Immune Dysfunction and Coinfection with Human Immunodeficiency Virus and Schistosoma japonicum in Yi People.

OBJECTIVE: To explore the association between infections with HIV and Schistosoma japonicum, and to determine the influences of the HIV-S. japonicum coinfections on the immune system of Yi people. METHODS: A block design study was conducted in a Yi county in southwestern China, one of the endemic areas of both HIV/AIDS and S. japonicum in China. All participants were screened for HIV antibodies and S. japonicum antibodies (SjAb) and were classified into four groups: HIV(+)/S. japonicum(-), HIV(-)/S. japonicum (+), HIV(+)/S. japonicum(+), and HIV(-)/S. japonicum(-). RESULTS: There were significant differences among the four groups in both CD4+ T lymphocytes and CD8+ T lymphocytes, but no significant difference in CD3+ T lymphocytes. Both the CD4+ T lymphocyte counts and the ratio of CD4+/CD8+ were lower in HIV-infected people compared with those uninfected. People infected with S. japonicum had increased CD4+ T lymphocyte counts but reduced CD8+ T lymphocyte counts. Similarly, the ratio of CD4+/CD8+ was higher in S. japonicum-infected people compared with those uninfected. People coinfected with HIV and S. japonicum had lower CD4+ T lymphocyte counts, lower ratio of CD4+/CD8+, and higher CD8+ T lymphocyte counts compared with those infected with HIV only or S. japonicum only. People infected with HIV only and those coinfected with HIV and S. japonicum had a higher level of IFN-γ compared with people with no infection. There were no significant differences between people infected with HIV only and with S. japonicum only in the levels of IFN-γ and IL-10. CONCLUSIONS: People coinfected with HIV and S. japonicum might have a suppressed immune function because of a decrease in CD4+ T lymphocyte counts, a lowered ratio of CD4+/CD8+, and an increase in CD8+ T lymphocyte counts. Coinfection with HIV and S. japonicum would alter the level of IFN-γ in plasma.

Three Gorges Dam: polynomial regression modeling of water level and the density of schistosome-transmitting snails Oncomelania hupensis.

BACKGROUND: Schistosomiasis remains a major public health concern in China. Oncomelania hupensis (O. hupensis) is the sole intermediate host of Schistosoma japonicum, and its change in distribution and density influences the endemic S. japonicum. The Three Gorges Dam (TGD) has substantially changed the downstream water levels of the dam. This study investigated the quantitative relationship between flooding duration and the density of the snail population. METHODS: Two bottomlands without any control measures for snails were selected in Yueyang City, Hunan Province. Data for the density of the snail population and water level in both spring and autumn were collected for the period 2009-2015. Polynomial regression analysis was applied to explore the relationship between flooding duration and the density of the snail population. RESULTS: Data showed a convex relationship between spring snail density and flooding duration of the previous year (adjusted R2, aR2 = 0.61). The spring snail density remained low when the flooding duration was fewer than 50 days in the previous year, was the highest when the flooding duration was 123 days, and decreased thereafter. There was a similar convex relationship between autumn snail density and flooding duration of the current year (aR2 = 0.77). The snail density was low when the flooding duration was fewer than 50 days and was the highest when the flooding duration was 139 days. CONCLUSIONS: There was a convex relationship between flooding duration and the spring or autumn snail density. The snail density was the highest when flooding lasted about four to 5 months.

Prevalence of hepatitis C virus infection and its correlates in a rural area of southwestern China: a community-based cross-sectional study.

