High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS₄₀₀. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS₁₀₀ and Fab-PAS₂₀₀, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS₁₀₀ and Fab-PAS₂₀₀. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS₁₀₀ vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.     

Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide ⁶⁸Ga (T_1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule Z_HER2:S1 targeting HER2. Affibody molecules were labeled with ⁶⁸Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1, as well as that of their ¹¹¹In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for ⁶⁸Ga-DOTA-Z_HER2:S1 (17.9±0.7%IA/g) was significantly higher than for both ⁶⁸Ga-NODAGA-Z_HER2:S1 (16.13±0.67%IA/g) and ⁶⁸Ga-NOTA-Z_HER2:S1 (13±3%IA/g) at 2 h after injection. ⁶⁸Ga-NODAGA-Z_HER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both ⁶⁸Ga-DOTA-Z_HER2:S1 (28±4) and ⁶⁸Ga-NOTA-Z_HER2:S1 (42±11). The tumor-to-liver ratio was also higher for ⁶⁸Ga-NODAGA-Z_HER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for ⁶⁸Ga than for ¹¹¹In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with ⁶⁸Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for ⁶⁸Ga and ¹¹¹In.     

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

Background

The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.

Methods and Findings

Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm² versus 12.8±0.9 N/cm²; PBS versus TAT).

Conclusions

These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.     

Evaluation of 89Zr-Labeled Human Anti-CD147 Monoclonal Antibody as a Positron Emission Tomography Probe in a Mouse Model of Pancreatic Cancer

Introduction

Pancreatic cancer is an aggressive cancer and its prognosis remains poor. Therefore, additional effective therapy is required to augment and/or complement current therapy. CD147, high expression in pancreatic cancer, is involved in the metastatic process and is considered a good candidate for targeted therapy. CD147-specfic imaging could be useful for selection of appropriate patients. Therefore, we evaluated the potential of a fully human anti-CD147 monoclonal antibody 059-053 as a new positron emission tomography (PET) probe for pancreatic cancer.

Methods

CD147 expression was evaluated in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1) and a mouse cell line A4 as a negative control. Cell binding, competitive inhibition and internalization assays were conducted with ¹²⁵I-, ⁶⁷Ga-, or ⁸⁹Zr-labeled 059-053. In vivo biodistribution of ¹²⁵I- or ⁸⁹Zr-labeled 059-053 was conducted in mice bearing MIA Paca-2 and A4 tumors. PET imaging with [⁸⁹Zr]059-053 was conducted in subcutaneous and orthotopic tumor mouse models.

Results

Among four pancreatic cancer cell lines, MIA Paca-2 cells showed the highest expression of CD147, while A4 cells had no expression. Immunohistochemical staining showed that MIA Paca-2 xenografts also highly expressed CD147 in vivo. Radiolabeled 059-053 specifically bound to MIA Paca-2 cells with high affinity, but not to A4. [⁸⁹Zr]059-053 uptake in MIA Paca-2 tumors increased with time from 11.0±1.3% injected dose per gram (ID/g) at day 1 to 16.9±3.2% ID/g at day 6, while [¹²⁵I]059-053 uptake was relatively low and decreased with time, suggesting that 059-053 was internalized into tumor cells in vivo and ¹²⁵I was released from the cells. PET with [⁸⁹Zr]059-053 clearly visualized subcutaneous and orthotopic tumors.

Conclusion

[⁸⁹Zr]059-053 is a promising PET probe for imaging CD147 expression in pancreatic cancer and has the potential to select appropriate patients with CD147-expressing tumors who could gain benefit from anti-CD147 therapy.     

nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation,Apoptosis, and Angiogenesis

Background

Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).

Methods and Results

To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31⁺ cells (13.7±2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3±1.4%), CD31 (97.2±2.5%), and VE-cadherin (93.7±2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5×10⁶ hESC-ECs treated for 24 hours with nicotine (10⁻⁸ M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 µg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).

Conclusions

This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways.     

CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

Background

Although the etiology of idiopathic pulmonary fibrosis (IPF) remains perplexing, adaptive immune activation is evident among many afflicted patients. Repeated cycles of antigen-induced proliferation cause T-cells to lose surface expression of CD28, and we hypothesized this process might also occur in IPF.

Methodology/Principal Findings

Peripheral blood CD4 T-cells from 89 IPF patients were analyzed by flow cytometry and cytokine multiplex assays, and correlated with clinical events. In comparison to autologous CD4⁺CD28⁺cells, the unusual CD4⁺CD28^null lymphocytes seen in many IPF patients had discordant expressions of activation markers, more frequently produced cytotoxic mediators perforin (2.4±0.8% vs. 60.0±7.4%, p<0.0001) and granzyme B (4.5±2.8% vs.74.9±6.5%, p<0.0001), produced greater amounts of many pro-inflammatory cytokines, and less frequently expressed the regulatory T-cell marker FoxP3 (12.9±1.1% vs. 3.3±0.6% p<0.0001). Infiltration of CD4⁺CD28^null T-cells in IPF lungs was confirmed by confocal microscopy. Interval changes of CD28 expression among subjects who had replicate studies were correlated with conterminous changes of their forced vital capacities (r_s = 0.49, p = 0.012). Most importantly, one-year freedom from major adverse clinical events (either death or lung transplantation) was 56±6% among 78 IPF patients with CD4⁺CD28⁺/CD4_total≥82%, compared to 9±9% among those with more extensive CD28 down-regulation (CD4⁺CD28⁺/CD4_total<82%) (p = 0.0004). The odds ratio for major adverse events among those with the most extensive CD28 down-regulation was 13.0, with 95% confidence intervals 1.6-111.1.

Conclusions/Significance

Marked down-regulation of CD28 on circulating CD4 T-cells, a result of repeated antigen-driven proliferations, is associated with poor outcomes in IPF patients. The CD4⁺CD28^null cells of these patients have potentially enhanced pathogenic characteristics, including increased productions of cytotoxic mediators and pro-inflammatory cytokines. These findings show proliferative T-cell responses to antigen(s) resulting in CD28 down-regulation are associated with progression and manifestations of IPF, and suggest assays of circulating CD4 T-cells may identify patients at greatest risk for clinical deterioration.     

PLGA nanoparticles for peptide receptor radionuclide therapy of neuroendocrine tumors: a novel approach towards reduction of renal radiation dose

Background

Peptide receptor radionuclide therapy (PRRT), employed for treatment of neuroendocrine tumors (NETs) is based on over-expression of Somatostatin Receptors (SSTRs) on NETs. It is, however, limited by high uptake and retention of radiolabeled peptide in kidneys resulting in unnecessary radiation exposure thus causing nephrotoxicity. Employing a nanocarrier to deliver PRRT drugs specifically to the tumor can reduce the associated nephrotoxicity. Based on this, ¹⁷⁷Lu-DOTATATE loaded PLGA nanoparticles (NPs) were formulated in the present study, as a potential therapeutic model for NETs.

Methodology and Findings

DOTATATE was labeled with Lutetium-177 (¹⁷⁷Lu) (labeling efficiency 98%; R_f∼0.8). Polyethylene Glycol (PEG) coated ¹⁷⁷Lu-DOTATATE-PLGA NPs (50∶50 and 75∶25) formulated, were spherical with mean size of 304.5±80.8 and 733.4±101.3 nm (uncoated) and 303.8±67.2 and 494.3±71.8 nm (coated) for PLGA(50∶50) and PLGA(75∶25) respectively. Encapsulation efficiency (EE) and In-vitro release kinetics for uncoated and coated NPs of PLGA (50∶50 & 75∶25) were assessed and compared. Mean EE was 77.375±4.98% & 67.885±5.12% (uncoated) and 65.385±5.67% & 58.495±5.35% (coated). NPs showed initial burst release between 16.64–21.65% with total 42.83–44.79% over 21days. The release increased with coating to 20.4–23.95% initially and 60.97–69.12% over 21days. In-vivo studies were done in rats injected with ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE-NP (uncoated and PEG-coated) by imaging and organ counting after sacrificing rats at different time points over 24 hr post-injection. With ¹⁷⁷Lu-DOTATATE, renal uptake of 37.89±10.2%ID/g was observed, which reduced to 4.6±1.97% and 5.27±1.66%ID/g with uncoated and coated ¹⁷⁷Lu-DOTATATE-NP. The high liver uptake with uncoated ¹⁷⁷Lu-DOTATATE-NP (13.68±3.08% ID/g), reduced to 7.20±2.04%ID/g (p = 0.02) with PEG coating.

