Histamine release “in vivo” and “in vitro” induced by hypnotics in normal and atopic patients

The “in vitro” test of histamine release induced in the leukocytes of atopic subjects selected according to specific criteria would seem to be much more accurate to study the histamine releasing characteristics of intravenous agents than the “in vivo” study in patients who have to be anaesthetised. Moreover, different concentrations of the test drug may be used, and thus the threshold for histamine release may be compared. It is the test which we recommend for investigating new drugs. However, it is lengthy and expensive; it can therefore not be recommended as a routine preoperative investigation.     

Interferon-γ-like immunoreactivity in developing rat spinal ganglia neuronsin vivo andin vitro

Summary Interferon--like immunoreactivity was observed in a subpopulation of 16-day-old embryonic rat spinal ganglion neurons using two monoclonal antibodies directed against different epitopes of recombinant interferon-. During ontogenesis bothin vivo andin vitro, it was found that the strong immunoreactivity was confined to small neurons when neurons become morphologically distinct on the basis of size.In vivo, the interferon--immunoreactive neurons started to express major histocompatibility complex class I antigens after the first postnatal week, whilein vitro no such antigen could be detected. A quantitative Elisa method was developed to determine the levels of major histocompatibility complex class I and interferon-in vitro, whereby increased amounts of major histocompatibility complex class I antigen was detected after exposing the cultures to recombinant interferon- and Sendai virus. Sendai virus also caused a small increase in interferon- with a peak about 12 hours after infection. Thein vitro system will be used to study further the role of the putative neuronal interferon--like molecule in the regulation of cell growth, for induction of major histocompatibility complex antigens and in virus infection of sensory neurons.     

Effects of the PPAR-β agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination

Background  

Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-β seems to play an important role in the regulation of central inflammation. In addition, PPAR-β agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-β agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-γ and LPS.     

Tumour necrosis factor α production by rat blood and itsex vivo pharmacological modulation

Rat blood was investigated as a suitable test system for the discovery of inhibitors of tumour necrosis factor (TNF) biosynthesis. Lipopolysaccharide (LPS) caused a concentration- and time-dependent stimulation of TNF production by heparinised rat blood with peak levels (1000–5000 U/ml; L929 bioassay) at 6h. Bioactive material was neutralised with a polyclonal rabbit anti-murine TNF antibody which cross-reacts with rat TNF. Dexamethasone, pentoxifylline and denbufylline inhibited TNF production with IC₅₀s of 6.0±2.0 nM, 20.6±8.00 M and 138.0 nM, respectively. When rats were dosed p.o. with dexamethasone or pentoxifylline or i.p. with denbufylline and 1.5h later TNF production was assessedex vivo by LPS-stimulated blood, a dose-related inhibition of TNF production occurred with ID₅₀s of approximately 0.08, 250.0 and 5.0 mg/kg, respectively. These results demonstrate that rat blood provides a useful test system for the detection andex vivo evaluation of inhibitors of TNF biosynthesis.     

“Inflammatory skin march” in atopic dermatitis and psoriasis

Background

Comorbidities of cardiovascular diseases (CVDs), metabolic syndrome and autoimmune diseases with systemic inflammation are recent topics in medicine. Inflammatory skin diseases such as atopic dermatitis and psoriasis are an active source of diverse proinflammatory cytokines and chemokines, which are readily detectable in the circulation and are likely to be involved in developing comorbidities.

Evidence

Both atopic dermatitis and psoriasis are frequently comorbid with CVD, metabolic syndrome and autoimmune diseases, the consequence of which is called “inflammatory skin march”, “psoriatic march” or “march of psoriasis”.

Conclusion

In this review, we summarize the epidemiological evidence and pathogenetic concepts regarding inflammatory skin march in atopic dermatitis and psoriasis.

     

“Afferent dysgraphia” in a patient and in normal subjects

We report a detailed single case study of a patient, VB, who showed a form of dysgraphia which Lebrun (1976) termed “afferent dysgraphia”. VB, who was not aphasic and showed preserved spelling knowledge in spelling aloud or arranging cardboard letters, tended to write only down the right-hand side of a page and made frequent errors involving the omission or repetition of letters or strokes. These errors occurred most often in repeated sequences of identical or similar letters or strokes. The same letter and stroke errors, occurring at much the same frequency, were observed when normal subjects were required to write without sight of their writing hand whilst simultaneously executing a continuous tapping sequence with the fingers of their left hand.