OBJECTIVES: Hepatitis C virus (HCV) infection is a major public health problem in southwestern China. Our aim of the study was to assess the prevalence of HCV infection and its correlates in the Yi population of this region. METHODS: A community-based survey was conducted to investigate sociodemographic characteristics and other associated factors for HCV infection in a rural area of southwestern China. Blood samples were collected and tested for antibodies to HCV. Anti-HCV positive samples were further assessed for HCV RNA. RESULTS: A total of 2558 participants aged ≥14 years were included in our analysis. Of them, 2.8% (95% CI 2.2% to 3.5%) were positive for HCV antibody. Multiple logistic regression analysis revealed that sex (male vs female: adjusted OR (aOR)=3.30, 95% CI 1.80 to 6.07), marital status (unmarried vs married: aOR=0.27, 95% CI 0.09 to 0.80), ever using injection drug (aOR=28.65, 95% CI 15.9 to 51.64) and ever having blood transfusion (aOR=7.64, 95% CI 1.94 to 30.16) were significantly associated with HCV infection (indicated by positive HCV antibody). Stratified analysis by HIV infection found that in HIV-negative individuals, sex (male vs female: aOR=3.84, 95% CI 1.88 to 7.85), ever using injection drug (aOR=22.15, 95% CI 8.45 to 58.04), having multiple sexual partners (aOR=2.57, 95% CI 1.26 to 5.23), and ever having blood transfusion (aOR=16.54, 95% CI 4.44 to 61.58) were significantly associated with HCV infection and in HIV-positive individuals, ever using injection drug (aOR=8.96, 95% CI 3.16 to 25.38) was associated with HCV infection. CONCLUSION: The data suggested a higher risk of HCV infection in this area when compared with the rest of China and some unique associated factors. Rapid scale-up of targeted interventions are needed to prevent further transmission and consequent morbidities.

Prevalence and Determinants of Cryptosporidium Infection in an Underdeveloped Rural Region of Southwestern China.

Few studies have focused on the epidemiology of Cryptosporidium in resource-challenged settings in China. We report a community-based cross-sectional study to investigate the prevalence of Cryptosporidium infection and its risk factors and associations with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infections. The prevalence of Cryptosporidium infection was 12.6% (95% confidence interval = 11.0-14.3). Individuals living in households with ≥ 5 family members and raising domestic pigs tended to have a greater risk of Cryptosporidium infection. In addition, Cryptosporidium infection was significantly associated with HBV infection. There were no significant associations of Cryptosporidium infection with HIV viral load and HBV viral load. Further studies are needed to determine the association of Cryptosporidium infection with HBV infection.

Prevalence and risk factors of Ascaris lumbricoides (Linnaeus, 1758), Trichuris trichiura (Linnaeus, 1771) and HBV infections in Southwestern China: a community-based cross sectional study.

BACKGROUND: Intestinal helminths do not cause severe diseases in general, however, when combined with other diseases such as immunodeficiency diseases, there would be massive complications. Infections with Hepatitis B Virus (HBV) may lead to immunological disturbances and defects of cellular immunity and there is a need of clarification whether HBV infections are associated with infections with intestinal helminths. METHODS: A community-based cross sectional study was conducted in Tezi town, Puge County of the Liangshan Prefecture, southwestern China from October 23rd to November 3rd, 2014. Four hundred and thirty eight people (median age = 37 years, IQR = 22-49) were enrolled in this study. Modified Kato-Katz thick smear was used to detect intestinal helminths. HBV DNA was quantified to confirm HBV infection. RESULTS: Among the 438 participants, 9.1%, 13.5% and 30.6% were infected with HBV, A. lumbricoides (L., 1758) and T. trichiura (L., 1771), respectively; 7.1% (30/438) were infected with both A. lumbricoides and T. trichiura and 2.3% (10/438) were co-infected with HBV and A. lumbricoides. The multivariate logistic regression analysis showed that age (21-30 years versus >50 years: OR = 6.66, 95% CI = 2.15-20.68), drug abuse (OR = 6.96, 95% CI = 1.11-43.90), A. lumbricoides infection (OR = 3.60, 95% CI = 1.48-8.75), fertilization with faeces after disposal (OR = 0.15, 95% CI = 0.04-0.47) and working on a farm (OR = 4.59, 95% CI = 1.44-14.63) were significantly associated with HBV infection. Having toilets at home was negatively related to A. lumbricoides infection (OR = 0.52, 95% CI = 0.27-0.98) and T. trichiura infection (OR = 0.48, 95% CI = 0.28-0.80). CONCLUSIONS: Ascaris lumbricoides was independently associated with HBV infection, and faeces might be the medium of HBV transmission. Improving hygiene conditions and habits are essential to reduce the risks of A. lumbricoides and T. trichiura infections.

