Evaluation of sampling locations in pregnant women and newborns for the detection of colonisation with antibiotic-resistant bacteria.

Up to now, little has been known about the prevalence and clinical relevance of colonisation of asymptomatic pregnant women with methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) or extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. In this two-centre cross-sectional study, we evaluated the performance and importance of screening at different times and different locations for colonisation in pregnant women and newborns. Between October 2013 and December 2015, four samples were collected from pregnant women, two from newborns at birth and three from 3-day-old newborns. Samples were screened on culturing media and were confirmed with molecular methods. MSSA was used as a surrogate for MRSA, as the two share most microbiologic characteristics and colonisation patterns. Of 763 pregnant women, 14.5% (111) were colonised with MSSA, 0.4% (3) with MRSA and 2.6% (20) with ESBL-producing E. coli. Of 658 newborns, 0.9% (10) were colonised with MSSA at birth and 13.1% (70) at 3 days old, 0.5% (3) were colonised with MRSA and 2.6% (17) with ESBL-producing E. coli. Nasal sampling identified 91.0% of MSSA-colonised pregnant women and 60.0% of newborns. In newborns, nasal and umbilical sampling at 3 days after birth discovered 84.0% of colonised cases. For ESBL-producing E. coli, the perianal region was positive in all colonised pregnant women and in 88.2% of colonised newborns. Combining nasal and perianal swabs is optimal when screening for antibiotic-resistant bacteria in pregnant women. Nasal, perianal and umbilical sample collection from 3-day-old newborns significantly increased the sensitivity compared to screening immediately after birth.

[Exposure of Pupils to Recreational Noise from Portable Listening Devices and Possible Preventive Measures]. / Freizeitlärmbelastung durch tragbare Musikabspielgeräte bei Schülern und Präventionsmöglichkeiten.

Background and Objectives: Exposure to recreational noise is becoming increasingly important due to a change in leisure behavior amongst children and adolescents. The aim of this pilot study was to assess exposure of 6th grade pupils to recreational noise from portable listening devices (PLD). Furthermore, preventive measures to reduce recreational noise exposure should be identified. Methods: In "Ohrkan Kids", 38 Bavarian pupils aged 11 to 14 were interviewed regarding their music listening behavior using a standardized questionnaire. In addition, measurements of commonly used volume settings on the children's portable listening devices were carried out. Furthermore, the German Social Accident Insurance (DGUV), health insurance companies as well as health and education ministries of the German federal states were surveyed regarding their activities in the prevention of recreational noise exposure. Results: Based on the questionnaire data for weekly usage, 10 out of 31 children (32.3%) exceeded the upper exposure value of 85 dB recommended by labor protection law. Taking actually measured values, 9 out of 31 children (29%) exceeded this level. The DGUV and some federal states carry out specific projects for the prevention of recreational noise exposure. Conclusion: The large number of children with hazardous music consumption indicates that measures for the prevention of noise-induced hearing loss are already required for 11 to 14 year olds. To maximize the number of children addressed, age-appropriate and target-group-specific preventive measures are needed. As there are only few studies which examined the effectiveness of awareness campaigns for the prevention of recreational noise, any future prevention projects should be evaluated with an increased focus on estimating their effectiveness.

[Taking Care of Asylum Seekers: Occupational Health Aspects with a Special Focus on Vaccination]. / Arbeitsmedizinische Aspekte und Impfschutz beim Umgang mit Asylbewerbern.

Employees and volunteers often feel insecure about the potential transmission of infectious diseases when taking care of asylum seekers. It could be shown that overall only a minor risk of infection emanates from asylum seekers. However, aspects of occupational health and vaccination should be kept in mind.Besides the standard vaccination the Standing Committee on Vaccination (STIKO) recommends for occupational indication, which is given for employees and volunteers in asylum facilities, vaccination against hepatitis A, hepatitis B, polio (if the last vaccination was more than 10 years before) as well as influenza (seasonal).According to the German Occupational Safety and Health Act taking care of the employer has to determine which exposures might occur at the workplace (risk assessment) and define necessary protection measures. Depending on task and exposure when taking care of asylum seekers different acts (e. g. biological agents regulation) and technical guidelines for the handling biological agents (e. g. TRBA 250 or TRBA 500) have to be applied.The Bavarian Health and Food Safety Authority (LGL) has published several information sheets regarding "asylum seekers and health management" for employees and volunteers from the non-medical as well as the medical area (www.lgl.bayern.de search term "Asylbewerber"). With theses publications insecurities in taking care of asylum seekers should be prevented. Furthermore the employer gets support in the implementation of legal obligations to ensure occupational safety for the employees.

