Characterization of the CCL21-mediated melanoma-specific immune responses and in situ melanoma eradication

Previous studies have shown that secondary lymphoid chemokine, CCL21, can be used for modulation of tumor-specific immune responses. Here, using B16F0 melanoma cells stably expressing CCL21 under the control of cytomegalovirus and ubiquitin promoters, we showed that CCL21-activated immune responses depend on the amount of melanoma-derived chemokine, which, in turn, depends on the strength of the promoter. We showed that ubiquitin promoter-driven expression of CCL21 enabled massive infiltration of tumors with CD4(+)CD25(-), CD8(+) T lymphocytes, and CD11c(+) dendritic cells, and consequent activation of cellular and humoral immune responses sufficient for complete rejection of CCL21-positive melanomas within 3 weeks in all tumor-inoculated mice. Mice that rejected CCL21-positive tumors acquired protective immunity against melanoma, which was transferable to naive mice via splenocytes and central memory T cells. Moreover, melanoma-derived CCL21 facilitated immune-mediated remission of preestablished, distant wild-type melanomas. Overall, these results suggest that elevated levels of tumor-derived CCL21 are required for the activation of strong melanoma-specific immune responses and generation of protective immunologic memory. They also open new perspectives for the development of novel vaccination strategies against melanoma, which use intratumoral delivery of the optimized CCL21-encoding vectors in conjunction with DNA-based vaccines.     

Point-of-care testing for community-acquired pneumonia: do we have all the answers?

Point-of-care tests (POCTs) are available for rapid, "bedside" diagnosis of some causes of community-acquired pneumonia. POCTs complement other laboratory investigations for pneumonia. Although their sensitivity and specificity are improving, they are generally less sensitive than nucleic acid amplification and culture techniques. Questions remain as to the most cost-effective use of POCTs in clinical practice. To ensure their maximum value for both individual patients and the public health system, POCTs are probably best used as part of laboratory-designed algorithms for investigating pneumonia. POCTs are a valuable tool for surveillance, for rapid investigation of outbreaks, and for use in laboratories with limited diagnostic facilities.     

A mobile laminar airflow unit to reduce air bacterial contamination at surgical area in a conventionally ventilated operating theatre

The aim of this study was to evaluate the efficacy of a mobile laminar airflow (LAF) unit in reducing bacterial contamination at the surgical area in an operating theatre supplied with turbulent air ventilation. Bacterial sedimentation was evaluated during 76 clean urological laparotomies; in 34 of these, a mobile LAF unit was added. During each operation, settle plates were placed at four points in the operating theatre (one at the patient area and three at the perimeter), a nitrocellulose membrane was placed on the instrument table and an additional membrane near the wound. During four operations, particle counting was performed to detect particles > or =0.5 microm. Mean bacterial sedimentation on the nitrocellulose membrane on the instrument table was 2730 cfu/m(2)/h under standard ventilation conditions, whereas it decreased significantly to a mean of 305 cfu/m(2)/h when the LAF unit was used, i.e. within the suggested limit for ultraclean operating theatres (P=0.0001). The membrane near the wound showed a bacterial sedimentation of 4031 cfu/m(2)/h without the LAF unit and 1608 cfu/m(2)/h with the unit (P=0.0001). Particle counts also showed a reduction when the LAF unit was used. No significant difference was found at the four points in the operating theatre between samplings performed with, and without, the LAF unit. Use of a mobile LAF unit with turbulent air ventilation can reduce bacterial contamination at the surgical area in high-risk operations (e.g. prosthesis implant).     

Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction.

Nanoparticles prepared by self-assembly from oligonucleotides (ONs), protamine free base, and human serum albumin ("ternary proticles") are spheres of diameters around 200 nm. Substitution of the protamine free base by protamine sulfate leads to proticles of only around 40 nm in diameter with otherwise unchanged properties. The availability of drug delivery systems of very similar composition but grossly different size may be advantageous when dealing with cells which show size-dependent particle uptake. These nanoparticles are promising candidates for ON delivery to cells because of the following reasons: (1) They are stable for several hours in solutions of up to physiological ionic strength; (2) they are efficiently taken up by cells; (3) after cellular uptake, they easily release the ONs even when these are present as phosphorothioates.     

