Autoantibodies against the exocrine pancreas in autoimmune pancreatitis: gene and protein expression profiling and immunoassays identify pancreatic enzymes as a major target of the inflammatory process.

OBJECTIVES: Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP. METHODS: To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis. RESULTS: Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen. CONCLUSIONS: These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.

Erythrocyte-mediated delivery of dexamethasone in patients with mild-to-moderate ulcerative colitis, refractory to mesalamine: a randomized, controlled study.

BACKGROUND AND AIM: Nearly 25% of patients with ulcerative colitis (UC) requiring steroids therapy become steroid-dependent after 1 yr, and virtually all develop steroid-related adverse events. We planned a controlled study to investigate the efficacy and safety of dexamethasone 21-P (Dex 21-P) encapsulated into erythrocytes (DEE). MATERIALS AND METHODS: Forty patients with mild-to-moderate UC, refractory to mesalamine, were randomly assigned to one of the following three treatments: two DEE infusions 14 days apart (group A, N = 20), oral prednisolone (0.5 mg/kg for 14 days followed by a 6 mg/weekly tapering (group B, N = 10), and sham infusions (group C, N = 10). The clinical, biochemical, and endoscopic parameters were monitored at inclusion and after 8 wk. RESULTS: In group A, a mean dose of 9.9 +/- 4.1 mg Dex 21-P was loaded into autologous erythrocytes at each infusion. At 8 wk, 15 patients in group A (75%), 8 in group B (80%), and 1 in group C (10%, P < 0.001 vs A and B) were in clinical and endoscopic remission. When compared with the baseline values, C-reactive protein (CRP) dropped in groups A (1.6 mg/dL vs 0.4 mg/dL, P= 0.006) and B (1.0 vs 0.5, P= 0.02), but not in group C. No steroid-related adverse events were apparent in the patient treated with DEE, compared with 8 out of 10 patients on oral steroids (P< or = 0.01). CONCLUSION: Low doses of Dex (mean total dose +/- 20 mg) loaded into autologous erythrocytes were significantly more effective than sham infusions in terms of symptoms relief, endoscopic, and biochemical improvements in UC patients refractory to mesalamine. In addition, in contrast to oral prednisolone (mean total dose +/- 1 g), no steroid-related adverse events were induced.

Prolonged islet allograft survival in diabetic mice upon macrophage depletion by clodronate-loaded erythrocytes.

Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4+/-0.89 and 20+/-2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25+/-1.9 days in the group treated with free-clodronate and 35+/-6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.

Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment.

Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

Erythrocyte-based drug delivery.

The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimising inevitable adverse side effects, is an extremely fascinating perspective. The behaviour of erythrocytes as a delivery system for several classes of molecules (i.e., proteins, including enzymes and peptides, therapeutic agents in the form of nucleotide analogues, glucocorticoid analogues) has been studied extensively as they possess several properties, which make them unique and useful carriers. Furthermore, the possibility of using carrier erythrocytes for selective drug targeting to differentiated macrophages increases the opportunities to treat intracellular pathogens and to develop new drugs. Finally, the availability of an apparatus that permits the encapsulation of drugs into autologous erythrocytes has made this technology available in many clinical settings and competitive with other drug delivery systems.

Macrophage depletion induced by clodronate-loaded erythrocytes.

Given the important role of macrophages in various disorders, the transient and organ specific suppression of their functions may benefit some patients. Until now, liposome-encapsulated bisphosphonate clodronate has been extensively proposed to this end. In this paper, we demonstrate that erythrocytes loaded with clodronate can also be effective in macrophage depletion. Here, clodronate was encapsulated in erythrocytes through hypotonic dialysis, isotonic resealing and reannealing to final concentrations of 4.1 +/- 0.4 and 10.1 +/- 0.8 micromol/ml of human and murine erythrocytes, respectively. The ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in vitro and in vivo. In vitro studies on human macrophages showed that a single administration of engineered erythrocytes was able to reduce cell adherence capacity in a time-dependent manner, reaching 50 +/- 4% reduction, 13 days post treatment. The administration of loaded erythrocytes to cultures of murine peritoneal macrophages was able to reduce macrophage adhesion 67 +/- 3%, 48 h post treatment. In vivo, the ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in Swiss and C57BL/6 mice. Swiss mice received 125 microg of clodronate through erythrocytes and 6 days post treatment 69 +/- 7% reduction in the number of adherent peritoneal macrophages and 75 +/- 5% reduction in number of spleen macrophages were observed. C57BL/6 mice received 220 microg clodronate by RBC and 3 and 8 days post treatment 65 +/- 7% reduction in the number of spleen macrophages and the complete depletion of liver macrophages were obtained. In summary, our results indicate that clodronate selectively targeted to the phagocytic cells by a single administration of engineered erythrocytes is able to deplete macrophages, even if not completely. The transient suppression of macrophage functions through clodronate-loaded erythrocytes can be used in many biomedical phenomena and research applications.