[Analysis of relationship between natural death of Oncomelania hupensis snails and water level in Eastern Dongting Lake district].

OBJECTIVE: To study the reasons of natural death of Oncomelania hupensis snails by comparing the differences of the indicator days covered with water (DCW) in snail marshland and non-snail marshland around the build of Three Gorges Dam in Eastern Dongting Lake areas. METHODS: Two marshlands were selected, one was a non-snail marshland (Qianliang Lake) and another was a snail marshland (Junshan Park). The measuring points were set through the mechanical sampling. The snails and elevation of the points were surveyed, and the data of the water levels from the hydrological station were collected, and then DCWs were calculated. RESULTS: From 1995 to 2013, DCWs of the marshland of natural death of snails were all more than that of the snail marshland (P < 0.01). In Qianliang Lake marshland, the difference between DCW before natural death and DCW from natural death until the dam was not significant (P = 0.23), while DCWs of the two stages both were more than that after the dam (P1 = 0.045, P2 = 0.002). Before the build of the dam, DCW of the Qianliang Lake marshland of natural death of snails was more than that after the build of the dam (P = 0.013), and there was the same situation in Junshan Park marshland (P = 0.005). The relationship between snail density and DCW was not significant in Junshan Park marshland (r(s) = 0.008, P = 0.914), and the reference range of DCW of all the measuring points was 76-251 days. CONCLUSION: In the eastern Dongting Lake district, the build of Three Gorges Dam and DCW may be not the direct factors affecting the natural death of snails and the latter may change the distribution of snails.

Single and double emulsion manufacturing techniques of an amphiphilic drug in PLGA nanoparticles: formulations of mithramycin and bioactivity.

Formulation of hydrophilic compounds in nanoparticles is problematic due to their escape to the external aqueous phase. The certain amphiphilic nature of mithramycin, utilized clinically in cancer, makes its incorporation into nanoparticles an interesting challenge, elucidating the formulation factors of amphiphilics in nanoparticles. We hypothesized that mithramycin nanoparticles could provide more effective therapy of restenosis due to its antiproliferating and potential monocyte inhibition properties. The nanoprecipitation technique (designed for lipophilic compounds) was found preferable, with better encapsulation efficiency, than the emulsification solvent diffusion (ESD) technique (79.3 +/- 3.1% and 40.8 +/- 1.1%, respectively). The double emulsion solvent diffusion (DESD) method, designed for hydrophilic compounds, yielded similar encapsulation efficiency (80%). Nanoparticles size was, 110 +/- 36, 130 +/- 30, and 160 +/- 31 nm, ESD, nanoprecipitation, and DESD techniques, respectively. Mithramycin solution and in nanoparticles significantly inhibited RAW264 macrophages and smooth muscle cells in a dose-dependent relationship, and reduced the number of circulating monocytes in rabbits. However, no inhibition of restenosis was obtained in the rat carotid model following i.v. administration of mithramycin nanoparticles. It can be concluded that PLGA-based polymeric nanoparticles of mithramycin can be formulated by techniques suitable for lipophilic/hydrophilic compounds. The ineffectiveness in the rat restenosis model is probably due to the short depletion period of circulating monocytes and lack of arterial targeting.

Predicting in vivo efficacy of potential restenosis therapies by cell culture studies: species-dependent susceptibility of vascular smooth muscle cells.

