Sustainable aquaculture through the One Health lens.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics - underpinned by evidence, policy and legislation - that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production - and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design.

Characterization of microsporidian Ameson herrnkindi sp. nov. infecting Caribbean spiny lobsters Panulirus argus.

The Caribbean spiny lobster Panulirus argus supports a large and valuable fishery in the Caribbean Sea. In 2007-2008, a rare microsporidian parasite with spore characteristics typical of the Ameson genus was detected in 2 spiny lobsters from southeast Florida (FL). However, the parasite species was not confirmed by molecular analyses. To address this deficiency, reported here are structural and molecular data on single lobsters displaying comparable 'cotton-like' abdominal muscle containing ovoid microsporidian spores found at different locations in FL in 2014 and 2018 and in Saint Kitts and Nevis Islands in 2017. In the lobster from 2014, multiple life stages consistent with an Ameson-like monokaryotic microsporidian were detected by transmission electron microscopy. A partial (1228 bp) small subunit (SSU) rRNA gene sequence showed each microsporidia to be identical and positioned it closest phylogenetically to Ameson pulvis in a highly supported clade also containing A. michaelis, A. metacarcini, A. portunus, and Nadelspora canceri. Using ecological, pathological, ultrastructural, and molecular data, the P. argus microsporidian has been assigned to a distinct species: Ameson herrnkindi.

Linking dendroecology and association genetics in natural populations: Stress responses archived in tree rings associate with SNP genotypes in silver fir (Abies alba Mill.).

Genetic association studies in forest trees would greatly benefit from information on the response of trees to environmental stressors over time, which can be provided by dendroecological analysis. Here, we jointly analysed dendroecological and genetic data of surviving silver fir trees to explore the genetic basis of their response to the iconic stress episode of the 1970s and 1980s that led to large-scale forest dieback in Central Europe and has been attributed to air pollution. Specifically, we derived dendrophenotypic measures from 190 trees in the Bavarian Forest that characterize the resistance, resilience and recovery during this growth depression, and in the drought year in 1976. By focusing on relative growth changes of trees and by standardizing the dendrophenotypes within stands, we accounted for variation introduced by micro- and macroscale environmental differences. We associated the dendrophenotypes with single nucleotide polymorphisms (SNPs) in candidate genes using general linear models (GLMs) and the machine learning algorithm random forest with subsequent feature selection. Most trees at our study sites experienced a severe growth decline from 1974 until the mid-1980s with minimum values during the drought year. Fifteen genes were associated with the dendrophenotypes, including genes linked to photosynthesis and drought stress. With our study, we show that dendrophenotypes can be a powerful resource for genetic association studies that permit to account for micro- and macroenvironmental variation when data are derived from natural populations. We call for a wider collaboration of dendroecologists and forest geneticists to integrate individual tree-level dendrophenotypes in genetic association studies.

Gemcitabine combined with the monoclonal antibody nimotuzumab is an active first-line regimen in KRAS wildtype patients with locally advanced or metastatic pancreatic cancer: a multicenter, randomized phase IIb study.

BACKGROUND: This randomized study was designed to investigate the superiority of gemcitabine (gem) plus nimotuzumab (nimo), an anti-epidermal growth factor receptor monoclonal antibody, compared with gem plus placebo as first-line therapy in patients with advanced pancreatic cancer. PATIENTS AND METHODS: Patients with previously untreated, unresectable, locally advanced or metastatic pancreatic cancer were randomly assigned to receive gem: 1000 mg/m2, 30-min i.v. once weekly (d1, 8, 15; q29) and nimo: fixed dose of 400 mg once weekly as a 30-min infusion, or gem plus placebo, until progression or unacceptable toxicity. The primary end point was overall survival (OS), secondary end points included time to progression, overall response rate, safety and quality of life. RESULTS: A total of 192 patients were randomized, with 186 of them being assessable for efficacy and safety (average age 63.6 years). One-year OS/progression-free survival (PFS) was 34%/22% for gem plus nimo compared with 19%/10% for gem plus placebo (HR = 0.69; P = 0.03/HR = 0.68; P = 0.02). Median OS/PFS was 8.6/5.1 months for gem plus nimo versus 6.0/3.4 mo in the gem plus placebo group (HR = 0.69; P = 0.0341/HR = 0.68; P = 0.0163), with very few grade 3/4 toxicities. KRAS wildtype patients experienced a significantly better OS than those with KRAS mutations (11.6 versus 5.6 months, P = 0.03). CONCLUSION: This randomized study showed that nimo in combination with gem is safe and well tolerated. The 1-year OS and PFS rates for the entire population were significantly improved. Especially, those patients with KRAS wildtype seem to benefit. The study was registered as protocol ID OSAG101-PCS07, NCT00561990 and EudraCT 2007-000338-38.

