The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of any symmetry.

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.

Suppression of immune responses by regulatory T cells (Tregs) is thought to limit late stages of pathogen-specific immunity as a means of minimizing associated tissue damage. We examined a role for Tregs during mucosal herpes simplex virus infection in mice, and observed an accelerated fatal infection with increased viral loads in the mucosa and central nervous system after ablation of Tregs. Although augmented interferon production was detected in the draining lymph nodes (dLNs) in Treg-deprived mice, it was profoundly reduced at the infection site. This was associated with a delay in the arrival of natural killer cells, dendritic cells, and T cells to the site of infection and a sharp increase in proinflammatory chemokine levels in the dLNs. Our results suggest that Tregs facilitate early protective responses to local viral infection by allowing a timely entry of immune cells into infected tissue.

The current study explored the predictive validity of the Global Deficit Score (GDS) approach in summarizing neuropsychological (NP) test results, and specifically in detecting HIV-associated cognitive impairment. A comprehensive NP test battery was administered to 88 HIV+ subjects and 61 healthy HIV- controls comparable for age, education, and ethnicity. Demographically corrected test data were converted to a GDS, which simulates clinicians' ratings by quantifying the number and degree of impaired performances throughout the test battery while attaching relatively less significance to superior performances and/or those within normal limits. Our results indicated that the GDS approach effectively discriminated HIV+ and normal control groups, and accurately classified HIV+ individuals with NP impairment based on the "gold standard" clinical rating approach. Consistent with previous studies using different subject samples and different NP test batteries, the GDS cutpoint of>>or=0.50 yielded optimal balance between sensitivity and specificity in classifying NP impairment, thus supporting the generalizability of the method. Moreover, the ability of the GDS to predict NP impairment across several cutpoints was quite strong, with positive predictive power values ranging from 0.71 to 1.00. These findings support the validity of the GDS as a clinically useful way of summarizing results on NP testing.

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

Broth-culture filtrates of Campylobacter pylori induced non-lethal cytopathic effects in vitro in 7 of 9 mammalian cell lines tested. Transmission electronmicroscopy revealed that the response consisted of intracellular vacuolisation. Intestine 407 cells were among the most responsive and were used for routine assay. About 55% of isolates of C. pylori tested, originating from four geographic regions worldwide, produced cytotoxic activity. The activity was neutralisable by specific antisera to broth-culture filtrates or to sonicated bacteria but not by antisera to other bacterial preparations. Cytotoxic activity was heat-labile (70 degrees C for 30 min), was protease-sensitive and ammonium-sulphate precipitable. It did not pass through an ultrafiltration membrane with a nominal mol.-wt limit of 100 X 10(3). It was concluded that C. pylori can produce a factor that alters cultured cells in vitro. The relevance of this factor to the pathogenesis of gastritis associated with C. pylori remains to be determined.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

The domestication of all major crop plants occurred during a brief period in human history about 10,000 years ago. During this time, ancient agriculturalists selected seed of preferred forms and culled out seed of undesirable types to produce each subsequent generation. Consequently, favoured alleles at genes controlling traits of interest increased in frequency, ultimately reaching fixation. When selection is strong, domestication has the potential to drastically reduce genetic diversity in a crop. To understand the impact of selection during maize domestication, we examined nucleotide polymorphism in teosinte branched1, a gene involved in maize evolution. Here we show that the effects of selection were limited to the gene's regulatory region and cannot be detected in the protein-coding region. Although selection was apparently strong, high rates of recombination and a prolonged domestication period probably limited its effects. Our results help to explain why maize is such a variable crop. They also suggest that maize domestication required hundreds of years, and confirm previous evidence that maize was domesticated from Balsas teosinte of southwestern Mexico.