We attribute certain of VB's dysgraphic symptoms to a general left-sided neglect which also affected her reading (Ellis, Young,&Flude, 1987). The letter and stroke errors (with the possible exception of failures to dot i's and to cross t's) are, however, given the same explanation as for the normal subjects, namely a failure to utilise visual and kinaesthetic feedback in the motor control of handwriting.     

Histamine release from rat peritoneal mast cells and human basophils induced by the free radical generator 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)

Inflammation Research -     

The role of mast cells in histologically “normal” appendices following emergency appendectomy in pediatric patients

Fifteen percent to 25% of appendices resected for a preoperative diagnosis of acute appendicitis have no neutrophilic infiltration, thus histologically “normal.” The discrepancy between clinical presentation and the lack of definite morphologic changes is confounding. It has been indicated that mast cells may play a role in the pathogenesis of the appendicitis-like pain in patients with histologically negative appendices (HNAs). To investigate whether mast cell density (MCD) is increased in pediatric HNAs, we retrieved 50 appendectomy cases (30 HNA and 20 control, ages 2 days-18 years) in our institute in the last 10 years. All cases were stained with mast cell tryptase by immunohistochemistry, and MCD (count/high-power field) was measured in mucosa, submucosa, muscularis, and serosa. Mast cells had the greatest density in the mucosa, followed by the submucosa, in all appendices. MCDs in all 4 layers were significantly higher in HNAs than in the normal controls (mucosa: 46 ± 9 vs 26 ± 11, P < .01; submucosa: 18 ± 5 vs 11 ± 5, P < .01; muscularis: 6 ± 3 vs 4 ± 2, P < .01; serosa: 6 ± 2 vs 4 ± 2, P < .01). This result suggests that mast cells play an important role in pathogenesis of HNA cases. In clinical practice, pathologists may order immunohistochemical stains for mast cells in cases with no classic histologic findings of acute appendicitis following emergency appendectomy. If increased MCD is noted, the case may be reported as “appendicitis with increased mast cells.” This assures surgeons that the appendectomy is the correct treatment and it is not necessary to look for other causes of acute abdomen. This is especially important in children.     

The initiation of smooth pursuit eye movements and saccades in normal subjects and in “express-saccade makers”

A vast knowledge exists about saccadic reaction times (RT) and their bi- or multimodal distributions with very fast (express) and regular RT. Recently, there has been some evidence that the smooth pursuit system may show a similar RT behavior. Since moving targets usually evoke a combined pursuit/saccade response, we asked which processes influence the initiation of pursuit and saccadic eye movements. Furthermore, we investigated whether and how the pursuit and saccadic system interact during the initiation of eye movements to moving targets. We measured the RT of the initial smooth pursuit (iSP) response and of the first corrective saccade and compared the RT behavior of both. Furthermore we compared the behavior of the corrective saccades to moving targets to that of saccades to stationary targets, known from the literature. The stimulus consisted of a target that moved suddenly at constant velocity (ramp). In addition, prior to the movement, a temporal gap, a position step or a combination of both could occur (gap-ramp, step-ramp, gap-step-ramp, respectively). Differently from most previous studies, we chose step and ramp with the same direction to provoke competition between the pursuit and saccade system. For the first time we investigated pursuit initiation in "express-saccade makers" (ES makers), a subject group known to produce an abnormally high percentage of short-latency saccades in saccade tasks. We compared their results with subject groups who were either naive or trained with respect to saccade tasks. The iSP started at approximately 100 ms, which corresponds to express saccade latencies. These short iSP-RT occurred reflex-like and almost independent of the experimental task. A bimodal frequency distribution of RT with a second peak of longer iSP-RT occurred exclusively in the ramp paradigm. The RT of the first corrective saccades in a pursuit task were comparable with that in a saccade task and depended on the stimulus. The ability of ES makers to produce a high number of express saccades was transferred to corrective saccades in the pursuit task, but not to pursuit initiation. In summary, short-latency pursuit responses differ from express saccades with respect to their independence of experiment and subject group. Therefore, a simple analogy to express saccades cannot be drawn, although some mechanisms seem to act similarly on both the pursuit and the saccade system (such as disengagement of attention with the gap effect). Furthermore, we found evidence that the initial pursuit response and the first corrective saccade are processed independently of each other. The first corrective saccades to moving targets behave like saccades to stationary targets. Normal pursuit but abnormal saccade RT of ES makers can be explained by recent theories of superior colliculus (SC) function in terms of retinal error handling.     

