Exploring the gap: attitudes, knowledge, and training needs in complementary and integrative medicine among healthcare professionals at German university hospitals.

Introduction: The use of Complementary and Integrative Medicine (CIM) is very popular among the general population in Germany. However, international studies show that nurses, physicians, and other health care professionals (HCPs) at hospitals often do not feel sufficiently informed about different CIM approaches. Moreover, they do not feel trained enough to counsel their patients appropriately. In the German-speaking context, particularly within university hospitals, research on this subject is scarce. Therefore, the aim of this explorative study was to evaluate attitudes, subjective knowledge, and needs regarding CIM among HCPs with direct patient interaction across all four university hospitals in the federal state of Baden-Württemberg, Germany (Tübingen, Ulm, Freiburg, Heidelberg). Methods: The multicenter, cross-sectional, anonymous full survey was conducted online using a self-developed, semi-structured, web-based questionnaire. Recruitment took place via all-inclusive e-mail distribution lists of all four university hospitals. Results: A total of n = 2,026 participants (response rate varied by location from about 5 to 14%) fully answered the questionnaire. Nurses constituted the largest professional group (n = 1,196; 59%), followed by physicians (n = 567; 28%), physiotherapists (n = 54), psychologists (n = 48), midwives (n = 37), and other professions (n = 124). More than two-thirds (71%, n = 1,437) of the participants were female and 14% (n = 286) reported additional training in CIM. The overall attitude toward CIM (10-point Likert scale, 10 = "very favorable") was clearly positive (M ± SD: 7.43 ± 2.33), with notable differences between professional groups: midwives (9.05 ± 1.18), physiotherapists (8.44 ± 1.74), and nurses (8.08 ± 1.95) expressed the highest support, whereas physicians (5.80 ± 2.39) the lowest. 42% of the participants incorporated CIM in patient care (from 33% of physicians to 86% of midwives). Overall, relaxation therapy (n = 1,951; 96%), external applications (n = 1,911; 94%), massage (n = 1,836; 91%), and meditation/mindfulness (n = 1,812; 89%) were rated as useful or rather useful for patients. The average self-assessed knowledge level about CIM was moderate (M ± SD: 5.83 ± 2.03). Most of the participants found CIM training at university hospitals important and saw research about CIM as one of the tasks of university hospitals. The participants expressed the highest interest in education for acupuncture/acupressure, relaxation therapies, and manual medicine. Discussion: This comprehensive survey of health care professionals (HCPs) at university hospitals in Germany reveals a clearly positive disposition toward CIM, aligning with findings from other hospital-based surveys and highlighting differences among professional groups. While most therapies deemed beneficial for patient care are supported by positive evidence, further research is required for others. Given the average self-reported knowledge of CIM, targeted education is essential to meet the needs of both HCPs and patients and to ensure the provision of evidence-based information on the risks and benefits of CIM.

Complementary medicine in Germany: a multi-centre cross-sectional survey on the usage by and the needs of patients hospitalized in university medical centers.

BACKGROUND: The results of recent surveys indicate that more than 50% of the German population has experience with complementary and alternative medicine (CAM) or uses CAM regularly. This study investigated the CAM usage and CAM-related needs of hospitalized patients at university medical centres in the state of Baden-Württemberg, Germany. METHODS: A multi-centre, paper-based, pseudonymous survey was carried out by the members of the Academic Centre for Complementary and Integrative Medicine. Patients of all ages, regardless of sex, diagnosis and treatment, who were hospitalized in the Department of Cardiology, Gastroenterology, Oncology, Gynaecology or Surgery at the university medical centres in Freiburg, Heidelberg, Tübingen and Ulm were eligible for inclusion. RESULTS: Of the 1275 eligible patients, 67% (n = 854) consented to participate in the survey. Forty-eight percent of the study participants stated that they were currently using CAM. The most frequently used therapies were exercise (63%), herbal medicine (54%) and dietary supplements (53%). Only 16% of the patients discussed CAM usage with their attending physician. Half of the patients (48%) were interested in CAM consultations. More than 80% of the patients desired reliable CAM information and stated that physicians should be better informed about CAM. CONCLUSIONS: The frequency of CAM usage and the need for CAM counselling among hospitalized patients at university medical centres in Baden-Württemberg are high. To better meet patients' needs, CAM research and physician education should be intensified. TRIAL REGISTRATION: German Clinical Trial register ( DRKS00015445 ).