Erythrocytes-mediated delivery of dexamethasone 21-phosphate in steroid-dependent ulcerative colitis: a randomized, double-blind Sham-controlled study.

BACKGROUND: Efficacy of erythrocyte-mediated delivery of dexamethasone 21-phosphate in patients with steroid-dependent ulcerative colitis. METHODS: Thirty-seven patients with steroid-dependent ulcerative colitis were randomized to infusions of dexamethasone 21-phosphate encapsulated into autologous erythrocytes (n = 19) or to sham infusions (n = 18). Each infusion was given monthly for 6 months. The primary endpoint was the proportion of patients able to discontinue oral corticosteroids during treatment while maintaining clinical remission or stable disease. Secondary endpoint was the proportion of patients with disappearance of steroid-related adverse events. RESULTS: At each infusion, a mean of 9.8 ± 4.6 mg dexamethasone 21-phosphate was administered at each infusion, which allowed steady-state plasma levels of 8 ng/mL for the following 28 days. Thirteen patients in the dexamethasone 21-phosphate group and 4 sham-treated patients attained the primary outcome of the study, i.e., maintaining a stable condition despite oral steroids withdrawal (P = 0.008). In the remaining patients (6 and 15 in the 2 experimental groups, respectively), the treatment was prematurely withdrawn because of clinical deterioration while tapering oral steroids. At endoscopy, mucosal healing was ascertained in 4 patients and 1 patient of the 2 experimental groups, respectively (P = 0.339). At inclusion, 14 and 13 patients in the 2 experimental groups complained of steroid-related adverse events; at end of the treatment, events were still present in 5 and 13 patients, respectively (P = 0.008). CONCLUSIONS: In patients with steroid-dependent ulcerative colitis, 6-month therapy with low dose of dexamethasone 21-phosphate allowed the withdrawal of oral steroids and the reversal of steroid-related adverse events in most patients while maintaining clinical remission (ClinicalTrials.gov number, NCT01171807).

Cytotoxic activity of 2-Fluoro-ara-AMP and 2-Fluoro-ara-AMP-loaded erythrocytes against human breast carcinoma cell lines.

Fludarabine phosphate (2-Fluoro-ara-AMP) is a purine analogue approved for the clinical treatment of haematological malignancies. This antimetabolite has also shown 'in vitro' antiproliferative activity against experimental models of solid mammary tumor. In this perspective, we have determined the cytotoxic effects of 2-Fluoro-ara-AMP against two human breast cancer cell lines (the ER-positive MCF-7 and the ER-negative MDA-MB-435), by adding the drug both in its free form and encapsulated into erythrocytes, as a strategy to modify the pharmacokinetic profile of the compound in order to increase its efficacy and decrease its toxicity. Similar antiproliferative activity of 2-Fluoro-ara-AMP in the two cell lines was obtained, reaching an almost complete abrogation of growth already after just 24 h of free drug exposure at all the tested doses. Meanwhile, encapsulated 2-Fluoro-ara-AMP was successfully released from erythrocytes into the culture media in a time-dependent manner with an efficacy comparable to that of the free drug treatment. This result suggests the possibility of administering 2-Fluoro-ara-AMP in patients with breast cancer using autologous erythrocytes as a system to slowly and constantly deliver 2-Fluoro-ara-A into circulation. In addition, possible mechanisms involved in the antiproliferative activity of 2-Fluoro-ara-AMP, such as the effects on cell cycle progression, p53 expression and STAT1 pathway activation in ER+ and ER- cancer cell lines, are proposed.

Observation of erythrocyte dynamics in the retinal capillaries and choriocapillaris using ICG-loaded erythrocyte ghost cells.