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Excitatory amino-acid transporters (EAATs) in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and may limit the activation of glutamate receptors. Here we report the cloning of a novel human aspartate/glutamate transporter, EAAT4, which is expressed predominantly in the cerebellum. The transport activity encoded by EAAT4 has high apparent affinity for L-aspartate and L-glutamate, and has a pharmacological profile consistent with previously described cerebellar transport activities. In Xenopus oocytes expressing EAAT4, L-aspartate and L-glutamate elicited a current predominantly carried by chloride ions. This chloride conductance was not blocked by components that block endogenous oocyte chloride channels. Thus EAAT4 combines the re-uptake of neurotransmitter with a mechanism for increasing chloride permeability, both of which could regulate excitatory neurotransmission.

Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1(-/-)) mice. CD4(+) and CD8(+) T cell responses were markedly enhanced in PD-L1(-/-) mice compared with wild-type mice in vitro and in vivo. PD-L1(-/-) dendritic cells stimulated greater wild-type CD4(+) T cell responses than wild-type dendritic cells, and PD-L1(-/-) CD4(+) T cells produced more cytokines than wild-type CD4(+) T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8(+) T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8(+) T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4(+) T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1(-/-) recipients led to exacerbated disease. Disease was even more severe in PD-L1(-/-) recipients of PD-L1(-/-) T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.

We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.

During photosynthesis, plants must control the utilization of light energy in order to avoid photoinhibition. We isolated an Arabidopsis mutant, pgr5 (proton gradient regulation), in which downregulation of photosystem II photochemistry in response to intense light was impaired. PGR5 encodes a novel thylakoid membrane protein that is involved in the transfer of electrons from ferredoxin to plastoquinone. This alternative electron transfer pathway, whose molecular identity has long been unclear, is known to function in vivo in cyclic electron flow around photosystem I. We propose that the PGR5 pathway contributes to the generation of a Delta(pH) that induces thermal dissipation when Calvin cycle activity is reduced. Under these conditions, the PGR5 pathway also functions to limit the overreduction of the acceptor side of photosystem I, thus preventing photosystem I photoinhibition.

OBJECTIVE

To prospectively compare the agreement of left ventricular volumes and ejection fraction by M-mode echocardiography (echo), 2D echo, radionuclide ventriculography and cardiovascular magnetic resonance performed in patients with chronic stable heart failure. It is important to know whether the results of each technique are interchangable, and thereby how the results of large studies in heart failure utilizing one technique can be applied using another. Some studies have compared cardiovascular magnetic resonance with echo or radionuclude ventriculography but few contain patients with heart failure and none have compared these techniques with the current fast breath-hold acquisition cardiovascular magnetic resonance.

RESULTS

Fifty two patients with chronic stable heart failure taking part in the CHRISTMAS Study, underwent M-mode echo, 2D echo, radionuclude ventriculography and cardiovascular magnetic resonance within 4 weeks. The scans were analysed independently in blinded fashion by a single investigator at three core laboratories. Of the echocardiograms, 86% had sufficient image quality to obtain left ventricular ejection fraction by M-mode method, but only 69% by 2D Simpson's biplane analysis. All 52 patients tolerated the radionuclude ventriculography and cardiovascular magnetic resonance, and all these scans were analysable. The mean left ventricular ejection fraction by M-mode cube method was 39+/-16% and 29+/-15% by Teichholz M-mode method. The mean left ventricular ejection fraction by 2D echo Simpson's biplane was 31+/-10%, by radionuclude ventriculography was 24+/-9% and by cardiovascular magnetic resonance was 30+/-11. All the mean left ventricular ejection fractions by each technique were significantly different from all other techniques (P<0.001), except for cardiovascular magnetic resonance ejection fraction and 2D echo ejection fraction by Simpson's rule (P=0.23). The Bland-Altman limits of agreement encompassing four standard deviations was widest for both cardiovascular magnetic resonance vs cube M-mode echo and cardiovascular magnetic resonance vs Teichholz M-mode echo at 66% each, and was 58% for radionuclude ventriculography vs cube M-mode echo, 44% for cardiovascular magnetic resonance vs Simpson's 2D echo, 39% for radionuclide ventriculography vs Simpson's 2D echo, and smallest at 31% for cardiovascular magnetic resonance-radionuclide ventriculography. Similarly, the end-diastolic volume and end-systolic volume by 2D echo and cardiovascular magnetic resonance revealed wide limits of agreement (52 ml to 216 ml and 11 ml to 188 ml, respectively).