Piperlongumine Acts as an Immunosuppressant by Exerting Prooxidative Effects in Human T Cells Resulting in Diminished TH17 but Enhanced Treg Differentiation.

Piperlongumine (PL), a natural small molecule derived from the Piper longum Linn plant, has received growing interest as a prooxidative drug with promising anticancer properties. Yet, the influence of PL on primary human T cells remained elusive. Knowledge of this is of crucial importance, however, since T cells in particular play a critical role in tumor control. Therefore, we investigated the effects of PL on the survival and function of primary human peripheral blood T cells (PBTs). While PL was not cytotoxic to PBTs, it interfered with several stages of T cell activation as it inhibited T cell/APC immune synapse formation, co-stimulation-induced upregulation of CD69 and CD25, T cell proliferation and the secretion of proinflammatory cytokines. PL-induced immune suppression was prevented in the presence of thiol-containing antioxidants. In line with this finding, PL increased the levels of intracellular reactive oxygen species and decreased glutathione in PBTs. Diminished intracellular glutathione was accompanied by a decrease in S-glutathionylation on actin suggesting a global alteration of the antioxidant response. Gene expression analysis demonstrated that TH17-related genes were predominantly inhibited by PL. Consistently, the polarization of primary human naïve CD4+ T cells into TH17 subsets was significantly diminished while differentiation into Treg cells was substantially increased upon PL treatment. This opposed consequence for TH17 and Treg cells was again abolished by thiol-containing antioxidants. Taken together, PL may act as a promising agent for therapeutic immunosuppression by exerting prooxidative effects in human T cells resulting in a diminished TH17 but enhanced Treg cell differentiation.

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Sulforaphane as anticancer agent: A double-edged sword? Tricky balance between effects on tumor cells and immune cells.

Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from cruciferous vegetables such as broccoli. It has been reported to inhibit the growth of a variety of cancers, such as breast, prostate, colon, skin, lung, gastric or bladder cancer. SFN is supposed to act primarily as an antioxidant due to the activation of the Nrf2-Keap1 signaling pathway. This enhances the activity of phase II detoxifying enzymes and the trapping of free radicals. Finally, SFN induces cell cycle arrest or apoptosis of tumor cells. Here, we discuss effects of SFN on the immune defense system. In contrast to the situation in tumor cells, SFN acts pro-oxidatively in primary human T cells. It increases intracellular ROS levels and decreases GSH, resulting in inhibition of T cell activation and T cell effector functions. Regarding the use of SFN as an "anticancer agent" we conclude that SFN could act as a double-edged sword. On the one hand it reduces carcinogenesis, on the other hand it blocks the T cell-mediated immune response, the latter being important for immune surveillance of tumors. Thus, SFN could also interfere with the successful application of immunotherapy by immune checkpoint inhibitors (e.g. CTLA-4 antibodies and PD-1/PD-L1 antibodies) or CAR T cells. Therefore, a combination of SFN with T cell-mediated cancer immunotherapies does not seem advisable.

Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione.

The activity and function of T-cells are influenced by the intra- and extracellular redox milieu. Oxidative stress induces hypo responsiveness of untransformed T-cells. Vice versa increased glutathione (GSH) levels or decreased levels of reactive oxygen species (ROS) prime T-cell metabolism for inflammation, e.g., in rheumatoid arthritis. Therefore, balancing the T-cell redox milieu may represent a promising new option for therapeutic immune modulation. Here we show that sulforaphane (SFN), a compound derived from plants of the Brassicaceae family, e.g., broccoli, induces a pro-oxidative state in untransformed human T-cells of healthy donors or RA patients. This manifested as an increase of intracellular ROS and a marked decrease of GSH. Consistently, increased global cysteine sulfenylation was detected. Importantly, a major target for SFN-mediated protein oxidation was STAT3, a transcription factor involved in the regulation of TH17-related genes. Accordingly, SFN significantly inhibited the activation of untransformed human T-cells derived from healthy donors or RA patients, and downregulated the expression of the transcription factor RORγt, and the TH17-related cytokines IL-17A, IL-17F, and IL-22, which play a major role within the pathophysiology of many chronic inflammatory/autoimmune diseases. The inhibitory effects of SFN could be abolished by exogenously supplied GSH and by the GSH replenishing antioxidant N-acetylcysteine (NAC). Together, our study provides mechanistic insights into the mode of action of the natural substance SFN. It specifically exerts TH17 prone immunosuppressive effects on untransformed human T-cells by decreasing GSH and accumulation of ROS. Thus, SFN may offer novel clinical options for the treatment of TH17 related chronic inflammatory/autoimmune diseases such as rheumatoid arthritis.

