Evidence for increased expression of eotaxin and monocyte chemotactic protein-4 in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with tissue eosinophilia and the activation of T lymphocytes. The novel eosinophil chemoattractants, eotaxin and monocyte chemotactic protein (MCP)-4, are up-regulated at sites of allergic inflammation, yet their contribution to the pathophysiologic mechanisms of AD remains to be determined. OBJECTIVE: We sought to investigate the expression of eotaxin and MCP-4 in acute and chronic lesions from patients with AD and to determine their relationship to the numbers of resident inflammatory cells. METHODS: With use of in situ hybridization, the expression of eotaxin and MCP-4 messenger RNA (mRNA) in skin biopsy specimens from patients with acute and chronic AD skin lesions was compared with that of uninvolved skin from these patients and skin from healthy volunteers. RESULTS: There was a constitutive expression of eotaxin and MCP-4 mRNA in skin biopsy specimens from healthy subjects. Positive signal for chemokine mRNA was observed both within the epidermis and inflammatory cells (macrophages, eosinophils, and T cells) of the subepidermis in AD skin lesions. Within the subepithelium acute and chronic skin lesions exhibited a significant increase in the numbers of eotaxin and MCP-4 mRNA-positive cells compared with uninvolved skin (P <.01), whereas the numbers of eotaxin and MCP-4 mRNA-positive cells were significantly higher in chronic AD compared with acute AD skin lesions (P <.005, P <.001, respectively). Correlations were observed between the expression of eotaxin and MCP-4 mRNA and the presence of eosinophils and macrophages, respectively, in AD lesions (r(2) = 0.84, r(2) = 0.94). CONCLUSION: There is an increased expression of eotaxin and MCP-4 in acute and chronic lesions, suggesting that these chemotactic factors play a major role in the pathophysiologic mechanisms of AD.     

Genetic epidemiology in the study of susceptibility/resistance to malaria in the human population

The development of genetic epidemiology methods using recent human genetic map together with the growing availability of candidate genes have led to substantial advances in the identification of host genes in human malaria. Investigation of these genes has progressed along two complementary ways: 1) The search for genes influencing the severe malaria clinical phenotype by means of population based case-control studies which showed the protective role of several red cell genetic defects (sickle cell anemia, a-thalassaemia ...) and that some polymorphisms of the TNF-alpha promoter region could predispose to cerebral malaria; 2) The investigation of the genetic regulation of malaria-related biological phenotypes (infection levels, immune response) by means of familial studies which underlined the influence of the 5q31-q33 chromosomal region in the control of Plasmodium falciparum blood parasitemia and the role of major histocompatibility complex (MHC) and non-MHC genes in the regulation of humoral and cellular response to various malarial antigens. Ongoing studies will precise the role of these genes and probably reveal the existence of other genes not identified yet. The impact of these findings on the understanding of malaria pathogenesis and on the design of future preventive and therapeutic strategies should be considerable.     

Stimulation of human monocyte beta-glucan receptors by glucan particles induces production of TNF-alpha and IL-1 beta.

beta-glucans are pharmacologic agents that rapidly enhance host resistance to a variety of biologic insults through mechanisms involving macrophage activation. To determine whether stimulation of the beta-glucan receptors on human monocytes resulted in cytokine production, monolayers of monocytes were incubated with purified yeast glucan particles and measured for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) mRNA and protein. By Northern blot analysis, TNF-alpha mRNA was detected within 30 min of incubation with glucan particles, peaked at 2 h, and remained elevated for at least 8 h. Glucan induction of IL-1 beta mRNA followed a similar time-course of initiation and accumulation. By enzyme-linked immunosorbent assays (ELISAs), significant levels of TNF-alpha and IL-1 beta were present in supernatants of glucan-treated cells within 1 h and plateau levels of both cytokines were approached within 4 h. At particle-to-cell ratios of from 0.4 to 18, glucan particles induced dose-dependent increases in TNF-alpha and IL-1 beta mRNA and corresponding increases in TNF-alpha and IL-1 beta proteins. Exposure of monocytes to glucan particles for 0-30 min and washing before continued incubation for 4 h in particle-free buffer induced production and secretion of TNF-alpha and IL-1 beta in a time-dependent fashion compatible with phagocytosis. The pretreatment of monocyte monolayers with trypsin reduced glucan-induced production of TNF-alpha and IL-1 beta in a dose-dependent manner with 5 micrograms/ml of trypsin effecting reductions of greater than 50%. Thus, glucan particles induce human monocyte production of TNF-alpha and IL-1 beta by a mechanism that is dependent on trypsin-sensitive beta-glucan receptors.     

Low sialic acid-bearing mouse thymocytes do not express helper T cell properties in vitro

Immature, presumably cortical, mouse thymocytes were isolated by removing mature thymocytes by agglutination with the sialic acid-specific lectin, lobster agglutinin 1 (LAg1). These immature cells do not respond to the mitogenic effects of concanavalin A (Con A), even in the presence of interleukin 2. Moreover, they do not exhibit two properties of helper T cells; they do not secrete interleukin 2 when stimulated with Con A, nor do they provide T help for an in vitro immune response by spleen B cells to the T-dependent antigen, sheep erythrocytes. LAg1-negative thymocytes fail to provide T help even though Con A is added to the cultures, regardless of the number of LAg1-negative thymocytes added per culture, and even in the presence of exogenous macrophages. Unseparated thymocytes, LAg1-positive thymocytes and cortisone-resistant thymocytes all provide T cell help under these conditions. These experiments indicate that immature, presumably cortical mouse thymocytes, isolated by virtue of their low levels of surface sialic acid, are inherently unable to provide T cell help in vitro.     

Genetic predisposition to leprosy: A major gene reveals novel pathways of immunity to Mycobacterium leprae

The elucidation of the genetic control of susceptibility to common infectious diseases is expected to provide new and more effective tools for prevention and control of some of the most pressings health needs on a global scale. A major advantage of whole genome based genetic approaches is that no a priori assumptions about mechanisms of pathogenesis need to be made in these studies. Hence, genetic studies can identify previously unrecognized pathways of disease susceptibility and tag critical pathogenic events for further biochemical, immunological or physiological analysis. We have applied this strategy to leprosy, a disease that still claims 400,000 new cases each year. We identified genetic variants in the shared promoter region of the PARK2 and PACRG genes as major risk factors of leprosy susceptibility. Both encoded proteins are part of the cellular ubiquitination system. Specifically, PARK2, the cause of early onset Parkinson's disease, is an E3 ligase that likely is involved in controlled proteolysis, the cellular anti-oxidants response and the regulation of innate immune responsiveness. In addition, numerous E3 ligases have recently been shown to be critical regulators of immunity. While the specific role of PARK2/PACRG in leprosy pathogenesis remains unknown, a number of experimentally testable scenarios can be developed to further explore the role of these proteins in anti-Mycobacterium leprae host responsiveness.     

Genetic and biochemical characterization of Malassezia pachydermatis with particular attention to pigment-producing subgroups.

The aim of the study was the characterization of Malassezia pachydermatis and its pigment-producing subgroup using biochemical tests and RAPD. It was of interest to determine whether particular RAPD patterns could be used to indicate pigment production, as well as a close genetic relatedness to Malassezia furfur. Therefore, 210 strains of M. pachydermatis were examined for morphology, catalase and ss-glucosidase activity, lipid and carbohydrate assimilation and the tryptophan-dependent synthesis of pigments. Of these, 114 strains were subjected to RAPD analyses. A multivariate logistic regression model was applied to classify M. pachydermatis isolates regarding their pigment production by using genetic and biological parameters. Biological and RAPD findings showed a high biological and genetic diversity within the species M. pachydermatis and within pigment producers. RAPD analysis revealed 28 genotypes within 114 strains tested. Pigment producing strains could not be assigned to a common RAPD profile, but a genetic relatedness of pigment-producing M. pachydermatis with M. furfur can be assumed. A particular RAPD pattern allowed statistically significant probability of pigment production (P<0.001) and might be used as a tool to rapidly detect pigment producing M. pachydermatis, e.g. in Malassezia-associated pityriasis versicolor. The reported method is useful for identification of pigment producing M. pachydermatis isolates and has advantages over established tests.     

The human lumbar anterior epidural space: morphological comparison in adult and fetal specimens

To increase our understanding of the clinical anatomy of the epidural space, the human lumbar anterior epidural space was studied morphologically and developmentally. Histological transverse sections of human lumbar spines were taken at the level of the intervertebral disc and the vertebral body in adult specimens and in fetuses aged 13, 15, 21, 32 and 39 weeks (menstrual age). At 13 weeks, connective tissue filled the epidural space. The dura mater was attached anteriorly to the posterior longitudinal ligament (PLL). The PLL was attached to the vertebral body beside the midline, whereas it adhered to the posterior edge of intervertebral disc. The anterior internal vertebral venous plexus was located anterolaterally and anteromedially. The vertebral canal was lined with connective tissue that differentiated in a periosteum in contact with the ossification centers. At 15 weeks, the PLL was composed of deep and superficial layers. At 21 weeks, the attachment between the dura mater and PLL was ligament-like at the level of the vertebral body. At 32 weeks, the dura mater was adherent to the superficial layer of PLL. At 39 weeks, groups of adipocytes were identified, and the dura mater was attached to the PLL by some ligaments. There were many more similarities between the adult and the 39-week fetus. In conclusion, some differences in the anatomy of the epidural space exist at each fetal stage studied. The structures of the epidural space are already formed in the fetus of 13 weeks, but they differentiate progressively within the connective tissue.     

Bioadhesion and biocompatibility evaluations of gelatin and polyacrylic acid as a crosslinked hydrogel in vitro

This study describes the potentiality of crosslinked hydrogels comprised of gelatin and polyacrylic acid (CHGP) as a biological glue for soft tissues and compares its bonding strength with that of fibrin glue. Water-soluble carbodimide (WSC) was used to crosslink the mixture of gelatin and polyacrylic acid (PAA). An addition of PAA to gelatin increases bonding strength and reduces the gelation time and WSC concentration. Increasing the gelatin, WSC and PAA concentration increases the bonding strength. There is a critical concentration to have a maximum bonding strength. The cured hydrogel exhibited sufficient adhesion to mouse skin with a higher bonding strength than fibrin glue. The in vitro test has been done for evaluating CHGP toxicity.     

Protein synthesis is required for the enhancement of long-term potentiation and long-term memory by spaced training

Spaced training is generally more effective than massed training for learning and memory, but the molecular mechanisms underlying this trial spacing effect remain poorly characterized. One potential molecular basis for the trial spacing effect is the differential modulation, by distinct temporal patterns of neuronal activity, of protein synthesis-dependent processes that contribute to the expression of specific forms of synaptic plasticity in the mammalian brain. Long-term potentiation (LTP) is a type of synaptic modification that may be important for certain forms of memory storage in the mammalian brain. To explore the role of protein synthesis in the trial spacing effect, we assessed the protein synthesis dependence of hippocampal LTP induced by 100-Hz tetraburst stimulation delivered to mouse hippocampal slices in either a temporally massed (20-s interburst interval) or spaced (5-min interburst interval) fashion. To extend our studies to the behavioral level, we trained mice in fear conditioning using either a massed or spaced training protocol and examined the sensitivity of long-term memory to protein synthesis inhibition. Larger LTP was induced by spaced stimulation in hippocampal slices. This improvement of synaptic potentiation following temporally spaced synaptic stimulation in slices was attenuated by bath application of an inhibitor of protein synthesis. Further, the maintenance of LTP induced by spaced synaptic stimulation was more sensitive to disruption by anisomycin than the maintenance of LTP elicited following massed stimulation. Temporally spaced behavioral training improved long-term memory for contextual but not for cued fear conditioning, and this enhancement of memory for contextual fear was also protein synthesis dependent. Our data reveal that altering the temporal spacing of synaptic stimulation and behavioral training improved hippocampal LTP and enhanced contextual long-term memory. From a broad perspective, these results suggest that the recruitment of protein synthesis-dependent processes important for long-term memory and for long-lasting forms of LTP can be modulated by the temporal profiles of behavioral training and synaptic stimulation.     

Daily running promotes spatial learning and memory in rats.

Previous studies on exercise have shown that physical activity improves learning and memory. Present study was performed to determine the effects of acute, chronic and continuous exercise with different periods on spatial learning and memory recorded as the latency and length of swim path in the Morris water maze testing in subsequent 8 days. Four rat groups were included as follows: (1) group C, controls which did not exercise; (2) group A, 30 days treadmill running before and 8 days during the Morris water maze testing period; (3) group B, 30 days exercise before the Morris water maze testing period only; (4) group D, 8 days exercise only during the Morris water maze testing period. The results showed that chronic (30 days) and continuous (during 8 days of Morris water maze testing days) treadmill training produced a significant enhancement in spatial learning and memory which was indicated by decreases in path length and latency to reach the platform in the Morris water maze test (p<0.05). The benefits in these tests were lost in 3 days, if the daily running session was abandoned. In group D with acute treadmill running (8 days exercise only) the difference between the group A disappeared in 1 week and benefit seemed to be obtained in comparison with the controls without running program. In conclusion the chronic and daily running exercises promoted learning and memory in Morris water maze, but the benefits were lost in few days without daily running sessions in adult rats.