Iron-rich silicates in the Earth's D'' layer

High-pressure experiments and theoretical calculations demonstrate that an iron-rich ferromagnesian silicate phase can be synthesized at the pressure-temperature conditions near the core-mantle boundary. The iron-rich phase is up to 20% denser than any known silicate at the core-mantle boundary. The high mean atomic number of the silicate greatly reduces the seismic velocity and provides an explanation to the low-velocity and ultra-low-velocity zones. Formation of this previously undescribed phase from reaction between the silicate mantle and the iron core may be responsible for the unusual geophysical and geochemical signatures observed at the base of the lower mantle.     

Prophylaxis of pouchitis onset with probiotic therapy: a double-blind,placebo-controlled trial

BACKGROUND & AIMS: We have recently documented the efficacy of a highly concentrated probiotic preparation (VSL#3) in the prevention of flare-up in patients with chronic pouchitis. The aim of this study was to compare probiotic therapy with VSL#3 versus placebo in the ability to prevent the onset of acute pouchitis during the first year after ileal pouch-anal anastomosis. METHODS: Forty consecutive patients who underwent ileal pouch-anal anastomosis for ulcerative colitis were randomized to receive either VSL#3 (1 packet containing 900 billion bacteria/day) (n = 20) or an identical placebo (n = 20) immediately after ileostomy closure for 1 year. The patients were assessed clinically, endoscopically, and histologically after 1, 3, 6, 9, and 12 months. Health-related quality of life was assessed using the Inflammatory Bowel Disease Questionnaire. RESULTS: Two of the 20 patients (10%) treated with VSL#3 had an episode of acute pouchitis compared with 8 of the 20 patients (40%) treated with placebo (log-rank test, z = 2.273; P < 0.05). Treatment with VSL#3 determined a significant improvement in Inflammatory Bowel Disease Questionnaire score, whereas this was not the case with placebo. CONCLUSIONS: Treatment with VSL#3 is effective in the prevention of the onset of acute pouchitis and improves quality of life of patients with ileal pouch-anal anastomosis.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr–Purcell–Meinboom–Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the “invisible” GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from ¹⁵N, ¹H_N, and ¹³C_methyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.Chaperone networks have evolved to correctly fold native proteins and protect against the damaging effects of misfolding and aggregation on protein homeostasis (1, 2). The chaperonins, a ubiquitous subclass of chaperones, are barrel-shaped, multisubunit assemblies composed of two ring cavities, transiently capped by either an extrinsic cochaperone or a built-in lid domain, which assist protein folding in an ATP-dependent manner (3–6). Although the encapsulation mechanism and accompanying allosteric transitions driven by ATP have been extensively studied, the details of how chaperonins fold proteins remain elusive (3, 6, 7). Further, encapsulation does not appear to be an absolute requirement for successful re/folding (8). Moreover, hydrogen/deuterium exchange experiments on several protein substrates (9–12) and fluorescence-based refolding experiments (13) suggest that the prototypical chaperonin GroEL may possess intrinsic unfoldase activity. Here we take advantage of a monomeric, nonaggregating, well-defined system—a triple mutant of the Fyn SH3 domain that exists in dynamic equilibrium between the major native state and a sparsely populated folding intermediate (14, 15)—to directly demonstrate, using NMR relaxation-based methods (16), the ability of apo GroEL to accelerate the interconversion between these two states by almost three orders of magnitude. Simultaneous analysis of lifetime line-broadening (17), dark state exchange saturation transfer (DEST) (18), and Carr–Purcell–Meinboom–Gill (CPMG) relaxation dispersion (19) data permitted us to determine the catalytic rate constants and ascertain the location of the GroEL binding site on the folding intermediate under conditions where the population of GroEL-bound native and intermediate states is less than 1%. Further, GroEL unfoldase/foldase activity is modulated by SH3 deuteration, indicating that catalysis of the exchange reaction between folded and intermediate states involves direct interaction of the substrate with the walls of the GroEL chambers. These results provide a basis for how chaperonins, in the absence of cofactors and encapsulation, may be able to passively protect the cell from the deleterious effects of misfolded protein accumulation.