[Preoperational study for the improvement of hygiene conditions in long-term care facilities]. / Modellprojekt zur Verbesserung der Hygiene in stationären Pflegeeinrichtungen.

Hygiene is becoming more and more important in long-term care facilities. Long-term care facilities are subject to monitoring by the Public Health Service (PHS) and other authorities. For the PHS in Bavaria the Bavarian Health and Food Safety Authority (Landesamt für Gesundheit und Lebensmittelsicherheit, LGL) published a hygiene monitoring concept and there exists an inspection guide developed by a specialist department for nursing homes and institutions for the handicapped (Fachstelle für Pflege und Behinderteneinrichtungen, FQA). Because inspections are performed in multiprofessional teams, it makes sense to use a coordinated inspection catalog. The aim was to integrate hygienic requirements specified in the Bavarian guidelines for hygiene by the LGL into the inspection guide published by the FQA to obtain a quality assured surveillance. The involved parties were questioned about the inspection guide and their hygiene management and then the hygiene criteria of the LGL were implemented into the inspection guide. Questions dealing with hygiene requirements concerning intensive care, management of multidrug resistant bacteria and interviews with the person responsible for infection control in the facility itself were developed for the first time and were integrated into the inspection guide. The revised inspection guide was tested for its applicability. With the revised inspection guide there now exists a tool which allows not only comprehensive inspections of the facilities including hygiene issues but also a good cooperation of the various parties involved. There are many actions which have to be conveyed into the future, especially programs to train staff to apply the inspection guide and to enhance the ability of all participants to act in cooperation. The guide will also allow the facilities to cooperate more easily and more closely, as the guide takes the respective problems and challenges of the different facilities into consideration. Additionally the development of legal guidelines regarding hygiene can support general healthcare of residents.

First detection of Borrelia burgdorferi-antibodies in free-living birds of prey from Eastern Westphalia, Germany.

Borrelia (B.) burgdorferi sensu lato, the causative agent of Lyme disease, is the most important arthropod-borne zoonosis-pathogen in the Northern hemisphere. Besides small mammals, birds, primarily Passeriformes and sea birds, play an important role in the transmission, distribution and maintenance of this disease. Previous studies on birds have focused mainly on the detection of Borrelia-infected ticks. However, the presence or absence of an infected tick cannot be taken as an indicator of the infective status of the avian host; to date this area of research has not been explored. In this study, serological analyses of blood collected from free-living birds of prey (n = 29) at the rehabilitation centre in Eastern Westphalia, Germany, highlights that birds of prey are also susceptible to B. burgdorferi and react immunologically to an infection. Increased antibody-levels could be found by using a modified Indirect Immunofluorescent-testing in two common buzzards, Buteo buteo, and two eagle owls, Bubo bubo. Further research regarding the serological diagnostics of B. burgdorferi within the avian host is required. In the future, it should be taken into account that birds of prey can be reservoirs for B. burgdorferi, as well as carriers of infected ticks; although at present their epidemiological importance is still to be confirmed.

Trends in the incidence of visual impairment certification secondary to diabetic retinopathy in the Leeds metropolitan area, 2005-2010.

