Nanoparticle-mediated targeting of MAPK signaling predisposes tumor to chemotherapy

The MAPK signal transduction cascade is dysregulated in a majority of human tumors. Here we report that a nanoparticle-mediated targeting of this pathway can optimize cancer chemotherapy. We engineered nanoparticles from a unique hexadentate-polyD,L-lactic acid-co-glycolic acid polymer chemically conjugated to PD98059, a selective MAPK inhibitor. The nanoparticles are taken up by cancer cells through endocytosis and demonstrate sustained release of the active agent, resulting in the inhibition of phosphorylation of downstream extracellular signal regulated kinase. We demonstrate that nanoparticle-mediated targeting of MAPK inhibits the proliferation of melanoma and lung carcinoma cells and induces apoptosis in vitro. Administration of the PD98059-nanoparticles in melanoma-bearing mice inhibits tumor growth and enhances the antitumor efficacy of cisplatin chemotherapy. Our study shows the nanoparticle-mediated delivery of signal transduction inhibitors can emerge as a unique paradigm in cancer chemotherapy.     

Diet- and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs

BACKGROUND & AIMS: Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. METHODS: Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1-2 days of age. RESULTS: Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. CONCLUSIONS: A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens.     

Gene therapy: targeting the myocardium

Effective clinical delivery of gene therapy to the heart requires understanding and design of complex biological systems to deliver therapeutic gene expression. The development of vectors that specifically target the myocardium, in particular bioengineered recombinant viruses, has improved the efficiency of gene delivery to the heart. These tools, coupled with advances in selection and design of the genetic payload, have led to effective cardiac gene therapy in preclinical models. This technology is currently translating to the clinic with a new wave of gene therapy trials for myocardial disease.     

Hypothyroidism delays ischemia-induced contracture and adenine nucleotide depletion in rat myocardium

Isolated perfused paced hearts from rats rendered hypothyroid by chronic administration of propylthiouracil have a delayed onset of ischemia-induced myocardial contracture in contrast to hearts from control rats. In addition, the time to reach maximum contracture is delayed, and the magnitude of the contracture pressure is reduced. Preischemia myocardial adenosine triphosphate (ATP) values in the hypothyroid rat hearts are similar to those of control, but the rate of decrease in ATP is slower in the hearts of hypothyroid rats. Thus, it appears that in the hypothyroid state the development of ischemic contracture is associated with a slower fall of ATP.     

Myocyte apoptosis and reduced SR gene expression precede the transition from chronically stunned to hibernating myocardium.

A systematic transition from chronic stunning to hibernation occurs as coronary flow reserve decreases to a critical level. Hibernating myocardium exhibits apoptosis-induced myocyte loss and a reduction in the expression of the sarcoplasmic reticulum (SR) Ca2+ ATPase but whether similar cellular changes occur in chronic stunning is unknown. Pigs with a chronic left anterior descending coronary artery (LAD) stenosis were studied one (n=9) or two (n=10) months after instrumentation. Anterior hypokinesis with normal levels of resting perfusion developed at each time-point, consistent with chronic stunning. After 1 month, sub-endocardial flow reserve was moderately reduced (adenosine/rest, LAD: 3.60+/-0.91 v Remote: 6.00+/-0.54, P<0.01) with no regional differences in SR protein expression, no increase in apoptosis (32+/-6 v 21+/-5 nuclei/10(6) myocyte nuclei, p-ns) and no regional myocyte loss (1976+/-44 v 1955+/-30 nuclei/mm2, p-ns). After 2 months, sub-endocardial flow reserve in chronically stunned myocardium was severely impaired (LAD: 1.41+/-0.21 v Remote: 5.59+/-0.96, P<0.01). There were small but significant reductions in LAD mRNA and protein levels for the SRCa2+ ATPase and phospholamban whereas calsequestrin was unchanged. In addition, regional myocyte apoptosis increased (127+/-24 v 55+/-9 nuclei/10(6) myocyte nuclei, P<0.01), resulting in the onset of myocyte loss (1293+/-50 v 1394+/-32 nuclei/mm2, P<0.01). Apoptosis-induced myocyte loss and reductions in SR protein expression are not invariably present in viable chronically dysfunctional myocardium. They are induced as the propensity of a region to develop reversible ischemia increases (as reflected by coronary flow reserve). The temporal progression indicates that alterations in SR protein expression and myocyte apoptosis precede the transition from chronically stunned to hibernating myocardium.     

