 18 samples, alpha angles were not reported in 7 samples, LI30 was not reported in 10 samples, and LI45 was not reported in 12 samples. Of the qualitative curves, 6 reflected normal mammalian curves, and the remainder were consistent with a hypocoaguable state. The results were markedly variable, with the majority of VCM tracings being hypocoagulable in comparison with reference intervals established for dogs and cats. Using these protocols, the VCM is not reliable in Amazon parrots. Future areas of investigation include altering the temperature during sample analysis, the use of activators, or an exchange of clotting reagents for an extrinsic pathway activator, which may contribute to the success of this device in avian species.

Epcoritamab induces potent anti-tumor activity against malignant B-cells from patients with DLBCL, FL and MCL, irrespective of prior CD20 monoclonal antibody treatment

Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.

Abstract A140: A T-cell utilizing bispecific anti-CD3/GD2 construct mediates superior in vitro efficacy compared to CH14.18 mAb in neuroblastoma patients after allogeneic SCT

Abstract Introduction: Whereas immunotherapy in high-risk/relapsed neuroblastoma with standard antibody formats targeting the tumor associated antigen GD2 is currently used after autologous/allogeneic stem cell transplantation (SCT), T-cells have rarely been addressed as effectors. Bispecific antibodies can bring T-cells into close proximity to tumor cells and activate them through the CD3 region, which ultimately leads to targeT-cell elimination. They lack the Fc-domain and cannot trigger ADCC or CDC as NK-cells. An example for bispecific antibodies is NG-CU (developed at the Department of Immunology, University of Tuebingen), which recognizes CD3 and GD2 as target antigens. The tumor antigen specific antibody domain, derived from a fab fragment of GD2 14.18 antibody and the T-cell recruiting antibody domain, derived from single chain variable fragment (scFv fragment) of UCHT-1 (CD3) are linked by modified CH2 domain. We investigated whether the bispecific antibody NG-CU might be an alternative to the therapeutic monoclonal antibody CH14.18, which mediates CDC and ADCC through NK-cells. Method: Different antibody concentrations and effector to target ratios (E:T) were evaluated using the xCELLigence RTCA system, PBMCs (healthy donors and patients after allogeneic SCT) and the neuroblastoma cell lines LS and LAN-1. CDC was evaluated using autologous serum.Results: NG-CU showed enhanced cytotoxicity compared to CH14.18. Median specific lysis (n=3) after 12/24/48 hrs (effectors: healthy PBMCs, E:T=5:1) with LS cells was: 55/73/77% (CH14.18, 1 µg/ml) vs. 70/100/100% (NG CU, 100ng/ml). P values with Mann-Whitney test: p=0,0244; p<0,0001; p<0,0001. With LAN-1 cells: 56/69/88% (CH14.18, 1 µg/ml) vs. 9/46/99% (NG-CU, 100 ng/ml). P values with Mann-Whitney test: p<0,0001; p=0,1139; p=0,0464.Taking CDC into account by adding autologous serum, cytotoxicity of CH14.18 was drastically increased. ADCC and CDC eradicated targets with 64% after 4 hrs and 93% after 48 hrs (E:T=5:1). However, using a lower E:T ratio (1:1) lysis of CH14.18 remained static, but not for NG-CU. Median lysis (n=3) after 12/24/48 hrs: 71/74/77% (CH14.18) vs. 25/42/67% (NG-CU). After >48 hrs targets were completely eradicated by NG-CU, while targets treated with CH14.18 still proliferated.Using patient PBMCs, significant lysis with both constructs was detected dependent on percentages and total numbers of T- and NK-cells. In patients early after SCT, NK-cells represented the majority of effectors. Here, CH14.18 produced higher lysis, whereas later on, associated with increasing T-cell counts, NG-CU was more effective. With both antibody constructs complete eradication of neuroblastoma cells (LS+LAN-1) was detectable. Conclusion: NG-CU showed enhanced cytotoxicity compared to CH14.18 even in lower concentrations and E:T ratios. The bispecific design represents an alternative to classical CH14.18 based therapies. Dependent on the differential recovery of effector cells post-transplant, either NK-cell based or T-cell based antibody constructs might result in optimal antitumor activity. Citation Format: Anne-Marie Lang, Gundram Jung, Ursula Seidel, Florian Heubach, Armin Rabsteyn, Patrick Schlegel, Christian Seitz, Emmanuelle Moraes Ribeiro, Rupert Handgretinger, Peter Lang. A T-cell utilizing bispecific anti-CD3/GD2 construct mediates superior in vitro efficacy compared to CH14.18 mAb in neuroblastoma patients after allogeneic SCT [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A140.