Conclusion

PLGA NPs were easily formulated and modified for desired release properties. PLGA 50∶50 NPs were a more suitable delivery vehicle for ¹⁷⁷Lu-DOTATATE than PLGA 75∶25 because of higher EE and slower release rate. Reduced renal retention of ¹⁷⁷Lu-DOTATATE and reduced opsonisation strongly advocate the potential of ¹⁷⁷Lu-DOTATATE-PLGA-PEG NPs to reduce radiation dose in PRRT.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to Phase 1 clinical studies

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake. Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Metabolic and Behavioral Compensations in Response to Caloric Restriction: Implications for the Maintenance of Weight Loss

Background

Metabolic and behavioral adaptations to caloric restriction (CR) in free-living conditions have not yet been objectively measured.

Methodology and Principal Findings

Forty-eight (36.8±1.0 y), overweight (BMI 27.8±0.7 kg/m²) participants were randomized to four groups for 6-months; Control: energy intake at 100% of energy requirements; CR: 25% calorie restriction; CR+EX: 12.5% CR plus 12.5% increase in energy expenditure by structured exercise; LCD: low calorie diet (890 kcal/d) until 15% weight reduction followed by weight maintenance. Body composition (DXA) and total daily energy expenditure (TDEE) over 14-days by doubly labeled water (DLW) and activity related energy activity (AREE) were measured after 3 (M3) and 6 (M6) months of intervention. Weight changes at M6 were −1.0±1.1% (Control), −10.4±0.9% (CR), −10.0±0.8% (CR+EX) and −13.9±0.8% (LCD). At M3, absolute TDEE was significantly reduced in CR (−454±76 kcal/d) and LCD (−633±66 kcal/d) but not in CR+EX or controls. At M6 the reduction in TDEE remained lower than baseline in CR (−316±118 kcal/d) and LCD (−389±124 kcal/d) but reached significance only when CR and LCD were combined (−351±83 kcal/d). In response to caloric restriction (CR/LCD combined), TDEE adjusted for body composition, was significantly lower by −431±51 and −240±83 kcal/d at M3 and M6, respectively, indicating a metabolic adaptation. Likewise, physical activity (TDEE adjusted for sleeping metabolic rate) was significantly reduced from baseline at both time points. For control and CR+EX, adjusted TDEE (body composition or sleeping metabolic rate) was not changed at either M3 or M6.

Conclusions

For the first time we show that in free-living conditions, CR results in a metabolic adaptation and a behavioral adaptation with decreased physical activity levels. These data also suggest potential mechanisms by which CR causes large inter-individual variability in the rates of weight loss and how exercise may influence weight loss and weight loss maintenance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00099151","term_id":"NCT00099151"}}NCT00099151     

DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N′-N′′-N′′′-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)₁-decalysine, (DOTA)₃-decalysine or (DOTA)₅-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with ¹⁷⁷Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]₂, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)₃-decalysine]₂ and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)₅-decalysine)]₂. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]₂ (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)₃ versus 11.7±1.4% ID/g (DOTA)₅, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)₃ versus 5.8±0.7 (DOTA)₅, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)₅-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

β-Lactam Effects on Mixed Cultures of Common Respiratory Isolates as an Approach to Treatment Effects on Nasopharyngeal Bacterial Population Dynamics

Background

Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae are bacteria present in the nasopharynx as part of normal flora. The ecological equilibrium in the nasopharynx can be disrupted by the presence of antibiotics.