Open-label, randomized study of individualized, pharmacokinetically (PK)-guided dosing of paclitaxel combined with carboplatin or cisplatin in patients with advanced non-small-cell lung cancer (NSCLC).

BACKGROUND: Variable chemotherapy exposure may cause toxicity or lack of efficacy. This study was initiated to validate pharmacokinetically (PK)-guided paclitaxel dosing in patients with advanced non-small-cell lung cancer (NSCLC) to avoid supra- or subtherapeutic exposure. PATIENTS AND METHODS: Patients with newly diagnosed, advanced NSCLC were randomly assigned to receive up to 6 cycles of 3-weekly carboplatin AUC 6 or cisplatin 80 mg/m(2) either with standard paclitaxel at 200 mg/m(2) (arm A) or PK-guided dosing of paclitaxel (arm B). In arm B, initial paclitaxel dose was adjusted to body surface area, age, sex, and subsequent doses were guided by neutropenia and previous-cycle paclitaxel exposure [time above a plasma concentration of 0.05 µM (Tc>0.05)] determined from a single blood sample on day 2. The primary end point was grade 4 neutropenia; secondary end points included neuropathy, radiological response, progression-free survival (PFS) and overall survival (OS). RESULTS: Among 365 patients randomly assigned, grade 4 neutropenia was similar in both arms (19% versus 16%; P = 0.10). Neuropathy grade ≥2 (38% versus 23%, P < 0.001) and grade ≥3 (9% versus 2%, P < 0.001) was significantly lower in arm B, independent of the platinum drug used. The median final paclitaxel dose was significantly lower in arm B (199 versus 150 mg/m(2), P < 0.001). Response rate was similar in arms A and B (31% versus 27%, P = 0.405), as was adjusted median PFS [5.5 versus 4.9 months, hazard ratio (HR) 1.16, 95% confidence interval (CI) 0.91-1.49, P = 0.228] and OS (10.1 versus 9.5 months, HR 1.05, 95% CI 0.81-1.37, P = 0.682). CONCLUSION: PK-guided dosing of paclitaxel does not improve severe neutropenia, but reduces paclitaxel-associated neuropathy and thereby improves the benefit-risk profile in patients with advanced NSCLC. CLINICAL TRIAL INFORMATION: NCT01326767 (https://clinicaltrials.gov/ct2/show/NCT01326767).

Disease will limit future food supply from the global crustacean fishery and aquaculture sectors.

Seafood is a highly traded food commodity. Farmed and captured crustaceans contribute a significant proportion with annual production exceeding 10 M metric tonnes with first sale value of $40bn. The sector is dominated by farmed tropical marine shrimp, the fastest growing sector of the global aquaculture industry. It is significant in supporting rural livelihoods and alleviating poverty in producing nations within Asia and Latin America while forming an increasing contribution to aquatic food supply in more developed countries. Nations with marine borders often also support important marine fisheries for crustaceans that are regionally traded as live animals and commodity products. A general separation of net producing and net consuming nations for crustacean seafood has created a truly globalised food industry. Projections for increasing global demand for seafood in the face of level or declining fisheries requires continued expansion and intensification of aquaculture while ensuring best utilisation of captured stocks. Furthermore, continued pressure from consuming nations to ensure safe products for human consumption are being augmented by additional legislative requirements for animals (and their products) to be of low disease status. As a consequence, increasing emphasis is being placed on enforcement of regulations and better governance of the sector; currently this is a challenge in light of a fragmented industry and less stringent regulations associated with animal disease within producer nations. Current estimates predict that up to 40% of tropical shrimp production (>$3bn) is lost annually, mainly due to viral pathogens for which standard preventative measures (e.g. such as vaccination) are not feasible. In light of this problem, new approaches are urgently required to enhance yield by improving broodstock and larval sourcing, promoting best management practices by farmer outreach and supporting cutting-edge research that aims to harness the natural abilities of invertebrates to mitigate assault from pathogens (e.g. the use of RNA interference therapeutics). In terms of fisheries losses associated with disease, key issues are centred on mortality and quality degradation in the post-capture phase, largely due to poor grading and handling by fishers and the industry chain. Occurrence of disease in wild crustaceans is also widely reported, with some indications that climatic changes may be increasing susceptibility to important pathogens (e.g. the parasite Hematodinium). However, despite improvements in field and laboratory diagnostics, defining population-level effects of disease in these fisheries remains elusive. Coordination of disease specialists with fisheries scientists will be required to understand current and future impacts of existing and emergent diseases on wild stocks. Overall, the increasing demand for crustacean seafood in light of these issues signals a clear warning for the future sustainability of this global industry. The linking together of global experts in the culture, capture and trading of crustaceans with pathologists, epidemiologists, ecologists, therapeutics specialists and policy makers in the field of food security will allow these issues to be better identified and addressed.