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-α/β and TNF-α in cultured endothelial cells

Background

Immune activation that results due to the aberrant proliferation of lymphocytes leads to inflammation and graft rejection in organ transplant recipients. We hypothesize that the cell cycle control and inflammation are parallel events, inhibition of cellular proliferation by cyclin kinase inhibitor specifically p21 will limit inflammation and prevent allograft rejection.

Methods

We performed in vitro and in vivo studies using lymphocytes, and rat heart transplant model to understand the role of cyclins and p21 on mitogen and allo-induced lymphocyte activation and inflammation. Lymphocyte proliferation was studied by ³H-thymidine uptake assay and mRNA expression was studied RT-PCR.

Results

Activation of allo- and mitogen stimulated lymphocytes resulted in increased expression of cyclins, IL-2 and pro-inflammatory cytokines, which was inhibited by cyclosporine. The over-expression of p21 prolonged graft survival in a completely mismatched rat heart transplant model resulted by inhibiting circulating and intra-graft expression of proinflammatory cytokines.

Conclusion

Cyclins play a significant role in transplant-induced immune activation and p21 over-expression has potential to inhibit T cell activation and inflammation. The results from this study will permit the design of alternate strategies by controlling cell cycle progression to achieve immunosuppression in transplantation.     

Prevalence of community-associated meticillin-resistant Staphylococcus aureus and Panton–Valentine leucocidin-positive S. aureus in general practice patients with skin and soft tissue infections in the northern and southern regions of The Netherlands

The purpose of this investigation was to determine the prevalence of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) and Panton–Valentine leucocidin (PVL)-positive S. aureus in general practice (GP) patients with skin and soft tissue infections (SSTI) in the northern (Groningen and Drenthe) and southern (Limburg) regions of The Netherlands. Secondary objectives were to assess the possible risk factors for patients with SSTI caused by S. aureus and PVL-positive S. aureus using a questionnaire-based survey. From 2007 to 2008, wound and nose cultures were obtained from patients with SSTI in general practice. These swabs were analysed for the presence of S. aureus and the antibiotic susceptibility was determined. The presence of the PVL toxin gene was determined by polymerase chain reaction (PCR) and the genetic background with the use of spa typing. A survey was performed to detect risk factors for S. aureus infection and for the presence of PVL toxin.S. aureus was isolated from 219 out of 314 (70%) patients with SSTI, of which two (0.9%) patients were MRSA-positive. In 25 (11%) patients, the PVL toxin gene was found. A higher prevalence of PVL-positive S. aureus of patients with SSTI was found in the northern region compared to the south (p < 0.05). Regional differences were found in the spa types of PVL-positive S. aureus isolates, and for PVL-negative S. aureus isolates, the genetic background was similar in both regions. The prevalence of CA-MRSA in GP patients with SSTI in The Netherlands is low. Regional differences were found in the prevalence of PVL-positive S. aureus isolates from GP patients with SSTI. Household contacts having similar symptoms were found to be a risk factor for SSTI with S. aureus.     

“ECG variability contour” method reveals amplitude changes in both ischemic patients and normal subjects during Dipyridamole stress: a preliminary report

To detect and quantify consistent ECG amplitude changes, the “ECG variability contour” (EVC) method was proposed. Using this method we investigated amplitude changes in subjects undergoing myocardial perfusion imaging (MPI) with Dipyridamole (Dp). Fifty-three patients having reversible perfusion defects and 19 normal subjects (NS) who were free of: perfusion defects on their MPI, standard ST–T changes during Dp stress, and a negative clinical follow up. Mean ∏ ¹ (〈∏ ¹〉) was similar for the NS and patient group (6.2 ± 6.1 vs. 6.3 ± 6.2, P = 0.95). 〈∏ ¹〉 was 4.6 ± 3.0 in patients not having ST–T changes during Dp stress (n = 42), whereas in patients having ST–T changes (n = 11) it was 13.1 ± 10.2 (P < 0.001). For both groups 〈∏ ^QRS〉 was smaller than 〈∏ ^ST〉, which in turn was smaller than 〈∏ ^T〉. The values of 〈∏ ^QRS〉, 〈∏ ^ST〉, and 〈∏ ^T〉 for the NS, patients without and with ST–T changes were: 26.8 ± 28.6, 42.6 ± 41.8, 44.9 ± 36.5; 19.6 ± 20.8, 26.4 ± 31.4, 38.7 ± 27.3; 51.0 ± 30.0, 71.0 ± 36.8, 75.1 ± 20.9, respectively (P < 0.05 for all comparisons of patients with versus without ST–T changes). This study showed that Dp stress, with or without hypoperfusion, had a clear effect on myocyte electrophysiology, expressed by consistent ECG amplitude changes, detected by the EVC method. The EVC method did not distinguish between NS and patients in this clinical setting.     

Allergen-specific immunoglobulin,histamine and T-cell responses induced by soybean glycinin and β-conglycinin in BALB/c mice of oral sensitisation

Glycinin and β-conglycinin are major soybean allergens involved in food hypersensitivity. However, the mechanism of immune responses induced by glycinin and β-conglycinin has not been fully understood. Balb/c mice were oral sensitised with different doses (0.1, 1.0 and 10 mg/day) of soybean glycinin and β-conglycinin for five weeks. Allergen-specific immunoglobulin (Ig), serum histamine and T-cell responses were tested to assess the allergenic activity of glycinin and β-conglycinin. Mice sensitised with 0.1 or 1.0 mg/day allergens induced high levels of specific IgE, IgG1 and serum histamine compared with mice treated with saline. Furthermore, specific T-cell proliferation and significant up-regulation of interleukin (IL)-4, IL-5 and interferon (IFN)-γ were observed in splenocytes from mice gavaged with 0.1 or 1.0 mg/day soybean proteins. Low doses of glycinin or β-conglycinin can induce allergic reactions in BALB/c mice, which might be associated with increased IgE and cytokine production.     

The study of heterogeneity of 16S rRNA gene and groESL operone in DNA samples of Anaplasma phagocytophilum, Ehrlichia muris, and “Candidatus Neoehrlichia mikurensis” determined in Ixodes persulcatus ticks on the territory of Ural, Siberia and Far East of Russia

A total of 3552 Ixodes persulcatus from Sverdlovsk, Chelyabinsk, Novosibirsk, Irkutsk regions and Khabarovsk Territory were examined on the Ehrlichia and Anaplasma presence by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris DNA were found in I. persulcatus in all studied regions — A. phagocytophilum was detected in 1.3–6.3% of ticks and E. muris — in 2.0–14.1% of ticks. Moreover, “Candidatus Neoehrlichia mikurensis” DNA was found in 8 ticks collected in Novosibirsk, Irkutsk Regions and Khabarovsk Territory. Partial nucleotide sequences of 16S rRNA gene and groESL operone (1240–1300 bp) were determined for 65 samples of A. phagocytophilum, 17 samples of E. muris and 4 samples of “Candidatus Neoehrlichia mikurensis”. Nucleotide sequences of 16S rRNA gene and groESL operone of E. muris and “Candidatus Neoehrlichia mikurensis” were shown to be highly conservative, and nucleotide sequences of groESL operone of both E. muris and “Candidatus Neoehrlichia mikurensis” differed from the sequences found previously in other species of Ixodid tick. On the basis of analysis of the 16S rRNA gene and groESL operone sequences it was concluded that all revealed samples A. phagocytophilum could be divided into 2 groups. GroESL operone sequences of A. phagocytophilum from the first group were identical to each other but significantly differed from the known groESL operone sequences (less than 98.2% of similarity), whereas their 16S rRNA gene sequences were identical to the sequence of widely distributed and pathogenic for human A. phagocytophilum genetic variant (CAHU-HGE1, GenBank AF093788) or differed from it by a single nucleotide substitution. The nucleotide sequences of groESL operone of A. phagocytophilum from the second group differed from each other by 1–4 nucleotides and were closely related (99.2–99.4% of similarity) to the sequences of groESL operone of A. phagocytophilum isolates found in Europe in Ixodes ricinus and roe deer. The nucleotide sequences of the 16S rRNA gene of A. phagocytophilum from the second group were most similar to the sequence of the rare A. phagocytophilum genetic variant previously found only in China (GenBank DQ342324).     