Targeting CD37 in B-Cell Malignancies Using the Novel Therapeutic Duohexabody-CD37 Results in Efficient Killing of Tumor B-Cells Ex Vivo Via Complement-Dependent Cytotoxicity, Even in Relapsed and/or Refractory Patient Samples

CD37 is a tetraspanin molecule expressed on mature B-cells, but absent on normal stem cells and plasma cells. Due to this cellular distribution, which is highly similar to CD20, CD37 has gained attention as a target for B-cell lymphoma, especially for rituximab-resistant and relapsed patients.The CD37-targeting antibody-based products that are currently being evaluated in the clinic are generally poor inducers of complement-dependent cytotoxicity (CDC) - a rapid and powerful Fc-mediated effector function. DuoHexaBody-CD37 is a bispecific IgG1 antibody targeting two non-overlapping epitopes in the large extracellular loop of CD37, with a hexamerization-enhancing mutation (E430G) in the IgG Fc domain. The E430G mutation increases the intrinsic capacity of IgG1 molecules to form hexameric antibody clusters upon binding to membrane-bound antigens, thereby facilitating binding of the hexavalent complement component C1q and subsequent CDC activity (Diebolder et al., Science 2014; de Jong et al., PLoS Biology 2016). The combination of dual epitope targeting and enhanced hexamerization of DuoHexaBody-CD37 upon binding to the cell surface significantly increases complement activation and CDC efficacy in CD37-positive B-cells.To assess its clinical relevance and potential, we tested the capacity of DuoHexaBody-CD37 to induce ex vivo CDC activity using a broad range of B-cell lymphoma and leukemia patient samples (Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Chronic Lymphocytic Leukemia, n=40) derived from both newly diagnosed (ND) (n=23) and relapsed and/or refractory (RR) patients (n=17), most of whom had received prior treatment with rituximab. The CDC activity of DuoHexaBody-CD37 was compared to the CDC activity of the CD20 antibodies rituximab, ofatumumab and obinutuzumab. Our results revealed that, independent of the B-cell malignancy subtype, DuoHexaBody-CD37 induced potent CDC activity with a median lysis of 82% (n=40, both RR and ND) at 10 µg/ml, and significantly outperformed all three tested CD20 antibodies (18%, 24% and 51% median lysis for rituximab, obinutuzumab and ofatumumab, respectively). Furthermore, CDC induced by DuoHexaBody-CD37 was shown to be less sensitive to the expression of complement regulatory proteins (CD46, CD55, CD59), compared to the tested CD20 antibodies, although the few patient samples that were resistant to DuoHexaBody-CD37 in the ex vivo CDC assays expressed significantly higher CD59 levels than the responsive samples.Importantly, while CDC induced by the tested CD20 antibodies was lower in samples derived from RR patients than from ND patients, DuoHexaBody-CD37 was equally effective in inducing CDC in samples derived from ND and RR patients that received prior treatment with rituximab. In a direct comparison to the strong CDC inducer ofatumumab, DuoHexaBody-CD37 induced significantly more potent CDC in samples from 17 RR CLL and B-cell lymphoma patients with a median lysis of 82% for DuoHexaBody-CD37 versus 29% for ofatumumab (p<0.001) (Fig. 1).In conclusion, our results indicate great therapeutic potential for DuoHexaBody-CD37 in B cell malignancies, based on its strong capacity to induce CDC, even in ex vivo samples from patients that had relapsed from or were refractory to rituximab-containing treatments.Figure1 Complement-dependent cytotoxicity induced by DuoHexaBody-CD37, rituximab, ofatumumab or obinutuzumab at 10 µg/mL in frozen CLL or lymphoma B-cells of relapsed and/ or refractory patients that received prior treatment with rituximab (n=17). Statistical analysis was performed using a non-parametric t-test (Wilcoxon). ***p < 0.001 and ****p < 0.0001. Data is shown as mean ± SD. [Display omitted] DisclosuresVan Der Horst:Genmab: Research Funding. Oostindie:Genmab: Employment, Equity Ownership. Overdijk:Genmab: Employment, Equity Ownership. Chamuleau:Gilead: Research Funding; Genmab: Research Funding; celgene: Research Funding; BMS: Research Funding. Breij:Genmab: Employment, Equity Ownership. Mutis:Celgene: Research Funding; Novartis: Research Funding; Gilead: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding.