Gallbladder metastasis from renal cell carcinoma mimicking acute cholecystitis.

Renal cell carcinoma constitutes about 3% of adult malignancies. It has a high metastatic potential associated with synchronous or metachronous metastatic disease. Further, it is known to metastasize mainly to the lung, bone, brain, liver, or adrenal glands. In very rare cases it can metastasize to the gallbladder mimicking acute cholecystitis on clinical exam. In this case we present a patient who developed a gallbladder metastasis five years after a renal cell carcinoma mimicking acute cholecystitis.

Lymphocyte reconstitution following allogeneic hematopoietic stem cell transplantation: a retrospective study including 148 patients.

Here we investigated the influence of parameters known before hematopoietic stem cell transplantation (HSCT) as well as the relevance of graft-versus-host disease (GvHD) and cytomegalovirus (CMV) reactivation on post transplant lymphocyte reconstitution in 148 patients treated in our institution between 1996 and 2003. Median patient age was 42 (19-68) years, HSCT followed standard high dose (n=91) or reduced-intensity conditioning regimens (n=57) with bone marrow (BM, n=67) or peripheral blood stem cells (PBSC, n=81) from related (n=71) or unrelated (n=77) donors. In the first months, we observed a partially faster reconstitution of CD3+4+, CD3+8+ and CD4+45RA+ T cells in patients following peripheral blood stem cell transplantation when compared to bone marrow transplantation. Prolonged CD3+4+ and CD4+45RA+ lymphopenia was noted after unrelated donor HSCT and GvHD prophylaxis containing anti-T-lymphocyte globulin. Lymphocyte subset counts in patients older than the median age were comparable to those in patients transplanted at a younger age and not influenced by the conditioning regimen. CD3+8+ T cell reconstitution was strongly correlated with CMV reactivation, but not significantly affected by CMV serostatus before HSCT. Incidence or extent of GvHD did not significantly influence lymphocyte reconstitution. Therefore, the source of graft is the most predictive parameter in early lymphocyte reconstitution, but the differences in lymphocyte recovery completely resolved within the first year after HSCT.

A phase I clinical and pharmacokinetic study of the camptothecin glycoconjugate, BAY 38-3441, as a daily infusion in patients with advanced solid tumors.

BACKGROUND: The aim of this study was to define the maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and pharmacokinetics of the camptothecin glycoconjugate BAY 38-3441, administered as an infusion for 30 min on two separate schedules every 3 weeks. PATIENTS AND METHODS: A total of 81 patients with advanced solid tumors were treated with BAY 38-3441 either at doses of 20, 40, 67, 100, 140, 210, 315, 470 and 600 mg/m2/day for 1 day every 3 weeks (single-dose schedule), or at doses of 126, 189, 246, 320 and 416 mg/m2/day once daily for three consecutive days every 3 weeks (3-day schedule). Plasma sampling was performed to characterize the pharmacokinetics of BAY 38-3441 and camptothecin with these schedules. RESULTS: DLTs included renal toxicity, granulocytopenia and thrombocytopenia on the single-day schedule at doses > or = 470 mg/m2/day, and diarrhea and thrombocytopenia on the 3-day schedule at doses > or = 320 mg/m2/day. Other non-DLTs were gastrointestinal, dermatological and hematological. Pharmacokinetics of BAY 38-3441 and camptothecin appear to be dose-dependent, but not linear. CONCLUSIONS: Renal toxicity was dose-limiting for BAY 38-3441 using 30-min infusions on the single-dose schedule. Dose escalation to 470 mg/m2/day is feasible using a 2-h infusion. However, because of the superior safety profile, we recommend the 3-day schedule for BAY 38-3441 at a dose of 320 mg/m2/day as 30-min infusions for further phase II studies.