Although drug-eluting stents (DES) are successfully utilized for restenosis therapy, the development of local and systemic therapeutic means including nanoparticles (NP) continues. Lack of correlation between in vitro and in vivo studies is one of the major drawbacks in developing new drug delivery systems. The present study was designed to examine the applicability of the arterial explant outgrowth model, and of smooth muscle cells (SMC) cultures for prescreening of possible drugs. Elucidation of different species sensitivity (rat, rabbit, porcine and human) to diverse drugs (tyrphostins, heparin and bisphsophonates) and a delivery system (nanoparticles) could provide a valuable screening tool for further in vivo studies. The anticipated sensitivity ranking from the explant outgrowth model and SMC mitotic rates (porcine>rat>>rabbit>human) do not correlate with the observed relative sensitivity of those animals to antiproliferative therapy in restenosis models (rat>/=rabbit>porcine>human). Similarly, the inhibitory profile of the various antirestenotic drugs in SMC cultures (rabbit>porcine>rat>>human) do not correlate with animal studies, the rabbit- and porcine-derived SMC being highly sensitive. The validity of in vitro culture studies for the screening of controlled release delivery systems such as nanoparticles is limited. It is suggested that prescreening studies of possible drug candidates for restenosis therapy should include both SMC cell cultures of rat and human, appropriately designed with a suitable serum.

Nanosuspensions of alendronate with gallium or gadolinium attenuate neointimal hyperplasia in rats.

Monocytes/macrophages play a pivotal role in the formation of neointinal hyperplasia following vascular injury. Transient depletion of circulating monocytes by particulate delivery systems containing bisphosphonates, such as alendronate, results in restenosis inhibition. We hypothesized that a self-suspendable nanoparticulate dosage form, with a minimum amount of expients, could be formulated by complexing the negatively charged alendronate with gallium or gadolinium. We further hypothesized that a synergistic biological effect could be obtained by nanosuspensions of alendronate with these counter ions. Nanosuspensions (150-250 nm) of alendronate-gallium and alendronate-gadolinium were successfully formulated with no additives except for the active agents and HCl for pH adjustment. Both nanosuspensions exhibited macrophage cell line growth inhibition in a dose-response relationship in comparison to the various agents in solution and in liposomes. A synergistic effect of the nanosuspensions was observed in the inhibition of raw264 macrophages, and in reducing IL-1beta and TNF-alpha secretion in cell culture. Single IV administration at the time of injury, of alendronate-gallium or alendronate-gadolinium nanosuspensions resulted in inhibition of neointimal hyperplasia and stenosis in the rat model of vascular injury. The results correlated with the significant reduction of circulating monocytes. The nanosuspensions possess the advantages of no additives for minimal provocation of side effects, and the potential of immunomodulating inflammatory disorders.

Nanospheres of a bisphosphonate attenuate intimal hyperplasia.

The present study explored a novel strategy for attenuation of restenosis after arterial injury by a bisphosphonate encapsulated in polymeric nanoparticles (NP) for transient selective depletion of macrophages. A bisphosphonate (BP), 2-(2-Aminopyrimidino) ethyldiene-1,1-bisphosphonic acid betaine (ISA), was successfully formulated in 400 nm sized polylactide/glycolide-based NP with high yield (69%) and entrapment efficiency (60% w/w). ISA NP, but not blank NP or free ISA, exhibited specific and significant cytotoxic effect on macrophages-like RAW 264 cells, in a dose-dependent manner, with no inhibitory effect on the growth of smooth muscle cells (SMCs). Fluorescent pyrene-labeled NP were shown to be taken up by RAW 264 cells, but not by SMCs. Intravenously (i.v.) administered ISA NP (15 mg/kg, single dose on day-1) resulted in a significant attenuation of neointima to media area ratio (N/M) by 40% and stenosis by 45% 14 days after rat carotid injury, in comparison to animals treated with free ISA, buffer or blank NP. However, the effect was not preserved 30 days post injury, and an insignificant reduction of neointimal formation was observed. Neointimal hyperplasia was also significantly suppressed after subcutaneous (SC) injection of ISA NP (15 mg/kg, single dose on day-1), reducing both N/M and stenosis. Intraperitoneal (i.p.) injection of silica, a known selective toxin for macrophages, (1000 mg/kg), also resulted in a significant inhibition of N/M and stenosis, which further reinforces the cause-effect relationship of macrophage-inactivation and the prevention of neointima formation. Biocompatible and biodegradable NP loaded with ISA characterized by high colloidal stability, reproducible activity, and high drug entrapment warrant further consideration for restenosis therapy, and may be useful in other disease processes involving monocytes/macrophages.