Methodology/Principal Findings

A computerized two-compartment pharmacodynamic model was used to explore β-lactam effects on the evolution over time of a bacterial load containing common pharyngeal isolates by simulating free serum concentrations obtained with amoxicillin (AMX) 875 mg tid, amoxicillin/clavulanic acid (AMC) 875/125 mg tid and cefditoren (CDN) 400 mg bid regimens over 24 h. Strains and MICs (µg/ml) of AMX, AMC and CDN were: S. pyogenes (0.03, 0.03 and 0.015), S. pneumoniae (2, 2 and 0.25), a β-lactamase positive H. influenzae (BL⁺; >16, 2 and 0.06) and a β-lactamase positive AMC-resistant H. influenzae (BLPACR, >16, 8 and 0.06). Mixture of identical 1∶1∶1∶1 volumes of each bacterial suspension were prepared yielding an inocula of ≈4×10⁶ cfu/ml. Antibiotic concentrations were measured both in bacterial and in bacteria-free antibiotic simulations. β-lactamase production decreased AMX concentrations and fT_>MIC against S. pneumoniae (from 43.2% to 17.7%) or S. pyogenes (from 99.9% to 24.9%), and eradication was precluded. The presence of clavulanic acid countered this effect of co-pathogenicity, and S. pyogenes (but not BL⁺ and S. pneumoniae) was eradicated. Resistance of CDN to TEM β-lactamase avoided this co-pathogenicity effect, and CDN eradicated S. pyogenes and H. influenzae strains (fT_>MIC >58%), and reduced in 94% S. pneumoniae counts (fT_>MIC ≈25%).

Conclusions/Significance

Co-pathogenicity seems to be gradual since clavulanic acid countered this effect for strains very susceptible to AMX as S. pyogenes but not for strains with AMX MIC values in the limit of susceptibility as S. pneumoniae. There is a potential therapeutic advantage for β-lactamase resistant cephalosporins with high activity against streptococci.     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

Measurement of Pulmonary Flow Reserve and Pulmonary Index of Microcirculatory Resistance for Detection of Pulmonary Microvascular Obstruction

Background

The pulmonary microcirculation is the chief regulatory site for resistance in the pulmonary circuit. Despite pulmonary microvascular dysfunction being implicated in the pathogenesis of several pulmonary vascular conditions, there are currently no techniques for the specific assessment of pulmonary microvascular integrity in humans. Peak hyperemic flow assessment using thermodilution-derived mean transit-time (T_mn) facilitate accurate coronary microcirculatory evaluation, but remain unvalidated in the lung circulation. Using a high primate model, we aimed to explore the use of T_mn as a surrogate of pulmonary blood flow for the purpose of measuring the novel indices Pulmonary Flow Reserve [PFR = (maximum hyperemic)/(basal flow)] and Pulmonary Index of Microcirculatory Resistance [PIMR = (maximum hyperemic distal pulmonary artery pressure)×(maximum hyperemic T_mn)]. Ultimately, we aimed to investigate the effect of progressive pulmonary microvascular obstruction on PFR and PIMR.

Methods and Results

Temperature- and pressure-sensor guidewires (TPSG) were placed in segmental pulmonary arteries (SPA) of 13 baboons and intravascular temperature measured. T_mn and hemodynamics were recorded at rest and following intra-SPA administration of the vasodilator agents adenosine (10–400 µg/kg/min) and papaverine (3–24 mg). Temperature did not vary with intra-SPA sensor position (0.010±0.009 v 0.010±0.009°C; distal v proximal; p = 0.1), supporting T_mn use in lung for the purpose of hemodynamic indices derivation. Adenosine (to 200 µg/kg/min) & papaverine (to 24 mg) induced dose-dependent flow augmentations (40±7% & 35±13% T_mn reductions v baseline, respectively; p<0.0001). PFR and PIMR were then calculated before and after progressive administration of ceramic microspheres into the SPA. Cumulative microsphere doses progressively reduced PFR (1.41±0.06, 1.26±0.19, 1.17±0.07 & 1.01±0.03; for 0, 10⁴, 10⁵ & 10⁶ microspheres; p = 0.009) and increased PIMR (5.7±0.6, 6.3±1.0, 6.8±0.6 & 7.6±0.6 mmHg.sec; p = 0.0048).

