Crystalline deposits in the cornea and various areas of the kidney as symptoms of an underlying monoclonal gammopathy: a case report.

BACKGROUND: Plasma cell dyscrasias (PCD) are characterized by an abnormal production of intact monoclonal immunoglobulins or parts such as heavy or light chains. In most cases, the monoclonal protein (also termed paraprotein) is produced by a clonal plasma cell population. The production of monoclonal proteins can result in deposits of various types and localization depending on the type, amount, and electrochemical properties of the paraprotein. One histopathologic presentation, albeit rare, are crystalline deposits. They can form in various organs and hence cause a wide spectrum of symptoms. CASE PRESENTATION: A 49-year-old man presented to the emergency department with eyestrain and foreign body sensation after overhead drilling. Examination of the eyes revealed crystalline deposits in the cornea of both eyes. After additional diagnostic testing, deposits were attributed to free light chains. Monoclonal gammopathy of undetermined significance (MGUS) was diagnosed according to serum electrophoresis and immunofixation. Four years later, new onset of proteinuria was detected. A percutaneous biopsy of the kidney showed severe light chain podocytopathy with secondary focal segmental glomerulosclerosis (FSGS) and light chain proximal tubulopathy (LCPT). In these lesions, crystalline deposits identical to the corneal deposits were found in ultrastructural and immunofluorescent analysis. The patient was diagnosed with monoclonal gammopathy of renal significance (MGRS), and a plasma cell directed therapy was initiated. CONCLUSIONS: PCD can present with a wide array of symptoms and are notoriously difficult to diagnose. Extrarenal manifestations such as crystalline deposits in the cornea are one possible manifestation. The case presented herein emphasizes the notion that extrarenal paraprotein deposits warrant a thorough search for the underlying clonal disease.

Characteristic Sizes of Life in the Oceans, from Bacteria to Whales.

The size of an individual organism is a key trait to characterize its physiology and feeding ecology. Size-based scaling laws may have a limited size range of validity or undergo a transition from one scaling exponent to another at some characteristic size. We collate and review data on size-based scaling laws for resource acquisition, mobility, sensory range, and progeny size for all pelagic marine life, from bacteria to whales. Further, we review and develop simple theoretical arguments for observed scaling laws and the characteristic sizes of a change or breakdown of power laws. We divide life in the ocean into seven major realms based on trophic strategy, physiology, and life history strategy. Such a categorization represents a move away from a taxonomically oriented description toward a trait-based description of life in the oceans. Finally, we discuss life forms that transgress the simple size-based rules and identify unanswered questions.

[Therapy Goals for Children and Adolescents with ADHD and their Primary Caregivers. A Content Analysis and Examination of Agreement of ADHD Patients and their Primary Caregivers]. / Therapieziele von Kindern und Jugendlichen mit ADHS und ihren Hauptbezugspersonen. Eine inhaltsanalytische Auswertung und Prüfung der Übereinstimmung zwischen Betroffenen und Bezugspersonen.

OBJECTIVE: The aim of this study was to identify individual therapy goals (ITGs) of children and adolescents with ADHD and their primary caregivers. METHODS: Within the evaluation of the selective contract for children and adolescents with ADHD in Bremerhaven, Germany, ITGs of 42 study participants (aged 8-17) and their primary caregivers were collected with the psychotherapy basis documentation for children and adolescents (Psy-BaDo-KJ). ITGs were analysed following the classification of categories for individual therapy goals (KITZ) and their modification for children and adolescents. Analysis focused on the most frequently named ITGs and the agreement of patients and primary caregivers ITGs on the individual level. RESULTS: 235 ITGs were named. The greatest proportion of ADHD patients and their caregivers (47%) focused on interactional, psychosocial conflicts. In 19% of the cases (n=8) patients and their caregivers had the same main goal. 38% of patients and of caregivers (n=16) named the other ones main goal in one of his/her ITGs as well. CONCLUSIONS: ADHD patients and their primary caregivers both pursue ITGs related to ADHD symptoms. Few ITGs address medication related aspects. In case of differences in the ITGs of a patient and his/her primary caregivers, therapists should check whether differing ITGs address the same problem from different perspectives.

Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany.

Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2-5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400-6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4',6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0-56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

Transient lymphocyte decrease due to adhesion and migration following catumaxomab (anti-EpCAM x anti-CD3) treatment in vivo

INTRODUCTION: In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro. MATERIALS AND METHODS: A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNFα. Adherent T cells were determined by flow cytometry. RESULTS: Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNFα, IFNγ). TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNFα. CONCLUSIONS: Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes (AU)

Trifunctional antibodies induce efficient antitumour activity with immune cells from head and neck squamous cell carcinoma patients after radio-chemotherapy treatment.

BACKGROUND Trifunctional antibodies, such as catumaxomab (anti-EpCAM×anti-CD3) and ertumaxomab (anti- HER-2/neu×anti-CD3), transiently link immune effector cells to tumour cells, which results in cellular cytotoxicity towards the tumour cells. A functional immune system is therefore essential for effective anti-tumour activity. However, the commonly observed haematotoxicity of chemotherapeutics and radiation therapy may be associated with some degree of immunosuppression. Combining chemotherapy and trifunctional antibodies in cancer treatment requires understanding of the impact of chemotherapeutics on immune cell function and, thus, on the activity of trifunctional antibodies. METHODS The effect of chemotherapeutic treatment on trifunctional antibody-mediated anti-tumour activity was assessed in vitro. Blood samples were collected from 12 head and neck squamous cell carcinoma patients after chemotherapy (5-fluorouracil, cisplatin) and radiotherapy, and from one healthy control donor. The immune cell status was analysed and mononuclear cells (MNC) were isolated. The potency of catumaxomab and ertumaxomab was assessed in a cytotoxicity assay using MNC isolated from each patient sample in co-culture with a tumour target cell line. The release of infl ammatory cytokines was also monitored in the cell culture supernatant. RESULTS Most patients included in this study had decreased immune cell counts during the course of chemotherapy. Nonetheless, an effective and concentration-dependent anti- tumour activity mediated by trifunctional antibodies was demonstrated using these patient immune effector cells. The immune response activity of the patient immune cells was not impaired one week after cisplatin administration or even three days after the last 5-fluorouracil treatment. CONCLUSION This study shows for the first time that immune effector cells from cancer patients undergoing standard chemotherapy and radiotherapy can be activated by trifunctional antibodies for efficient killing of tumour cells.

Trifunctional antibodies induce efficient antitumour activity with immune cells from head and neck squamous cell carcinoma patients after radio-chemotherapy treatment

BACKGROUND Trifunctional antibodies, such as catumaxomab (anti-EpCAM×anti-CD3) and ertumaxomab (anti- HER-2/neu×anti-CD3), transiently link immune effector cells to tumour cells, which results in cellular cytotoxicity towards the tumour cells. A functional immune system is therefore essential for effective anti-tumour activity. However, the commonly observed haematotoxicity of chemotherapeutics and radiation therapy may be associated with some degree of immunosuppression. Combining chemotherapy and trifunctional antibodies in cancer treatment requires understanding of the impact of chemotherapeutics on immune cell function and, thus, on the activity of trifunctional antibodies. METHODS The effect of chemotherapeutic treatment on trifunctional antibody-mediated anti-tumour activity was assessed in vitro. Blood samples were collected from 12 head and neck squamous cell carcinoma patients after chemotherapy (5-fluorouracil, cisplatin) and radiotherapy, and from one healthy control donor. The immune cell status was analysed and mononuclear cells (MNC) were isolated. The potency of catumaxomab and ertumaxomab was assessed in a cytotoxicity assay using MNC isolated from each patient sample in co-culture with a tumour target cell line. The release of infl ammatory cytokines was also monitored in the cell culture supernatant. RESULTS Most patients included in this study had decreased immune cell counts during the course of chemotherapy. Nonetheless, an effective and concentration-dependent anti- tumour activity mediated by trifunctional antibodies was demonstrated using these patient immune effector cells. The immune response activity of the patient immune cells was not impaired one week after cisplatin administration or even three days after the last 5-fluorouracil treatment. CONCLUSION This study shows for the first time that immune effector cells from cancer patients undergoing standard chemotherapy and radiotherapy can be activated by trifunctional antibodies for efficient killing of tumour cells (AU)

Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive.

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.