Alendronate-loaded nanoparticles deplete monocytes and attenuate restenosis.

Systemic transient depletion of monocytes and macrophages by liposome-encapsulated bisphosphonates (BPs), reduces neointimal formation in experimental restenosis. The aim of this study was to examine the antirestenotic effect of a polymeric nanoparticulate formulation containing the BP alendronate (ALN). The BP was successfully formulated in polylactide-co-glycolide (PLGA) nanoparticles (NP). ALN NP with negative charge, size of 223+/-64 nm, and high entrapment efficiency (55.1%) have been formulated. ALN NP exhibited a significant cytotoxic effect, in a dose-response relationship, on macrophage-like RAW264 cells in cell culture. Subcutaneously (SC) administrated ALN NP (1.5 mg/kg on days -1 and +6) resulted in a significant attenuation of neointima to media ratio (N/M) by 52.7% and stenosis by 39.7% 28 days after balloon injury in the hypercholesterolemic rabbit model. Moreover, a good correlation was found between macrophage abundance in the injured arteries and the extent of stenosis. ALN NP treatment resulted in the reduction of both interleukin-1beta and matrix metalloproteinases (2 and 9). It is concluded that a particulated dosage form of polymeric NP loaded with ALN reduce neointimal formation in vivo by systemic transient depletion of monocytes.

Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries.

Local delivery of antiproliferative drugs encapsulated in biodegradable nanoparticles (NP) has shown promise as an experimental strategy for preventing restenosis development. A novel PDGFRbeta-specific tyrphostin, AGL-2043, was formulated in polylactide-based nanoparticles and was administered intraluminally to the wall of balloon-injured rat carotid and stented pig coronary arteries. The disposition and elimination kinetics within the vessel wall, as well as the antirestenotic potential of the novel drug and delivery system, were evaluated. The efficacy and the local drug elimination kinetics were affected by the size of the NP and the drug-carrier binding mode. Despite similar arterial drug levels 90 min after delivery in rats, small NP were more efficacious in comparison to large NP (90 and 160 nm, respectively). AGL-2043 selectively inhibited vascular SMC in a dose-dependent manner. The antiproliferative effect of nanoencapsulated tyrphostin was considerably higher than that of surface-adsorbed drug. In the pig model, intramural delivery of AGL-2043 resulted in reduced in-stent neointima formation in the coronary arteries over control despite similar degrees of wall injury. The results of this study suggest that locally delivered tyrphostin AGL-2043 formulated in biodegradable NP may be applicable for antirestenotic therapy independent of stent design or type of injury.

The role of oxygen-free radical in the apoptosis of enterocytes in scalded rats after delayed resuscitation.