Factors Associated with Long-Term Outcome in 191 Patients with Ilio-Femoral DVT Treated With Catheter-Directed Thrombolysis

To identify factors associated with long-term treatment success after catheter-directed thrombolysis (CDT) for acute deep venous thrombosis (DVT) involving the ilio-femoral vein. This was a non-randomised observational cohort study. From 1999 to 2013, 191 consecutive patients (203 limbs) attending a tertiary vascular centre at Gentofte University Hospital, Denmark underwent CDT. All patients had ultrasonically verified acute ilio-femoral DVT with open distal popliteal vein and calf veins. Patients were seen in the outpatient clinic 6 weeks, 3, 6, and 12 months, and annually thereafter following the DVT. Successful outcome was defined as patent deep veins without reflux on Duplex ultrasound scanning (DUS). Data were collected prospectively as per protocol and analysed retrospectively. Median age was 27 years (range 14-74 years) and overall median lysis time was 56 h (range 22-146 h). A stent was placed in 106 limbs (52%). Six patients had major bleeding. The median follow-up was 5 years (range 1 month-14.3 years). The cumulative rate of patients with deep patent veins without reflux at 7 years was 79%. Multivariate Cox regression analyses showed that symptom duration >2 weeks (hazard ratio (HR) 2.78, 95% CI 1.14-6.73) and chronic post-thrombotic lesions (HR 19.3, 95% CI 7.29-51.2) were significantly associated with poorer outcome, while the pulse-spray technique (HR 0.15, 95% CI 0.05-0.48) was associated with better outcome. Age, gender, side, IVC atresia, stenting, and lysis duration did not affect outcome. In this observational study of CDT for ilio-femoral DVT it was demonstrated that symptom duration less than 2 weeks, absence of chronic post-thrombotic lesions and use of the pulse-spray technique for CDT resulted in better primary patency including normal valve function in the long term.

Editor's Choice – Factors Associated with Long-Term Outcome in 191 Patients with Ilio-Femoral DVT Treated With Catheter-Directed Thrombolysis

To identify factors associated with long-term treatment success after catheter-directed thrombolysis (CDT) for acute deep venous thrombosis (DVT) involving the ilio-femoral vein. This was a non-randomised observational cohort study. From 1999 to 2013, 191 consecutive patients (203 limbs) attending a tertiary vascular centre at Gentofte University Hospital, Denmark underwent CDT. All patients had ultrasonically verified acute ilio-femoral DVT with open distal popliteal vein and calf veins. Patients were seen in the outpatient clinic 6 weeks, 3, 6, and 12 months, and annually thereafter following the DVT. Successful outcome was defined as patent deep veins without reflux on Duplex ultrasound scanning (DUS). Data were collected prospectively as per protocol and analysed retrospectively. Median age was 27 years (range 14-74 years) and overall median lysis time was 56h (range 22-146h). A stent was placed in 106 limbs (52%). Six patients had major bleeding. The median follow-up was 5 years (range 1 month-14.3 years). The cumulative rate of patients with deep patent veins without reflux at 7 years was 79%. Multivariate Cox regression analyses showed that symptom duration >2 weeks (hazard ratio (HR) 2.78, 95% CI 1.14-6.73) and chronic post-thrombotic lesions (HR 19.3, 95% CI 7.29-51.2) were significantly associated with poorer outcome, while the pulse-spray technique (HR 0.15, 95% CI 0.05-0.48) was associated with better outcome. Age, gender, side, IVC atresia, stenting, and lysis duration did not affect outcome. In this observational study of CDT for ilio-femoral DVT it was demonstrated that symptom duration less than 2 weeks, absence of chronic post-thrombotic lesions and use of the pulse-spray technique for CDT resulted in better primary patency including normal valve function in the long term.

