Incidence and risk factors for herpes zoster following heart transplantation.

BACKGROUND: Data on the incidence, timing, and risk factors for herpes zoster (HZ) in heart transplant (HT) recipients are limited. METHODS: We determined HZ incidence rates and actuarial estimates of time to first HZ episode in 314 HT recipients at our institution from 1995 to 2010. We developed Cox models to assess potential risk factors for HZ in HT. RESULTS: Median age at HT was 54 (range, 17-71) years; 237 (76%) were male. There were 60 episodes of HZ in 51 patients, with an overall incidence rate of 31.6 cases (95% confidence interval [CI], 23.5-41.6)/1000 person-years. Although most cases occurred during the first post-HT year, cumulative HZ incidence was 0.078 at 1, 0.15 at 5, and 0.20 at 10 years. Many patients had substantial HZ morbidity, including 14% with HZ ophthalmicus and 45% with post-herpetic neuralgia. Adjusting for age, gender, and acute cellular rejection episodes, exposure to mycophenolate mofetil (MMF) was an independent risk factor for HZ (adjusted hazard ratio [HR] 2.18; 95% CI, 1.20-3.96; P = 0.01), while ganciclovir-based cytomegalovirus prophylaxis reduced HZ risk (adjusted HR 0.09; 95% CI, 0.01-0.71; P = 0.02). Although age and female gender increased HZ risk, the magnitude of their effect was not statistically significant in Cox models. CONCLUSIONS: HZ is common and morbid after HT, particularly with MMF exposure. Ganciclovir prophylaxis is effective in reducing the short-term risk of HZ, but the steady incidence of cases for years post HT makes long-term HZ prevention challenging. Augmenting varicella zoster virus immunity post HT with vaccines warrants further exploration.

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components.

We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the resulting fluorescent nuclei was used to define the relationship between cytoplasmic release of nucleic acids and gene expression. Endothelial cells were stained after i.v. administration, and epithelial cells were stained after i.t. administration. The delivery of nucleic acids was also more homogeneous with i.v. administration of lipoplex than with i.t. administration. After i.t. administration, it was notable that high concentrations of fluorescent nuclei correlated with low GFP expression. This suggested that toxicity was associated with high local concentrations of cationic lipoplexes. The ratio of GFP-expressing cells to fluorescent nuclei indicated that capillary endothelial cells were more efficient in gene expression per delivery event than were pulmonary epithelial cells. Thus, the greater gene expression efficiency of i.v. administered lipoplexes was due not only to the initial distribution but also to the greater efficiency of the vascular endothelial cells to appropriately traffic and express the foreign gene.

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.

Simultaneous mucinous cystadenoma of ovary and mucinous cystadenocarcinoma of pancreas.

Mucinous cystic tumors were discovered synchronously in the tail of the pancreas and in the right ovary of an adult female. Both tumors were amenable to surgical resection. The pancreatic tumor was a noninvasive mucinous cystadenocarcinoma and the ovarian tumor was a mucinous cystadenoma. We feel these tumors represent two primaries, an uncommon occurrence, and not a single primary tumor with metastasis.

Lipoplex-mediated gene delivery to the lung occurs within 60 minutes of intravenous administration.

We have defined the critical time period for gene delivery in the lung after intravenous administration of cationic lipoplex. We accomplished this through the displacement of intravenously injected cationic lipoplexes from the lungs by the subsequent administration of anionic liposomes. When reporter gene-bearing lipoplexes were injected intravenously and followed by anionic liposomes 5 min later, reporter gene expression was reduced up to 400-fold compared with animals into which lipoplex alone was administered. Administration of anionic liposomes 60-90 min after lipoplex injection yielded no significant reduction in lung transfection. When lipoplexes were disrupted 5 min after administration, the pulmonary distribution of the cationic lipid and DNA components was reduced by 80%. Lipids subsequently accumulated primarily in the liver, while the plasmid DNA constituent distributed into the blood and liver. As the interval between lipoplex and anionic liposome injection increased, the degree of lipoplex displacement from the lung decreased to such a point that, 60 min after lipoplex injection, the anionic liposome injection did not displace significant quantities of the lipoplex. We conclude that cationic lipid-DNA complexes can be disrupted in vivo via the administration of anionic liposomes; moreover, we have employed this phenomenon to demonstrate that transfectionally active DNA is taken up within 60 min of systemic lipoplex administration.

Simulated lung transfection by nebulization of liposome cDNA complexes using a cascade impactor seeded with 2-CFSME0-cells.