BACKGROUND: This study aimed to evaluate the relation between apoptosis of enterocytes and oxygen-free radical injury in scalded rats with delayed resuscitation as well as the role of antioxidants in the prevention of enterocyte apoptosis. METHODS: For this study, 150 male Wistar rats were divided randomly into four groups representing early resuscitation (ER), delayed resuscitation (DR), N-acetylcysteine (NAC) treatment, and allopurinol (Allo) treatment. The animals were subjected to a 30% total body surface area, full-thickness scald. Fluid therapy was started 6 hours after the injury in the DR and treatment groups. Apoptosis of enterocytes was identified by DNA fragmentation (ap%), DNA agarose gel electrophoresis, and terminal deoxynucleotidyl transferace (TdT)-mediated dUPT-biotin nick end labeling (TUNEL). The contents of malondialdehyde (MDA), total sulfhydryl (TSH), and nonprotein sulfhydryl (NPSH) and the activity of xanthine oxidase in intestinal mucosa were determined after the burn in the four groups. RESULTS: Apoptosis of enterocytes increased significantly in all the groups. The animals in the DR group showed an earlier and greater increase in ap% than the animals in the ER group. Similar results were seen for electrophoresis, TUNEL assay, and levels of MDA, xanthine oxidase (XO), TSH, and NPSH. Treatment with NAC was associated with a decrease in ap% and MDA, but not XO, as compared with the levels in the DR group, whereas treatment with Allo was associated with a decrease in MDA and XO, but not ap%. Delayed resuscitation was associated with significant decreases in TSH and NPSH, as compared with the levels in the ER group, whereas both the NAC and Allo groups had significantly higher levels of TSH and NPSH than the DR group. CONCLUSIONS: Significant apoptosis of enterocytes was induced by oxidative stress in the intestinal mucosa after a burn in rats. The findings show that NAC blunted intestinal apoptosis induced by oxygen-free radical, which was generated in the process of ischemia-reperfusion injury after a burn because of delayed resuscitation.

[The effects of early escharectomy on resting energy expenditure in the severely burned patients].

OBJECTIVE: To investigate the effect of early escharectomy on resting energy expenditure (REE) in severely burned patients dynamically with the metabolic monitoring and diagnostic system. METHODS: Fifty-six adult male patients with severe burns were divided into early escharectomy (group A, n = 39, escharectomy within 5 PBDs) and non-early escharectomy (group B, n = 17, escharectomy after 5 PBDs) groups. The wounds of full thickness and deep partial thickness burn in the two groups were all excised and covered with allogeneic skin and autologous micro-skin in the first operation. The changes in REE were observed dynamically at the bedside of the patients with the metabolic monitoring and diagnostic system. The plasma contents of IL-6, IL-8, TNF-alpha and LPS from 9 patients in group A and 7 in group B were also determined dynamically. RESULTS: All patients survived. The REE in both groups was elevated markedly, but REE in group A was lower compared with group B before and after escharectomy within 14 days. (P < 0.05). The plasma level of IL-6, IL-8, TNF-alpha and LPS in group A were obviously lower than those in group B (P < 0.05). CONCLUSION: The hypermetabolic response of burn patients with severe burns could be lowered by early escharectomy, and it seemed to be related to the decrease of the release of proinflammatory mediators.

Delivery and expression of pDNA embedded in collagen matrices.

Collagen matrices can be used as non-viral biocompatible gene carriers for localized implantable gene therapy. Collagen matrices embedding pDNA with enhanced binding through condensing agent linkage to the matrix or to the pDNA have been formulated, and characterized in various systems. pDNA and condensed pDNA were released intact from the matrices within 1-2 days. In vitro transfection with collagen matrices containing pDNA (luciferase encoding), pDNA in liposome (LIP), and pDNA with polyethylenimine (PEI) resulted in significantly higher expression levels in comparison to naked pDNA. pDNA-LIP matrices exhibited a dose response transfection of NIH 3T3, 293, MDA-MB-231 and smooth muscle cells (SMCs) in cell cultures. Subdermal implantations of collagen-polylysine-pDNA matrices in rats resulted in significantly higher gene expression levels in comparison to non-condensed pDNA matrices. Perivascular treatment with pDNA matrix and of naked pDNA solution in balloon-injured rat carotid arteries resulted in significant expression. In conclusion, a facile method for embedding cationic formulations of pDNA in collagen matrices was developed. These bioactive matrices seem to be suitable for tissue engineering and local gene therapy strategies.

