Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome.

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all ß-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2-3 kcal mol-1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both ß-sheets, rather than by the location of the terminal strands in the ribosome tunnel.

Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins.

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2-like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2-like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder.

Promiscuous and Selective: How Intrinsically Disordered BH3 Proteins Interact with Their Pro-survival Partner MCL-1.

The BCL-2 family of proteins plays a central role in regulating cell survival and apoptosis. Disordered BH3-only proteins bind promiscuously to a number of different BCL-2 proteins, with binding affinities that vary by orders of magnitude. Here we investigate the basis for these differences in affinity. We show that eight different disordered BH3 proteins all bind to their BCL-2 partner (MCL-1) very rapidly, and that the differences in sequences result in different dissociation rates. Similarly, mutation of the binding surface of MCL-1 generally affects association kinetics in the same way for all BH3 peptides but has significantly different effects on the dissociation rates. Importantly, we infer that the evolution of homologous, competing interacting partners has resulted in complexes with significantly different lifetimes.

Protein-peptide association kinetics beyond the seconds timescale from atomistic simulations.

Understanding and control of structures and rates involved in protein ligand binding are essential for drug design. Unfortunately, atomistic molecular dynamics (MD) simulations cannot directly sample the excessively long residence and rearrangement times of tightly binding complexes. Here we exploit the recently developed multi-ensemble Markov model framework to compute full protein-peptide kinetics of the oncoprotein fragment 25-109Mdm2 and the nano-molar inhibitor peptide PMI. Using this system, we report, for the first time, direct estimates of kinetics beyond the seconds timescale using simulations of an all-atom MD model, with high accuracy and precision. These results only require explicit simulations on the sub-milliseconds timescale and are tested against existing mutagenesis data and our own experimental measurements of the dissociation and association rates. The full kinetic model reveals an overall downhill but rugged binding funnel with multiple pathways. The overall strong binding arises from a variety of conformations with different hydrophobic contact surfaces that interconvert on the milliseconds timescale.

Affinity of IDPs to their targets is modulated by ion-specific changes in kinetics and residual structure.

Intrinsically disordered proteins (IDPs) are characterized by a lack of defined structure. Instead, they populate ensembles of rapidly interconverting conformations with marginal structural stabilities. Changes in solution conditions such as temperature and crowding agents consequently affect IDPs more than their folded counterparts. Here we reveal that the residual structure content of IDPs is modulated both by ionic strength and by the type of ions present in solution. We show that these ion-specific structural changes result in binding affinity shifts of up to sixfold, which happen through alteration of both association and dissociation rates. These effects follow the Hofmeister series, but unlike the well-established effects on the stability of folded proteins, they already occur at low, hypotonic concentrations of salt. We attribute this sensitivity to the marginal stability of IDPs, which could have physiological implications given the role of IDPs in signaling, the asymmetric ion profiles of different cellular compartments, and the role of ions in biology.

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1.

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye-Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies.

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.

De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of ß-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.

Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein.

Protein-protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including ß-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.

Coupled folding and binding of the disordered protein PUMA does not require particular residual structure.

Many cellular proteins are 'disordered' in isolation. A subset of these intrinsically disordered proteins (IDPs) can, upon binding another molecule, fold to a well-defined three-dimensional structure. In the structurally heterogeneous, unbound ensemble of these IDPs, conformations are likely to exist that, in part, resemble the final bound form. It has been suggested that these conformations, displaying 'residual structure', could be important for the mechanism of such coupled folding and binding reactions. PUMA, of the BCL-2 family, is an IDP in isolation but will form a single, contiguous α-helix upon binding the folded protein MCL-1. Using the helix-breaking residue proline, we systematically target each potential turn of helix of unbound PUMA and assess the binding to MCL-1 using time-resolved stopped-flow techniques. All proline-containing mutants bound, and although binding was weaker than the wild-type protein, association rate constants were largely unaffected. We conclude that population of particular residual structure, containing a specific helical turn, is neither required for the binding nor for fast association of PUMA and MCL-1.

Football for life versus antidoping for the masses: ethical antidoping issues and solutions based on the extenuating experiences of an elite footballer competing while undergoing treatment for metastatic testicular cancer.

It is thankfully rare for extenuating circumstances to fully test the processes and procedures enshrined in national and world antidoping authorities' rules and laws. It is also thankfully very rare that a failed drugs test can have some positive implications. Antidoping laws are undoubtedly focused on ensuring fair competition, however, there are occasions when honest athletes discover medical diagnoses through failed antidoping tests. The purpose of this paper is to broadly discuss antidoping considerations encountered, based on the four principles of medical ethics and to propose simple solutions to these problems. Unfortunately, extreme medical circumstances will often test the limits of antidoping and medical processes and with open channels for feedback, these systems can improve. Performance enhancement seems an illogical concept if an athlete's medical treatment and disease are more inherently performance harming than unintended potential doping, but needs to be carefully managed to maintain fair sport.