The aim of this study was to devise a simulation aerosol system for quasirealistic gene transfection that could eventually be used to study the characteristics of aerosol delivery, stability, delivery efficiency, and expression efficacy of gene products. It consisted of (1) a PARI aerosol generator and PARI LL jet; (2) an Andersen cascade impactor with a calibrated vacuum pump, fitted with a glass "throat," nebulizer in which stages were seeded with pulmonary cells of interest (e.g., 2-CFSME0-); and (3) a hot room set to 37 degrees C and approximately 70% relative humidity. Cell viability remained at 95% to 99%. A prostaglandin G/H synthase (PGH)- and a human alpha 1-antitrypsin (AAT)-expressing plasmid, respectively, driven by a cytomegalovirus promoter (pCMV4-PGH, pCMV4-AAT) and a heat-insensitive placental alkaline phosphatase (PAP)-expressing plasmid driven by a Rous sarcoma virus promoter (pRSV-PAP) were employed; cationic liposomes consisted of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol/DOPE (DC-Chol/ DOPE). The fluorescent dye Toto-1 was used to visualize aerosol distribution and to monitor cellular uptake. Alternatively, pCMV4-PGH deposited onto impactor stages covered with nitrocellulose membranes was hybridized with random primer-32P-radiolabeled pCMV4-PGH and autoradiographed. The mass median aerodynamic diameter (MMAD) of the plasmid, liposomes, and liposome-plasmid complexes and their effect on the mass output were monitored. A majority of gene product was delivered to stages 1 through 5, corresponding to an area ranging from the pharynx to the terminal bronchi, excluding the alveolar space. A corresponding, although very low, transfection of cells with pRSV-PAP was found, with the majority of transfected cells on stages 4 and 5. The MMAD was significantly affected by the presence of the DNA constructs alone or by DNA constructs complexed with cationic liposomes; the control phosphate buffered saline (PBS) MMAD of 2.3 microns increased to 3.5 microns for DC-Chol liposomes and 4.5 microns for the DC-Chol/PGH complex; DOTMA-based liposomes and liposome DNA complexes precipitated during aerosolization. Mass output was reduced for cationic liposomes from 0.61 g/min (PBS control) to 0.35 g/min. Large plasmid (pRSV-PAP, 10.1 kb) was more rapidly degraded by aerosolization than smaller plasmid (pCMV4-AAT, 6.2 kb) although complexation with cationic liposomes provided some protection in both cases.

Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.

The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.

Influence of phospholipid composition on antibody responses to liposome encapsulated protein and peptide antigens.

The effect of phospholipid composition on mouse IgG antibody responses to liposomal bovine serum albumin (BSA), murine monoclonal antibody GK1.5 (anti-CD4) or a 21 amino acid peptide from the second conserved domain of HIV gp120 after s.c. administration, and on the IgA, IgE, and IgG antibody response to liposomal Shistosoma mansoni glutathione-S-transferase (Sm28GST) after oral administration, was determined. Antibody responses were compared with alum-adsorbed and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-antigen mixtures. For the s.c. route, dipalmitoylphosphatidylcholine (DPPC)/dimyristoylphosphatidylglycerol (DMPG) liposomes induced 54-60% IgG1 and 35-44% IgG(2a+2b). DPPC/dipalmitoylphosphatidylethanolamine (DPPE) liposomes induced 73-78% IgG1 and 15-25% IgG(2a+2b). DPPC/ phosphatidylserine (PS) liposomes induced 86-89% IgG1 and 8-12% IgG(2a+2b). Alum and MDP induced 79-91% IgG1 and 4-17% IgG(2a+2b). The rank order of adjuvanticity for induction of IgG antibody was DPPC/DMPGDPPC/PE > > alum > > MDPDPPC/PS for all three antigens. DPPC/DMPG liposomes were the only effective adjuvant for the induction of secretory IgA and circulatory IgE and IgG antibodies against Sm28GST after oral administration. The failure of liposome-antigen mixtures to elicit an antibody response showed that liposomal incorporation of the antigens was obligatory for adjuvant activity. These results demonstrate that the correlation between phospholipid composition and adjuvanticity is independent of liposome charge, antigen, or route of administration.

Modulation of murine macrophage nitric oxide synthesis by liposomal phospholipids: correlation with liposome immune adjuvant activity.