CONCLUSIONS

These results suggest that ejection fraction measurements by various techniques are not interchangeable. The conclusions and recommendations of research studies in heart failure should therefore be interpreted in the context of locally available techniques. In addition, there are very wide variances in volumes and ejection fraction between techniques, which are most marked in comparisons using echocardiography. This suggests that cardiovascular magnetic resonance is the preferred technique for volume and ejection fraction estimation in heart failure patients, because of its 3D approach for non-symmetric ventricles and superior image quality.

The sense of smell is typically thought of as a 'slow' sense, but the true temporal constraints on the accuracy of olfactory perception are not known. It has been proposed that animals make finer odor discriminations at the expense of additional processing time. To test this idea, we measured the relationship between the speed and accuracy of olfactory discrimination in rats. We found that speed of discrimination was independent of odor similarity, as measured by overlap of glomerular activity patterns. Even when pushed to psychophysical limits using mixtures of two odors, rats needed to take only one sniff (<200 ms at theta frequency) to make a decision of maximum accuracy. These results show that, for the purpose of odor quality discrimination, a fully refined olfactory sensory representation can emerge within a single sensorimotor or theta cycle, suggesting that each sniff can be considered a snapshot of the olfactory world.

This paper reviews the factors affecting the fatty acid composition of adipose tissue and muscle in pigs, sheep and cattle and shows that a major factor is the total amount of fat. The effects of fatty acid composition on meat quality are also reviewed. Pigs have high levels of polyunsaturated fatty acids (PUFA), including the long chain (C20-22) PUFA in adipose tissue and muscle. The full range of PUFA are also found in sheep adipose tissue and muscle whereas cattle 'conserve' long chain PUFA in muscle phospholipid. Linoleic acid (18:2n-6) is a major ingredient of feeds for all species. Its incorporation into adipose tissue and muscle in relation to the amount in the diet is greater than for other fatty acids. It is deposited in muscle phospholipid at a high level where it and its long chain products eg aracidonic acid (20:4n-6) compete well for insertion into phospholipid molecules. Its proportion in pig adipose tissue declines as fat deposition proceeds and is an index of fatness. The same inverse relationships are not seen in ruminant adipose tissue but in all species the proportion of 18:2n-6 declines in muscle as fat deposition increases. The main reason is that phospholipid, where 18:2n-6 is located, declines as a proportion of muscle lipid and the proportion of neutral lipid, with its higher content of saturated and monounsaturated fatty acids, increases. Oleic acid (18:1cis-9), formed from stearic acid (18:0) by the enzyme stearoyl Co-A desaturase, is a major component of neutral lipid and in ruminants the same enzyme forms conjugated linoleic acid (CLA), an important nutrient in human nutrition. Like 18:2n-6, α-linolenic acid (18:3n-3) is an essential fatty acid and is important to ruminants since it is the major fatty acid in grass. However it does not compete well for insertion into phospholipid compared with 18:2n-6 and its incorporation into adipose tissue and muscle is less efficient. Greater biohydrogenation of 18:3n-3 and a long rumen transit time for forage diets also limits the amount available for tissue uptake compared with 18:2n-6 from concentrate diets. A positive feature of grass feeding is that levels of the nutritionally important long chain n-3 PUFA are increased ie EPA (20:5n-3) and DHA (22:6n-3). Future research should focus on increasing n-3 PUFA proportions in lean carcasses and the use of biodiverse pastures and conservation processes which retain the benefits of fresh leafy grass offer opportunities to achieve this. The varying fatty acid compositions of adipose tissue and muscle have profound effects on meat quality. Fatty acid composition determines the firmness/oiliness of adipose tissue and the oxidative stability of muscle, which in turn affects flavour and muscle colour. Vitamin E is an essential nutrient, which stabilises PUFA and has a central role in meat quality, particularly in ruminants.

