Cross-reactive CD4+ T cells against one immunodominant tumor-derived epitope in melanoma patients

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.     

A heat shock protein 90 binding domain in endothelial nitric-oxide synthase influences enzyme function

Previous reports suggest heat shock protein 90 (hsp90) associates with endothelial nitric-oxide synthase (eNOS) to increase nitric oxide (*NO) generation. Ansamycin inhibition of chaperone-dependent activity increases eNOS generation of superoxide anion (O(2)(*)) upon enzyme activation. In the present study we identify where hsp90 binds to eNOS using overlapping decoy peptides based on the amino acid (aa) sequence of eNOS (291-420). B1, B2, and B3 peptides inhibited hsp90 association with eNOS in cell lysates from proliferating bovine aortic endothelial cells. B2 (aa 301-320), common to both B1 and B3, decreased stimulated *NO production and hsp90 association in bovine aortic endothelial cells. The B2/B3 peptide was redesigned to TSB2 that includes a TAT protein transduction domain and shortened to 14 aa. TSB2 impaired vasodilation of isolated facialis arteries in vitro and in vivo and increased eNOS-dependent O(2)(*) generation in native endothelial cells on mouse aortas, whereas a control peptide, TSB(Ctr), which has the four glutamic acids in TSB2 substituted with alanine, showed no such effects. Site-directed mutagenesis of eNOS at 310, 314, 318, and 323 Glu to Ala yields an eNOS mutant that exhibited reduced hsp90 association and generated O(2)(*) rather than *NO upon activation. Together, these data demonstrate that hsp90 associates with eNOS at aa 310-323. Moreover, a decoy peptide based on this sequence is sufficient to displace hsp90 from eNOS and uncouple eNOS activity from *NO generation. Thus, Glu-310, Glu-314, Glu-318, and Glu-323 in eNOS, although each does not do much by itself, synergistically they increase "cooperativity" in the association step that is critical for maintaining hsp90-eNOS interactions and promoting coupled eNOS activity. Such chaperone-dependent signaling may play an important role in modulating the balance of *NO and O(2)(*) generation from eNOS and, therefore, vascular function.     

Extreme dives by free-ranging emperor penguins

We examined the incidence of extreme diving in a 3-year overwintering study of emperor penguins Aptenodytes forsteri in East Antarctica. We defined extreme dives as very deep (> 400 m) and/or very long (> 12 min). Of 137364 dives recorded by 93 penguins 264 dives reached depths > 400 m and 48 lasted > 12 min. Most (65%) very long dives occurred in winter (May–August) while 83% of the very deep dives took place in spring (September–November). The two most extreme dives (564 m depth, 21.8 min duration) were separate dives and were performed by different individual penguins. Penguins diving extremely deeply may have done so as part of their foraging strategy whereas penguins diving for very long times may have been forced to do so by changes in the sea-ice conditions.     

Mitochondria and ageing: winning and losing in the numbers game

Mitochondrial dysfunction has long been considered a key mechanism in the ageing process but surprisingly little attention has been paid to the impact of mitochondrial number or density within cells. Recent reports suggest a positive association between mitochondrial density, energy homeostasis and longevity. However, mitochondrial number also determines the number of sites generating reactive oxygen species (ROS) and we suggest that the links between mitochondrial density and ageing are more complex, potentially acting in both directions. The idea that increased density, especially when combined with mitochondrial dysfunction, might accelerate ageing is supported by a negative correlation between mitochondrial density and maximum longevity in an interspecies comparison in mammals, and by evidence for an intimate interconnection between cellular ROS levels, mitochondrial density and cellular ageing. Recent data suggest that retrograde response, which activates mitochondrial biogenesis, accompanies cellular ageing processes. We hypothesise that increased mitochondrial biogenesis, and possibly also impaired degradation and segregation of mitochondria, if occurring as adaptation to pre-existing mitochondrial dysfunction, might aggravate ROS production and thus actively contribute to ageing.     

Testing evolutionary theories of menopause

Why do women cease fertility rather abruptly through menopause at an age well before generalized senescence renders child rearing biologically impossible? The two main evolutionary hypotheses are that menopause serves either (i) to protect mothers from rising age-specific maternal mortality risks, thereby protecting their highly dependent younger children from death if the mother dies or (ii) to provide post-reproductive grandmothers who enhance their inclusive fitness by helping to care and provide for their daughters'' children. Recent theoretical work indicates that both factors together are necessary if menopause is to provide an evolutionary advantage. However, these ideas need to be tested using detailed data from actual human life histories lived under reasonably ‘natural’ conditions; for obvious reasons, such data are extremely scarce. We here describe a study based on a remarkably complete dataset from The Gambia. The data provided quantitative estimates for key parameters for the theoretical model, which were then used to assess the actual effects on fitness. Empirically based numerical analysis of this nature is essential if the enigma of menopause is to be explained satisfactorily in evolutionary terms. Our results point to the distinctive (and perhaps unique) role of menopause in human evolution and provide important support for the hypothesized evolutionary significance of grandmothers.     

