Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo.

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.

Therapeutic efficacy of a novel membrane-targeted complement regulator in antigen-induced arthritis in the rat.

OBJECTIVE: Complement system activation is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis. The objective of this study was to explore the therapeutic potential and local retention of a novel membrane-targeting complement regulatory protein, derived from human complement receptor 1, in the experimental setting of rat antigen-induced arthritis. METHODS: Sensitized animals were treated at the time of arthritis induction with a single intraarticular (IA) dose of the membrane-targeting regulator APT070, a non-membrane-targeting control regulator (APT898), or vehicle control, and disease was assessed clinically and histologically. In addition, immunocytochemical analysis was performed on sections from normal rat knee joints at various time points after IA injection with APT070. RESULTS: Animals treated with APT070 showed a dose-dependent therapeutic effect, with significantly milder clinical and histologic disease compared with both other treatment groups (P < 0.008 at the higher dose) and minimal evidence of erosive disease at study end in the active treatment group. Immunoperoxidase and immunofluorescence studies demonstrated local retention of APT070 on cell surface membranes within the normal joint up to 48 hours after IA injection. CONCLUSION: These results show that IA complement inhibition represents an effective therapeutic strategy in experimental arthritis, by demonstrating that the exogenous delivery of a membrane-targeting complement regulator can result in prolonged synovial cell surface binding and significant clinical benefit in vivo. Complement inhibitory strategies of this type should be considered as novel therapies in human inflammatory arthritis.

Novel aspects of the transport of organic anions by the malpighian tubules of Drosophila melanogaster.

Para-aminohippuric acid (PAH) is a negatively charged organic ion that can pass across the epithelium of Malpighian tubules. Its mode of transport was studied in Malpighian tubules of Drosophila melanogaster. PAH transport was an active process, with a K(m) of 2. 74 mmol l(-)(1) and a V(max) of 88.8 pmol min(-)(1). Tubules had a low passive permeability to PAH, but PAH transport rates (832 nmol min(-)(1 )mm(2)) and concentrative ability ([PAH](secreted fluid):[PAH](bath)=81.2) were the highest measured to date for insects. Competition experiments indicated that there were two organic anion transporters, one that transports carboxylate compounds, such as PAH and fluorescein, and another that transports sulphonates, such as amaranth and Indigo Carmine. PAH transport appears to be maximal in vivo because the rate of transport by isolated tubules is not increased when these are challenged with cyclic AMP, cyclic GMP, leucokinin I or staurosporine. Basolateral PAH transport was inhibited by ouabain and dependent on the Na(+) gradient. The Malpighian tubules appeared not to possess an organic acid/ &agr; -keto acid exchanger because PAH accumulation was not affected by low concentrations (100 micromol l(-)(1)) of &agr; -keto acids ( &agr; -ketoglutarate, glutarate, citrate and succinate) or the activity of phosphokinase C. PAH transport may be directly coupled to the Na(+) gradient, perhaps via Na(+)/organic acid cotransport. Fluorescence microscopy showed that transport of the carboxylate fluorescein was confined to the principal cells of the main (secretory) segment and all the cells of the lower (reabsorptive) segment. Organic anions were transported across the cytoplasm of the principal cells both by diffusion and in vesicles. The accumulation of punctate fluorescence in the lumen is consistent with exocytosis of the cytoplasmic vesicles. Apical PAH transport was independent of the apical membrane potential and may not occur by an electrodiffusive mechanism.

The nitrogen requirements and dietary nitrogen utilization for the gecarcinid land crab Gecarcoidea natalis.

The nitrogen requirements for tissue maintenance, moulting, and oogenesis were determined experimentally for the herbivorous land crab Gecarcoidea natalis. The maintenance nitrogen requirements for intermoult animals was very low (4.83+/-1.68 mmol N kg-1 dry body wt d-1), but during oogenesis the total requirement was much higher (8. 6 mmol N kg-1 dry body wt d-1). Gecarcoidea natalis could potentially assimilate enough nitrogen from rain forest leaf litter or leaves of Ficus or Erythrina to satisfy not only the maintenance nitrogen requirements but the observed rate of incorporation of nitrogen into the ovaries during oogenesis. The ovaries developed slowly over a period of 2 mo (mid-July to late September) and had a final nitrogen content of 359+/-15.9 (n=18) mmol kg-1 dry body wt. This was equivalent to 9.3%+/-0.4% of the total body nitrogen. A substantial nitrogen debt was incurred during ecdysis (658+/-126 mmol kg-1 dry body wt). This nitrogen debt could be satisfied slowly, from leaf litter, over a period of 1-3 mo. After ecdysis, the majority of the nitrogen and urate within the animal prior to moulting was retained within the soft crab (85.0%+/-1.2% total nitrogen, 82.0%+/-1.2% nonurate nitrogen and 99.56% urate), while only a minority was lost with the exuviae (18.0%+/-1.2% total nitrogen, 14.7%+/-1.2% nonurate nitrogen, and 0.4%+/-0.4% urate). The urate deposits in G. natalis were not mobilized as a source of nitrogen in animals maintained on a nitrogen-free diet.

Function of the factor I modules (FIMS) of human complement component C6.

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.

Role of the midgut gland in purine excretion in the robber crab, Birgus latro (Anomura: Coenobitidae).

