Tsetse GmmSRPN10 has anti-complement activity and is important for successful establishment of trypanosome infections in the fly midgut

The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission

Trastuzumab for HER2-positive early stage breast cancer : a meta-analysis of individual patient data from 13,864 women from seven randomised trials

Background: Trastuzumab targets the extracellular domain of the HER2 protein. Adding trastuzumab to chemotherapy for patients with early-stage, HER2-positive breast cancer reduces the risk of recurrence and death, but is associated with cardiac toxicity. We investigated the long-term benefits and risks of adjuvant trastuzumab on breast cancer recurrence and cause-specific mortality. Methods: We did a collaborative meta-analysis of individual patient data from randomised trials assessing chemotherapy plus trastuzumab versus the same chemotherapy alone. Randomised trials that enrolled women with node-negative or node-positive, operable breast cancer were included. We collected individual patient-level data on baseline characteristics, dates and sites of first distant breast cancer recurrence and any previous local recurrence or second primary cancer, and the date and underlying cause of death. Primary outcomes were breast cancer recurrence, breast cancer mortality, death without recurrence, and all-cause mortality. Standard intention-to-treat log-rank analyses, stratified by age, nodal status, oestrogen receptor (ER) status, and trial yielded first-event rate ratios (RRs). Findings: Seven randomised trials met the inclusion criteria, and included 13 864 patients enrolled between February, 2000, and December, 2005. Mean scheduled treatment duration was 14·4 months and median follow-up was 10·7 years (IQR 9·5 to 11·9). The risks of breast cancer recurrence (RR 0·66, 95% CI 0·62 to 0·71; p<0·0001) and death from breast cancer (0·67, 0·61 to 0·73; p<0·0001) were lower with trastuzumab plus chemotherapy than with chemotherapy alone. Absolute 10-year recurrence risk was reduced by 9·0% (95% CI 7·4 to 10·7; p<0·0001) and 10-year breast cancer mortality was reduced by 6·4% (4·9 to 7·8; p<0·0001), with a 6·5% reduction (5·0 to 8·0; p<0·0001) in all-cause mortality, and no increase in death without recurrence (0·4%, –0·3 to 1·1; p=0·35). The proportional reduction in recurrence was largest in years 0–1 after randomisation (0·53, 99% CI 0·46 to 0·61), with benefits persisting through years 2–4 (0·73, 0·62 to 0·85) and 5–9 (0·80, 0·64 to 1·01), and little follow-up beyond year 10. Proportional recurrence reductions were similar irrespective of recorded patient and tumour characteristics, including ER status. The more high risk the tumour, the larger the absolute reductions in 5-year recurrence (eg, 5·7% [95% CI 3·1 to 8·3], 6·8% [4·7 to 9·0], and 10·7% [7·7 to 13·6] in N0, N1–3, and N4+ disease). Interpretation: Adding trastuzumab to chemotherapy for early-stage, HER2-positive breast cancer reduces recurrence of, and mortality from, breast cancer by a third, with worthwhile proportional reductions irrespective of recorded patient and tumour characteristics. Funding: Cancer Research UK, UK Medical Research Council

Molecular mechanisms of vaspin action: from adipose tissue to skin and bone, from blood vessels to the brain

Visceral adipose tissue derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that  were specifically expressed or overexpressed in the intra abdominal or visceral adipose tissue  (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral  obesity, insulin resistance, hyperinsulinemia and ‐glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type  2 diabetes.  The follow-up study reporting the cloning, expression and functional characterization of  vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain

N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewisx. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M−1s−1 for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M−1s−1 for the corresponding PNGase F-treated forms. The 7–8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA

Increasing the dose intensity of chemotherapy by more frequent administration or sequential scheduling: a patient-level meta-analysis of 37 298 women with early breast cancer in 26 randomised trials