Prognosis of older patients with acute myeloid leukemia receiving either induction or noncurative treatment: a single-center retrospective study.

Since few studies focus on prognostic factors in unselected elderly acute myeloid leukemia (AML) patients, a retrospective analysis of 138 consecutive patients aged >55 years (median age: 67, range: 56-89) with AML diagnosed at a single center over an 8-year period was performed: 69% had de novo AML and 31% secondary (s) AML; 67% of the patients were karyotyped. Of the patients, 73 (53%) were treated with standard induction therapy protocols and 65 (47%) received palliative treatment only. Univariate and multivariate analyses of the effects of the following factors on overall survival (OS) were performed: sex, age > or = vs <65 years, de novo vs sAML, serum (s) lactate dehydrogenase (LDH) > or = vs <400 U/l, leukocytes > or = vs <50,000/ microl, induction therapy, and karyotype. Additionally, in patients receiving induction therapy, complete remission (CR) rates and survival from CR were analyzed. CR rate was 47% [95% confidence interval (35%, 59%)], 53% (39%, 66%) in de novo AML, and 21% (5%, 51%) in sAML. After a median follow-up of 4 years, 130 deaths were observed (94%). In a univariate analysis, significant factors for longer OS were induction therapy, age <65 years, sLDH <400 U/l, and de novo AML. In a multivariate analysis, significant factors for longer OS were sLDH <400 U/l and induction therapy. However, the difference between treatment outcome may also be due to selection criteria not captured, such as performance status, comorbid conditions, wish of the patient, etc. The effects of intensive and nonintensive treatment in this patient group need to be investigated in prospective, randomized trials in which these clinical parameters of high relevance for treatment decisions in older patients are also considered.

Paraneoplastic cerebellar degeneration and nephrotic syndrome preceding Hodgkin's disease: case report and review of the literature.

A patient presented with symptoms of cerebellar degeneration and nephrotic syndrome. A work-up at that time failed to reveal an underlying disease; however, 20 months later Hodgkin's disease was diagnosed. Hodgkin's lymphadenopathy developed 2 wk after prednisone therapy for the nephrotic syndrome had been discontinued. Systemic polychemotherapy resulted in complete remission of both Hodgkin's disease and nephrotic syndrome, while the neurological deficit persisted. Patients with unexplained cerebellar degeneration and/or nephrotic syndrome demand extensive evaluation for the presence of Hodgkin's disease, and steroid therapy may delay diagnosis.

Comparison of 1-hour and 3-hours paclitaxel infusion pharmacokinetics: results from a randomized trial.