AIM: This study reports the incidence of visual impairment certification due to diabetic retinopathy in Leeds between 2008 and 2010 and makes a comparison with data from 2005, immediately before the introduction of a comprehensive screening service. METHODS: The primary causes of visual impairment certification between 2008 and 2010 were collected and reviewed. Mid-year population estimates and a diabetes prevalence model were used to determine the incidence of certification secondary to diabetic retinopathy. RESULTS: Diabetic retinopathy was the primary cause of visual impairment certification in 33 of 446 (7.4%) certificates in 2008, 34 of 410 (8.3%) certificates in 2009 and 24 of 392 (6.1%) in 2010. For the total population in 2008, 2009 and 2010, the combined incidence of either sight impairment or severe sight impairment due to diabetic retinopathy was 42.3, 43.2 and 30 per million per year, respectively. For the population with diagnosed diabetes mellitus, the combined incidence of either sight impairment or severe sight impairment secondary to diabetic retinopathy was 1227, 1192 and 796 per million per year, respectively. For each year, the incidence of visual impairment was lower than the corresponding figure for 2005. CONCLUSION: Following the introduction of a comprehensive retinal screening service, the incidence of visual impairment certification secondary to diabetic retinopathy in the Leeds Metropolitan area appears to be decreasing. However, a multifaceted approach, addressing all the avoidable risk factors, may be required to maintain this trend in view of the increasing prevalence of Type 2 diabetes.

The predictive value of OCT characteristics for the visual outcome in neovascular AMD.

BACKGROUND: The intravitreal injection of an anti-VEGF compound is the current standard of care in neovascular AMD. The response to this therapy varies greatly. To date it was not possible to determine clear predictive factors in regard to therapy response, thus making it difficult when counselling patients regarding the probability for visual improvement. The aim of this study was to evaluate baseline OCT characteristics in regard to their predictive value on the outcome of visual acuity (VA) after 12 months. PATIENTS AND METHODS: A retrospective analysis of 75 eyes with neovascular AMD treated with intravitreal anti-VEGF injections at the University Hospital of Zurich with a documented follow-up of at least 12 months. Measurement and comparison of the following OCT structures were undertaken: central retinal thickness (CRT), integrity of the IS/OS junction, and presence of intra- or subretinal fluid. VA at baseline and after 12 months was evaluated. OCT findings were compared between eyes that gained ≥ 5 letters ETDRS (group 1) and eyes that did not (group 2). RESULTS: 75 eyes with a mean baseline VA of 57.2 ± 15 letters and a mean baseline CRT of 430 ± 226 µm were analyzed. Although baseline VA did not differ statistically significantly, eyes in group 2 had a higher VA than eyes in group 1 (60.2 ± 16.2 vs. 54.9 ± 13.6, p = 0.123). In group 1 the change of VA after 12 months was 12.6 ± 8.0 letters while it was -5.0 ± 7.8 letters in group 2. No statistically significant differences between the two groups with respect to the analyzed OCT parameters were found. None of the analyzed OCT factors had a predictive value regarding the VA outcome at month 12. CONCLUSIONS: Our study was not able to find baseline OCT parameters that could predict the course of VA after 12 months. However, eyes with a thicker central retinal thickness at presentation showed a greater reduction in CRT during the analyzed period. This was associated with a more favourable course in VA. Perhaps this might be due to a less pronounced initial morphological retinal damage.

"Walking" in the newborn.

Brief daily exercise of the walking and placing reflexes in the newborn leads to a high rate of responding by 8 weeks and to an earlier onset of walking alone. There appears to be a critical period during which the walking response can be transformed intac from a reflexive to an instrumental action.

Thyroid testing in Sloughis.

BACKGROUND: Thyroid hormone concentrations were found to be different in Greyhounds and Whippets compared with nonsight hound dogs. HYPOTHESIS: In Sloughis, thyroid hormone concentration is lower than in nonsight hounds and comparable to Greyhounds. ANIMALS: Fifty-one Sloughis with no evidence of disease and a mean age of 4 years (range, 1-12 years). METHODS: Thyroid profiles consisting of total thyroxine (tT4), free thyroxine (fT4), free thyroxine after equilibrium dialysis (fT4 after ED), canine thyroid stimulation hormone (cTSH), and thyroglobulin antibodies as well as CBC and serum biochemistry results of Sloughis were compared with those of normal dogs. In 8 Sloughis, TSH stimulation tests were performed. RESULTS: In Sloughis, tT4 concentrations and fT4 concentrations measured by chemiluminescence were lower than those of controls (1.13+/-0.65 microg/dL compared with 2.9+/-0.8 microg/dL, P< .0001 and 11+/-4.3 pmol/L compared with 16.7+/-5.2 pmol/L, P< .0001, respectively). Concentrations of fT4 after ED and TSH were increased in Sloughis, when compared with controls (41.3+/-26.9 pmol/L compared with 20.98+/-10.29 pmol/L, P< .0001 and 0.22 +/- 0.15 pmol/L compared with 0.15+/-0.13 pmol/L, P= .0138, respectively). T4 concentration after TSH stimulation increased from 1.5 microg/dL (range, 0.2-2.7 microg/dL) to 2.7 microg/dL (range, 1.2-4.7 microg/dL); the recommended post-TSH T4 concentration was achieved by only 3 of 8 Sloughis. Hemoconcentration was found in 84.3% and hypoglobulinemia in 80.3%. CONCLUSIONS AND CLINICAL IMPORTANCE: When evaluating Sloughis for hypothyroidism, veterinarians should be aware that these dogs have different thyroid hormone concentrations than nonsight hound dogs.