Background Increasing the dose intensity of cytotoxic therapy by shortening the intervals between cycles, or by giving individual drugs sequentially at full dose rather than in lower-dose concurrent treatment schedules, might enhance efficacy. Methods To clarify the relative benefits and risks of dose-intense and standard-schedule chemotherapy in early breast cancer, we did an individual patient-level meta-analysis of trials comparing 2-weekly versus standard 3-weekly schedules, and of trials comparing sequential versus concurrent administration of anthracycline and taxane chemotherapy. The primary outcomes were recurrence and breast cancer mortality. Standard intention-to-treat log-rank analyses, stratified by age, nodal status, and trial, yielded dose-intense versus standard-schedule first-event rate ratios (RRs). Findings Individual patient data were provided for 26 of 33 relevant trials identified, comprising 37 298 (93%) of 40 070 women randomised. Most women were aged younger than 70 years and had node-positive disease. Total cytotoxic drug usage was broadly comparable in the two treatment arms; colony-stimulating factor was generally used in the more dose-intense arm. Combining data from all 26 trials, fewer breast cancer recurrences were seen with dose-intense than with standard-schedule chemotherapy (10-year recurrence risk 28·0% vs 31·4%; RR 0·86, 95% CI 0·82–0·89; p<0·0001). 10-year breast cancer mortality was similarly reduced (18·9% vs 21·3%; RR 0·87, 95% CI 0·83–0·92; p<0·0001), as was all-cause mortality (22·1% vs 24·8%; RR 0·87, 95% CI 0·83–0·91; p<0·0001). Death without recurrence was, if anything, lower with dose-intense than with standard-schedule chemotherapy (10-year risk 4·1% vs 4·6%; RR 0·88, 95% CI 0·78–0·99; p=0·034). Recurrence reductions were similar in the seven trials (n=10 004) that compared 2-weekly chemotherapy with the same chemotherapy given 3-weekly (10-year risk 24·0% vs 28·3%; RR 0·83, 95% CI 0·76–0·91; p<0·0001), in the six trials (n=11 028) of sequential versus concurrent anthracycline plus taxane chemotherapy (28·1% vs 31·3%; RR 0·87, 95% CI 0·80–0·94; p=0·0006), and in the six trials (n=6532) testing both shorter intervals and sequential administration (30·4% vs 35·0%; RR 0·82, 95% CI 0·74–0·90; p<0·0001). The proportional reductions in recurrence with dose-intense chemotherapy were similar and highly significant (p<0·0001) in oestrogen receptor (ER)-positive and ER-negative disease and did not differ significantly by other patient or tumour characteristics. Interpretation Increasing the dose intensity of adjuvant chemotherapy by shortening the interval between treatment cycles, or by giving individual drugs sequentially rather than giving the same drugs concurrently, moderately reduces the 10-year risk of recurrence and death from breast cancer without increasing mortality from other causes. Funding Cancer Research UK, Medical Research Council

Potential Use of a Serpin from Arabidopsis for Pest Control

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC50 = 637 µg ml−1). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

<p>Abstract</p> <p>Background</p> <p>Mosquitoes of the <it>Anopheles gambiae </it>species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect <it>Plasmodium </it>development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution.</p> <p>Methods</p> <p>Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the <it>An. gambiae </it>species complex in both East and West Africa.</p> <p>Results</p> <p>Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes.</p> <p>Conclusion</p> <p>It is well known that phylogenetic and population history in the <it>An. gambiae </it>complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the <it>An. gambiae </it>genome are discussed.</p

20-Year Risks of Breast-Cancer Recurrence after Stopping Endocrine Therapy at 5 Years

The administration of endocrine therapy for 5 years substantially reduces recurrence rates during and after treatment in women with early-stage, estrogen-receptor (ER)-positive breast cancer. Extending such therapy beyond 5 years offers further protection but has additional side effects. Obtaining data on the absolute risk of subsequent distant recurrence if therapy stops at 5 years could help determine whether to extend treatment