Neutrophil Granulocytes in Ovarian Cancer - Induction of Epithelial-To-Mesenchymal-Transition and Tumor Cell Migration.

BACKGROUND: Ovarian cancer (OvCa) is a highly aggressive malignoma with a tumor-promoting microenvironment. Infiltration of polymorphonuclear neutrophils (PMN) is frequently seen, raising the question of their impact on tumor development. In that context, effects of PMN on human ovarian cancer cells were assessed. METHODS: Human epithelial ovarian cancer cells were incubated with human PMN, lysate of PMN, or neutrophil elastase. Morphological alterations were observed by time-lapse video-microscopy, and the underlying molecular mechanism was analyzed by flow cytometry and Western blotting. Functional alternations were assessed by an in vitro wound healing assay. In parallel, a large cohort of n=334 primary OvCa tissue samples of various histological subtypes was histologically evaluated. RESULTS: Co-cultivation of cancer cells with either PMN or PMN lysate causes a change of the polygonal epithelial phenotype of the cells towards a spindle shaped morphology, causing a cribriform cell growth. The PMN-induced alteration could be attributed to elastase, a major protease of PMN. Elastase-induced shape change was most likely due to the degradation of membranous E-cadherin, which results in loss of cell contacts and polarity. Moreover, in response to elastase, epithelial cytokeratins were downmodulated, in parallel with a nuclear translocation of ß-catenin. These PMN-elastase induced alterations of cells are compatible with an epithelial-to-mesenchymal transition (EMT) of the cancer cells. Following EMT, the cells displayed a more migratory phenotype. In human biopsies, neutrophil infiltration was seen in 72% of the cases. PMN infiltrates were detected preferentially in areas with low E-cadherin expression. CONCLUSION: PMN in the microenvironment of OvCa can alter tumor cells towards a mesenchymal and migratory phenotype.

A new model for the aerobic metabolism of yeast allows the detailed analysis of the metabolic regulation during glucose pulse.

The onset of aerobic fermentation (the so-called Crabtree effect) in yeast has long been of interest. However, the underlying mechanisms at the metabolic level are not yet fully understood. We developed a detailed kinetic model of the aerobic central metabolism of Saccharomyces cerevisiae comprising glycolysis, TCA cycle and major transport reactions across the mitochondrial membrane to investigate this phenomenon. It is the first one of this extent in the literature. The model is able to reproduce experimental steady state fluxes and time-course behavior after a glucose pulse. Due to the lack of parameter identifiability in the model, we analyze a model ensemble consisting of a set of differently parameterized models for robust findings. The model predicts that the cooperativity of pyruvate decarboxylase with respect to pyruvate and the capacity difference between alcohol dehydrogenase and the pyruvate dehydrogenase bypass play a major role for the onset of the Crabtree effect.

An in-silico model of lipoprotein metabolism and kinetics for the evaluation of targets and biomarkers in the reverse cholesterol transport pathway.

High-density lipoprotein (HDL) is believed to play an important role in lowering cardiovascular disease (CVD) risk by mediating the process of reverse cholesterol transport (RCT). Via RCT, excess cholesterol from peripheral tissues is carried back to the liver and hence should lead to the reduction of atherosclerotic plaques. The recent failures of HDL-cholesterol (HDL-C) raising therapies have initiated a re-examination of the link between CVD risk and the rate of RCT, and have brought into question whether all target modulations that raise HDL-C would be atheroprotective. To help address these issues, a novel in-silico model has been built to incorporate modern concepts of HDL biology, including: the geometric structure of HDL linking the core radius with the number of ApoA-I molecules on it, and the regeneration of lipid-poor ApoA-I from spherical HDL due to remodeling processes. The ODE model has been calibrated using data from the literature and validated by simulating additional experiments not used in the calibration. Using a virtual population, we show that the model provides possible explanations for a number of well-known relationships in cholesterol metabolism, including the epidemiological relationship between HDL-C and CVD risk and the correlations between some HDL-related lipoprotein markers. In particular, the model has been used to explore two HDL-C raising target modulations, Cholesteryl Ester Transfer Protein (CETP) inhibition and ATP-binding cassette transporter member 1 (ABCA1) up-regulation. It predicts that while CETP inhibition would not result in an increased RCT rate, ABCA1 up-regulation should increase both HDL-C and RCT rate. Furthermore, the model predicts the two target modulations result in distinct changes in the lipoprotein measures. Finally, the model also allows for an evaluation of two candidate biomarkers for in-vivo whole-body ABCA1 activity: the absolute concentration and the % lipid-poor ApoA-I. These findings illustrate the potential utility of the model in drug development.

