A systems biology analysis of the Drosophila phagosome.

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.

Heart-lung interactions: the sigh and autonomic control in the bronchial and coronary circulations.

1. The Darwin hypothesis that human and animal expressions of emotion are the product of evolution and are tied to patterns of autonomic activity specified to progress the emotion remains under challenge. 2. The sigh is a respiratory behaviour linked with emotional expression in animals and humans from birth to death. The aim of the present study was to explore Darwin's hypothesis with respect to tied autonomic activity underlying sigh-induced changes in the bronchial and coronary circulations. 3. Awake dogs were prepared using pulsed ultrasonic flow probes on the right bronchial artery, parent intercostal artery and brachial artery, or on the right, circumflex and anterior descending coronary arteries. Central venous (CVP) and arterial pressures (AP) were measured; heart rate and flow conductances were derived. Three spontaneous sighs were monitored before and during random blockade of individual and combinations of cholinoceptors, alpha-adrenoceptors and beta-adrenoceptors using methscopolamine, phentolamine and propranolol infusions. The data were subject to a 2(3) factorial analysis. 4. A spontaneous sigh is marked by a transient fall and return (< 3 s) in CVP of 18 mmHg (from 4 +/- 1 to -14 +/- 2 mmHg), usually followed by apnoea lasting 23 +/- 2 s. There is an immediate tachycardia and small rise in AP (phase 1) then, during apnoea, bradycardia and a fall in AP (phase 2). During phase 2, bronchial and coronary blood flow and conductance rise two- to three-fold over 30s (peak at 8s). The vascular changes are absent in parent intercostal and brachial beds.

Baroreflex control of coronary blood flow varies regionally in awake dogs.

1. Baroreflex responses to changes in aortic pressure were measured simultaneously in three main coronary regions of awake dogs. 2. Pulsed Doppler flow probes were mounted at prior surgery on the right, circumflex and anterior descending coronary arteries; the animals were placed in complete heart block and the left ventricle was paced. After 2-4 weeks recovery, baroreflexes were evoked by inflating a balloon catheter placed in the mid-thoracic aorta via the femoral arteriotomy. Flow and pressure data were collected at rest, and during acute (8s) and steady-state (25s) baroreflex challenge. 3. Changing ventricular rate alone caused a fall in aortic pressure at low rates; however, over the range 60 to 180 b.p.m., circumflex and anterior descending coronary flow and conductance changed directly with ventricular rate, but right coronary flow and conductance remained unchanged. 4. Acute aortic pressure elevation increased flow at 8s in all beds at all rates. Conductance effects at 60 b.p.m. were negligible in all three beds, but rose at 100 and 180 b.p.m. in the right and circumflex beds. 5. Sustained aortic pressure elevation (25s) caused flow to return towards control in all beds ventricular rates, but in the right coronary at 60 b.p.m. flow fell below control. Conductance at this time was unchanged at all rates in the anterior descending bed, fell modestly in the circumflex, and decreased to below resting in the right coronary bed. 6. Baroreflex control of coronary flow and conductance thus varies between territories, and within territories, depending on ventricular rate. The right coronary bed appears to be regulated by a bidirectional, baroreflex-linked mechanism, which is functionally opposite in action to that found in most vascular beds.

Appearance of IgG and IgA antibodies in human bile after tetanus toxoid immunization.

Humans immunized intramuscularly with one dose of tetanus toxoid exhibited IgG, and in some cases IgA antibody, in their bile as well as serum. Both isotypes appeared in bile transiently with titres declining after about day 10 for both classes. These kinetics resembled those of the serum IgA response but were markedly different to those for IgG antibody in serum. Measured IgG titres in bile were between 0.07 and 4.2% of those in paired sera, and IgA titres were between 6.8 and 124% of sera. Peak responses in bile, while generally of smaller size, exceeded those of paired sera when expressed as antibody/mg of IgG or IgA present. This calculation showed that during the peak response bile was up to nineteen-fold more abundant in IgG antibody than was serum taken at the same time, and up to forty-five-fold more for IgA. Enrichment of antibody in bile is not consistent with the Ig of bile being solely conferred by plasma, and may mean the involvement of local synthesis too. This study indicates that tetanus toxoid immunization of humans results in biliary antibody and raises the possibility of intra-hepatic antibody production for export to the intestinal tract in man.

Regulation of IgA secretion in polyclonally induced in vitro human lymphocyte cultures: the function of T and B cells from mesenteric lymph nodes and peripheral blood.

Human gut-associated immunoregulatory events were studied in a pokeweed mitogen (PWM)-stimulated culture system using lymphocytes obtained from the mesenteric lymph nodes (MLN) of female subjects undergoing gastroplasty for obesity. Compared with peripheral blood lymphocytes, lymphocytes obtained from MLN secreted IgG, IgA and IgM isotypes that differ in pattern and distribution despite similar proportions of T cells and B cells expressing isotype-specific surface membrane immunoglobulin (SmIg). Among the isotypes secreted, IgA appeared to be increased relatively to other isotypes in MLN cultures. Crossover coculture experiments using T and B cells isolated from both MLN and blood by E-rosetting and cell panning procedures demonstrated that IgA was particularly sensitive to help and suppression exerted by MLN T cells and T cell subsets defined by monoclonal antibodies OKT4 and OKT8 respectively, when compared with similar subsets isolated from blood. The results presented provide a basis for study of gut handling of ingested antigen in man, and of disturbed immunoregulatory events in inflammatory and neoplastic disease of the human gut.

Study of lymphocyte interaction in human mesenteric lymph nodes in vitro.

The immune function of lymphocytes isolated from human mesenteric lymph nodes (MLN) has been examined. Large yields of viable lymphocytes were obtained from MLN without the need for using enzyme or mucolytic agents. In comparison to gut mucosal lymphocytes, MLN lymphocytes (MLNL) were technically easier to manipulate and to use in providing a more quantitative analysis of T-B cell interaction. The results presented indicate that the pattern of immunoglobulin secretion and immunoregulation of lymphocytes isolated from MLN was similar to that in cells isolated from the lamina propria, supporting that MLN constitute an important component of gut-associated lymphoid tissue (GALT).

Perforated peptic ulcer long-term follow-up.

A series of 349 survivors of perforated peptic ulcer was followed for periods of up to 23 years. Almost nine out of every 10 patients suffered from dyspepsia during follow-up. Subsequent elective gastroduodenal surgery was required in more than a quarter of the cases. The surgery rate for gastric ulcer was more than one and a half times that for pyloroduodenal ulcer, and for females almost double that for males. The highest rate of all was for females with gastric ulcer, of whom almost one half came for surgery. One in five patients bled during follow-up. One in eight developed stenosis of the stomach of duodenum, and one in 11 perforated again. There was a significantly increased incidence of subsequent perforation and stenosis in those with an initial perforation of 5 mm or more in diameter. Gastric carcinoma occurred in less than 2% of cases and was restricted to cases of pyloroduodenal perforation. When complications occurred, the majority did so within five years. Only 15% of the 262 patients about whom complete information was available had no complications on follow-up. The indications for definitive surgery at perforation should be extended to include perforated gastric ulcer in the female, particularly if the ulcer is large.