The influence of alum and liposomal phospholipids on interferon-gamma-(IFN-gamma), IFN-gamma/N-acetylmuramyl-L-alanyl-D-isoglutamine- (MDP) or IFN-gamma/tumor necrosis factor-alpha- (IFN-gamma/TNF-alpha) induced macrophage nitric oxide (NO) synthesis has been investigated. IFN-gamma induced NO synthesis in a dose-dependent manner. TNF-alpha and MDP did not induce NO synthesis, but interacted synergistically with sub-optimal doses of IFN-gamma. Alum strongly inhibited IFN-gamma-induced NO synthesis (ID50 25 microgram/ml). Liposomes composed of dipalmitoylphosphatidylcholine (DPPC) had no effect on IFN-gamma-induced NO synthesis. IFN-gamma-induced NO synthesis was stimulated by DPPC/dimyristoylphosphatidylglycerol (DMPG) liposomes (9:1 mol ratio, ED50 45 nmol phospholipid/ml), and inhibited by DPPC/dipalmitoylphosphatidylethanolamine (DPPE) liposomes (9:1 mol ratio, ID50 > 500 nmol phospholipid/ml), and DPPC/phosphatidylserine (PS) liposomes (7:3 mol ratio, ID50 150 nmol phospholipid/ml). Alum, DPPC/PE and DPPC/PS liposomes also inhibited IFN-gamma/MDP- and IFN-gamma/TNF-alpha-induced NO synthesis. Neither alum or the liposome preparations had significant toxicity towards macrophages in vitro at concentrations that induced maximal inhibition or stimulation of IFN-gamma-induced NO synthesis. Immunization of mice with alum-adsorbed and liposome-incorporated bovine serum albumin (BSA) demonstrated that enhancement or reduction of both IgG antibody and the proportion of IgG2a/IgG2b was correlated with stimulation or inhibition of IFN-gamma-induced NO synthesis.

Immunoliposome targeting to murine CD4+ leucocytes is dependent on immune status.

Immunoliposomes were prepared from dipalmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol, biotinylated PE, and surface-linked avidin-biotinylated GK1.5 monoclonal rat (anti-mouse CD4) IgG or F(ab)2. Anti-CD4 immunoliposomes bound quantitatively to CD4+ PBMC's in vitro. Intravenous injection of mice with 1 mumol anti-CD4 immunoliposomes resulted in targeting to a significant proportion (> 75%) of CD4+ PBMCs. Repeated administration of anti-CD4 immunoliposomes induced significant levels of anti-GK 1.5 IgG Ab, with concomitant reductions in immunoliposome plasma t1/2 and targeting efficacy, and increased volumes of distribution. The immunogenicity of the immunoliposome was enhanced if GK1.5 F(ab)2 instead of IgG was used as the targeting ligand. Lipophilic poly(ethylene glycol) derivatives incorporated into the immunoliposomes did not affect targeting efficiency but significantly enhanced immunogenicity. These results demonstrate that IgG or F(ab)2 immunoliposomes are highly immunogenic and that targeting in vivo is conditional on the immune status of the host.

The importance of dose scheduling with mitoxantrone, 5-fluorouracil and leucovorin in metastatic breast cancer.

We have studied a mitoxantrone, 5-fluorouracil (5-FU) and leucovorin chemotherapy regimen in metastatic breast cancer. 8 patients received mitoxantrone 10 mg/m2 on day 1, leucovorin 200 mg/m2 and 5-FU 300 mg/m2 on days 1-5 by intravenous bolus every 28 days in a pilot study. Grades 3-4 granulocytopenia followed 55% of the courses, with 2 patients admitted for febrile neutropenia. Only a 29% objective response rate was seen in a subsequent phase II trial using reduced mitoxantrone doses. Comparison with other trials suggested that 5-day bolus 5-FU administration adversely affects the combination's therapeutic index.

Protein kinase C as a modulator of response amplification in vascular smooth muscle.

The amplification of alpha-adrenoceptor-mediated vasoconstriction by angiotensin II was studied in femoral artery rings from rabbits. Threshold concentrations of angiotensin II (0.1 nM) increased the maximal response to clonidine to 139 +/- 8% of control and produced a 3.2-fold increase in sensitivity. These effects of angiotensin II were reversed when tissues were pretreated with staurosporine (50 nM), an inhibitor of protein kinase C. The amplification of the alpha-adrenoceptor-mediated vasoconstrictor effects of thrombin and norepinephrine by angiotensin II were also reversed by pretreatment with staurosporine. Angiotensin II induced a response amplification in vascular smooth muscle known to be a nonspecific phenomenon, implying postreceptor interaction at intracellular transducer systems. Our findings suggest that upon activation of protein kinase C by angiotensin II, arterial responses to alpha-adrenoceptor agonists are amplified. This provides for nonspecific changes in vascular sensitivity by tonic alterations in postsynaptic modulation by enzyme systems known to regulate Ca2(+)-dependent phenomena, e.g. those related to vascular excitation-contraction mechanisms.