Ca2+ dynamics correlates with phenotype and function in primary human neutrophils.

Central to the immune defense function of neutrophils is to sense, to move and to kill. Neutrophils acquire distinct cellular states necessary to fulfill these functions each associated with a particular phenotype. The cells constituting the neutrophil population are presumably not synchronized with respect to their actual state, e.g. due to maturity or preactivation. It is also likely that they exhibit a different degree of phenotypic plasticity (that is, the ability to switch to a particular state). Calcium is known to play a crucial role in neutrophils such as for cell motility. The present study focuses on characterizing the cell-to-cell variability at the morphological as well as at the level of calcium dynamics by studying single primary human neutrophils. We apply long-term multivariate live cell imaging to (i) characterize neutrophil phenotypes of different functional states, (ii) analyze the distribution of cells being in these states and, (iii) study the individual intracellular calcium response simultaneously with shape changes. We are able to differentiate the five distinct subpopulations of neutrophils based on quantitative parameters of cell morphology and motility. As a major result, we demonstrate that the calcium dynamics of individual cells correlates with their respective functional state. Finally, we see a number of cells that undergo spontaneous phenotypic changes from one cellular state to another. These events are preceded either by exhibiting the calcium dynamics of the future state or by switching to the respective calcium dynamics in parallel to switching the morphology. Based on our results we conclude that specific calcium dynamics carries crucial information for the function and phenotype of neutrophils.

Simplification of biochemical models: a general approach based on the analysis of the impact of individual species and reactions on the systems dynamics.

BACKGROUND: Given the complex mechanisms underlying biochemical processes systems biology researchers tend to build ever increasing computational models. However, dealing with complex systems entails a variety of problems, e.g. difficult intuitive understanding, variety of time scales or non-identifiable parameters. Therefore, methods are needed that, at least semi-automatically, help to elucidate how the complexity of a model can be reduced such that important behavior is maintained and the predictive capacity of the model is increased. The results should be easily accessible and interpretable. In the best case such methods may also provide insight into fundamental biochemical mechanisms. RESULTS: We have developed a strategy based on the Computational Singular Perturbation (CSP) method which can be used to perform a "biochemically-driven" model reduction of even large and complex kinetic ODE systems. We provide an implementation of the original CSP algorithm in COPASI (a COmplex PAthway SImulator) and applied the strategy to two example models of different degree of complexity - a simple one-enzyme system and a full-scale model of yeast glycolysis. CONCLUSION: The results show the usefulness of the method for model simplification purposes as well as for analyzing fundamental biochemical mechanisms. COPASI is freely available at http://www.copasi.org.

In vivo binding of Orc2 to a region of the chicken lysozyme GAS41 origin containing multiple Sp1-binding sites.

Most known DNA replication origins in vertebrate genomes have been found to occur close to transcriptional promoters. The origin of bidirectional DNA replication of the chicken lysozyme GAS41 locus was identified in a CpG island covering the GAS41 gene promoter. In this study, we generated an α-Orc2 antibody from rabbits immunized with the C-terminal half of Orc2 for studying in vivo Orc2 binding to the lysozyme-GAS41 origin. Using the chromatin immunoprecipitation technique and quantitative real-time polymerase chain reaction, we were able to map the Orc2-binding site to a region of the lysozyme GAS41 origin that contains multiple Sp1/Sp3-binding sites co-mapping with two DNase I hypersensitive sites. Further, knockdown of endogenous Sp1 by RNA interference reduced specific Orc2 binding to the lysozyme GAS41 origin. These results suggest that Sp1 participates in recruiting Orc2 to the origin.

Metannogen: annotation of biological reaction networks.

