Whole body-element composition of Atlantic salmon Salmo salar influenced by migration direction and life stage in three distinct populations.

Body-element content was measured for three life stages of wild Atlantic salmon Salmo salar from three distinct Newfoundland populations as individuals crossed between freshwater and marine ecosystems. Life stage explained most of the variation in observed body-element concentration whereas river of capture explained very little variation. Element composition of downstream migrating post-spawn adults (i.e. kelts) and juvenile smolts were similar and the composition of these two life stages strongly differed from adults migrating upstream to spawn. Low variation within life stages and across populations suggests that S. salar may exert rheostatic control of their body-element composition. Additionally, observed differences in trace element concentration between adults and other life stages were probably driven by the high carbon concentration in adults because abundant elements, such as carbon, can strongly influence the observed concentrations of less abundant elements. Thus, understanding variation among individuals in trace elements composition requires the measurement of more abundant elements. Changes in element concentration with ontogeny have important consequences the role of fishes in ecosystem nutrient cycling and should receive further attention.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Residency time, migration route and survival of Atlantic salmon Salmo salar smolts in a Canadian fjord.

Atlantic salmon Salmo salar smolts (n = 181) from two rivers were surgically implanted with acoustic transmitters and released to determine migration route, residency time and survival in a 50 km long estuarine fjord located on the south coast of Newfoundland, Canada. Data obtained from automated receivers placed throughout the Bay d'Espoir fjord indicated that migrating smolts used different routes to reach the outer areas of the fjord. The duration of time that smolts spent in the immediate estuary zone also differed between the two localities (7 and 17 days) although the total time smolts were resident in the fjord was similar and extensive (40 days). Many smolts were resident for periods of 4-8 weeks moving back and forth in the outer part of the fjord where maximum water depths range from 300 to 700 m. Survival in the estuary zone was greater for smolts with prolonged residency in estuarine habitat. Overall smolt survival to the fjord exit was moderately high (54-85%), indicating that the initial phase of migration did not coincide with a period of unusually high mortality.

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells.

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo-high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL-2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental regulation of a mucinlike glycoprotein selectively expressed on natural killer cells.

Natural killer (NK) cells are CD3:TCR-, CD16+, CD56+ large granular lymphocytes capable of recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. Two functionally distinct populations of peripheral blood NK cells can be differentiated by their surface expression of an isoform of the neural cell adhesion molecule (CD56). CD56bright NK cells have the attributes of an undifferentiated cell, in that they proliferate in response to exogenous cytokines, but exert poor cytolytic activity. CD56dim NK cells have the attributes of a more differentiated cell, in that they proliferate poorly in response to exogenous cytokines, but are potent cytolytic effector cells. Here we describe the molecular characterization of a NK cell restricted epitope (PEN5) that is selectively expressed on the functionally differentiated CD56dim NK cells. PEN5+ NK cells proliferate poorly in response to interleukin 2 (IL-2), but are potent cytolytic effectors, whereas PEN5- NK cells proliferate in response to IL-2, but are poor cytolytic effectors. Biochemical and immunochemical analyses reveal the PEN5 epitope to be an unusual sulfated poly-N-lactosamine carbohydrate related to keratan sulfate glycosaminoglycans. Immunoprecipitates prepared using a monoclonal antibody reactive with PEN5 include two polydisperse membrane-bound glycoproteins, PEN5 alpha (120-170 kD) and PEN5 beta (210-245 kD). Enzymatic deglycosylation reduces the apparent molecular weight of both PEN5 isoforms by 80-90%, and classifies PEN5 beta as a mucinlike glycoprotein. The surface expression of the PEN5 epitope is downmodulated by stimuli that induce NK cell proliferation, and it is absent from leukemic NK cells of patients with granular lymphocyte proliferative disorder. Taken together, these results indicate that PEN5 is a developmentally regulated poly-N-lactosamine epitope associated with a mucin-type glycoprotein, whose expression is restricted to the population of nonproliferative NK cells fully committed to cytolytic effector function.

Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF.

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex-unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study.

BACKGROUND: We previously reported results of the phase 2, multicenter PINNACLE study, which confirmed the substantial single-agent activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma (MCL). MATERIALS AND METHODS: We report updated time-to-event data, in all patients and by response to treatment, after extended follow-up (median 26.4 months). RESULTS: Median time to progression (TTP) was 6.7 months. Median time to next therapy (TTNT) was 7.4 months. Median overall survival (OS) was 23.5 months. In responding patients, median TTP was 12.4 months, median duration of response (DOR) was 9.2 months, median TTNT was 14.3 months, and median OS was 35.4 months. Patients achieving complete response had heterogeneous disease characteristics; among these patients, median TTP and DOR were not reached, and median OS was 36.0 months. One-year survival rate was 69% overall and 91% in responding patients. Median OS from diagnosis was 61.1 months, after median follow-up of 63.7 months. Activity was seen in patients with refractory disease and patients relapsing following high-intensity treatment. Toxicity was generally manageable. CONCLUSIONS: Single-agent bortezomib is associated with lengthy responses and notable survival in patients with relapsed or refractory MCL, with considerable TTP and TTNT in responding patients, suggesting substantial clinical benefit.

Effectiveness of a publicly-funded demonstration program to promote management of dryland salinity.

Community and catchment-based approaches to salinity management continue to attract interest in Australia. In one such approach, Catchment Demonstration Initiative (CDI) projects were established by the Western Australian (WA) Government in 2000 for targeted investment in large-scale catchment-based demonstrations of integrated salinity management practices. The aim was to promote a process for technically-informed salinity management by landholders. This paper offers an evaluation of the effectiveness of one CDI project in the central wheatbelt of WA, covering issues including: its role in fostering adoption of salinity management options, the role of research and the technical requirements for design and implementation of on-ground works, the role of monitoring and evaluation, the identification and measurement of public and private benefits, comparison and identification of the place and value of plant-based and engineering-based options, reliance on social processes and impacts of constraints on capacity, management of governance and administration requirements and an appreciation of the value of group-based approaches. A number of factors may reduce the effectiveness of CDI-type approaches in facilitating landholder action to address salinity, many of these are socially-based. Such approaches can create considerable demands on landholders, can be expensive (because of the planning and accountability required) on the basis of dollars per hectare impacted, and can be difficult to garner ownership from all involved. An additional problem could be that few community groups would have the capacity to run such programs and disseminate the new knowledge so that the CDI-type projects can impact outside the focus catchment. In common with many publicly-funded approaches to salinity, we found that direct benefits on public assets are smaller than planned and that results from science-based requirements of monitoring and evaluation have long lead times, causing farmers to either wait for the information or act sooner and take risks based on initial results. We also found that often it is a clear outline of the process that is of most importance in decision making as opposed to the actual results. We identified limitations in regulatory processes and the capacity for local government to engage in the CDI. The opportunities that CDI-type approaches provide centre around the value of its group-based approach. We conclude that they can overcome knowledge constraints in managing salinity by fostering group-based learning, offer a structured process of trialling options so that the costs and benefits can be clearly and transparently quantified, and avoid the costly mistakes and "learning failures" of the past.

Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2.

