IL-10 dependent adaptation allows macrophages to adjust inflammatory responses to TLR4 stimulation history.

During an infection, innate immune cells must adjust nature and strength of their responses to changing pathogen abundances. To determine how stimulation of the pathogen sensing TLR4 shapes subsequent macrophage responses, we systematically varied priming and restimulation concentrations of its ligand KLA. We find that different priming strengths have very distinct effects at multiple stages of the signaling response, including receptor internalization, MAPK activation, cytokine and chemokine production, and nuclear translocation and chromatin association of NFκB and IκB members. In particular, restimulation-induced TNF-α production required KLA doses equal to or greater than those used for prior exposure, indicating that macrophages can detect and adaptively respond to changing TLR4 stimuli. Interestingly, while such adaptation was dependent on the anti-inflammatory cytokine IL-10, exogenous concentrations of IL-10 corresponding to those secreted after strong priming did not exert suppressive effects on TNF-α without such prior priming, confirming the critical role of TLR4 stimulation history.

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex.

Regulation of N-methyl-D-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor-associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling.

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.

Localization of phospholipid-binding sites of caldesmon.

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C-terminal part of caldesmon (so-called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606-756), H7 (566-710) and H2 (626-710) form tight complexes with phosphatidylserine, whereas fragments H8 (658-737), H9 (669-737) and fragment H4 (566-624) interact with phospholipids less effectively. It is concluded that the phospholipid-binding site is located in the sequence 626-710 of caldesmon. This sequence contains calmodulin-binding sites and serine residues phosphorylated by protein kinase C and pro-directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon-phospholipid interaction described earlier.

Howell-Jolly bodies in idiopathic steatorrhoea.

Large numbers of Howell-Jolly bodies were found in the bone marrow and peripheral blood, both before and after corrective haematinic therapy, in three patients with idiopathic steatorrhoea with associated splenic atrophy, who presented with megaloblastic anaemia. The proportion of erythrocytes containing Howell-Jolly bodies was much higher in these patients when compared with the number found in post-splenectomy subjects. Red cell survival studies of patients' own red cells, sensitized with a potent anti-D serum, showed an abnormally prolonged survival in the three patients with splenic atrophy when compared with those in two splenectomized subjects, in four patients with coeliac disease, and in seven normal controls. There appears to be a relationship between atrophy of the reticuloendothelial system and Howell-Jolly body production in idiopathic steatorrhoea.

Plasma lactate dehydrogenase in megaloblastic anaemia.

Raised plasma lactate dehydrogenase (LDH) values were found in 26 patients with marked megaloblastic anaemia due either to vitamin B(12) or folic-acid deficiency or a combination of these factors.Minor megaloblastic changes were not usually accompanied by plasma LDH elevation. Serial LDH estimations were as valuable as serial reticulocyte counts in assessing the response to physiological doses of folic acid and therefore in deciding whether megaloblastic anaemia is partially or completely due to folic acid deficiency.

Effect of new plastics and leucocyte contamination on in vitro storage of platelet concentrates.

Platelet concentrates were prepared for in vitro storage in either Fenwal PL-732 or Cutter CLX platelet packs. The units were stored at 22 degrees C for seven days with either horizontal or tumbler agitation. Measurement of pH, hypotonic shock response and serotonin uptake indicated in vitro viability was well maintained during 5-7 days storage using either type of pack with either mode of agitation. The longer storage interval did not effect either plasma fibrinogen concentrations or binding of monoclonal antibody, AN51. However, gross contamination of the units with leucocytes caused increased glucose consumption, substantial fall in pH and loss of in vitro viability after five days storage. The work suggests the shelf-life of platelet concentrates can be extended to five days and that they are clinically effective providing the leucocyte contamination is minimised.

Intestinal motility changes in experimental large bowel obstruction.

Bipolar electrodes for recording electrical discharges of colon smooth muscle and strain gages for recording associated contractions of circular muscle were implanted in six rhesus monkeys. After recovery, baseline records were made. The animals then had an obstruction device implanted in sigmoid colon which resulted in progressive compromise of the lumen terminating in complete obstruction after 13 +/- 2 days. Recording were made daily during development of obstruction. As obstruction became more complete, contraction frequency decreased in right colon, increased in left colon proximal to the obstruction, and was unchanged in left colon distal to the obstruction. The frequency of distentions increased in colon proximal to the obstruction but was unchanged distally. Simultaneous mass actions, a complex of nonperistaltic high amplitude contractions and distentions occurring nearly simultaneously throughout the colon which is not seen in normal colon, appeared in colon both proximal and distal to the obstruction and became more frequent as the degree of obstruction progressed. Colon obstruction results in abnormal motility complexes, but not in hyperperistalsis. Mass actions probably are the basis for colic and rushing bowel sounds noted clinically in colon obstruction.

Longitudinal muscle of muscularis externa in human and nonhuman primate colon.

The idea that the longitudinal muscle of the muscularis externa of the colon is confined to the colonic teniae in the monkey and human is a common conceptual error. Forty-six colon specimens taken from human and nonhuman primates were reviewed histologically and the distribution and structure of the longitudinal muscle were evaluated. We found that the longitudinal muscle forms a complete coat around the colon although it is considerably thicker in the region of the teniae coli. In addition to its anatomic importance, this observation has implications for the physiology of colon muscle and for certain current hypotheses concerning the etiology of diverticulitis.

A device to stimulate the mechanical component of malignant bowel obstruction.

A predictable and progressive experimental intestinal obstruction, simulating a malignancy but without neoplastic change has been produced by application of a silicone rubber ring around the bowel of 20 rats, seven dogs, and five monkeys. Serosal reaction produced fibrous tissue that resulted in an "apple core" obstruction. An easily assembled extraluminal balloon device of silicone rubber can be used to produce acute and progressive experimental obstruction, especially of the large bowel.

Circumcision using bipolar diathermy scissors: a simple, safe and acceptable new technique.

BACKGROUND: Circumcision may be performed by a variety of techniques. Postoperative haemorrhage is a recognised complication of the procedure. METHOD: A method of circumcision using bipolar diathermy scissors is described. Foreskin vessels are coagulated as the foreskin is cut away. RESULTS: Thirty patients underwent this procedure without complication. CONCLUSION: Circumcision using bipolar scissors can be a safe and simple operation.

Spontaneous rupture of the pharynx.

A case of spontaneous rupture of the pharynx is described occurring during the act of vomiting. Comparisons are made with the only known previously recorded case of spontaneous pharyngeal rupture.

Osteomyelitis of the temporomandibular joint in patients with malignant otitis externa.

Malignant (invasive) otitis externa is an infection involving the external ear canal, often in elderly diabetic patients, which carries a high morbidity and mortality. It may involve widespread areas of soft tissue around the skull base, and in more advanced cases, may give rise to osteomyelitis and cranial neuropathy. We describe two patients who were treated for malignant otitis externa complicated by destructive osteomyelitis of the temporomandibular joint (TMJ). For both patients, diagnosis was made using magnetic resonance imaging (MRI), and repeat scans were employed during follow-up. Improved scan appearances mirrored improvements in clinical condition in both cases.