PURPOSE Romidepsin (depsipeptide or FK228) is a member of a new class of antineoplastic agents active in T-cell lymphoma, the histone deacetylase inhibitors. On the basis of observed responses in a phase I trial, a phase II trial of romidepsin in patients with T-cell lymphoma was initiated. PATIENTS AND METHODS The initial cohort was limited to patients with cutaneous T-cell lymphoma (CTCL), or subtypes mycosis fungoides or Sézary syndrome, who had received no more than two prior cytotoxic regimens. There were no limits on other types of therapy. Subsequently, the protocol was expanded to enroll patients who had received more than two prior cytotoxic regimens. Results Twenty-seven patients were enrolled onto the first cohort, and a total of 71 patients are included in this analysis. These patients had undergone a median of four prior treatments, and 62 patients (87%) had advanced-stage disease (stage IIB, n = 15; stage III, n= 6; or stage IV, n = 41). Toxicities included nausea, vomiting, fatigue, and transient thrombocytopenia and granulocytopenia. Pharmacokinetics were evaluated with the first administration of romidepsin. Complete responses were observed in four patients, and partial responses were observed in 20 patients for an overall response rate of 34% (95% CI, 23% to 46%). The median duration of response was 13.7 months. CONCLUSION The histone deacetylase inhibitor romidepsin has single-agent clinical activity with significant and durable responses in patients with CTCL.

BACKGROUND

Supporting 21st century health care and the practice of evidence-based medicine (EBM) requires ubiquitous access to clinical information and to knowledge-based resources to answer clinical questions. Many questions go unanswered, however, due to lack of skills in formulating questions, crafting effective search strategies, and accessing databases to identify best levels of evidence.

METHODS

This randomized trial was designed as a pilot study to measure the relevancy of search results using three different interfaces for the PubMed search system. Two of the search interfaces utilized a specific framework called PICO, which was designed to focus clinical questions and to prompt for publication type or type of question asked. The third interface was the standard PubMed interface readily available on the Web. Study subjects were recruited from interns and residents on an inpatient general medicine rotation at an academic medical center in the US. Thirty-one subjects were randomized to one of the three interfaces, given 3 clinical questions, and asked to search PubMed for a set of relevant articles that would provide an answer for each question. The success of the search results was determined by a precision score, which compared the number of relevant or gold standard articles retrieved in a result set to the total number of articles retrieved in that set.

RESULTS

Participants using the PICO templates (Protocol A or Protocol B) had higher precision scores for each question than the participants who used Protocol C, the standard PubMed Web interface. (Question 1: A = 35%, B = 28%, C = 20%; Question 2: A = 5%, B = 6%, C = 4%; Question 3: A = 1%, B = 0%, C = 0%) 95% confidence intervals were calculated for the precision for each question using a lower boundary of zero. However, the 95% confidence limits were overlapping, suggesting no statistical difference between the groups.

CONCLUSIONS

Due to the small number of searches for each arm, this pilot study could not demonstrate a statistically significant difference between the search protocols. However there was a trend towards higher precision that needs to be investigated in a larger study to determine if PICO can improve the relevancy of search results.

As new assay methods for quantitative reverse transcription-polymerase chain reaction (RT-PCR), such as real time RT-PCR techniques, approach theoretical limits of per reaction sensitivity, further increments in the sensitivity of measurements of viral load can only be achieved by increasing the amount of input RNA per reaction. We describe a robust, convenient, rapid integrated approach for specimen preparation and real time RT-PCR assay for plasma simian immunodeficiency virus (SIV) RNA viral load that provides a threshold sensitivity of 10 copy Eq/ml, and tolerates less than optimally processed specimens. The method provides accurate quantitation of viral load for the SIV virus isolates in common use for non-human primate studies. We demonstrate the utility of the method in sensitively tracking viral load in an animal showing effective control of viral replication to levels below the threshold for quantitation in conventional assays.