White fecal strands of Birgus latro are composed of small spherules of uric acid with a mean diameter of 1.6 +/- 0.6 microm. Large numbers of membrane-bound spherules with concentric lamellae are present in the R cells of the midgut gland, so we suggest that lengths of white feces are produced by coordinated secretion of these spherules into the lumen of the midgut gland tubules. There are four cell types in the tubules with embryonic (E) cells at the distal tip, B cells in a narrow band at the distal end and R cells making up the bulk of the tubules and gland. F cells are sparsely scattered among the R cells. Midgut gland tissue was assayed for activities of xanthine dehydrogenase and xanthine oxidase, the two forms of xanthine oxidoreductase. Contrary to previous reports, we found that the midgut gland of B. latro contains only high activities of xanthine dehydrogenase. If proteinase inhibitors were omitted from the assays, however, significant activity of xanthine oxidase was measured, a result we regard as an artifact attributable to the partial conversion of xanthine dehydrogenase to xanthine oxidase by endogenous proteinases. R cells were demonstrated to contain peroxisomes, which may be involved in lipid metabolism rather than synthesis of uric acid.

Contributions of K+:Cl- cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster.

Mechanisms of Na+ and K+ transport across the basolateral membrane of isolated Malpighian tubules of Drosophila melanogaster were studied by examining the effects of ion substitution and putative inhibitors of specific ion transporters on fluid secretion rates, basolateral membrane potential and secreted fluid cation composition. Inhibition of fluid secretion by [(dihydroindenyl)oxy]alkanoic acid (DIOA) and bumetanide (10(-)4 mol l-1) suggested that a K+:Cl- cotransporter is the main route for K+ entry into the principal cells of the tubules. Differences in the effects of bumetanide on fluxes of K+ and Na+ are inconsistent with effects upon a basolateral Na+:K+:2Cl- cotransporter. Large differences in electrical potential across apical (>100 mV, lumen positive) and basolateral (<60 mV, cell negative) cell membranes suggest that a favourable electrochemical gradient for Cl- entry into the cell may be used to drive K+ into the cell against its electrochemical gradient, via a DIOA-sensitive K+:Cl- cotransporter. A Na+/K+-ATPase was also present in the basolateral membrane of the Malpighian tubules. Addition of 10(-)5 to 10(-)3 mol l-1 ouabain to unstimulated tubules depolarized the basolateral potential, increased the Na+ concentration of the secreted fluid by 50-73 % and increased the fluid secretion rate by 10-19 %, consistent with an increased availability of intracellular Na+. We suggest that an apical vacuolar-type H+-ATPase and a basolateral Na+/K+-ATPase are both stimulated by cyclic AMP. In cyclic-AMP-stimulated tubules, K+ entry is stimulated by the increase in the apical membrane potential, which drives K+:Cl- cotransport at a faster rate, and by the stimulation of the Na+/K+-ATPase. Fluid secretion by cyclic-AMP-stimulated tubules was reduced by 26 % in the presence of ouabain, suggesting that the Na+/K+-ATPase plays a minor role in K+ entry into the tubule cells. Malpighian tubules secreted a Na+-rich (150 mmol l-1) fluid at high rates when bathed in K+-free amino-acid-replete saline (AARS). Secretion in K+-free AARS was inhibited by amiloride and bafilomycin A1, but not by bumetanide or hydrochlorothiazide, which inhibit Na+:Cl- cotransport. There was no evidence for a Na+ conductance in the basolateral membrane of unstimulated or cyclic-AMP-stimulated tubules. Possible mechanisms of Na+ entry into the tubule cells include cotransport with organic solutes such as amino acids and glucose.

Complement activation and inhibition in experimental models of arthritis.

Complement activation has been implicated as a pathological process in a number of inflammatory and autoimmune disorders including chronic rheumatoid arthritis (RA). Animal models of experimental arthritis have been widely used to investigate the pathogenesis of RA and also in the development of novel therapies. Many of these models are complement-dependent and both incidence and progression of disease can be influenced by complement inhibition. In certain situations, local inhibition is of greater therapeutic benefit than systemic decomplementation. An increasing awareness and availability of a wide range of naturally occurring complement regulatory proteins can now offer a more targeted approach to complement inhibition while the availability of novel engineering strategies has also improved the efficiency of this process. The success of complement inhibition in the experimental models described should offer a novel therapeutic approach to the treatment of human inflammatory arthritis.

Evidence for persistent Na+ current in apical dendrites of rat neocortical neurons from imaging of Na+-sensitive dye.

Evidence for a persistent Na+ current (I(NaP)) in the apical dendrite of neocortical neurons was sought with the use of fluorescence imaging to measure changes in intradendritic Na+ concentration. Neurons in neocortical brain slices were filled iontophoretically through an intracellular recording microelectrode with the Na+-sensitive dye benzofuran isophthalate (SBFI), and fluorescence images were recorded with a cooled charge-coupled device camera system using 380-nm illumination. In the presence of Ca2+ and K+ channel blockers, a short depolarizing current pulse evoked a single action potential followed by a plateau depolarization (PD) lasting >1 s. This tetrodotoxin (TTX)-sensitive PD is known to be maintained by I(NaP). A single action potential caused no detectable SBFI fluorescence change, whereas the PD was associated with an SBFI fluorescence change in the soma and apical dendrite indicating increased intracellular Na+ concentration. Determination of the full spatial extent of the dendritic fluorescence change was prevented by our inability to detect the dim fluorescence signal in the distal regions of the apical dendrite. In each experiment the fluorescence change extended into the apical dendrite as far as dye could be visualized (50-300 microm). A slow, depolarizing voltage-clamp ramp that activated I(NaP) caused similar fluorescence changes that were eliminated by TTX, indicating that the SBFI fluorescence changes are caused by Na+ influx due to I(NaP) activation. We conclude that I(NaP) can be generated by the apical dendritic membrane to at least 300 microm from the soma.