PD-1/PD-L1 Blocking Enhances CD33/CD3-Bispecific BiTE® Antibody (AMG 330) Mediated Lysis of Primary AML Cells

Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). In our previous work, the potency of the CD33/CD3-bispecific BiTE® antibody AMG 330 to activate and redirect T-cells to AML cells was evaluated in a long-term culture system using unmanipulated peripheral blood (PB) or bone marrow (BM) samples from AML patients. We were able to show effective elimination of AML cells by AMG 330-activated and -expanded residual autologous T-cells (n=13, Krupka et. al, Blood 2014 123(3):356-65). The goal of the present study was to identify and manipulate factors that interfere with AMG 330-mediated lysis of primary AML cells.In our ex-vivo long-term culture system (n= 32 primary diagnosis, 3 relapse), we observed that successful elimination of primary AML cells was predominantly influenced by the initial effector:target (E:T) ratio. Reduced short-term lysis efficacy was observed for patient samples with low E:T ratios (median lysis: E:T ratio <1:10 23.5% vs >1:10 99.3%) after 4 days of culture. However, after 12 days of culture this effect was less prominent as effector T-cell numbers increased and lysis was observed even in cultures with very low initial E:T ratios (up to an E:T ratio of 1:80).Considering the ubiquitous expression pattern of CD33 within the myeloid compartment, the number of AMG 330 treatment days will be limited due to expected hematotoxicity. Therefore, AMG 330 efficacy might benefit by increasing the E:T ratio within a short time period. Programmed cell death-1 (PD-1) and its primary ligand PD-L1 are immune checkpoints that limit T-cell responses and may contribute to slower lysis kinetics during AMG 330 treatment. Therefore, we assessed PD-1 expression on T-cells and PD-L1 expression on primary AML cells. No constitutive expression of PD-1/PD-L1 on T-cells and corresponding AML cells was found at primary diagnosis (PD-1: n=23; PD-L1: n=193). Upon the addition of AMG 330 to our primary ex-vivo AML cultures, we observed a strong upregulation of PD-1 on activated T-cells, which correlated with the extent of T-cell proliferation (10/10). This was most prominent within the subpopulation of CD4+/CD45RA-/CCR7- effector memory T-cells. Furthermore, in response to AMG 330-mediated T-cell activation, we observed an upregulation of PD-L1 on primary AML cells (16/19). Data obtained from AML cell lines confirmed an upregulation of PD-L1 after co-cultivation with T-cells and AMG 330. This phenomenon was cytokine-mediated as the addition of IFNγ and TNFα also induced PD-L1 expression (n=6). Interestingly, we also observed a PD-L1 upregulation on T-cells upon activation with AMG 330, but to a much lower extent compared to primary AML cells (n=17; mean MFI ratio: T-cells: 4.7; AML cells: 12.1). Blockade of the PD-1/PD-L1 interaction through the addition of an inhibitory antibody induced an increase in T-cell proliferation in ex-vivocultures resulting in enhanced cytotoxicity against primary AML cells (lysis on day 18: with/without PD-1 blocking antibody: 75 vs 44%; fold change T-cell expansion: 6 vs 3). This was accompanied by a significant increase in IFNγ production (with/without PD-1 blocking antibody: 280 pg/ml vs 81 pg/ml). Complimentary experiments using primary AML cells and allogeneic T-cells at defined E:T ratios demonstrated that this effect is most prominent in co-cultures with a low percentage of T-cells within the primary sample (lysis on day 7 with/without PD-L1 blocking antibody: E:T ratio 1:1: 100% vs 94.5%; E:T ratio 1:5: 96.6% vs 82.7%; fold change T-cell expansion: E:T ratio 1:1: 3.1 vs 3.1; E:T ratio 1:5: 23.4 vs 9.8). Intriguingly, upon addition of lenalidomide to our primary AML cultures, we were also able to circumvent the immune inhibitory effect of the PD-1/PD-L1 interaction by a decrease in PD-L1 upregulation on primary AML cells.Our data show that AMG 330-mediated lysis of primary AML cells can be enhanced by inhibiting the PD-1/PD-L1 axis on T-cells and corresponding AML cells. The modulation of the PD-1/PD-L1 axis increased T-cell proliferation and accelerated attaining a beneficial E:T ratio. We hypothesize that PD-L1 upregulation on primary AML cells is a relevant immune escape mechanism employed by AML cells to escape cytokine-mediated immune responses. Prospective clinical trials will be needed to assess the relevance of our finding in AMG 330-treated AML patients. DisclosuresKrupka:AMGEN Inc.: Research Funding. Kufer:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Kischel:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Zugmaier:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Sinclair:AMGEN Inc.: Employment, Equity Ownership. Newhall:AMGEN Inc.: Employment, Equity Ownership. Frankel:AMGEN Inc.: Employment, Equity Ownership. Baeuerle:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership. Subklewe:AMGEN Inc.: Research Funding.

