SUMMARYThe effector mechanisms of T cell-dependent acute glomerular injury were studied in autologous phase anti-GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti-rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4 þ and CD8 þ T cells. T cell depletion, using an anti-CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell-dependent. Inhibition of T helper cell function using an anti-CD4 MoAb prevented proteinuria and glomerular macrophage and CD4 þ T cell influx, but not accumulation of CD8 þ T cells. Depletion of CD8 þ T cells also prevented proteinuria and the influx of macrophages and CD8 þ T cells, but not accumulation of CD4 þ T cells. Macrophage depletion, using micro-encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4 þ or CD8 þ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell-dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti-GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell-mediated injury (cellular cytotoxicity) is not involved.

Macrophages release proinflammatory cytokines in response to infection that play a critical role in the pathophysiology of septic shock. We propose that targeting cytokine-neutralizing antibodies using albumin microspheres to macrophages will be more beneficial than the soluble form in reducing mortality related to peritonitis. In this study, we compared the distribution pattern of microspheres in infected and noninfected animals, evaluated the amount of microsphere taken up by peritoneal macrophages in vitro, and compared the efficacy of soluble and microsphere forms of cytokine-neutralizing antibodies in preventing lethality caused by Escherichia coli-induced peritonitis. The results indicate that twice the amount of microspheres accumulates near the site of infection (the peritoneal cavity), and 70% of the microspheres exposed to peritoneal macrophages were phagocytosed in 1 h. Treatment with the microsphere form of cytokine-neutralizing antibodies was more efficacious than using the soluble form in preventing lethality induced by E. coli. Immediate treatment was more efficacious than delayed treatment in the absence of gentamicin, whereas immediate and delayed treatment were equally efficacious in the presence of gentamicin. The combination of microspheres containing neutralizing antibodies to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) protected 100% of the animals, whereas either one alone protected only 60%-90% of the animals from lethality caused by E. coli-induced peritonitis. In conclusion, the microsphere form of neutralizing antibodies to TNF-alpha IL-1beta may be an effective therapeutic agent in the treatment of septic shock caused by peritonitis.

In this study, we formulated a microparticulate melanoma cancer vaccine via the transdermal route. The vaccine was delivered using microneedle-based Dermaroller® which is available for cosmetic purposes. Unlike subcutaneous injections, administration using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The vaccine microparticles were taken up by the antigen presenting cells which demonstrated a strong IgG titre level of 930 ug/mL in serum samples. The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size range of around 0.63-1.4 µm which acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 8 weeks duration, followed by challenge with live tumour cells which showed protection after transdermal vaccination.

NF-kappaB is an ideal target for inhibition of proinflammatory cytokines. The purpose of this study was to determine if microencapsulated antisense oligomer to NF-kappaB can inhibit proinflammatory cytokine release in response to Escherichia coli endotoxin and bacteria. Microencapsulation takes advantage of the phagocytic function of the macrophage to deliver the oligomer intracellularly and enhance the effect. Albumin microcapsules 1 microm in size were prepared by a nebulization method containing antisense oligomers to NF-kappaB. E. coli endotoxin was incubated in 1 ml aliquots of whole blood. Microencapsulated antisense to NF-kappaB was given, and the inhibition of tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and IL-8 was compared with similar amounts of oligomer in solution. Endotoxic shock was produced in rats using E. coli endotoxin (15 mg/kg). Peritonitis was induced by injecting 10(10) CFU E. coli. Cytokines were measured after simultaneous and delayed (4 h) administration of antisense to NF-kappaB in microcapsules and solution form. TNF was suppressed by 81% in whole blood, 56% in the endotoxic shock model, 89% in the peritonitis model (simultaneous treatment), and 56% in the delayed treatment group. Survival was 70% in the endotoxic shock group, 80% in the simultaneous peritonitis group, and 70% in the delayed treatment group. Microcapsule treatment using antisense to NF-kappaB suppressed TNF and IL-1 levels and mortality significantly better than all solution treatment groups in the whole blood model, endotoxic shock model, and peritonitis model.

Microparticles are widely employed as carriers of biologically active compounds with many possible applications. For targeted drug delivery and sustained release purposes, biopolymers (i.e. polysaccharides and proteins) have been proposed. In this study, microsphere formulations of vancomycin were prepared by the spray-drying method. Bovine serum albumin (BSA) was used as a polymer matrix and was cross-linked with glutaraldehyde after microsphere preparation. The product yield obtained from the spray-drying method was approximately 75%. The mean particle size was 5 +/- 1.6 microm, with the majority of particles between 4 and 8 microm. The extent of cross-linking affected the release of vancomycin from microspheres. Moreover, both rate and extent of vancomycin release from microspheres decreased with increasing glutaraldehyde concentration. Encapsulation of vancomycin did not alter the bioactivity of the drug and it was more effective in killing Staphylococcus aureus than the solution form.

A macrophage plays an important role in mediating the inflammatory response. Cytokines released by activated macrophages contribute to inflammation in glomerulonephritis (GN). Clodronate, a biphosphonate, causes macrophage depletion when administered in an encapsulated form in liposomes. We used albumin as the polymer matrix to microencapsulate clodronate to the microspheres (MS) in the 1-micron size range. The purpose of this study was to (a) determine macrophage depletion by clodronate MS, (b) determine the effect of clodronate MS on endotoxin-induced cytokine release in vitro, and (c) assess the effect of clodronate MS on macrophage infiltration in experimental antiglomerular basement membrane nephritis. Macrophage depletion by clodronate MS was assessed by staining for the EDI marker. The results indicate greater than 95% depletion of macrophages from the spleen, liver, kidney, and blood. In the whole blood model, clodronate MS attenuated endotoxin-induced tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) release, and the attenuation by the microencapsulated form of clodronate was also more effective than the free (solution) form of clodronate. Macrophage infiltration into the glomerulus in experimental GN was also blocked very effectively by pretreatment with clodronate MS. In conclusion, macrophage depletion by clodronate MS may be beneficial in reducing cytokine release and renal damage in GN.

The aim of this investigation was to prepare and evaluate microsphere formulations of gentamicin using bovine serum albumin (BSA) as a polymer matrix and glutaraldehyde as a cross-linker. Microsphere formulations of gentamicin were prepared using a spray dryer and were evaluated for product yield, encapsulation efficiency, particle size and in vitro drug release. The anti-microbial testing was performed using a modified Kirby-Bauer technique which showed that encapsulated gentamicin had an equivalent anti-microbial activity against E. coli bacteria as compared to gentamicin solution. Since it was the goal to deliver a high drug load intra-cellularly, the formulation with the least burst release profile in PBS was evaluated for its pharmacokinetic performance in rats. The in vivo pharmacokinetic evaluation on rats demonstrated increased bioavailability with microsphere formulation in comparison to the traditional solution form. The significant increase in bioavailability shall enable one to reduce the frequency of gentamicin administration and would effectively reduce the dose related side effects of gentamicin such as ototoxicity and nephrotoxicity.

The purpose of this study was to evaluate the possibility of lectin-coupled microspheres to improve the targeted delivery of protein antigens to the lymphoid tissues of mucosal surfaces. Bovine serum albumin containing acid phosphatase model protein and polystyrene microspheres were coupled with mouse M-cell-specific Ulex europaeus lectin. The coupling efficiency, physical characteristics and the binding capabilities of the microspheres to the follicle associated epithelium of the Peyer's patches were evaluated in vitro and ex vivo in mice intestine. The results showed that coupling of lectin to albumin microspheres did not significantly affect the bioactivity of the encapsulated acid phosphatase model protein. It was also shown that there was preferential binding of the lectin-coupled microspheres to the follicle-associated epithelium. It was concluded from the results of the study that coupling of ligands such as lectin specific to cells of the follicle associated epithelium can increase the targeting of encapsulated candidate antigens for delivery to the Peyer's patches of the intestine for improved oral delivery.