Jürgen Luhm

Anti‐myeloma activity of natural killer lymphocytes

Summary. Natural killer (NK) cells are assumed to contribute to a graft‐versus‐leukaemia effect. In vitro experiments have shown that many leukaemic cells are NK‐cell sensitive. Nevertheless, no data concerning the influence of purified NK cells on malignant myeloma (MM) cells exist. We co‐incubated NK cells with three different MM cell lines and fresh bone marrow samples of nine MM patients. The proportion of vital MM cells was determined before and after co‐cultivation by a flow‐cytometry‐based assay. All MM cells tested, with the exception of one cell line (NCI H929), were susceptible to a NK‐cell attack even without exogenous interleukin 2 (IL‐2). The mean killing of the native MM samples was 23·1 ± 5·4% and 34·5 ± 6·5% at 10:1 and 20:1 effector:target ratio respectively, This corresponded to about 2/3 of those values obtained with the highly sensitive line K562. In contrast, CD34‐positive haematopoietic stem cells as well as peripheral mononuclear cells were completely resistant under similar experimental conditions (1·3% killing). To elucidate the underlying triggering mechanisms, we measured human leucocyte antigen (HLA)‐class I expression of the MM cells. No evidence for HLA loss, which could have explained the NK‐cell recognition if it occurred, was demonstrated. These findings may contribute to the understanding of in vivo NK‐cell activation and encourage clinical applications of NK cells for MM patients.

Probability of anti-D development in D- patients receiving D+ RBCs.

BACKGROUND: In some situations, the administration of D+ RBCs to D– patients is necessary. The probability of a subsequent anti‐D formation is assumed to be around 80 percent, a figure based primarily on studies in healthy volunteers. It was hypothesized that patients requiring blood transfusion have a much lower probability of developing antibodies.

Large-scale generation of natural killer lymphocytes for clinical application.

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.

Early alterations in the number of circulating lymphocyte subpopulations and enhanced proinflammatory immune response during opioid-based general anesthesia.

The balance between proinflammatory and anti-inflammatory processes is of key importance in the reaction of the body to infection, injury, and surgical trauma. Drugs commonly used in anesthesia and intensive care may modulate immunological reactions by influencing intercellular communication through modification of cytokine response and fluctuation of peripheral immune cells such as natural killer (NK) cells, B cells, and T lymphocyte subpopulations (CD4+ and CD8+ cells). To examine the effects of general anesthesia with the hypnotic agent propofol and the opioid fentanyl, 30 patients undergoing minor elective orthopedic surgery were studied before and 20 min after application of the anesthetic drugs, but before the start of surgery. We found a significant enhancement of TNF-&agr; and IL-1&bgr; release in lipopolysaccharide (LPS)-stimulated whole blood cultures after induction of anesthesia. Similar results were observed with interferon-&ggr; (IFN-&ggr;) in cultures stimulated with phytohemagglutinin (PHA). Conversely, synthesis of the anti-inflammatory cytokine interleukin 10 (IL-10) decreased significantly in LPS-stimulated cultures. During general anesthesia, we found a decrease of circulating lymphocytes, characterized by a significant increase in the percentage of T lymphocytes in favor of CD4+ cells, increased B lymphocytes, and a significant decrease of NK cells. These data suggest that anesthesia with propofol and fentanyl promotes proinflammatory immune responses and influences peripheral lymphocyte composition in patients, which may subsequently affect pathophysiological processes during opioid-based anesthesia.

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.

A novel RT-PCR for reliable and rapid HCV RNA screening of blood donations

BACKGROUND: The objective of this work was to develop a novel and highly sensitive RT‐PCR method that is suitable for HCV RNA screening of blood donations according to the criteria released by the Paul Ehrlich Institute, the federal licensing agency of Germany, for routine HCV NAT.

Prolactin induces enhanced interferon gamma release in peripheral whole blood after stimulation with either PHA or LPS.

A number of recent studies have demonstrated the importance of prolactin as a key mediator in immune-neuroendocrine communication. Using a whole blood assay and various concentrations of prolactin, we stimulated cell cultures with either the plant lectin PHA or the endotoxin LPS, a widespread agent in common infectious diseases. Studying 15 healthy blood donors we found that human recombinant prolactin, at concentrations from 5 ng/ml to 100 ng/ml, significantly amplified IFN-gamma yields after stimulation with either PHA or LPS. PHA-stimulated cultures revealed a significant dose-dependent enhancement of IFN-gamma release. Our results indicate that prolactin can upregulate IFN-gamma secretion from immune cells in whole blood cell cultures in response to both PHA or LPS. Since IFN-gamma is suspected to play a key role in the cytokine cascade, amplifying the toxic effect of other pro-inflammatory cytokines and ultimately leading to augmented inflammatory tissue damage, our findings point to a modulatory role of prolactin in infection. Special interest should therefore be directed towards any naturally occurring hyperprolactinemia, caused for instance by stress, a number of drugs, and some chronic diseases.

Evaluation of algorithms for the diagnostic assessment and the reentry of blood donors who tested reactive for antibodies against hepatitis B core antigen

BACKGROUND: Screening of blood donations for antibodies against hepatitis B core antigen (anti‐HBc) is an accepted method to prevent some transfusion‐transmitted hepatitis B virus (HBV) infections. However, anti‐HBc testing may result in donor loss due to unspecific results in the currently available anti‐HBc tests. Algorithms to distinguish true‐positive from false‐positive results and for reentry of those donors who tested false anti‐HBc positive were evaluated retrospectively.