Partial nitritation at elevated loading rates: design curves and biofilm characteristics

There is a need to develop low operational intensity, cost-effective, and small-footprint systems to treat wastewater. Partial nitritation has been studied using a variety of control strategies, however, a gap in passive operation is evident. This research investigates the use of elevated loading rates as a strategy for achieving low operational intensity partial nitritation in a moving bed biofilm reactor (MBBR) system. The effects of loading rates on nitrification kinetics and biofilm characteristics were determined at elevated, steady dissolved oxygen concentrations between 5.5 and 7.0 mg O₂/L and ambient temperatures between 19 and 21 °C. Four elevated loading rates (3, 4, 5 and 6.5 g NH₄⁺-N/m² days) were tested with a distinct shift in kinetics being observed towards nitritation at elevated loadings. Complete partial nitritation (100% nitrite production) was achieved at 6.5 g NH₄⁺-N/m² days, likely due to thick biofilm (572 µm) and elevated NH₄⁺-N load, which resulted in suppression of nitrite oxidation.

     

Improving Retention of Very Old Participants in Longitudinal Research: Experiences from the Newcastle 85+ Study

Background

People aged 85 and over are often excluded from research on the grounds of being difficult to recruit and problematic to retain. The Newcastle 85+ study successfully recruited a cohort of 854 85-year-olds to detailed health assessment at baseline and followed them up over 3 phases spanning 5 years. This paper describes the effectiveness of its retention strategies.

Methods

Primary retention strategies involved meticulous management of contact information and active maintenance of contact with participants between research visits and between phases of the study. For statistical analysis, data on post-inclusion attrition over the 3 follow-up phases was separated into ‘death’ and ‘withdrawal’ categories, with sub-categories ‘health’ and ‘non-health’ reasons created for ‘withdrawal’. Multinomial logistic regression was used to determine if particular socio-demographic and health characteristics were associated with post-inclusion attrition due to withdrawal at each of the 3 phase-to-phase transition points.

Results

For both sexes, at successive follow-up phases there was a decrease in attrition due to withdrawal and an increase due to death. Withdrawal was most prevalent between baseline and phase 2. Across the 5 years of the study total post-inclusion (post-baseline) attrition due to death accounted for a 40% (344/854) loss to cohort and total post-inclusion attrition due to withdraw a 19% (166/854) loss to cohort, with health reasons for withdrawal becoming more dominant over time. Adjusting for sex, parsimonious modelling showed only occupational class (National Statistics Socio-economic Classification) to be associated with withdrawal and only between baseline and phase 2 (routine/manual compared to managerial (OR 3.41; 95% CI [1.23 to 9.44]).

Conclusion

Following successful recruitment, we retained a high proportion of participants from a very old age group over 5 years of longitudinal research. No strong predictors of post-inclusion attrition due to withdrawal were found, suggesting the general effectiveness of our retention strategies.     

Interferon-alpha in tumor immunity and immunotherapy

Interferon-alpha (IFN-alpha) is a pleiotropic cytokine belonging to type I IFN, currently used in cancer patients. Early studies in mouse tumor models have shown the importance of host immune mechanisms in the generation of a long-lasting antitumor response to type I IFN. Recent studies have underscored new immunomodulatory effects of IFN-alpha, including activities on T and dendritic cells, which may explain IFN-induced tumor immunity. Reports on new immune correlates in cancer patients responding to IFN-alpha represent additional evidence on the importance of the interactions of IFN-alpha with the immune system for the generation of durable antitumor response. This knowledge, together with results from studies on genetically modified tumor cells expressing IFN-alpha, suggest novel strategies for using these cytokines in cancer immunotherapy and in particular the use of IFN-alpha as an immune adjuvant for the development of cancer vaccines.     

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.     

The Ruegeria pomeroyi acuI gene has a role in DMSP catabolism and resembles yhdH of E. coli and other bacteria in conferring resistance to acrylate

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH(-) mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH(-) mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide "added protection" for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway.