Local proliferation of brain macrophages in central nervous system tissue cultures.

Mouse central nervous system tissue cultured for different lengths of time was analyzed for the proliferation of macrophages. These cells were identified and characterized by ultrastructural features, cell surface determinants and their ability to ingest latex particles and bacteria. Under the experimental conditions chosen brain macrophages were derived from perivascular cells which in short term cultures remained attached to blood vessels and later differentiated into brain macrophages with a typical ultrastructural appearance. Identical results were obtained when intravascular cells were largely removed by extensive saline perfusion before culturing. Macrophages assembled around stab wounds of the central nervous system or obtained from peritoneal lavage showed comparable cytological characteristics and cell membrane determinants.

IL-4 Signaling Mechanisms in Inflammatory Bowel Disease Mononuclear Phagocytes.

: In inflammatory bowel disease (IBD), intestinal mononuclear cells secrete increased amounts of proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), as well as nonspecific effector molecules (i.e., superoxide anions) in vitro and in vivo. Interleukin-4 (IL-4) is an important contrainflammatory cytokine to limit monocyte and macrophage activation. Data obtained with peripheral monocytes indicate that IL-4-mediated downregulation of activation may be impaired in IBD. High IL-4 concentrations are able to overcome the impairment in downregulation of proinflammatory cytokines and superoxide anions, respectively. We investigated molecular events involved in IL-4-induced signal transduction and regulation in IBD mononuclear phagocytes. Peripheral blood mononuclear cells were isolated by densitygradient centrifugation, intestinal lamina propria mononuclear cells by collagenase digestion. Proinflammatory cytokine mRNA levels were assessed by semiquantitative polymerase chain reaction using internal standards. IL-4 receptor expression was investigated by radiolabeled ligand binding studies and IL-4 receptor signal transduction by specific induction of signal transducer and activator of transcription 6 (Stat 6). Downregulation of TNF-α and IL-1ß mRNA levels, respectively, in IBD mononuclear phagocytes is impaired in comparison with normal cells. However, no differences between IBD and normal control mononuclear phagocytes were seen in IL-4 receptor surface expression and signal transduction by IL-4induced generation of Stat 6. Impaired downregulation of TNF-α and IL-1ß secretion by IL-4 in IBD mononuclear phagocytes is also seen on the mRNA level. The mechanism of IL-4 resistance may be located in elements of IL-4 receptor signal transduction downstream of Stat 6.

Anticipation in inflammatory bowel disease: a phenomenon caused by an accumulation of confounders.

Inflammatory bowel disease (IBD) has a definite genetic component as documented by epidemiological and linkage evidence. It shows an earlier onset of disease in children of affected patients than in their parents. This has lead to speculations about genetic anticipation in this disorder. 2,007 IBD patients with sporadic disease and 472 multiplex familial cases (including 103 affected parents and 99 children of affected patients) were evaluated with a multi-item questionnaire as part of a study of inflammatory bowel disease genetics. The Mann-Whitney U-test and the general linear model were used for analysis. Clinical characteristics such as presence of fistulae, stenoses, extraintestinal manifestations, and other parameters, which are related to the severity of the disease, were found to be similar between familial and sporadic cases of IBD (corrected P > or = 0.31 for all tests). The mean-age-of onset in children of affected patients was 19.4 years earlier than in their parents. However, the age of the parental cohort was significantly higher (27 years) and the diagnostic interval also longer (1.7 years). If these confounders are corrected in a general linear model, no significant difference is evident for the age-of-onset between the groups (P > or = 0.52). There is no evidence for genetic anticipation in inflammatory bowel disease. The absence of genetic anticipation is consistent with the clinical similarity of familial and sporadic inflammatory bowel disease. This finding justifies the primary genetic analysis of familial disease under the assumption that their genetic background will be representative for all presentations of IBD.

Mesalazine (5-aminosalicylic acid) micropellets show similar efficacy and tolerability to mesalazine tablets in patients with ulcerative colitis--results from a randomized-controlled trial.

BACKGROUND: Formulations containing 5-aminosalicylic acid, such as mesalazine, are the gold standard of treatment for mild-to-moderate ulcerative colitis. Current oral regimens require the use of large tablets and frequent dosing to reach the recommended treatment dose. Mesalazine micropellets were designed to allow less frequent dosing in an easier to swallow formulation. AIM: To compare the efficacy of mesalazine micropellets with the tablet formulation in patients with mild-to-moderate ulcerative colitis. METHODS: This phase 2, double-blind, active-controlled, parallel-group, multiple dose clinical trial randomized 362 patients to either mesalazine micropellets or tablets, at a dosage of 3 g/day. The primary efficacy end-point was the incidence of clinical remission within 8 weeks, defined as the sum of clinical activity index components 1-4 (CAI(C1-4)) < or = 2. RESULTS: CAI(C1-4) decreased significantly in both treatment groups within 8 weeks. The micropellet formulation showed confirmatory non-inferiority with statistical significance compared with the tablet formulation, with regard to the incidence of clinical remission (odds ratio in according-to-protocol population 1.008; 95% CI: 0.623-1.632). There was no significant difference in the incidence of adverse events. CONCLUSIONS: The mesalazine micropellet formulation is as effective as tablets in patients with mild-to-moderate ulcerative colitis, enabling a larger dose to be taken comfortably and conveniently, thereby potentially improving patient compliance, treatment response and quality of life.

Mycophenolate mofetil: lack of efficacy in chronic active inflammatory bowel disease.

BACKGROUND: Mycophenolate mofetil (MMF) is a new immunosuppressant with pharmacodynamic properties comparable to azathioprine. Recent reports found MMF to be effective in inflammatory bowel disease (IBD). METHODS: An open-label prospective and uncontrolled multicentre 6 month trial of MMF in combination with steroids was conducted in 24 chronic active IBD patients. A daily steroid demand of >/= 10 mg prednisone in the preceding 2 months and a Crohn's disease activity index (CDAI) > 150, or moderate to severe activity according to Truelove, served as criteria for chronic activity. The treatment consisted of a steroid pulse and tapering protocol in combination with MMF 2 g/day. A prednisone dose of 5 mg/day was maintained during months 4-6. The primary end-point was induction and maintenance of remission. RESULTS: Only 10 of 24 patients had achieved remission after 3 months. All but one Crohn's disease patient had relapsed by the end of the study at 6 months. Depression and migraine necessitated drug withdrawal in two patients. CONCLUSION: In conclusion, MMF 2 g/day was unable to induce and maintain remission for a period of 6 months in 23 of 24 chronic active IBD patients. Further controlled investigations are required in view of recent conflicting reports.

The use of lectins to study normal differentiation and malignant transformation.

Lectins are polypeptides that specifically recognize carbohydrate residues of glycoproteins and glycolipids. They can be extracted from plants, invertebrates and vertebrates. The binding between lectins and carbohydrate moieties can be blocked by the inhibitory sugar for which the lectin is specific. In analogy to antibodies, lectins can be used to analyse cell surface determinants. Thus, differentiation of cells, in particular of immunocytes, has been studied and lectin receptors are now important markers for distinguishing different developmental stages of T-lymphocytes. In consequence, premature, but already committed, T-cells can be eliminated from human bone marrow by means of lectins prior to transplantation in order to avoid graft-versus-host reactions. Moreover, it has been shown that malignant cells can be distinguished from their non-malignant counterparts by the profile of lectin receptors on their surface. This had led to the use of lectin binding sites as tumour markers in lymphomas and carcinomas.

N alpha-carboxyacyl analogues of CCK with a substituted Gly: interaction with pancreatic and gallbladder CCK receptors.