Germline variants in IL4, MGMT and AKT1 are associated with prostate cancer-specific mortality: An analysis of 12,082 prostate cancer cases.

BACKGROUND: Prostate cancer (PCa) is a leading cause of mortality and genetic factors can influence tumour aggressiveness. Several germline variants have been associated with PCa-specific mortality (PCSM), but further replication evidence is needed. METHODS: Twenty-two previously identified PCSM-associated genetic variants were genotyped in seven PCa cohorts (12,082 patients; 1544 PCa deaths). For each cohort, Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals for risk of PCSM associated with each variant. Data were then combined using a meta-analysis approach. RESULTS: Fifteen SNPs were associated with PCSM in at least one of the seven cohorts. In the meta-analysis, after adjustment for clinicopathological factors, variants in the MGMT (rs2308327; HR 0.90; p-value = 3.5 × 10-2) and IL4 (rs2070874; HR 1.22; p-value = 1.1 × 10-3) genes were confirmed to be associated with risk of PCSM. In analyses limited to men diagnosed with local or regional stage disease, a variant in AKT1, rs2494750, was also confirmed to be associated with PCSM risk (HR 0.81; p-value = 3.6 × 10-2). CONCLUSIONS: This meta-analysis confirms the association of three genetic variants with risk of PCSM, providing further evidence that genetic background plays a role in PCa-specific survival. While these variants alone are not sufficient as prognostic biomarkers, these results may provide insights into the biological pathways modulating tumour aggressiveness.

Magnetic resonance imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits.

BACKGROUND: Based on the observation that ultrasmall superparamagnetic particles of iron oxides (USPIOs) are phagocytosed by cells of the mononuclear phagocytic system, the purpose of this study was to evaluate their use as a marker of atherosclerosis-associated inflammatory changes in the vessel wall before luminal narrowing is present. METHODS AND RESULTS: Experiments were conducted on 6 heritable hyperlipidemic and 3 New Zealand White rabbits. 3D MR angiography (MRA) of the thoracic aorta was performed on all rabbits by use of a conventional paramagnetic contrast agent that failed to reveal any abnormalities. One week later, all rabbits except 1 of the hyperlipidemic animals were injected with a USPIO contrast agent (Sinerem, Guerbet) at a dose of 1 mmol Fe/kg. 3D MRA data sets collected over the subsequent 5 days showed increasing signal in the aortic lumen. Whereas the aortic wall of the control rabbits remained smooth and bright, marked susceptibility effects became evident on day 4 within the aortic walls of hyperlipidemic rabbits. Ex vivo imaging of aortic specimens confirmed the in vivo results. Histopathology documented marked Fe uptake in macrophages embedded in atherosclerotic plaque of the hyperlipidemic rabbits. Electron microscopy showed multiple cytoplasmic Fe particles in macrophages. No such changes were seen in control rabbits or in the hyperlipidemic rabbit that had not received Sinerem. CONCLUSIONS: USPIOs are phagocytosed by macrophages in atherosclerotic plaques of the aortic wall of hyperlipidemic rabbits in a quantity sufficient to cause susceptibility effects detectable by MRI.

Clinical characteristics of prostate cancer in an analysis of linkage to four putative susceptibility loci.