Macroautophagy (referred to hereafter as autophagy) is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell. Autophagy contributes to an array of normal and pathological processes, and has recently emerged as a key regulator of multiple aspects of cancer biology. The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. This complexity is illustrated by the identification of settings where autophagy acts potently to either promote or inhibit tumorigenesis. In this review, I discuss the underlying basis for these opposing functions and propose a model suggesting a dynamic role for autophagy in malignancy. Collectively, the data point to autophagy as serving as a barrier to limit tumor initiation. Once neoplastic lesions are established, it appears that adaptive changes occur that now result in positive roles for autophagy in malignant progression and in subsequent tumor maintenance. Remarkably, constitutive activation of autophagy is critical for continued growth of some tumors, serving to both reduce oxidative stress and provide key intermediates to sustain cell metabolism. Autophagy is also induced in response to cancer therapies where it can function as a survival mechanism that limits drug efficacy. These findings have inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. It is now apparent that aberrant control of autophagy is among the key hallmarks of cancer. While much needs to be learned about the regulation and context-dependent biological functions of autophagy, it seems clear that modulation of this process will be an attractive avenue for future cancer therapeutic approaches.

Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

Multidrug-resistant Acinetobacter baumannii is recognized to be among the most difficult antimicrobial-resistant gram-negative bacilli to control and treat. Increasing antimicrobial resistance among Acinetobacter isolates has been documented, although definitions of multidrug resistance vary in the literature. A. baumannii survives for prolonged periods under a wide range of environmental conditions. The organism causes outbreaks of infection and health care-associated infections, including bacteremia, pneumonia, meningitis, urinary tract infection, and wound infection. Antimicrobial resistance greatly limits the therapeutic options for patients who are infected with this organism, especially if isolates are resistant to the carbapenem class of antimicrobial agents. Because therapeutic options are limited for multidrug-resistant Acinetobacter infection, the development or discovery of new therapies, well-controlled clinical trials of existing antimicrobial regimens and combinations, and greater emphasis on the prevention of health care-associated transmission of multidrug-resistant Acinetobacter infection are essential.

A previous study reported the increased expression of the cytokine TNF in the adipose tissue of genetically obese rodents. To examine this paradigm in humans, we studied TNF expression in lean, obese, and reduced-obese human subjects. TNF mRNA was demonstrated in human adipocytes and adipose tissue by Northern blotting and PCR. TNF protein was quantitated by Western blotting and ELISA in both adipose tissue and the medium surrounding adipose tissue. Using quantitative reverse transcriptase PCR (RT-PCR), TNF mRNA levels were examined in the adipose tissue of 39 nondiabetic subjects, spanning a broad range of body mass index (BMI). There was a significant increase in adipose TNF mRNA levels with increasing adiposity. There was a significant correlation between TNF mRNA and percent body fat (r = 0.46, P < 0.05, n = 23). TNF mRNA tended to decrease in very obese subjects, but when subjects with a BMI>> 45 kg/m2 were excluded, there was a significant correlation between TNF mRNA and BMI (r = 0.37, P < 0.05, n = 32). In addition, there was a significant decrease in adipose TNF with weight loss. In 11 obese subjects who lost between 14 and 66 kg (mean 34.7 kg, or 26.6% of initial weight), TNF mRNA levels decreased to 58% of initial levels after weight loss (P < 0.005), and TNF protein decreased to 46% of initial levels (P < 0.02). TNF is known to inhibit LPL activity. When fasting adipose LPL activity was measured in these subjects, there was a significant inverse relationship between TNF expression and LPL activity (r = -0.39, P < 0.02, n = 39). With weight loss, LPL activity increased to 411% of initial levels. However, the magnitude of the increase in LPL did not correlate with the decrease in TNF. Thus, TNF is expressed in human adipocytes. TNF is elevated in most obese subjects and is decreased by weight loss. In addition, there is an inverse relationship between TNF and LPL expression. These data suggest that endogenous TNF expression in adipose tissue may help limit obesity in some subjects, perhaps by increasing insulin resistance and decreasing LPL.

The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory M1-like phenotype and in facilitating CD4(+) and CD8(+) T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8(+) T-cell-dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.