PURPOSE: Aim of this study was to characterize the difference in pharmacokinetics (PK) of paclitaxel (PAC) after 1-h and 3-h infusion in humans and to define a pharmacodynamic relationship between PAC PK and myelotoxicity. PATIENTS AND METHODS: PAC PK were studied during the first PAC application in the first treatment cycle (1 treatment cycle = 6 PAC applications) in 25 patients. This patient group represents a subgroup of a large clinical study with neurotoxicity as primary endpoint. These 25 patients were those patients who were willing to give additional blood samples. The group size was sufficient for a full description of the PK of PAC. PAC was administered at 100 mg/m(2) weekly by 1-h (n = 12) or 3-h (n = 13) infusion to patients with advanced cancer (lung, breast, ovarian, cervix, and head and neck). Total PAC was quantified by high-performance liquid chromatography (HPLC). Pharmacokinetic parameters were calculated by noncompartmental and model-dependent methods. The leukocyte and neutrophil decrease during a 6-week treatment period was calculated by the percentage in decrease of white blood cell count (WBC) and absolute neutrophil count (ANC) as well as the area over the curve (AOC) of WBC and ANC. RESULTS: The area under the curve (AUC), the plasma clearance (Clp), the volume of distribution at steady state (V(ss)), the mean residence time (MRT) and the distribution half-life (t(1/2)) of PAC(tot) were not different in the two application modes. The elimination half-life (t1/2) and maximum plasma concentration C(max) were significantly different. No significant differences in the percentage of reduction of WBC and ANC were seen. Calculation of AOC of WBC showed a borderline significant difference (p = 0.0547) in case of WBC and no significant difference in case of ANC between the two PAC schedules. A considerable variance of AOC was observed. CONCLUSION: The pharmacokinetic study of total PAC of the two schedules investigated showed significant differences in the elimination half-life, which is longer in case of the 1-h infusion of PAC and in the maximum plasma concentration, which is higher in case of the 1-h infusion. The two schedules showed a similar myelotoxicity with a trend of less toxicity in the 1-h procedure.

Paraneoplastic polyneuropathy preceding the diagnosis of Hodgkin's disease and non-small cell lung cancer in a patient with concomitant Borrelia burgdorferi infection.

A patient with painful peripheral neuropathy is presented, whose symptoms were thought to result from an infection with Borrelia burgdorferi sensu lato. Investigations of the cerebrospinal fluid for signs of inflammation and borrelial antibodies were negative, and the patient did not benefit from repeated antibiotic treatment. Electrophysiologic studies and sural nerve biopsy showed axonal neuropathy consistent with a paraneoplastic syndrome. Further workup revealed mediastinal Hodgkin's disease (HD; nodular sclerosing subtype) Ann Arbor stage II and non-small cell cancer of the lung (stage T1N0M0). Surgical resection of the lung cancer and combined chemo- and radiotherapy for HD resulted in complete remission of both malignancies. While the preexisting neurologic symptoms persisted during treatment, neurography showed some improvement of the distal nerves. During radiation therapy the patient developed transient left-sided brachial plexopathy. This case illustrates that the diagnosis of borreliosis in patients with isolated painful peripheral neuritis cannot be based solely upon positive IgG titers and supports the requirement for a thorough workup for an underlying--potentially curable--disease. In addition, singular pulmonary lesions in the setting of HD should be suspected to have a separate cause.

Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy.

Ex vivo culture of hematopoietic progenitor cells for autologous transplantation has generated world-wide interest, since it offers the prospect of using a limited cell number, and may allow more efficient gene transfer and passive elimination of contaminating tumor cells. In this study, we expanded bone marrow (BM) cells from 10 breast cancer patients to determine whether small BM aliquots can durably restore hematopoiesis, and whether thrombopoietin (TPO) improves hematopoietic reconstitution after myeloablative chemotherapy. We used the AastromReplicell System (ARS), performing a computer-controlled, stromal-based cell expansion process with frequent medium, cytokine and gas exchange. For the inoculation of 9 x 10(8) MNC, a median BM volume of 97.8 ml (range, 72.4-272) was harvested. We found a median 4.5-fold nucleated cell expansion, an 18-fold CFU-GM expansion, and 69% of input LTC-IC numbers. Nucleated and Lin-/CD34+ cells were infused with a median of 43.5 x 10(6)/kg (range, 34.1-71.7) and 2.8 x 10(5)/kg (range, 0.95-5.9), respectively. Despite tumor cell detection by immunocytochemical staining in 3/10 patients before expansion, tumor cells were not detectable in 9/10, and in one patient 1 log reduced post ARS culture. Following high-dose STAMP V chemotherapy, all patients received 12-day expanded BM cells. The median time to engraftment was 17 days (range, 11-20) for WBC >1000/microl, and 28 days (range, 21-55) for platelets >20,000/microl. A correlation between post-expansion Lin-/CD34+ cells and engraftment for ANC >500/microl, WBC >1000/microl and platelets >20,000/microl was observed. Hematopoiesis has been maintained for a median of 15 (range, 6-24) months. Our results demonstrate that transplantation of ex vivo expanded small BM aliquots allows hematopoietic reconstitution after myeloablative chemotherapy. Ex vivo generated ARS cells can reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which only small stem cell doses are available, eg when using cord blood transplants or in non-mobilizing patients.