Liposomal alendronate inhibits systemic innate immunity and reduces in-stent neointimal hyperplasia in rabbits.

BACKGROUND: Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation. METHODS AND RESULTS: Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment. Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting. Monocyte counts were reduced by >90% 24 to 48 hours after a single injection of liposomal alendronate, returning to basal levels at 6 days. This treatment significantly reduced intimal area at 28 days, from 3.88+/-0.93 to 2.08+/-0.58 and 2.16+/-0.62 mm2. Lumen area was increased from 2.87+/-0.44 to 3.57+/-0.65 and 3.45+/-0.58 mm2, and arterial stenosis was reduced from 58+/-11% to 37+/-8% and 38+/-7% in controls, rabbits treated with 3 mg/kg, and rabbits treated with 6 mg/kg, respectively (mean+/-SD, n=8 rabbits/group, P<0.01 for all 3 parameters). No drug-related adverse effects were observed. Reduction in neointimal formation was associated with reduced arterial macrophage infiltration and proliferation at 6 days and with an equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury. Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation. CONCLUSIONS: Systemic transient depletion of monocytes and macrophages, by a single liposomal bisphosphonates injection concurrent with injury, reduces in-stent neointimal formation and arterial stenosis in hypercholesterolemic rabbits.

Systemic depletion of macrophages by liposomal bisphosphonates reduces neointimal formation following balloon-injury in the rat carotid artery.

OBJECTIVES: Macrophage depletion by liposomal clodronate inhibits neointimal formation after balloon-injury. The present study examined bisphosphonates (BPs) potency-effect relationship and the role of systemic versus local monocytes in vascular repair. METHODS AND RESULTS: Liposomal preparations of clodronate, pamidronate, alendronate, and ISA-13-1 inhibited RAW-264 macrophages growth in a dose-response manner. Administration to balloon-injured rats suppressed neointimal growth. Neointima to media ratio (N/M) at 14 days was reduced from 1.35 +/- 0.22 (control) to 0.4 +/- 0.1 and 0.9 +/- 0.17 by liposomal alendronate (1.5 mg/kg, i.v.) and liposomal ISA-13-1 (15 mg/kg), respectively (n = 8-10, P < 0.05). Suppression of neointimal formation was preserved at 30 days. Subcutaneous administration of liposomal BP (LBP) was also effective in suppressing neointimal formation, while short local intraluminal application had no effect. Immunostaining for ED-1 and ED-2 revealed no resident macrophages in the arterial wall, and reduced macrophage infiltration in LBP-treated animals. Arterial PDGF-B chain and PDGF-beta receptor activation were reduced in LBP-treated animals and up-regulation of the PDGF receptor was noted. CONCLUSIONS: Systemic transient inactivation of monocytes and macrophages by LBPs reduced macrophage infiltration and neointimal formation in the rat carotid injury model. The findings demonstrate a BP potency-effect relationship, and highlight the role of circulating monocytes in vascular injury and repair.

[Translocation of intestinal endotoxin and bacteria induced by the apoptosis of enterocytes in scald rats after delayed resuscitation].