It has been reported that certain N alpha-carboxyacyl analogues of CCK-8 and of CCK-7 with a substituted Gly in position 3 or 4 of the peptide possess higher potencies at stimulating pancreatic enzyme secretion than at stimulating gallbladder contraction, suggesting that these analogues are able to differentiate subtypes of CCKA receptors. However, no studies examined directly the interaction of these peptides with the CCK receptors in both tissues. In the present study, CCK-8 and various N alpha-carboxyacyl analogues of CCK-7 and of CCK-8 were prepared by solid phase synthesis using Fmoc chemistry and were purified by HPLC; molecular weight and sufficient sulfation were determined by mass spectrometry. [125I]Bolton-Hunter(BH)-CCK-8 binding to sections of the guinea pig pancreas and gallbladder was determined under identical conditions; amylase release from pancreatic acini and contraction of gallbladder muscle strips were measured in vitro. Each peptide stimulated amylase release (EC50): CCK-8 (0.09 nM) > Suc[Sar3]CCK-7 (0.23 nM) > des(SO3)CCK-8 (28 nM) > Suc[D-Trp4]CCK-8 (32 nM) > Suc[D-Trp3]CCK-7 (53 nM) > Pht[D-Trp3]CCK-7 (180 nM) > Glt[D-Trp3]CCK-7 (220 nM). The same relative potencies were found for stimulation of gallbladder contraction, and for the inhibition of [125I]BH-CCK-8 binding to pancreas and gallbladder sections. These data demonstrate that the CCKA receptors in the pancreas and on gallbladder smooth muscle possess similar affinities for the various N alpha-carboxyacyl analogues of CCK-7 and CCK-8 with a substituted Gly and provide further evidence that the CCKA receptors in gallbladder and pancreas cannot be distinguished pharmacologically.

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction.

In gallbladder smooth muscle, carbachol interacts with M3 receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [3H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates and contraction were measured at various times (0-90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 micrograms/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [32P]ADP-ribosylation of a approximately 40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates. The time course of [3H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [3H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [3H]inositol trisphosphates and on contraction were abolished by atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic study of early neurogenesis in rat neocortex.

The early neurogenesis of rat neocortex was analysed by means of light and electron microscopic autoradiography. It was found that the very first preneurons originate probably as early as ED 11. They are the horizontal cells of Cajal-Retzius. The peak of their formation is on ED 13 (surface index estimated on ED 17 after injection of 3H-thymidine on ED 13:21, after injection on ED 12:4, after injection on ED 15:5), WHereas no Cajal-Retzius cells could be found to have originated after ED 15. Theses cells are the developmentally most advanced of the neocortex. The cells second in date of origin and maturation are preneurons which presumably correspond to the presumptive neurons of Layer VII (VI b), and begin to originate on ED 12. The end of their formation could not be defined owing to a lack of ultrastructural differences to other, younger preneurons in later gestational stages. These two cell types are the first cellular components of the primordial plexiform layer (Marin-Padilla, 1978) or pallial anlage (Rickmann, 1977), demonstrating an outside-in gradient within this layer, and are separated by the formation of the cortical plate. This could be proven by their simultaneous labelling above and below the cortical plate after administration of 3H-thymidine before ED 15. These results confirm the hypothesis of a dual origin of the mammalian neocortex (Marin-Padilla, 1978).

Varying expressions of lectin receptors within embryonic cell layers of murine cerebral cortex.

The ability of prenatal cerebral tissue to bind different lectins was analyzed using cryostat sections of mouse brains. It was shown that the immature cells within the embryonic cell layers possess receptors for different lectins in varying amounts. Of all lectins tested, only PNL, RCL and LPL were bound on the outer cell membranes to a considerable degree. Although the labeling patterns of PNL and RCL are similar, the latter is additionally well detectable on the wall of cerebral blood vessels. Cells of the ventricular layer are moderately labeled by PNL, which recognizes beta-D-galactosyl (1-3)-N-acetyl-D-galactosamine, but heavily labeled by LPL which binds to terminal sialic acid residues. Cells of the intermediate layer, on the other hand, are heavily stained PNL and only faintly by LPL. Hence it is suggested that the process of migration might be correlated to the removal of terminal sialic acid moieties from cell surface glycoconjugates, resulting in an exposure of the penultimate galactosyl residues.

The median ventricular formation. A distinct structure at the mesencephalic apex.