Tautomer interconversion of 2,4-pentanedione during gas chromatography on an oxidized cyano-modified capillary column.

The tautomerization of 2,4-pentanedione (acetylacetone) is examined on a microbore column containing an acid-modified stationary phase made by oxidizing a commercially available cyano-modified column. This stationary phase is found to provide separation of the two tautomers, which allows the kinetic and thermodynamic properties of the on-column interconversion to be investigated. The enol-to-keto tautomerization is found to occur primarily in the stationary phase, being enthalpically driven. By treating the column as a reactor, the interconversion is investigated as a function of temperature. Monitoring the loss of the more gas-stable 'enol' tautomer makes it possible to extract an energy of activation for the net tautomerization (42.7 kj/mol), because the reaction is found to obey pseudo first-order kinetics. Simple peak-shape analysis of the major component (enol), which is used commonly in treatments of peak tailing, provides insight into the nature of the retention processes of the two tautomers as well as information on chromatographic optimization.

Neurohumoral activation in percutaneous coronary interventions: apropos of ten vasoactive substances during and immediately following coronary rotastenting.

BACKGROUND: Ischemia, left ventricular dysfunction, endothelial damage and hemodynamic changes during percutaneous coronary intervention can lead to neurohumoral activation. This may partly explain the frequent episodes of coronary spasm, hypotension and bradycardia which occur during the procedure. Rotastenting, by employing the two basic mechanisms for coronary interventions-debulking and dilatation-epitomizes percutaneous coronary interventions in general. We sought to investigate the neurohumoral changes during and immediately following coronary rotastenting. METHODS AND RESULTS: Eighteen patients undergoing elective rotablator atherectomy followed by balloon predilatation and stenting for chronic stable angina were studied. Four femoral vein blood samples were drawn from each patient at the start of the intervention (baseline), and 2 (postdebulking-2), 10 (postdebulking-10) and 60 (postdebulking-60) minutes. respectively, after the first complete passage of the rotablation burr across the whole length of lesion. Levels of 10 neurohormones, namely, endothelin-1, bradykinin, arginine vasopressin, norepinephrine, dopamine, epinephrine, angiotensin II, serum angiotensin-converting enzyme activity. atrial natriuretic peptide and kininogen were estimated in each sample. Endothelin-1 and bradykinin attained their peak levels in the postdebulking-2 samples. and the rise from 0.34+/-0.07 pmol/ml and 235.8+/-17.7 pg/ml to 0.42+/-0.06 pmol/ml and 337.2+/-41.0 pg/ml, respectively, was statistically significant (p<0.05). The level of arginine vasopressin showed a significant (p<0.05) rise from baseline (108.5+/-31.8 pg/ml) to postdebulking-60 samples (136.5+/-39.4 pg/ml). The other neurohormones did not show significant changes. CONCLUSIONS: The results suggest a definite but differential neurohumoral activation during and immediately following rotastenting. These neurohumoral changes may have a role in untoward intra- and postprocedural vasomotor and hemodynamic effects. This study establishes the concept of neurohumoral activation during percutaneous coronary interventions.

Increased susceptibility to apoptosis in circulating lymphocytes of critically ill patients.

BACKGROUND AND AIMS: Lymphocyte apoptosis may influence immune responsiveness in systemic inflammation. Therefore, we investigated whether early signs of apoptosis (i.e., annexin-V binding and cell shrinkage) in peripheral lymphocytes were different among patients with severe sepsis, critically ill, nonseptic patients after major surgery, and healthy individuals. PATIENTS/METHODS: Ten patients with severe sepsis and ten critically ill, nonseptic patients after major surgery admitted to a surgical intensive care unit in a university hospital were included in the study. In addition, ten healthy blood donors were included for comparison. We investigated early signs of apoptosis using flow cytometric measurement of annexin-V binding to the cell surface and cell shrinkage of peripheral lymphocytes. RESULTS: The percentage of apoptotic lymphocytes determined as annexin-V positive and propidium iodide negative cells was increased in freshly prepared cells of patients with severe sepsis (11.4 +/- 0.5%) and critically ill, nonseptic patients after major surgery (18.5 +/- 2.0%) relative to healthy blood donors (4.4 +/- 0.5%) (P < 0.05). No significant difference between patients with severe sepsis and patients after major surgery were found. Annexin-V binding increased significantly after OKT-3 stimulation of lymphocytes in patients with severe sepsis (34.4 +/- 1.6%), patients after major surgery (33.8 +/- 3.4%), and healthy blood donors (21.1 +/- 2.8%). No significant difference among groups was detected following OKT-3 stimulation. Furthermore, freshly isolated peripheral lymphocytes of patients with severe sepsis and critically ill, nonseptic patients after major surgery revealed a significantly higher proportion of cell shrinkage than in healthy blood donors (55.0 +/- 2.2%, 21.5 +/- 2.4% vs 3.6 +/- 0.7%; P < 0.05). CONCLUSION: Circulating lymphocytes of critically ill patients show a high degree of early signs of cellular apoptosis. This may contribute to hyporesponsiveness of immune cells in systemic inflammation.