Endogenous fibrinolytic potential in tissue‐plasminogen activator‐modified thromboelastography analysis is significantly decreased in dogs suffering from diseases predisposing to thrombosis

In people, studies have shown that resistance to fibrinolysis could be a contributing factor to thrombosis. Tissue-plasminogen-activated (t-PA) thromboelastography (TEG) has been used to evaluate endogenous fibrinolytic potential. In dogs, TEG has been used for the diagnosis of various hemostatic disorders, but studies evaluating fibrinolysis are limited. Investigations into the potential of t-PA-modified TEG to monitor endogenous fibrinolytic potential are lacking in both healthy dogs and dogs with diseases predisposing to development of thrombosis. The aim of this study was to compare 3 t-PA-modified TEG assays and compare the endogenous fibrinolytic potential in dogs suffering from diseases associated with thrombosis with a group of healthy dogs. Three different TEG assays, such as native, tissue factor-activated, and kaolin-activated, were modified with t-PA and used to compare whole blood samples from 16 healthy control dogs and 20 diseased dogs. Thromboelastography lysis variables were significantly affected by addition of t-PA in all 3 assays. Lysis results in diseased dogs were comparable to those in healthy dogs prior to addition of t-PA. After addition of t-PA, lysis results were significantly decreased in the diseased group compared with healthy dogs. The lowest median lysis levels were found in dogs with systemic inflammation and protein-losing disorders. Addition of t-PA activates fibrinolysis in TEG of blood from both healthy dogs and dogs with diseases predisposing to thrombosis. The significantly decreased fibrinolysis in diseased dogs suggests that this may be a potential prothrombotic risk factor in dogs.

Activation and Expansion of Natural Killer (NK) Cells with Potent Cytotoxicity for Multiple Myeloma