心功能不全大鼠心肌诱生型一氧化氮合酶基因的表达

目的 探讨心功能不全大鼠心肌一氧化氮合酶（ｍｉｔｒｉｃ ｏｘｉｄｅ ｓｙｎｔｈａｓｅ,ＮＯＳ）活性变化的机制。方法 复制容量超负荷心功能不全大鼠模型,用原位核酸分子杂交技术检测心肌诱生型一氧化氮合酶（ｉｎｄｕｃｉｂｌｅ ｎｉｔｒｉｃ ｏｘｉｄｅ ｓｙｎｔｈａｓｅ,ｉＮＯＳ）ｍＲＮＡ的表达,用免疫组化技术检测心肌ｉＮＯＳ。结果 容量超负荷心功能不全大鼠心肌ｉＮＯＳ基因有达。结论 心功能不全心肌一氧化     

Noninvasive gene targeting to the brain

Gene therapy of the brain is hindered by the presence of the blood-brain barrier (BBB), which prevents the brain uptake of bloodborne gene formulations. Exogenous genes have been expressed in the brain after invasive routes of administration, such as craniotomy or intracarotid arterial infusion of noxious agents causing BBB disruption. The present studies describe the expression of an exogenous gene in brain after noninvasive i.v. administration of a 6- to 7-kb expression plasmid encoding either luciferase or beta-galactosidase packaged in the interior of neutral pegylated immunoliposomes. The latter are conjugated with the OX26 mAb to the rat transferrin receptor, which enables targeting of the plasmid DNA to the brain via the endogenous BBB transferrin receptor. Unlike cationic liposomes, this neutral liposome formulation is stable in blood and does not result in selective entrapment in the lung. Luciferase gene expression in the brain peaks at 48 h after a single i.v. administration of 10 microg of plasmid DNA per adult rat, a dose that is 30- to 100-fold lower than that used for gene expression in rodents with cationic liposomes. beta-Galactosidase histochemistry demonstrated gene expression throughout the central nervous system, including neurons, choroid plexus epithelium, and the brain microvasculature. In conclusion, widespread gene expression in the brain can be achieved by using a formulation that does not employ viruses or cationic liposomes, but instead uses endogenous receptor-mediated transport pathways at the BBB.     

Ileal resection/dysfunction in childhood predisposes to lithogenic bile only after puberty.

Children with ileal resection/dysfunction since infancy have bile that is not supersaturated with cholesterol. Five sexually mature subjects (age 16-19 years) who had been previously investigated in childhood (age 4-9 years) were studied. Gallstones were found in one. Bile rich duodenal aspirates were analyzed for lipid content (molar fraction) and the cholesterol saturation index was calculated. In the postpubertal subjects, a significantly higher proportion of biliary cholesterol (22.0% +/- 4.8% vs. 3.2% +/- 0.6% and 5.7% +/- 0.5%, P less than 0.005) and significantly lower bile acids (58.1% +/- 3.9% vs. 79.7% +/- 2.3% and 78.2% +/- 1.9%, P less than 0.005) were found compared with the initial (prepubertal) samples or in samples from 20 healthy young adults. The cholesterol saturation index was significantly higher (3.1 +/- 0.7 vs. 0.6 +/- 0.1 and 1.1 +/- 0.1, P less than 0.005) whereas phospholipid content did not change (19.9% +/- 1.6% vs. 17.1% +/- 1.8% and 16.6% +/- 1.6%) as compared with themselves before puberty and as healthy young adults, respectively. It was concluded that children with ileal resection/dysfunction do not appear at risk for cholesterol cholelithiasis before puberty; however, the development of biliary cholesterol supersaturation after puberty may predispose them to gallstone formation in adulthood.     

A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability

BACKGROUND: The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital to methylation reactions. A polymorphic variant (TT) has been linked to reduced levels of plasma folate, aberrant DNA methylation in leucocytes, and increased risk of colorectal cancer (CRC) under conditions of low folate intake. The cystathionine beta-synthase (CBS) enzyme reduces homocysteine levels and thus may protect against CRC. The CBS gene has a variant, 844ins68, that has been linked with increased activity. These variants may be involved in the development of the subgroup of CRC displaying aberrant DNA methylation and frequently associated with microsatellite instability (MSI). AIM: To investigate the frequencies of the TT and 844ins68 genotypes in CRC patients with MSI+ tumours compared with those with MSI- tumours and a control population. SUBJECTS: Patients with CRC (n=501) and healthy control subjects (n=1207) were studied. CRC cases were classified as MSI+ (n=75) or MSI- (n=426) based on deletions within the BAT-26 mononucleotide repeat. METHODS: Subjects were genotyped for MTHFR using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) techniques, and for CBS using PCR. RESULTS: The MTHFR TT genotype was more frequent in older CRC patients (>or=70 y) compared with equivalent aged controls (p=0.03), was associated with a significantly later age of diagnosis in patients with proximal colon tumours (p=0.02), and was almost twice as frequent in MSI+ than in MSI- tumours (p=0.05). Compared with normal controls, the 844ins68 variant of CBS was less frequent in patients with proximal tumours (p=0.02). CONCLUSIONS: The TT genotype of MTHFR is associated with an increased risk of CRC in older populations, possibly due to age related disturbances in folate metabolism. The TT genotype appears to predispose to CRC that is MSI+. This may reflect the involvement of aberrant DNA methylation frequently associated with MSI+. The 844ins68 CBS polymorphism may protect against proximal tumours.     