Conclusions

Thermodilution-derived mean transit time can be accurately and reproducibly measured in the pulmonary circulation using TPSG. Mean transit time-derived PFR and PIMR can be assessed using a TPSG and adenosine or papaverine as hyperemic agents. These novel indices detect progressive pulmonary microvascular obstruction and thus have with a potential role for pulmonary microcirculatory assessment in humans.     

An Experimental Study on 131I-CHIBA-1001: A Radioligand for α7 Nicotinic Acetylcholine Receptors

Objective

The α7 nicotinic acetylcholine receptors (nAChRs) play a vital role in the pathophysiology of neuropsychiatric diseases such as Alzheimer’s disease and depression. However, there is currently no suitable positron emission tomography (PET) or Single-Photon Emission Computed Tomography (SPECT) radioligands for imaging α7 nAChRs in brain. Here our aim is to radiosynthesize a novel SPECT radioligand ¹³¹I-CHIBA-1001 for whole body biodistribution study and in vivo imaging of α7 nAChRs in brain.

Method

¹³¹I-CHIBA-1001 was radiosynthesized by chloramine-T method. Different conditions of reaction time and temperature were tested to get a better radiolabeling yield. Radiolabeling yield and radiochemical purities of ¹³¹I-CHIBA-1001 were analyzed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) system. Whole body biodistribution study was performed at different time points post injection of ¹³¹I-CHIBA-1001 in KM mice. Monkey subject was used for in vivo SPECT imaging in brain.

Result

The radiolabeling yield of ¹³¹I-CHIBA-1001 reached 96% within 1.5∼2.0 h at 90∼95°C. The radiochemical purity reached more than 99% after HPLC purification. ¹³¹I-CHIBA-1001 was highly stable in saline and fresh human serum in room temperature and 37°C separately. The biodistribution data of brain at 15, 30, and 60 min were 11.05±1.04%ID/g, 8.8±0.04%ID/g and 6.28±1.13%ID/g, respectively. In experimental SPECT imaging, the distribution of radioactivity in the brain regions was paralleled with the distribution of α7 nAChRs in the monkey brain. Moreover, in the blocking SPECT imaging study, the selective α7 nAChR agonist SSR180711 blocked the radioactive uptake in the brain successfully.

Conclusion

The CHIBA-1001 can be successfully radiolabeled with ¹³¹I using the chloramine-T method. ¹³¹I-CHIBA-1001 can successfully accumulate in the monkey brain and image the α7 acetylcholine receptors. ¹³¹I-CHIBA-1001 can be a candidate for imagingα7 acetylcholine receptors, which will be of great value for the diagnosis and treatment of mental diseases.     

Risk Factors for Death among Children Less than 5 Years Old Hospitalized with Diarrhea in Rural Western Kenya, 2005–2007: A Cohort Study

Background

Diarrhea is a leading cause of childhood morbidity and mortality in sub-Saharan Africa. Data on risk factors for mortality are limited. We conducted hospital-based surveillance to characterize the etiology of diarrhea and identify risk factors for death among children hospitalized with diarrhea in rural western Kenya.