PURPOSE: To find evidence of retinal vasomotion and to examine the relationship between erythrocyte dynamics and previously observed high-frequency pulsatile blood flow through the choriocapillaris. METHODS: An osmotic shock technique was used to encapsulate indocyanine green (ICG) dye in erythrocyte ghost cells at a concentration that produced maximum cell fluorescence. By obviating the plasma staining that results from aqueous ICG's high affinity for plasma proteins, high contrast was maintained between reinjected ICG-loaded erythrocytes and their plasma background. High-speed, high-magnification ICG angiograms showing individual cell movement were recorded from the intact eyes of four monkeys and three rabbits for periods up to 30 seconds. RESULTS: In monkey retinal perifoveal capillaries, numerous erythrocytes were seen to pause for as long as 20 seconds and then resume transit. Similar pausing behavior was observed in the subfoveal choriocapillaris. Individual erythrocytes also were seen to pause in the rabbits' choriocapillaries below the medullary rays, where visualization of the cells was uninhibited by overlying retinal vasculature or dense pigment. CONCLUSIONS: Reinjected ICG-loaded erythrocytes permit routine visualization of retinal capillary and choriocapillaris hemodynamics of the intact eye. It is speculated that erythrocyte-pausing in both microcirculations facilitates metabolic exchange across capillary walls. In retinal capillaries, pausing is presumed to result from vasomotion-which has been postulated as necessary for the inhibition of retinal edema-and in choriocapillaries, to result from the shifting distributions of local perfusion pressures within the network of capillary vessel segments that comprise each lobular area of the choriocapillaris vascular plexus.

Inhibition of HIV-1 replication in macrophages by a heterodinucleotide of lamivudine and tenofovir.

OBJECTIVES: (i) To generate a new heterodinucleotide (3TCpPMPA) comprising the drugs lamivudine and tenofovir which have been shown to act synergistically and (ii) to protect macrophages from 'de novo' HIV-1-infection through its administration. METHODS: 3TCpPMPA was obtained by coupling the morpholidate derivative of tenofovir with the mono n-tri-butylammonium salt of lamivudine 5'-monophosphate. Stability and metabolism were evaluated in vitro and in vivo in mice. 3TCpPMPA was encapsulated into autologous erythrocytes by a procedure of hypotonic dialysis, isotonic resealing and reannealing. 3TCpPMPA-loaded erythrocytes were modified to increase their phagocytosis by human macrophages. Macrophages were infected by HIV-1(Ba-L) and inhibition of HIV-1 replication was assessed by HIV p24(gag) quantification. RESULTS: Pharmacokinetic studies in mice revealed a rapid disappearance of the heterodinucleotide from circulation (t(1/2)=15 min) without any advantage compared with the administration of single drugs. Adding free 3TCpPMPA to macrophages (18 h), a 90% inhibition of viral replication up to 35 days post-treatment was achieved, while only a 60% inhibition was obtained by the combined treatment 3TC and (R)PMPA. When 3TCpPMPA was selectively targeted to the macrophage compartment by a single addition of loaded erythrocytes, the protection of macrophages from 'de novo' infection (99% protection 3 weeks post-treatment) was nearly complete. CONCLUSIONS: Erythrocytes loaded with 3TCpPMPA and modified to increase their phagocytosis are able to protect macrophages from 'de novo' HIV-1 infection. 3TCpPMPA acts as an efficient antiviral pro-drug that, once inside macrophages, can be slowly converted into 3TCMP and (R)PMPA protecting these cells for a longer period of time.

Inhibition of murine AIDS by a heterodinucleotide of azidothymidine and 9-(R)-2-(phosphonomethoxypropyl)adenine.

Tenofovir [9-(R)-2-(phosphonomethoxypropyl)adenine (PMPA)] and zidovudine [azidothymidine (AZT)] are potent anti-HIV agents that have shown a strong synergy in in vitro studies. In this paper we have investigated both the potentiality of this synergy in vivo and the possibility to administer AZT and PMPA simultaneously as a single drug AZTpPMPA. The pharmacokinetic studies reported here have shown that AZTpPMPA administered intraperitoneally in mice performs as a prodrug, providing a slow delivery of AZT and PMPA in circulation. C57BL/6 mice infected with the retroviral complex LP-BM5 were used to evaluate the efficacy of AZTpPMPA in inhibiting disease progression. Furthermore, the effectiveness of the heterodinucleotide was compared with that of AZT and PMPA, administered as single drugs, or as a combination (AZT plus PMPA). The results obtained showed that AZTpPMPA is able to reduce lymphoadenopathy (88%), splenomegaly (64%), lymph node BM5 proviral DNA content (49%) and hypergammaglobulinaemia (40%). However, upon AZT plus PMPA administration, similar (splenomegaly and lymphoadenopathy reduction) or better results (64% hypergammaglobulinaemia reduction and 75% lymph node BM5 proviral DNA content inhibition) were obtained. Furthermore, these results overlapped those obtained upon PMPA administration. Thus, no synergy between PMPA and AZT was observed in murine AIDS and administration of AZT does not improve the antiviral results obtained by PMPA administration.

Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean.

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is still quite poor. Therefore, we carried out an ultrastructural morphometrical and immunocytochemical study on hepatocytes from mice fed on GM soybean, in order to investigate eventual modifications of nuclear components of these cells involved in multiple metabolic pathways related to food processing. Our observations demonstrate significant modifications of some nuclear features in GM-fed mice. In particular, GM fed-mice show irregularly shaped nuclei, which generally represents an index of high metabolic rate, and a higher number of nuclear pores, suggestive of intense molecular trafficking. Moreover, the roundish nucleoli of control animals change in more irregular nucleoli with numerous small fibrillar centres and abundant dense fibrillar component in GM-fed mice, modifications typical of increased metabolic rate. Accordingly, nucleoplasmic (snRNPs and SC-35) and nucleolar (fibrillarin) splicing factors are more abundant in hepatocyte nuclei of GM-fed than in control mice. In conclusion, our data suggest that GM soybean intake can influence hepatocyte nuclear features in young and adult mice; however, the mechanisms responsible for such alterations remain unknown.

Pharmacokinetic and antiretroviral activity in mice of oral [P(1),P(2)-bis[2-(adenin-9-yl)ethoxymethyl]phosphonate], a prodrug of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is an antiviral drug with activity against herpes viruses, Epstein-Barr virus and retroviruses, including the human immunodeficiency virus. Unfortunately, oral PMEA administration, as required for long-term therapy, is hindered by its low bioavailability. In the present study, the synthesis, oral bioavailability and antiretroviral activity of a new prodrug of PMEA, consisting of two molecules of PMEA bound together by a P-O-P bond (Bis-PMEA), are reported. Pharmacokinetic experiments in mice showed that the oral bioavailabilities of PMEA following oral gavage of Bis-PMEA or PMEA (at a dose equivalent to 28 mg of PMEA/kg) were 50.8 and 13.5%, respectively. These results correlate with the antiviral efficacy of Bis-PMEA administered orally at a dose equivalent to 50 mg/kg of PMEA in C57 BL/6 mice infected with the retroviral complex LP-BM5. Oral treatment with Bis-PMEA proved to be more effective than oral treatment with PMEA given at equimolar doses. Moreover, oral Bis-PMEA was more effective than intraperitoneal PMEA (50 mg/kg) in reducing lymphoadenopathy, hypergammaglobulinaemia and lymph node proviral DNA content, overall in the first weeks post virus inoculation. Bis-PMEA thus appears to be an efficient oral prodrug of PMEA without significant toxicity, at least in this mouse model.

Low doses of dexamethasone constantly delivered by autologous erythrocytes slow the progression of lung disease in cystic fibrosis patients.

OBJECTIVE: To evaluate the safety and efficacy of the administration of low doses of glucocorticoids in patients with cystic fibrosis (CF) by using autologous erythrocytes loaded with dexamethasone 21-phosphate. STUDY DESIGN: Nine consecutive CF patients (patients nos. 1-9) received autologous erythrocytes loaded with increasing amounts of dexamethasone 21-phosphate to obtain a slow delivery of dexamethasone in circulation. The appearance of possible adverse effects, the reproducibility of the procedure, and the dexamethasone pharmacokinetics were evaluated. Subsequently, patient no. 9 and eight additional patients (patient nos. 10-17) received dexamethasone 21-phosphate-loaded erythrocytes at 1-month intervals to evaluate the efficacy of continuous release in circulation of low doses of dexamethasone. RESULTS: Erythrocytes from CF patients can be processed to be loaded with increasing dexamethasone 21-P concentrations. Once reinfused in respective donors, a slow and prolonged delivery of dexamethasone in the blood stream was measured up to 28 days. Repeated administrations of drug-loaded erythrocytes at 4-week intervals for 15 months showed that very low doses of glucocorticoids provide significant improvement in FEV1 values and significant reduction of infective relapses due to Pseudomonas aeruginosa without adverse effects. CONCLUSIONS: The administration of very low doses of glucocorticoids using autologous erythrocytes is possible, with benefits for patients and without side effects. This method is likely to be extended to other chronic diseases.