PURPOSE: Hereditary prostate cancer is an etiologically heterogeneous disease with six susceptibility loci mapped to date. We aimed to describe a collection of high-risk prostate cancer families and assess linkage to multiple markers at four loci: HPC1 (1q24-25), PCaP (1q42.2-43), HPCX (Xq27-28), and CAPB (1p36). EXPERIMENTAL DESIGN: Medical record data on 505 affected men in 149 multiply-affected prostate cancer families were reviewed, and correlations of clinical traits within each family were calculated. Logarithm of odds (LOD) score and nonparametric (NPL) linkage analyses were performed; white families were stratified by age of diagnosis, grade and stage of disease, and evidence of linkage to the other loci to increase genetic homogeneity. RESULTS: Age at diagnosis was the most correlated clinical trait within families. A maximum NPL score of 2.61 (P = 0.007) appeared to confirm HPC1 linkage for families that had a prevalence of high-grade or advanced-stage prostate cancer and which were not likely to be linked to PCaP, HPCX, or CAPB. Because the NPL scores improved when families more likely to be linked to the other loci were excluded, HPC1 may act independently of the other loci. The relationship of HPC1 and aggressive disease was strongest in families with median age at diagnosis > or =65 years (NPL, 3.48; P = 0.0008). CONCLUSIONS: The current results suggest that HPC1 linkage may be most common among families with more severe prostate cancer. Stratification by clinical characteristics may be a useful tool in prostate cancer linkage analyses and may increase our understanding of hereditary prostate cancer.

Comparison of the activity profiles of two fixed combinations of regular/NPH human insulin (recombinant DNA) of different compositions with a fixed regular/NPH porcine insulin combination (PPI) in insulin-dependent diabetic individuals.

In a randomized double-blind crossover study, 10 insulin-dependent diabetic individuals were treated with a commercial brand of neutral, highly purified porcine insulin (PPI) with 30% regular and 70% NPH fractions (Mixtard, Nordisk, Denmark), and with human insulins (recombinant DNA) with 20% regular and 80% NPH fractions, and with 30% regular and 70% NPH fractions. Each of the preparations was given for 4 consecutive days. The human insulin combination with the 20% regular and 80% NPH components showed a similar activity profile as the PPI 30/70 combination. In contrast, the human insulin combination with the 30% regular and 70% NPH fractions produced substantially lower blood sugar concentrations with the associated risk of hypoglycemia.

Confirmation of prostate cancer susceptibility genes using high-risk families.

Data from many types of studies support the hypothesis that strong familial components are involved in the etiology of prostate cancer. One way to access such genes is through the study of families with multiple affected family members and, in particular, families with individuals affected comparatively early in life. Several prostate cancer susceptibility loci have been described to date. Confirmation of the linkage and estimation of the proportion of families who are linked in large independent datasets is essential to understanding the significance of susceptibility genes. We explore the methodology used to perform such studies and the factors that can limit the ability to confirm linkage results. We report specifically the example of the HPC1 gene on 1q24-25.

Identification of a T cell chemotactic factor in the cerebrospinal fluid of HIV-1-infected individuals as interferon-gamma inducible protein 10.

Central nervous system (CNS) involvement is a prominent feature of human immunodeficiency virus (HIV-1) infection. Monocytes and CD4+ T cells traverse the blood brain barrier (BBB), and serve as vehicles for the virus and perpetrators for brain pathology by their production of neurotoxins. In the present study cerebrospinal fluid (CSF) samples from HIV-1-infected patients were analyzed for the presence of chemotactic factors. All 36 CSF samples from the patients were positive for the CXC chemokine interferon-gamma inducible protein (IP-10), which was not detected in CSF samples of 14 controls. The IP-10 concentrations were higher in HIV-1-infected patients with HIV-1 associated neurologic disorders than in those without neurological deficits. In contrast to IP-10, other chemotactic factors including the CC chemokines MCP-1, MIP-1alpha, MIP-1beta and RANTES and the cytokines IL-15 and IL-16 were either not detected or increased in only less than 30% of the patients. Unlike the CSF samples of controls, all CSF samples from HIV-1-infected patients induced chemotaxis of T cells activated with IL-2. The significance of IP-10 as a T cell chemotactic cytokine in HIV-1-infected CSF is shown by (1) the correlation of the IP-10 levels with the extent of T cell chemotaxis, (2) the neutralization of T cell chemotaxis by anti-IP-10 antibodies and (3) the correlation of the chemotactic response of CSF samples on activated T cells and the CSF white cell count in the patients. Our data provide evidence that IP-10 contributes to the accumulation of activated T cells in the CSF compartment in HIV-1-infected individuals.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in viral meningitis: upregulation of MMP-9 and TIMP-1 in cerebrospinal fluid.