Differentiation associated modulation of the cytokine and chemokine expression pattern in human myeloid cell lines.

Hematopoietic progenitor cell differentiation is associated with the expression of different sets of genes including those encoding membrane bound molecules and cytokines. While expression of the former has meticulously been linked to both lineage specificity and maturation stages and is routinely used in the diagnosis of human leukemias, the production of cytokines has not systematically been analyzed in this respect. Secretion of cyto- and chemokines by HPC has been discussed as a key element of autocrine regulation of cell differentiation and proliferation in normal and malignant hematopoietic cells. Hematopoietic cell lines and their in vitro generated mature progeny were used as a model to investigate the cytokine and chemokine expression pattern prior to and after induction of differentiation. We show that a variety of cytokines are produced by these cells either constitutively or upon stimulation. Low levels of TNF-alpha and IL-8 were widely expressed by immature and mature cells, while peak values of TNF-alpha were detected in promyelocytic NB4 cells, as reported previously. Induction of monocytic differentiation by various agents was associated with upregulation of IL-1 beta and IL-1ra expression, while a differentiation shift to the granulocytic lineage in the presence of retinoic acid (RA) led to a marked increase of macrophage chemoattractant protein-1 (MCP-1) producing cells. These data indicate that lineage determination as well as maturation of hematopoietic cells may not only be associated with expression of specific surface molecules but also with a distinct cytokine expression pattern. Further studies are necessary to show if this holds true for primary leukemic and normal hematopoietic cells.

Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology.

Cell therapy with antigen-specific T cells holds promise for various diseases including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)-tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surface affinity matrix technology that allows direct identification and enrichment of life antigen-specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8(+) T cells directed against the HLA-A(*)0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generated by combining stimulation of peptide-pulsed autologous dendritic cells (DC) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies (MoAb). Antigen-specific cytotoxic T lymphocyte (CTL) were detected based on stimulation-induced interferon (IFN)-gamma and interleukin (IL)-4 secretion and enriched > 100-fold using the cell surface affinity matrix technology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan-A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine-driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.

Allogeneic bone marrow transplantation from unrelated donors using in vivo anti-T-cell globulin.

Despite improvements in HLA typing, graft-versus-host disease (GVHD) continues to impair the results after volunteer unrelated donor bone marrow transplantation (VUD-BMT) in adult patients compared with matched sibling BMT. Here, the outcome after VUD-BMT using a specific regimen with high-dose anti-T-lymphocyte globulin (ATG) was analysed. Fifty-five adult patients, median age 34 years (range 17-55 years), with acute or chronic leukaemia or myelodysplastic syndrome (MDS) were transplanted in first complete remission (CR1)/first chronic phase (CP1) (early disease) (n = 21) or in advanced (CR2/CP2, no remission) disease (n = 34) from an unrelated marrow donor. GVHD prophylaxis consisted of ATG-S (Fresenius) 60-90 mg/kg b.w. prior to transplantation, in addition to cyclosporin A and short-course methotrexate. Graft failure did not occur and white blood cell count (WBC) > 1.0 x 10(9)/l was reached at median day +16. The cumulative incidence of acute (a)GVHD grade II-IV was 15% [95% CI (8%, 28%)] and of chronic GVHD was 51% [95% CI (38%, 68%)]. The cumulative incidence of relapse within 1 year was 0% [95% CI (0%, 19%)] and 21% [95% CI (11%, 40%)] for patients with early and advanced disease respectively. With a median follow-up of 28 months (range 16-45 months), 2-year disease-free and overall survival for patients transplanted in CR1/CP1 was 81% and 81% [95% CI (64%, 98%)], respectively, and for patients with advanced disease was 33% [95% CI (17%, 50%)] and 40% [95% CI (23%, 57%)] respectively. Complete and persistent donor chimaerism was seen in 77.5% of 40 patients evaluated. All 14 chronic myeloid leukaemia (CML)-CP1 patients became bcr-abl negative within 250 d. High-dose ATG pretransplant results in a low incidence of severe aGVHD without compromising donor chimaerism or elimination of minimal residual disease. Our results are similar to data obtained after matched sibling donor transplantation.

Pure red cell aplasia after allogeneic stem cell transplantation with reduced conditioning.

Conditioning regimens with reduced intensity are used increasingly for allogeneic stem cell transplantation in elderly or extensively pretreated patients. Two cases of pure red cell aplasia after fludarabine-based conditioning and during immunosuppression with cyclosporin are described. Both patients received ABO-mismatched stem cells and had anti-donor isoagglutinins. Red cell recovery occurred after extended immunosuppression when isoagglutinins had disappeared. Colony assays indicated serologic suppression of the erythrocyte lineage in one patient. Since reduced conditioning permits donor cell engraftment primarily by suppression of host T cells, antibody-mediated immunological complications may occur more frequently than after 'classical' conditioning.

Generation and purification of CD8+ melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy.

Tumor antigens that might serve as potential targets for adoptive T-cell therapy have been defined in different tumor entities, especially in malignant melanoma. To generate conditions to induce primary T-cell responses against different HLA-A*0201-restricted melanoma peptides and to allow further expansion of peptide-specific T cells for adoptive transfer, CD8+-purified T cells from healthy donors were stimulated with Melan-A-pulsed autologous dendritic cells. Dendritic cells were generated in vitro from monocytes with granulocyte macrophage colony-stimulating factor, interleukin-4, and transforming growth factor-beta1. After 3-4 weekly stimulation cycles with Melan-A-pulsed DCs, we were able to induce a strong peptide-specific CTL response in vitro. MHC-peptide tetramer staining revealed a frequency of up to 3.5% CD8+/Melan-A+ T cells. Additional antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies together with interleukin-2 gave rise to 600-fold expansion of CD8+ CTLs that maintained Melan-A specificity and were able to efficiently lyse Melan-A-expressing melanoma cells. To enrich antigen-specific T cells in vitro, we used a recently established technology for analysis and sorting of live cells according to secreted cytokines. In the present study, we demonstrated that Melan-A-specific T cells can be purified by magnetic separation according to secreted IFN-gamma. These cells revealed a very potent monospecific CTL response, even at low E:T ratios, against Melan-A-pulsed and Melan-A-expressing target cells. Altogether, our study demonstrated that we have developed an efficient method for generating large numbers of peptide-specific T cells in vitro that may be used for adoptive T-cell transfer in tumor immunotherapy.

Quantitative lymphocyte subset reconstitution after allogeneic hematopoietic transplantation from matched related donors with CD34+ selected PBPC grafts unselected PBPC grafts or BM grafts.

CD34+ cell selection of PBPC after harvest from G-CSF-treated allogeneic donors results in a more than 200-fold depletion of T lymphocytes in the graft and has been used to reduce the incidence of acute GVHD post transplant. Since transplantation with T cell-depleted BM grafts is associated with a delay in immune reconstitution and an increase of opportunistic infections, we evaluated the immunological reconstitution of patients with hematologic malignancies after therapy followed by CD34+-selected PBPC34 transplantation from matched related donors. Lymphocyte subset reconstitution over the first 12 months post transplant and the incidence of infections were evaluated in 12 patients receiving PBPC34 grafts and compared to that of patients after transplantation with PBPC without CD34+ enrichment (n = 20) or unmanipulated bone marrow grafts (BM; n = 15). PBPC34 grafts contained 264-fold fewer T lymphocytes (median 0.53 x 10(6) kg/body weight) than PBPC grafts and 36-fold fewer than BM grafts (140 x 10(6)/kg and 19 x 10(6)/kg, respectively). Despite a two log depletion of T cells in the PBPC34 grafts, T lymphocyte reconstitution appeared comparable among the three transplant groups over the first 12 months. A positive patient CMV serostatus pretransplant was correlated with a faster T cell reconstitution in all transplant groups. GVHD prophylaxis with methylprednisolone delayed B lymphocyte reconstitution. The incidence of infections post transplant did not appear to be increased in the PBPC34 group compared with the PBPC and BMT groups. It remains to be shown in larger prospective trials, whether these promising preliminary data of lymphocyte reconstitution and the clinical course after transplantation with PBPC34 can be confirmed.