Introduction: Immunotherapy with innately competent natural killer (NK) cells can potentially synergize with chemotherapy by eradicating chemo-refractory residual disease. We recently reported that killer immunoglobulin-like receptor (KIR)-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary myeloma (MM) cells in vitro. In a pilot trial complete remission was achieved in 50% of patients with advanced MM. Although the initial clinical results were encouraging virtually all patients relapsed, suggesting that the number of allo-reactive NK cells transfused was too low to eradicate all MM cells taking into account that MM patients may harbor a tumor burden as large as 109–10 MM cells. This provided the rationale for expanding and activating NK cells from healthy donors with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562 transfectants), as described by Imai et al (Blood 2005; 106:376–383).Methods: PBMNC from 6 healthy donors were co-cultured for 14 days with irradiated K562 cell transfectants. The cultures were then analyzed for fold expansion, gene expression using the Affymetrix U133Plus2.0 microarray platform, immunophenotyping and cytotoxicity.Results: There was an average of 166-fold (range 54–267) expansion of NK cells in the 12 experiments from 6 healthy donors studied after 14 days of co-culture with K562 transfectants without expansion of T-cells. Comparison of gene expression profiles of purified, non-expanded and expanded NK cells of healthy donors showed up-regulation of the expression of activation molecules (e.g. NKG2D, NCRs, DNAM-1), death receptors, granzymes and adhesion molecules on activated/expanded NK cells all consistent with increased proclivity to MM killing. Up regulation of activating receptors was confirmed by flow cytometry. In contrast to non-expanded NK cells, inhibitory KIR receptors were not up regulated on activated NK cells. Using the same number of NK cells, the median lysis of primary MM by expanded NK cells was 62% (range: 49%–70%, n=6) at a 10:1 E:T ratio compared to non-expanded NK cells (median: 23%; range: 15%–34%, p<0.001) (Fig 1). There was no killing of patient normal cells. Blocking experiments demonstrated that antibodies to NKG2D, NCRs, DNAM-1, CD27 and CD69 could partially abrogate NK cell-mediated lysis of MM cells. We also observed that after stimulation with modified K562 cells, the non-alloreactive NK cell population, which under non-activated conditions is inhibited by HLA-C on target cells, could be recruited to kill MM cell lines thus further increasing the number of NK cells with anti-MM activity.Conclusion: K562 transfectants stimulated vigorous expansion of NK cells without expanding T lymphocytes. The expanded/activated NK cells were highly active and killed patient MM cells better than non-expanded NK cells without significant lysis of normal cells. Optimal activation of NK cells can overcome inhibitory signals via KIR and other inhibitory receptors and induce killing of MM by previously inhibited NK cells [Display omitted]

Immediate and early follow-up results of purse spray thrombolysis in patients with peripheral ischaemia.

Pulse spray thrombolysis is a technique of accelerated peripheral thrombolysis which has been evaluated in 38 patients, 29 with threatened limb viability and nine with a viable but critically ischaemic limb. The median length of occlusion was 26.5 (range 3-65) cm. The lytic agent used was recombinant tissue plasminogen activator (rtPA), injected manually via a pulse spray catheter at a concentration of 0.33 mg/ml and a bolus size of 0.2 ml. The median total dose of rtPA was 18 (range 5-35)mg. Patency and flow were completely restored in 34 of 38 patients and clinical success at 30 days was maintained in 27 of 38. The median lysis time was 120 (range 35-1125) min, compared with a median duration of 1545 (range 42-5760) min in 120 consecutive patients previously treated with conventional low-dose infusion thrombolysis using rtPA. This represents a 12-fold reduction in lysis time (P < 0.001). The overall estimated cumulative limb salvage rate for the 38 patients who entered the study was 84 per cent and the cumulative patency rate for the 34 patients in whom patency was restored was 74 per cent at 18 months follow-up. Pulse spray thrombolysis rapidly restores patency with good limb salvage at 30 days; the benefit is sustained even in patients with limbs at immediate risk of irreversible ischaemic injury who are not considered suitable for conventional thrombolysis.

Deep venous insufficiency: The relationship between lysis and subsequent reflux

Purpose: Although venous valvular insufficiency is well recognized as the most important etiologic mechanism in the development of the postthrombotic syndrome, the factors contributing to valve incompetence after deep venous thrombosis remain obscure.Methods: To establish the relationship between recanalization and valve competence, 113 patients with acute deep venous thrombosis were studied with serial duplex ultrasonography.Results: Median lysis times for segments developing reflux (214 to 474 days) were 2.3 to 7.3 times longer than for corresponding segments not developing reflux (65 to 130 days) for all except the posterior tibial vein. In the posterior tibial vein, median lysis times for those with and without reflux were nearly identical (72 vs 80 days). The median time to onset of reflux was significantly less than the median lysis time in the mid and distal superficial femoral veins and was simultaneous with recanalization in all other segments.Conclusions: Early recanalization is important in preserving valve integrity for all but the posterior tibial segment. However, the small number of patients with reflux despite early lysis (< 1 month) or without reflux despite relatively late lysis (> 9 to 12 months) suggests that other factors may also contribute to the development of valvular incompetence. These factors may be particularly important in the posterior tibial vein, in which lysis time has little relationship to the ultimate development of reflux.

Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease

The high rate of severe cytomegalovirus (CMV) disease after bone marrow transplantation (BMT) is related to the profound immunodeficiency posttransplant. Because cytotoxic T lymphocytes (CTL) have been implicated in resistance to viral infections, we examined the restoration of the CMV-specific CTL response in 20 patients who received bone marrow from HLA-matched, CMV-seropositive donors. Blood specimens were obtained from patients at 1, 2, and 3 months after BMT and from the donors on a single occasion. Peripheral blood mononuclear cells were cocultured with CMV-infected donor-derived fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected and uninfected autologous or HLA-mismatched fibroblasts. Cytolytic activity was detected in all 20 donors, with specificity for autologous CMV- infected targets demonstrable in 17 (median CMV-specific lysis at an effector:target ratio of 15:1 was 32%, range 18% to 83%). Specific lysis was mediated by CD8+, class I-restricted T cells, as shown by inhibition with anti-class I monoclonal antibody and by selective depletion of effector cells. By contrast, CMV-specific CTL were detected in only 10 of 20 patients after BMT (median lysis 29% at 3 months post-BMT). None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after BMT died with CMV pneumonia. These results demonstrate that restoration of CMV-specific CTL responses may require an extended time period after BMT in some patients, and that such patients are at increased risk of developing severe CMV disease. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV- specific CD8+ CTL clones derived from the bone marrow donor are now being pursued.

Cytotoxic T-Lymphocyte Response to Cytomegalovirus After Human Allogeneic Bone Marrow Transplantation: Pattern of Recovery and Correlation With Cytomegalovirus Infection and Disease

AbstractThe high rate of severe cytomegalovirus (CMV) disease after bone marrow transplantation (BMT) is related to the profound immunodeficiency posttransplant. Because cytotoxic T lymphocytes (CTL) have been implicated in resistance to viral infections, we examined the restoration of the CMV-specific CTL response in 20 patients who received bone marrow from HLA-matched, CMV-seropositive donors. Blood specimens were obtained from patients at 1,2, and 3 months after BMT and from the donors on a single occasion. Peripheral blood mononuclear cells were cocultured with CMV-infected donor-derived fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected and uninfected autologous or HLA-mismatched fibroblasts. Cytolytic activity was detected in all 20 donors, with specificity for autologous CMV-infected targets demonstrable in 17 (median CMV-specific lysis at an effector:target ratio of 15:1 was 32%, range 18% to 83%). Specific lysis was mediated by CD8+, class l-restricted T cells, as shown by inhibition with anticlass I monoclonal antibody and by selective depletion of effector cells. By contrast, CMV-specific CTL were detected in only 10 of 20 patients after BIVJT (median lysis 29% at 3 months post-BMT). None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after BMT died with CMV pneumonia. These results demonstrate that restoration of CMV-specific CTL responses may require an extended time period after BMT in some patients, and that such patients are at increased risk of developing severe CMV disease. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV-specific CD8+ CTL clones derived from the bone marrow donor are now being pursued.

Cytotoxic antibodies in congenital hypothyroidism.

Antibody-dependent cell-mediated cytotoxicity was determined in serum samples from 61 patients with congenital hypothyroidism (CH) and 46 of their mothers, using a 51chromium release assay with human thyroid cells. Such cytotoxic antibodies were found in 31% of the children and 24% of their mothers. In the 12 (of 37) newborn infants with hypothyroidism (group 1) who had positive tests, the median cell lysis was 20.1% compared to 4% in healthy newborns. In the 7 (of 29) mothers who had positive tests, the median lysis was 21.3%. In 24 older children with CH (group 2), cytotoxic activity was found in 6, and in them the median lysis was 19.7%; 4 of their mothers had increased lytic activity (median, 20.6%). In 96% of the mother-infant pairs concordance existed between the cytotoxic activity in mothers and their children. Increased thyroid cell lysis was found in 6 of 20 children with athyrosis (30%) and in 6 of 23 children with an ectopic gland (50%), but in only 1 of 14 children with a normal gland. Thyroglobulin antibodies were detected in 2 and antithyroid microsomal antibodies in 3 children-mother pairs. TSH receptor antibodies were present in 5 children and 3 of their mothers. The results indicate that intrauterine autoimmune thyroiditis could be involved in the pathogenesis of CH in some patients.