A hallmark of viral meningitis is the invasion of monocytes, lymphocytes and, in the initial phase of the disease, neutrophils into the subarachnoidal space. By their degradation of different macromolecular components in the extracellular connective tissue, matrix metalloproteinases (MMPs) may be essential for the breakdown of the vessel wall in the meninges and the choroid plexus. In this study, the occurrence of MMP-1, MMP-2, MMP-3 and MMP-9 and the two tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, was monitored in the cerebrospinal fluid (CSF) from patients with viral meningitis. Of the proteinases, MMP-9 was found in 13 of 39 (33%) patients, but not in controls; the levels being correlated with the neutrophil cell number in CSF. The CSF concentration of TIMP-1 was increased three-fold compared to the control group (median 233 ng/ml; range 9.4-1252.5 ng/ml) and was correlated to the levels of total protein in CSF. Of the other MMPs and TIMPs assayed, MMP-2 and TIMP-2 were constitutively expressed and not upregulated in viral meningitis. High levels of MMP-9 and MMP-2, as measured by ELISA, was associated with high proteolytic activity detected in CSF by zymography. In conclusion, invasion of the leukocytes into the CSF compartment in viral meningitis may involve MMP-9, its proteolytic effect likely being controlled by expression of TIMP-1.

Influence of buffer zone concentrations on efficiency in partial filling micellar electrokinetic chromatography.

The potential of counter pressure-moderated partial filling micellar electrokinetic chromatography (PF-MEKC) was investigated in this work. Plate numbers of homologous omega-phenylalcohols were measured in a two-plug PF-MEKC system varying the concentrations and hence the ionic strengths of the background buffer compared to the sodium dodecyl sulfate-containing separation buffer and the counter pressure on the cathodic buffer reservoir. It was observed that plate numbers are strongly influenced by both the buffer concentrations and the counter pressure. Highest plate numbers were obtained with a buffer system where the concentrations are adjusted such that the electroosmotic flow velocities in both zones are equal. Differences in the local electroosmotic flow velocities of the zones caused by different buffer concentrations are responsible for tremendously reduced plate numbers. The efficiency drop is explained in several models by the formation of an intersegmental pressure which produces a parabolically shaped laminar flow component in both zones. Thus, the electroosmotic plug-like flow profile is distorted and the efficiency is reduced. The effect of counter pressure on efficiency turned out to be very complex in dependence on the buffer system applied.

The fermenting bacterium Malonomonas rubra is phylogenetically related to sulfur-reducing bacteria and contains a c-type cytochrome similar to those of sulfur and sulfate reducers.

Malonomonas rubra is a microaerotolerant fermenting bacterium which can maintain its energy metabolism for growth by decarboxylation of malonate to acetate. 16S rRNA sequence analysis revealed that M. rubra is closely related to the cluster of mesophilic sulfur-reducing bacteria within the delta subclass of the Proteobacteria, with the fermenting bacterium Pelobacter acidigallici and the sulfur reducers Desulfuromusa kysingii, D. bakii and D. succinoxidans as closest relatives. The cells contain high amounts (up to 12% of the total cell protein content) of a c-type cytochrome which is present mainly (> 60%) in the cytoplasm and to minor parts in the periplasm (> 20%) and associated with the membrane fraction (> 10%), independent of the growth substrate. This cytochrome is a tetraheme cytochrome of 13,700 Da molecular mass with a midpoint redox potential of -0.210 V.M. rubra does not reduce sulfur or ferric iron compounds. Since this cytochrome appears not to be involved in the energy metabolism it is concluded that it is a remnant of sulfur-reducing ancestors of this bacterium, without a conceivable physiological function in its present energy metabolism.

Morphological and functional analysis of PTA granules in human NK cells.

Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3-, CD16+, CD56+) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNalpha, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation.