Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5–15 × 10⁶ leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts.     

Determinants that shape public attitudes towards the mentally ill

BACKGROUND: The stigmatisation of the mentally ill is considered a well-established fact. To improve negative attitudes among the general public, we need to identify the factors that cause them. Drawing from previous studies, we combined a variety of variables to examine a comprehensive explanative model. OBJECTIVES: We examined a sample of the Dutch public on their willingness to interact with mental patients. We examined a number of determinants concerning their influence on levels of social distance: demographical characteristics of the public, their beliefs about stereotypes of mental patients, their beliefs about causes of mental problems, their familiarity with mental illness. METHODS: We employed a questionnaire survey among two sub-samples of the Dutch public (n = 812, response 33%). RESULTS: Attributing psychiatric problems to structural causes (i.e. causes beyond patients' control and responsibility, such as genetic transmission) is associated with less social distance. Conversely, attribution to individual factors (e.g. drug abuse) related to more distant attitudes. Stereotypical beliefs about mental patients (e.g. untrustworthiness, aggressiveness, causing disturbances) relate to more social distance from mental patients. CONCLUSIONS: Results implied that our comprehensive model explains only a modest amount of variance, but shows that to improve public mental health literacy and attitudes should first deal with the most negative stereotypical beliefs.     

Phenotypic differences in murine chondrocyte cell lines derived from mature articular cartilage

OBJECTIVE: To obtain well characterized immortalized murine chondrocyte cell lines. The cell lines were obtained from mature articular chondrocytes, instead of embryonal cells which are used in most other studies. METHODS: Pieces of articular cartilage were cut from murine patellae and femoral heads. Chondrocytes were isolated by digestion with collagenase. These cells were cultured in monolayer and immortalized by transfection of the SV40 large T antigen gene. To preserve the differentiated phenotype, the resulting clones were cultured in three-dimensional carriers, alginate beads. The phenotypes of the cells were characterized using the following parameters: Cell morphology (light microscopy), messenger RNA (RT-PCR) and protein (immunohistochemistry) levels of extracellular matrix molecules. Moreover, responsiveness to interleukin-1(IL-1) was determined by measuring production of proteoglycans ((35)S-sulfate incorporation) and of nitric oxide (Griess reaction). RESULTS: Sixteen clones were obtained, ten (P1 to P10) derived from patellar cartilage, and six (H1 to H6) from femoral head cartilage. In seven cell lines (P2, P5, H1, H3, H4, H5, H6) high production of type II collagen corresponded with high levels of mRNA of type II collagen (and prevalence of the IIB type) and with high IL-1-induced suppression of proteoglycan synthesis. Like intact murine articular cartilage, all cell lines produced type I and type X collagens, but mRNA levels of both types of collagen were never higher in the cell lines as compared with intact cartilage. CONCLUSION: Our results demonstrate that it is possible to immortalize mature murine articular chondrocytes. Each of the obtained chondrocyte cell lines appeared to have a stable phenotype. Both relatively differentiated and relatively dedifferentiated chondrocyte cell lines could be identified.     

Identification of the epidermal growth factor–TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue

Objective

EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)–TM7 family of adhesion class 7‐span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST).

Methods

ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage– and activation‐specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2‐ and CD97‐specific multivalent fluorescent probes.

Results

EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor α. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast‐like synoviocytes.

Conclusion

The EGF‐TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

     

Herpes simplex virus type 1 and respiratory disease in critically-ill patients: real pathogen or innocent bystander?

Herpes simplex virus type 1 (HSV-1) has been associated with pulmonary disease, mostly in severely immunocompromised patients. After reactivation and shedding in the oropharynx, the virus may reach the lower respiratory tract by aspiration or by contiguous spread. HSV-1 can be detected in clinical specimens by virus culture or quantitatively by nucleic acid amplification techniques. With these techniques, HSV-1 is often detected in the respiratory secretions of critically-ill patients. However, a clear diagnosis of HSV-1 pneumonia is difficult to establish because clinical criteria, radiological features and laboratory findings all lack specificity. Lower respiratory tract HSV-1 infections have not been associated with specific risk-factors. There is also an absence of consistent data concerning the effect of antiviral treatment on the outcome of critically-ill patients. Further studies are needed to better define the pathogenic role of HSV-1 in the lower respiratory tract of these patients, to improve the diagnosis, and, especially, to assess the need for antiviral treatment in the individual patient.     

Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro.

Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3)-dependent multipotent hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day-old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation.     

The effect of chronic paracetamol administration to rats on the glycosaminoglycan content of patellar cartilage

Male Wistar rats were treated with paracetamol (200 mg/kg twice a day) for 2, 3, 4 and 9 weeks. During the first four weeks of paracetamol administration the serum sulfate concentration was significantly decreased. However, during the fourth until the ninth week, the serum sulfate concentration was only diminished to a small and insignificant extent. The paracetamol administration did not lead to serious liver or renal toxicity, as determined by alanine aminotransferase and creatinine levels in the serum of the rats. The paracetamol-induced serum sulfate depletion, observed during the first four weeks of the experiment, led to a significantly lower glycosaminoglycan content of the patellar cartilage of the rats after three and four weeks paracetamol treatment. When after the fourth week the serum sulfate concentration rose to nearly normal levels also the glycosaminoglycan content in the rat patellar cartilage reached control levels. These data indicate that the serum sulfate depletion might be the causative factor for the observed reduction in glycosaminoglycan content of rat patellar cartilage.     

In vivo protection against interleukin-1-induced articular cartilage damage by transforming growth factor-beta 1: age-related differences.

OBJECTIVES--Transforming growth factor-beta (TGF-beta) has been shown to antagonise interleukin-1 (IL-1) effects in different systems. Investigations were carried out to study whether TGF-beta 1 modulates IL-1 induced inflammation and IL-1 effects on articular cartilage in the murine knee joint. METHODS--IL-1, TGF-beta 1 or both factors together were injected into the knee joint. Inflammation was studied in whole knee histological sections. Patellar cartilage proteoglycan synthesis was measured using 35S-sulphate incorporation while patellar cartilage glycosaminoglycan content was determined with automated image analysis on joint sections. RESULTS--Co-injection of TGF-beta 1 and IL-1 resulted in synergistic attraction of inflammatory cells. In contrast, TGF-beta 1 counteracted IL-1 induced suppression of articular cartilage proteoglycan synthesis. Proteoglycan depletion was similar shortly after the last injection of IL-1 or IL-1/TGF-beta 1, but accelerated recovery was found with the combination at later days. This protective effect of TGF-beta 1 could not be demonstrated in older mice. CONCLUSIONS--TGF-beta 1 aggravates IL-1 induced knee joint inflammation, but counteracts the deleterious effects of IL-1 on articular cartilage proteoglycan synthesis and content. The data indicate that TGF-beta 1 could play an important part in articular cartilage restoration after IL-1 induced proteoglycan depletion.