On-line bioprocess monitoring with a multi-wavelength fluorescence sensor using multivariate calibration.

Cultivations of Pseudomonas fluorescens were monitored with a multi-wavelength on-line fluorescence sensor. The multi-wavelength fluorometer used excitation light from 270 to 550 nm with 20 nm steps and measured fluorescence emission from 310 to 590 nm. The fluorescence, on-line exhaust gas measurements and off-line analysis of nitrate, succinate, optical density and protein were compared chemometrically by multivariate calibration, i.e. computing partial least square (PLS) regression models. Based on the multivariate regression models, it was possible to determine CO2 and O2 composition in the exhaust gas (the correlation coefficients, R2 between the predicted values by the PLS model and the measured values was 0.97 for CO(2) and 0.97 for O2, respectively). Also to make quantitative determinations of succinate (R(2) = 0.97), protein (R(2) = 0.94), optical density (R(2) = 1.0) and nitrate (R(2) = 0.98) in the medium based on the fluorescence spectra. Only a limited data set was available but the results indicated that the sensor could indirectly determine non-fluorescent compounds, i.e. nitrate and succinate, which probably is due to the stoichiometric relationship between fluorescent cellular components and non-fluorescent compounds. Consequently multi-wavelength fluorescence is an interesting technique for a wide range of applications.

Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella.

Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420-431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 microm of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (+/-1.4) x 10(-7) dyne x cm (2.6 x 10(-14) N x m) and the apparent stiffness was 4.3 (+/-1.3) x 10(-10) dyne x cm(2) (4.3 x 10(-19) N x m(2)). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions.

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells.

We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells. SF91m3 combines several promising features. The vector backbone lacks viral coding sequences and AUG-start codons 5' of the MDR1 cDNA. A point mutation of a cryptic splice acceptor of the MDR1 cDNA increases the probability of transferring an intact provirus. The titer of a PG13 packaging cell clone containing a single proviral integration is high (>2 x 10(6) particles/ml from frozen stocks of serum-free vector harvests). Human hematopoietic cells transduced with SF91m3 reliably express MDR1 before and after passage through NOD/SCID mice, as shown by quantitative PCR and efflux assays with rhodamine 123 or Hoechst 33342. Finally, SF91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 x IC(50)). Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells.

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the half-life of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression with d2EGFP was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

MDR1 gene expression in NOD/SCID repopulating cells after retroviral gene transfer under clinically relevant conditions.

We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.

Retroviral transduction of T lymphocytes for suicide gene therapy in allogeneic stem cell transplantation.

Transplantation of suicide gene modified allogeneic T lymphocytes is an approach to prevent T cell mediated GVHD while preserving the 'graft-versus-leukemia' (GVL) effect of an allograft. A prerequisite for such a therapy is the efficient transduction of T cells with suitable vectors. Since existing techniques allow only insufficient transduction of T cells, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 10(6) cells/ml) by retroviral infection. The presented protocol allowed us to obtain transduction rates of more than 70% of CD3+ cells after two cycles of infection. It is based on the use of FBS-free media for both the production of retrovirus-containing supernatant, as well as the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large scale conditions, it may easily be adapted to clinical needs and 'good manufacturing practice' (GMP) standards.

Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1).

Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells. As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines. This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system. The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo. Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m. Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line. Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells. Results from the production runs and the applied biosafety concept are described.