The prenatal ontogenesis of the median ventricular formation (MVF)--a cell group at the seam between both sides of the mesencephalic roof--was analyzed ultrastructurally, autoradiographically and for the expression of intracytoplasmatic structures, i.e. glial filament antigen. As compared with other regions of the mesencephalic roof it was found that from embryonal day 12 onwards DNA synthesis of ventricular cells in the dorsal midline is significantly reduced. This reduction is more pronounced at later developmental stages. On the other hand, the MVF gains drastically in width during ontogenesis. It was shown that this increase may be caused by an immigration of postmitotic neighbouring ventricular cells. The characteristic morphological feature of MVF cells is their extension from the ventricular lining to the pial basement membrane. Their dorsal processes are joined to a thin fibre bundle and predominantly display microtubules as well as filaments and glycogen within their electrolucent cytoplasm. They also contain intracellular structures that react with antibodies against glial filaments as revealed by an enzyme-coupled immunolabelling. The perikarya of MVF cells, on the other hand, are almost all situated at the same level within the ventral third of the mesencephalic roof, thus bulging concentrically at the lateral sides of the MVF. Characteristically, a subfraction of MVF cells exhibits vast amounts of rough ER. The nature and function of the MVF cells is discussed in the light of the concept of guidance of preneurons by radial glia (Sidman and Rakic 1973).

Evidence for contrasuppression in patients with Crohn's disease.

Patients with Crohn's disease (CD) have elevated numbers of Vicia villosa agglutinin (VVA) binding cells in the peripheral blood. These cells represent a major subset of activated peripheral T cells. VVA binding T lymphocytes express either the T8 or the T4 determinant on their cell surface. In contrast in normal controls only a minor subset of peripheral T cell expresses binding sites for VVA. The majority of these cells coexpress T8. VVA binding T cells display no helper activity. Only in a subfraction of patients with CD and not in normal controls these cells mediate contrasuppressor activity for Ig and in particular for IgA. This subgroup of patients is characterized by the lack of extramucosal manifestations. It has now been shown that VVA binding T cells in their majority do not possess phenotypic features of helper inducer cells. This further supports the hypothesis of their involvement in contrasuppression. Moreover it was shown that IgA produced in the presence of VVA binding T cells is IgA1 and IgA2 (ratio 2:1) which are both modulated by VVA binding T cells.

Activated Fc alpha T cells in Crohn's disease are involved in regulation of IgA.

Activated peripheral T cells (APT) of patients with Crohn's disease (CD) have been analysed for the expression of IgA-Fc receptors and for competence of IgA regulation. It was found that within the subset of APT an increased number of cells express binding sites for IgA (IgA-Fc), that was not found in other diseases with elevated numbers of APT. Moreover the number of IgA-Fc receptor expressing T cells was found to be increased in the inflamed mucosa too. Cocultures with autologous B cells revealed that isolated IgA-Fc receptor bearing T cells of patients with CD suppress IgA secretion. These data support the hypothesis that APT are involved in the immunopathogenesis of CD.

Immunology of ulcerative colitis.

Although much work has been done on the analysis of immunopathogenesis of UC, more mysteries remain to be solved in the future. One of the most interesting questions is (i) what initiates the immune response in the GALT of patients with UC. We are far from understanding whether inflammation starts from intrinsic or extrinsic factors. Valuable information has recently been assembled about (ii) antigen presentation and even more about (iii) regulation of immune response in the mucosa. Attractive models have been put forward concerning the integrity of GALT and in particular the role of Ig isotype and Ig subclasses. Further progress can be expected in particular from an analysis of homing of GALT-lymphocytes in disease. Thanks to improved methods, elucidation of regulation of immune response in patients with UC can be expected in the near future. This is less true for the effector functions of the immune response. In particular the action of mediators of acute and chronic inflammation, i.e. prostaglandins, leukotrienes and the broad spectrum of cytokines, represents a wide field of future research.

Assessment of in vivo activated T cells in patients with Crohn's disease.

Besides clinical indices, acute phase reactants and measurement of permeability of the gut immunological parameters have been proposed for assessment of clinical activity in Crohn's disease (CD). The latter refers in particular to the number of activated peripheral T cells (APT) which are found to be increased in patients with CD and ulcerative colitis. Further analysis of the subset of APT revealed that in CD their number is correlated to the histopathological ratings and the number of activated T cells of the affected mucosa. A major subset of APTs in CD and ulcerative colitis expresses receptors for IgA (Fc-alpha-R). This T cell subset seems to be characteristic of patients with inflammatory bowel diseases, exhibiting a specificity of 88% and a sensitivity of 92% for CD as compared with non-inflammatory bowel diseases. Methodological complexity turned out to be the major disadvantage of assessment of APT and their subsets.