The immunologic consequences of prolonged infusions of rIL-2 in doses that produce physiologic serum concentrations of this cytokine were investigated. rIL-2 in doses of 0.5-6.0 x 10(6) U/m2 per d (3.3-40 micrograms/m2 per d) was administered by continuous intravenous infusion for 90 consecutive days to patients with advanced cancer. IL-2 concentrations (25 +/- 25 and 77 +/- 64 pM, respectively) that selectively saturate high-affinity IL-2 receptors (IL-2R) were achieved in the serum of patients receiving rIL-2 infusions of 10 micrograms/m2 per d and 30 micrograms/m2 per d. A gradual, progressive expansion of natural killer (NK) cells was seen in the peripheral blood of these patients with no evidence of a plateau effect during the 3 mo of therapy. A preferential expansion of CD56bright NK cells was consistently evident. NK cytotoxicity against tumor targets was only slightly enhanced at these dose levels. However, brief incubation of these expanded NK cells with IL-2 in vitro induced potent lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Infusions of rIL-2 at 40 micrograms/m2 per d produced serum IL-2 levels (345 +/- 381 pM) sufficient to engage intermediate affinity IL-2R p75, which is constitutively expressed by human NK cells. This did not result in greater NK cell expansion compared to the lower dose levels, but did produce in vivo activation of NK cytotoxicity, as evidenced by lysis of NK-resistant targets. There was no consistent change in the numbers of CD56- CD3+ T cells, CD56+ CD3+ MHC-unrestricted T cells, or B cells during infusions of rIL-2 at any of the dosages used. This study demonstrates that prolonged infusions of rIL-2 in doses that saturate only high affinity IL-2R can selectively expand human NK cells for an extended period of time with only minimal toxicity. Further activation of NK cytolytic activity can also be achieved in vivo, but it requires concentrations of IL-2 that bind intermediate affinity IL-2R p75. Clinical trials are underway attempting to exploit the differing effects of various concentrations of IL-2 on human NK cells in vivo.

Macrophage activation syndrome and other systemic inflammatory conditions after BMT.

Autologous hematopoietic cell transplantation (HCT) is being used to treat autoimmune diseases refractory to conventional therapy, including rheumatoid arthritis. Macrophage activation syndrome (MAS) is a descriptive term for a systemic inflammatory disorder that has been described in patients with juvenile rheumatoid arthritis (JRA). This case report describes a young adult with systemic JRA (sJRA) who developed MAS on day # 12 post-autologous transplantation. The patient developed high fever, laboratory evidence of disseminated intravascular coagulation (DIC), hepatocellular injury, pancytopenia and hyper-ferritinemia. All viral, bacterial and fungal studies were negative and the patient improved with high-dose glucocorticosteroid and cyclosporine therapy. Extreme elevation of serum ferritin was documented and helpful in monitoring response to therapy. A number of systemic inflammatory syndromes have been described in association with HCT. These include DIC, 'engraftment syndrome,' infection-associated hemophagocytic syndrome and familial hemophagocytic lymphohistiocytosis. Macrophage activation syndrome presents with features of DIC and is closely related or identical to infection-associated hemophagocytic syndrome. The diagnosis needs to be established in a timely fashion because early and appropriate treatment may improve outcome.

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex.

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.

Prevention of graft-versus-host disease by selective depletion of CD6-positive T lymphocytes from donor bone marrow.

PURPOSE: Acute and chronic graft-versus-host disease (GVHD) continues to be the major causes of morbidity and mortality after allogeneic bone marrow transplantation (BMT). In this study, we have evaluated the clinical effects of selective in vitro T-cell depletion of donor allogeneic bone marrow by using a single monoclonal antibody ([MoAb] anti-T12, CD6) and rabbit complement. This antibody recognizes mature T cells, but not other cellular elements such as natural-killer (NK) cells, B cells, and myeloid precursors. PATIENTS AND METHODS: From August 1983 to April 1991, 112 consecutive adult patients with hematologic malignancies underwent BMT with bone marrow from HLA-identical sibling donors. Marrow was harvested and depleted of mature T lymphocytes ex vivo by the use of three rounds of incubation with an anti-T12 antibody and rabbit complement. The preparative regimen consisted of cyclophosphamide and fractionated total body irradiation (TBI) in 108 patients. No patients received prophylactic immune suppression post-BMT. Purgation by anti-T12 was used as the only method for the prevention of GVHD. RESULTS: Twenty patients (18%) developed acute GVHD (grade 2 to 4); only eight patients developed chronic GVHD. The incidence of GVHD did not increase significantly with age. Only three of 112 patients (2.7%) exhibited acute graft failure. One patient developed late graft failure that was associated with cytomegalovirus (CMV) infection. Within the subset of 50 patients who had not previously undergone unsuccessful conventional therapy (acute leukemia in first remission or chronic myelogenous leukemia [CML] in stable phase), we estimated by the Kaplan-Meier method that the probability of disease-free survival was 50% at 3 years post-BMT, with a median follow-up of 44 months. The treatment-related mortality rate in this group was only 14% and was independent of patient age. CONCLUSIONS: We conclude that selective in vitro T-cell depletion with an anti-T12 monoclonal antibody effectively reduces the incidence of both acute and chronic GVHD after allogeneic BMT without compromising engraftment. Moreover, depletion of CD6-positive cells from donor marrow obviates the need to administer immune suppressive medications to the majority of patients. This approach reduces the morbidity and mortality of allogeneic BMT and permits the BMT of older patients.

Autologous bone marrow transplantation in poor-prognosis intermediate-grade and high-grade B-cell non-Hodgkin's lymphoma in first remission: a pilot study.

PURPOSE: Using high-dose therapy and autologous bone marrow transplantation (ABMT) to overcome cellular resistance and eradicate minimal disease, we initiated a pilot study during first remission in patients with non-Hodgkin's lymphoma (NHL) to examine whether the long-term disease-free survival (DFS) rate can be improved for patients with poor-prognosis intermediate/high-grade NHL. PATIENTS AND METHODS: Twenty-six patients with advanced-stage diffuse intermediate/high-grade B-cell NHL (including 16 patients with diffuse small cleaved-cell [DSC]) were selected at presentation by histologic and clinical characteristics to have less than a 25% probability of long-term DFS with conventional treatment. After induction chemotherapy, 16 patients were in complete remission (CR) and 10 were in a minimal disease state. Patients were then treated with high-dose cyclophosphamide, total-body irradiation (TBI), and anti-B-cell monoclonal antibody-purged ABMT. RESULTS: Following ABMT, no acute in-hospital treatment deaths occurred, and engraftment of granulocytes and platelets was significantly faster than for patients undergoing ABMT who were in second or subsequent remission. Of 26 patients, 21 remain in CR maintained without continued therapy, three relapsed in sites of prior nodal disease (4.8, 5.4, and 28 months post-ABMT), and two died in remission. The DFS rate is estimated to be 85% at 28 months and thereafter. The median follow-up period for the 21 patients who are alive and disease-free is 32 months. CONCLUSION: This pilot study suggests that consolidation of first remission with ABMT may improve the long-term DFS rate for diffuse intermediate/high-grade NHL patients at high risk for relapse.

Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model.

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

Immunological effects of interleukin 12 administered by bolus intravenous injection to patients with cancer.

The immunological effects of recombinant human interleukin 12 (rhIL-12) administration were examined during the conduct of a Phase I clinical trial. Forty patients with advanced cancer received bolus i.v. injections of rhIL-12 in doses ranging between 3 and 1000 ng/kg. Dose-dependent increases in serum IFN-gamma levels were seen during rhIL-12 therapy. Significant lymphopenia was observed 24 h after single i.v. injections of rhIL-12 at each dose level. The degree of lymphopenia was dose dependent, and a plateau effect was seen with rhIL-12 doses of 100 ng/kg and higher. Lymphocyte counts reached nadir levels at approximately 10 h after rhIL-12 injection and returned to baseline within 14 days postinjection. Rebound lymphocytosis, as seen after interleukin 2 therapy, was not observed after recovery from rhIL-12-induced lymphopenia. rhIL-12-induced lymphopenia involved all major lymphocyte subsets, although natural killer (NK) cell numbers were the most profoundly affected, and CD4 T-cell numbers were the least affected. CD2, LFA-1, and CD56 were transiently up-regulated on the surface of NK cells exposed to rhIL-12 in vivo. Peripheral blood mononuclear cells obtained from cancer patients before rhIL-12 therapy exhibited defective NK cell cytotoxicity and T-cell-proliferative responses. Peripheral blood mononuclear cells obtained after lymphocyte recovery following the administration of a single 500 ng/kg dose of rhIL-12 displayed augmented NK cell cytolytic activity in four of four patients tested and enhanced T-cell proliferation in three of four patients tested. These studies confirm that doses of rhIL-12 resulting in significant immunological activity can be administered with acceptable toxicity to cancer patients. Furthermore, rhIL-12 therapy can reverse defects in NK cell and T-cell function that are associated with advanced cancer in humans.

Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies.

A Phase I dose escalation trial of i.v. administered recombinant human interleukin 12 (rhIL-12) was performed to determine its toxicity, maximum tolerated dose (MTD), pharmacokinetics, and biological and potential antineoplastic effects. Cohorts of four to six patients with advanced cancer, Karnofsky performance >/=70%, and normal organ function received escalating doses (3-1000 ng/kg/day) of rhIL-12 (Genetics Institute, Inc.) by bolus i.v. injection once as an inpatient and then, after a 2-week rest period, once daily for five days every 3 weeks as an outpatient. Therapy was withheld for grade 3 toxicity (grade 4 hyperbilirubinemia or neutropenia), and dose escalation was halted if three of six patients experienced a dose-limiting toxicity (DLT). After establishment of the MTD, eight more patients were enrolled to further assess the safety, pharmacokinetics, and immunobiology of this dose. Forty patients were enrolled, including 20 with renal cancer, 12 with melanoma, and 5 with colon cancer; 25 patients had received prior systemic therapy. Common toxicities included fever/chills, fatigue, nausea, vomiting, and headache. Fever was first observed at the 3 ng/kg dose level, typically occurred 8-12 h after rhIL-12 administration, and was incompletely suppressed with nonsteroidal anti-inflammatory drugs. Routine laboratory changes included anemia, neutropenia, lymphopenia, hyperglycemia, thrombocytopenia, and hypoalbuminemia. DLTs included oral stomatitis and liver function test abnormalities, predominantly elevated transaminases, which occurred in three of four patients at the 1000 ng/kg dose level. The 500 ng/kg dose level was determined to be the MTD. This dose, administered by this schedule, was associated with asymptomatic hepatic function test abnormalities in three patients and an onstudy death due to Clostridia perfringens septicemia but was otherwise well tolerated by the 14 patients treated in the dose escalation and safety phases. The T1/2 elimination of rhIL-12 was calculated to be 5.3-9.6 h. Biological effects included dose-dependent increases in circulating IFN-gamma, which exhibited attenuation with subsequent cycles. Serum neopterin rose in a reproducible fashion regardless of dose or cycle. Tumor necrosis factor alpha was not detected by ELISA. One of 40 patients developed a low titer antibody to rhIL-12. Lymphopenia was observed at all dose levels, with recovery occurring within several days of completing treatment without rebound lymphocytosis. There was one partial response (renal cell cancer) and one transient complete response (melanoma), both in previously untreated patients. Four additional patients received all proposed treatment without disease progression. rhIL-12 administered according to this schedule is biologically and clinically active at doses tolerable by most patients in an outpatient setting. Nonetheless, additional Phase I studies examining different schedules and the mechanisms of the specific DLTs are indicated before proceeding to Phase II testing.

Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia.

Cell line NKL was established from the the peripheral blood of a patient with CD3-CD16+CD56+ large granular lymphocyte (LGL) leukemia. The neoplastic LGL of this patient mediated natural killing and antibody-dependent cellular cytotoxicity (ADCC) and exhibited proliferative responses similar to normal CD16+CD56dim natural killer (NK) cells. The Morphology of NKL cells resembles that of normal activated NK cells. The karyotype of NKL is 47, XY, add (1) (q42), +6 del (6) (q15 q23), del (17) (p11). NKL cells express CD2, CD6, CD11a, CD26, CD27, CD29, CD38, CD43, CD58, CD81, CD94, CD95, class II MHC, and the C1.7.1 antigen, but do not express detectable levels of CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD28, alpha/beta or gamma/delta T cell receptors on the cell surface. The density of the CD16, CD56, and CD57 antigens declined markedly on NKL cells during prolonged im vitro culture. Nevertheless, NKL cells can mediate ADCC as well as natural killing. NKL cells are strictly dependent on interleukin-2 (IL-2) for sustained growth and die if deprived of IL-2 for more than 7 days. NKL cells proliferate in response to concentrations of IL-2 as low as 1 pM, but an optimal proliferative response requires approximately 100 pM IL-2. NKL cells growing in the presence of IL-2 express abundant IL-2R alpha with little or no detectable IL-2 beta or gamma chain on the cell surface; NKL cells deprived of IL-2 express high levels of both IL-2R alpha and beta. IL-4, IL-7, and IL-12, unlike IL-2, do not maintain the viability of NKL cells. Furthermore, IL-1, IL-4, IL-6, IL-7, IL-12, tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha) and IFN-gamma do not support the growth of NKL cells. The NKL cell line may prove useful for studies of human NK cell biology.

Relationship of ECMO duration with outcomes after pediatric cardiac surgery: a multi-institutional analysis.

BACKGROUND: There are very sparse data on the outcomes of children receiving prolonged extracorporeal membrane oxygenation (ECMO) after cardiac surgery. This study was aimed to evaluate the association of ECMO duration with outcomes in children undergoing surgery for congenital heart disease using the Pediatric Health Information System (PHIS) database. METHODS: Patients aged ≤18 years receiving ECMO after pediatric cardiac surgery (with or without cardiopulmonary bypass) at a PHIS-participating hospital (2004-2013) were included. De-identified data obtained from retrospective, observational dataset included demographic information, baseline characteristics, pre-ECMO risk factors, operation details, patient diagnoses, and center data. Outcomes evaluated included in-hospital mortality, length of mechanical ventilation, length of ICU stay, length of hospital stay, and hospital charges. Cox proportional hazards models were fitted to study the probability of study outcomes as a function of ECMO duration. RESULTS: Nine hundred ninety-eight patients from 37 hospitals qualified for inclusion. The median duration of ECMO run was 4 days (IQR: 1.7). After adjusting for patient and center characteristics, there was 12% increase in the odds of mortality for every 24 hours increase in ECMO duration (OR: 1.12, 95% CI: 1.07-1.18, P<0.001). Patients receiving longer duration of ECMO were associated with longer length of mechanical ventilation, longer length of ICU stay, longer length of hospital stay, and higher hospital charges. CONCLUSION: Data from this large multicenter database suggest that longer duration of ECMO support after pediatric cardiac surgery is associated with worsening outcomes.

Flt3 ligand antitumor activity in a murine breast cancer model: a comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action.

We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing Flt3L (C3Lt-Flt3L) and GM-CSF (C3L5-GMCSF) by injecting 1 x 10(4) cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 x 10(3) cells; or low, 1 x 10(3) cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5-Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.

Molecular cloning of the canine angiotensin II receptor. An AT1-like receptor with reduced affinity for DuP753.

Canine aortic strip studies revealed insensitivity of angiotensin II (AII)-induced aortic contraction to inhibition by the non-peptide antagonist DuP753 (pKB = 6.7 +/- 0.1). In order to determine the origin of this phenomenon we cloned the canine homologue of the AT1 AII receptor. Expression of this cDNA in COS-7 cells indicated a low affinity of DuP753 for the cloned receptor (KD = 92 nM). The predicted amino acid sequence is highly homologous to other mammalian AT1 receptors; sequence comparisons suggest the pharmacological difference may be the result of a threonine residue in position 163 in the IVth transmembrane domain.