OBJECTIVE: To investigate the role of enterocyte apoptosis in translocation of intestinal endotoxin and bacteria after delayed resuscitation in scalded rats. METHODS: One hundred and ten male Wistar rats were divided randomly into two groups: group A, early resuscitation, n=60; group B, delayed resuscitation, n=50. All animals were subjected to 30% total body surface area (TBSA) full-thickness scald. In group A, saline resuscitation was begun immediately after the injury. Saline resuscitation later than 6 hours after scalding was referred as delayed resuscitation. Apoptosis of enterocytes was identified by DNA fragmentation (ap%), DNA agarose gel electrophoresis, TdT-mediated dUTP nick end labeling (TUNEL) method and electron microscope (EM). The levels of endotoxin in portal vein and systemic circulation were determined by limulus amebocyte lysate technique. The amount of bacteria in mesenteric lymph nodes (MLN) was detected by a quantitative bacteria culture of biopsy. RESULTS: The ap% of enterocytes was increased significantly in groups A and B, peaking at 12 hours postburn. The increased ap% in the group B occurred much earlier and higher than in group A from 3 hours to 48 hours postburn (P<0.05 or P<0.01). This was corroborated by the results observed in electrophoresis, TUNEL method and EM. The portal endotoxin was much higher in group B than in group A at the same postburn timepoints. So were the endotoxin levels in systemic circulation. A significant positive relationship existed between the portal endotoxin levels and the ap% of intestinal epithelial cells in groups A and B (group A: r=0.936, P<0.01; group B: r=0.899, P<0.05). The frequency of bacteria translocation of MLN in group B was higher than that in group A. CONCLUSION: Significant pathologic apoptosis of enterocytes is induced by delayed resuscitation after thermal injury in rats. This may lead to a compromise of intestinal barrier. It may be one of the major causes of translocation of endotoxin and bacteria postburn.

The influence of apoptosis of mucosal epithelial cells on intestinal barrier integrity after scald in rats.

OBJECTIVE: To study the influence of apoptosis of intestinal epithelial cells occurring as a result of reperfusion after burn shock on the intestinal barrier. METHODS: Fifty Wistar rats were subjected to a 30% TBSA full thickness burn, and normal saline (40 ml/kg) was given intraperitoneally immediately after the injury (group A). Ten rats served as a sham control group. The experimental group B consisted of 50 rats with identical injuries, but the normal saline was not given until 6h after the injury. Apoptosis of intestinal epithelial cells was verified by DNA fragmentation, DNA agarose gel electrophoresis, TUNEL and electron microscope (EM), and DNA fragmentation rate was expressed as ap%. The D-lactic acid in portal vein blood and intestinal diamine oxidase (DAO) were determined to evaluate the permeability and integrity of intestinal mucosal epithelium. RESULTS: The ap% of intestinal epithelium group B was higher than in that of group A (P<0.05 or 0.01), and its amplitude peaked at 12h for both groups. Typical DNA ladder pattern was seen in electrophoresis in both groups. Apoptotic cells were discerned on the tips of the ileal villi at 3h postscald by TUNEL and EM in the group B, and they appeared earlier than in the group A. There was a significant positive correlation between the ap% and the level of D-lactic acid (group A: r=0.817, P<0.05; group B: r=0.727, P<0.05). On the other hand, a significant negative correlation was found between the ap% and the DAO values (group A: r=-0.937, P<0.01; group B: r=-0.836, P<0.05). CONCLUSION: Apoptosis occurred in enterocytes after scald injury this pathological change might contribute to a breach of integrity of intestinal epithelium, leading to a compromise in its barrier function. Delayed fluid resuscitation might lead to an earlier and higher degree of apoptosis of the intestinal epithelial cells.

Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits.

BACKGROUND: Inflammation is critical to vascular repair after mechanical injury. Excessive inflammation enhances neointimal formation and restenosis. We examined whether transient systemic inactivation of macrophages at the time of vascular intervention could attenuate the degree of expected restenosis. METHODS AND RESULTS: Liposomal clodronate (LC) inhibited the growth of cultured macrophages but had no effect on endothelial or smooth muscle cells and suppressed neointimal hyperplasia in hypercholesterolemic rabbits and rats after intravenous administration of LC, with no adverse effects. LC treatment reduced the number of blood monocytes and decreased macrophage infiltration in the injured arteries as well as smooth muscle cell proliferation, interleukin-1beta transcription, and production and matrix metalloproteinase-2 activity. CONCLUSIONS: Macrophages play a pivotal role in vascular repair after mechanical arterial injury. Systemic inactivation and depletion of monocytes and macrophages by LC reduce neointimal hyperplasia and restenosis.