Transition between Mycobacterium tuberculosis and nontuberculous mycobacteria in recurrent “tuberculosis” patients

European Journal of Clinical Microbiology & Infectious Diseases - Recurrence of tuberculosis (TB) is still a key issue in the control of tuberculosis. The presence of nontuberculous...     

MicroRNA-155 suppresses the catabolic effect induced by TNF-α and IL-1β by targeting C/EBPβ in rat nucleus pulposus cells

Aim: miR-155 is a pro-inflammatory or anti-inflammatory factor depending on the cell type in which it is expressed. miR-155 controls apoptosis and matrix degradation in nucleus pulposus (NP) cells in vitro. The aim of this study is to explore the effect of miR-155 in vivo and further investigate the mechanism of miR-155 in vitro. Methods: MRI, hematoxylin–eosin staining, or Collagen-II immunochemistry were performed to observe intervertebral disk degeneration in conditional miR-155 overexpression mice and miR-155 knockout mice. In vitro, a dual luciferase reporter assay, real-time PCR and western blot experiments were performed to demonstrate the effect of miR-155 on the expression of catabolic genes induced by inflammatory cytokines and determine the role of β-catenin and C/EBPβ in the miR-155-mediated modulation of the expression of catabolic genes. Results: Degeneration was observed in the lumbar disks of 1-year-old miR-155 knockout mice but not in the conditional miR-155 overexpression mice. miR-155 overexpression repressed the catabolic effect induced by TNF-α or IL-1β in vitro. Furthermore, specifically in NP cells, miR-155 overexpression suppressed the expression of C/EBPβ but not of β-catenin. Additionally, in the loss-of-function experiments using C/EBPβ siRNA, C/EBPβ knockdown repressed the expression of catabolic genes induced by TNF-α and IL-1β, which is consistent with the miR-155 results. Conclusion: miR-155 is a sustainable factor for intervertebral disk and suppresses the expression of catabolic genes induced by TNF-α and IL-1β by targeting C/EBPβ in rat NP cells.     

MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1β induced in human endometriosis stromal cells in vitro

Objective: Now there are more and more evidences that Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis of endometriosis (EMs). Vascular endothelial growth factor (VEGF) has a potent angiogenic activity. However, it is worth studying about the regulating mechanism of COX-2/COX-1 and VEGF in the development of human endometriosis in vitro. The current study was designed to investigate the effect of 4 cytokines on COX-2/COX-1 expression and the effect of IL-1β on VEGF release in human endometriosis stromal cells (ESC), and to explore the related signaling pathways involved in vitro. Methods: Isolation, culture and identification of ESC. Cells were treated with 4 cytokines, and the inhibitor mitogen-activated protein-Erk (MEK) and the inhibitor p38 mitogen-activated protein kinase (MAPK) prior to adding cytokine IL-1β. COX-2 protein expression was measured by western blot and VEGF secretion was determined by ELISA. Results: Among four kinds of cytokines, IL-1β treatment increased COX-2 protein expression and VEGF release in three ESC, and TNF-α had the same effect on COX-2 protein level as IL-1β only in ectopic and eutopic ESC, and MCSF had only slight effect on ectopic ESC. In contrast, cytokines had no effect on COX-1 expression. We also demonstrated that MAPK reduced the synthesis of COX-2 by IL-1β induced. COX-2 inhibitor reduced VEGF release by IL-1β induced. Conclusions: i) In human ESC in vitro, IL-1β up-regulated the COX-2 expression through the activation of p38 MAPK pathway, and not to COX-1. ii) Up-regulation of VEGF level by IL-1β treatment was found in human endometriosis stromal cell and COX-2 inhibitor was involved in this process.