MOTIVATION: Semantic annotations of the biochemical entities constituting a biological reaction network are indispensable to create biologically meaningful networks. They further heighten efficient exchange, reuse and merging of existing models which concern present-day systems biology research more often. Two types of tools for the reconstruction of biological networks currently exist: (i) several sophisticated programs support graphical network editing and visualization. (ii) Data management systems permit reconstruction and curation of huge networks in a team of scientists including data integration, annotation and cross-referencing. We seeked ways to combine the advantages of both approaches. RESULTS: Metannogen, which was previously developed for network reconstruction, has been considerably improved. From now on, Metannogen provides sbml import and annotation of networks created elsewhere. This permits users of other network reconstruction platforms or modeling software to annotate their networks using Metannogen's advanced information management. We implemented word-autocompletion, multipattern highlighting, spell check, brace-expansion and publication management, and improved annotation, cross-referencing and team work requirements. Unspecific enzymes and transporters acting on a spectrum of different substrates are efficiently handled. The network can be exported in sbml format where the annotations are embedded in line with the miriam standard. For more comfort, Metannogen may be tightly coupled with the network editor such that Metannogen becomes an additional view for the focused reaction in the network editor. Finally, Metannogen provides local single user, shared password protected multiuser or public access to the annotation data. AVAILABILITY: Metannogen is available free of charge at: http://www.bioinformatics.org/strap/metannogen/ or http://3d-alignment.eu/metannogen/. CONTACT: christoph.gille@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Applications and trends in systems biology in biochemistry.

Systems biology has received an ever increasing interest during the last decade. A large amount of third-party funding is spent on this topic, which involves quantitative experimentation integrated with computational modeling. Industrial companies are also starting to use this approach more and more often, especially in pharmaceutical research and biotechnology. This leads to the question of whether such interest is wisely invested and whether there are success stories to be told for basic science and/or technology/biomedicine. In this review, we focus on the application of systems biology approaches that have been employed to shed light on both biochemical functions and previously unknown mechanisms. We point out which computational and experimental methods are employed most frequently and which trends in systems biology research can be observed. Finally, we discuss some problems that we have encountered in publications in the field.

HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology.

We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds.

Sp1 and Sp3 regulate transcription of the chicken GAS41 gene.

The 5'-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3' end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene.

Computational lipidology: predicting lipoprotein density profiles in human blood plasma.

Monitoring cholesterol levels is strongly recommended to identify patients at risk for myocardial infarction. However, clinical markers beyond "bad" and "good" cholesterol are needed to precisely predict individual lipid disorders. Our work contributes to this aim by bringing together experiment and theory. We developed a novel computer-based model of the human plasma lipoprotein metabolism in order to simulate the blood lipid levels in high resolution. Instead of focusing on a few conventionally used predefined lipoprotein density classes (LDL, HDL), we consider the entire protein and lipid composition spectrum of individual lipoprotein complexes. Subsequently, their distribution over density (which equals the lipoprotein profile) is calculated. As our main results, we (i) successfully reproduced clinically measured lipoprotein profiles of healthy subjects; (ii) assigned lipoproteins to narrow density classes, named high-resolution density sub-fractions (hrDS), revealing heterogeneous lipoprotein distributions within the major lipoprotein classes; and (iii) present model-based predictions of changes in the lipoprotein distribution elicited by disorders in underlying molecular processes. In its present state, the model offers a platform for many future applications aimed at understanding the reasons for inter-individual variability, identifying new sub-fractions of potential clinical relevance and a patient-oriented diagnosis of the potential molecular causes for individual dyslipidemia.

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus.

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Deltaabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Deltaabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Deltaabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.

KN-62, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, inhibits the lysozyme pre-mRNA splicing in myelomonocytic HD11 cells.

The lysozyme primary transcript has been shown to be slowly spliced, particularly in LPS-activated myelomonocytic HD11 cells. In this study, Northern blot analysis shows that the splicing of lysozyme pre-mRNA in LPS-activated cells is significantly delayed by treatment with KN-62, a selective inhibitor of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but not with Gö 6976 and herbimycin A, inhibitors of Ca(2+)-dependent PKC and PTK, respectively. In vitro kinase assay using autocamtide 2 as specific substrate for CaMKII demonstrates that KN-62, when added to the extract from HD11 cells, inhibits selectively CaMKII activity. Treatment of HD11 cells with cycloheximide, a potent inhibitor of protein synthesis, results in a transient increase in lysozyme pre-mRNA levels, whilst the mature mRNA levels are not increased. Moreover, neither cycloheximide nor KN-62 has any effect on the glyceraldehyde-3-phosphate dehydrogenase pre-mRNA splicing. Together, our results indicate that phosphorylation by CaMKII, and probably new protein synthesis may be required for the lysozyme pre-mRNA processing.