Methods and Findings

We enrolled all children <5 years old, hospitalized with diarrhea (≥3 loose stools in 24 hours) at two district hospitals in Nyanza Province, western Kenya. Clinical and demographic information was collected. Stool specimens were tested for bacterial and viral pathogens. Bivariate and multivariable logistic regression analyses were carried out to identify risk factors for death. From May 23, 2005 to May 22, 2007, 1,146 children <5 years old were enrolled; 107 (9%) children died during hospitalization. Nontyphoidal Salmonella were identified in 10% (118), Campylobacter in 5% (57), and Shigella in 4% (42) of 1,137 stool samples; rotavirus was detected in 19% (196) of 1,021 stool samples. Among stools from children who died, nontyphoidal Salmonella were detected in 22%, Shigella in 11%, rotavirus in 9%, Campylobacter in 5%, and S. Typhi in <1%. In multivariable analysis, infants who died were more likely to have nontyphoidal Salmonella (adjusted odds ratio [aOR] = 6·8; 95% CI 3·1–14·9), and children <5 years to have Shigella (aOR = 5·5; 95% CI 2·2–14·0) identified than children who survived. Children who died were less likely to be infected with rotavirus (OR = 0·4; 95% CI 0·2–0·8). Further risk factors for death included being malnourished (aOR = 4·2; 95% CI 2·1–8·7); having oral thrush on physical exam (aOR = 2·3; 95% CI 1·4–3·8); having previously sought care at a hospital for the illness (aOR = 2·2; 95% CI 1·2–3·8); and being dehydrated as diagnosed at discharge/death (aOR = 2·5; 95% CI 1·5–4·1). A clinical diagnosis of malaria, and malaria parasites seen on blood smear, were not associated with increased risk of death. This study only captured in-hospital childhood deaths, and likely missed a substantial number of additional deaths that occurred at home.

Conclusion

Nontyphoidal Salmonella and Shigella are associated with mortality among rural Kenyan children with diarrhea who access a hospital. Improved prevention and treatment of diarrheal disease is necessary. Enhanced surveillance and simplified laboratory diagnostics in Africa may assist clinicians in appropriately treating potentially fatal diarrheal illness. Please see later in the article for the Editors'' Summary     

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background

High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated.

Methodology/Principal Findings

To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-¹³C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized.

Conclusions/Significance

High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.     

Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

Background

Glycerol monolaurate (GML), a 12 carbon fatty acid monoester, inhibits Staphylococcus aureus growth and exotoxin production, but is degraded by S. aureus lipase. Therefore, dodecylglycerol (DDG), a 12 carbon fatty acid monoether, was compared in vitro and in vivo to GML for its effects on S. aureus growth, exotoxin production, and stability.

Methodology/Principal Findings

Antimicrobial effects of GML and DDG (0 to 500 µg/ml) on 54 clinical isolates of S. aureus, including pulsed-field gel electrophoresis (PFGE) types USA200, USA300, and USA400, were determined in vitro. A rabbit Wiffle ball infection model assessed GML and DDG (1 mg/ml instilled into the Wiffle ball every other day) effects on S. aureus (MN8) growth (inoculum 3×10⁸ CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-α (TNF-α) concentrations and mortality over 7 days. DDG (50 and 100 µg/ml) inhibited S. aureus growth in vitro more effectively than GML (p<0.01) and was stable to lipase degradation. Unlike GML, DDG inhibition of TSST-1 was dependent on S. aureus growth. GML-treated (4 of 5; 80%) and DDG-treated rabbits (2 of 5; 40%) survived after 7 days. Control rabbits (5 of 5; 100%) succumbed by day 4. GML suppressed TNF-α at the infection site on day 7; however, DDG did not (<10 ng/ml versus 80 ng/ml, respectively).

Conclusions/Significance

These data suggest that DDG was stable to S. aureus lipase and inhibited S. aureus growth at lower concentrations than GML in vitro. However, in vivo GML was more effective than DDG by reducing mortality, and suppressing TNF-α, S. aureus growth and exotoxin production, which may reduce toxic shock syndrome. GML is proposed as a more effective anti-staphylococcal topical anti-infective candidate than DDG, despite its potential degradation by S. aureus lipase.     

Detection of Pancreatic Carcinomas by Imaging Lactose-Binding Protein Expression in Peritumoral Pancreas Using [18F]Fluoroethyl-Deoxylactose PET/CT

Background

Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[¹⁸F]fluoroethyl-D-glucopyranose ([¹⁸F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [¹⁸F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells.

Methodology/Principal Findings

Dynamic PET/CT imaging demonstrated rapid accumulation of [¹⁸F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [¹⁸F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [¹⁸F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm.

Conclusion/Significance

We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [¹⁸F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [¹⁸F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.