Keratin mutation in transgenic mice predisposes to Fas but not TNF-induced apoptosis and massive liver injury

Hepatocytes express keratins 8 and 18 (K8/18) as their only cytoskeletal intermediate filament (IF) proteins, and K8/18 mutations predispose their carriers to liver cirrhosis. Transgenic mice that overexpress mutant human K18 (Arg89-->Cys [R89C]) develop mild chronic hepatitis, hepatocyte fragility, keratin filament disruption, and increased susceptibility to drug-induced liver injury. K18 is a major caspase substrate during apoptosis, and K8- or K18-null mice are significantly predisposed to Fas- and possibly tumor necrosis factor (TNF)-mediated apoptosis in the liver. Here we tested the potential role of the K18 R89C mutation on Fas- or TNF-mediated apoptotic liver injury by injecting Fas antibody (Ab) or TNF-alpha plus actinomycin D into mice that overexpress wild-type (WT) human K18 (with intact filament network, termed TG2 mice) or into K18 R89C mice (with disrupted filament network). K18 R89C mice are significantly more susceptible to Fas-mediated liver injury compared with nontransgenic and TG2 mice. This included differences in lethality, histology, apoptosis, and serum transaminase levels. In contrast, K18 WT and R89C mice manifest similar sensitivity to TNF-induced injury. Both Fas- and TNF-induced apoptosis in liver tissues are associated with caspase-mediated K18 degradation and increased keratin phosphorylation on several but not all sites. In conclusion, transgenic mouse K18 mutation and its consequent keratin filament disruption predispose hepatocytes to Fas- but not TNF-mediated apoptotic injury. This supports the association of keratin mutations with cirrhosis in patients with liver disease and suggests that keratins modulate apoptosis induced by Fas but not TNF.     

The H63D variant in the HFE gene predisposes to arthralgia, chondrocalcinosis and osteoarthritis

OBJECTIVES: To investigate the relation between the HFE C282Y and H63D variants with arthralgia and joint pathology in the population-based Rotterdam Study. METHODS: From a cohort of 7983 people aged 55 years and over, 2095 randomly drawn subjects were genotyped for C282Y and H63D variants. We compared the frequency of arthralgia, and the presence of chondrocalcinosis, osteophytes, joint space narrowing and radiographic osteoarthritis in hand, hip and knee joints, and Heberden's nodes in carriers of HFE variants with that in non-carriers. RESULTS: Overall, there was a significantly higher frequency of arthralgia (odds ratio 1.6; 95% CI 1.0 to 2.6), oligoarthralgia (2.3; 1.2 to 4.4) and Heberden's nodes (2.0; 1.1 to 3.8) in H63D homozygotes compared with non-carriers. In subjects aged 65 years or younger, H63D homozygotes had significantly more often polyarthralgia (3.1; 1.3 to 7.4), chondrocalcinosis in hip or knee joints (4.7; 1.2 to 18.5), and more hand joints with osteophytes (6.1+/-1.0 vs 4.4+/-0.3), space narrowing (2.8+/-0.5 vs 1.0+/-0.1), radiographic osteoarthritis (4.4+/-0.7 vs 2.0+/-0.2) and Heberden's nodes (3.1; 1.3 to 12.8) than non-carriers. We found no relation of arthralgia or joint pathology to C282Y, but compound heterozygotes had a significantly higher frequency of arthralgia (2.9; 1.0 to 9.3), chondrocalcinosis in hip joints (6.5; 1.8 to 22.3), and an increased number of osteophytes in knee (6.9+/-1.2, n = 5 vs 2.4+/-0.1) joints at a later age (>65 years). CONCLUSIONS: The HFE H63D variant may explain, at least in part, the prevalence of arthralgia in multiple joints sites, chondrocalcinosis, and hand osteoarthritis in the general population.