Antioxidants for female subfertility.

BACKGROUND: A couple may be considered to have fertility problems if they have been trying to conceive for over a year with no success. This difficulty with conception may affect up to a quarter of all couples planning a child. The reported prevalence of subfertility has increased significantly over the past twenty years. It is estimated that for 40% to 50% of couples, subfertility may be a result of female problems, including ovulatory disorders, poor egg quality, fallopian tube damage and endometriosis. Antioxidants are thought to reduce the oxidative stress brought on by these conditions. Currently, limited evidence suggests that antioxidants improve fertility, and trials have explored this area with varied results. This review assessed the evidence for the effectiveness of different antioxidants in female subfertility. OBJECTIVES: To determine whether supplementary oral antioxidants compared with placebo, no treatment/standard treatment or another antioxidant improve fertility outcomes for subfertile women. SEARCH METHODS: We searched the following databases (from inception to April 2013) with no language restrictions applied: Cochrane Menstrual Disorders and Subfertility Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO, CINAHL, LILACS and OpenSIGLE. We also searched conference abstracts and citation lists in the ISI Web of Knowledge. Ongoing trials were searched in the Trials Registers. Reference lists were checked, and a search on Google was performed. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared any type, dose or combination of oral antioxidant supplement with placebo, no treatment or treatment with another antioxidant, among women attending a reproductive clinic. Trials comparing antioxidants with fertility drugs alone and trials that exclusively included fertile women attending a fertility clinic because of male partner infertility were excluded. DATA COLLECTION AND ANALYSIS: Three review authors independently screened 2127 titles and abstracts, and 67 of these potentially eligible trials were appraised for inclusion and quality through review of full texts and contact with authors. Three review authors were involved in data extraction and assessment of risk of bias. Review authors also collected data on adverse events as reported from the trials. Studies were pooled using fixed-effect models; however, if high heterogeneity was found, a random-effects model was used. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for the dichotomous outcomes of live birth, clinical pregnancy and adverse events. Analyses were stratified by type of antioxidant, by indications for subfertility and by those women also undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection techniques (ICSIs). The overall quality of the evidence was assessed by applying GRADE criteria. MAIN RESULTS: A total of 28 trials involving 3548 women were included in this review. Investigators compared oral antioxidants, including combinations of antioxidants, pentoxifylline, N-acetyl-cysteine, melatonin, L-arginine, vitamin E, myo-inositol, vitamin C, vitamin D+calcium and omega-3-polyunsaturated fatty acids with placebo, with no treatment/standard treatment or another antioxidant.Antioxidants were not associated with an increased live birth rate compared with placebo or no treatment/standard treatment (OR 1.25, 95% CI 0.19 to 8.26, P = 0.82, 2 RCTs, 97 women, I(2) = 75%, very low-quality evidence). This suggests that among subfertile women with an expected live birth rate of 37%, the rate among women taking antioxidants would be between 10% and 83%.Antioxidants were not associated with an increased clinical pregnancy rate compared with placebo or no treatment/standard treatment (OR 1.30, 95% CI 0.92 to 1.85, P = 0.14, 13 RCTs, 2441 women, I(2)= 55%, very low-quality evidence). This suggests that among subfertile women with an expected clinical pregnancy rate of 23%, the rate among women taking antioxidants would be between 22% and 36%.Only one trial reported on live birth in the antioxidant versus antioxidant comparison, and two trials reported on clinical pregnancy in this comparison. Only subtotals were used in this analysis, and meta-analysis was not possible as each trial used a different antioxidant.Pentoxifylline was associated with an increased clinical pregnancy rate compared with placebo or no treatment (OR 2.03, 95% CI 1.19 to 3.44, P = 0.009, 3 RCTs, 276 women, I(2) = 0%).Adverse events were reported by 14 trials in the meta-analysis and included miscarriage, multiple pregnancy, ectopic pregnancy and gastrointestinal effects. No evidence revealed a difference in adverse effects between antioxidant groups and control groups, but these data were limited.The overall quality of evidence was 'very low' to 'low' because of poor reporting of outcomes, the number of small studies included, high risk of bias within studies and heterogeneity in the primary analysis. AUTHORS' CONCLUSIONS: The quality of the evidence in the 'antioxidant versus placebo/no treatment' and in the 'antioxidant versus antioxidant' comparisons was assessed to be 'very low'. Antioxidants were not associated with an increased live birth rate or clinical pregnancy rate. There was some evidence of an association of pentoxifylline with an increased clinical pregnancy rate; however, there were only three trials included in this comparison. Future trials may change this result. Variation in the types of antioxidants given meant that we could not assess whether one antioxidant was better than another. There did not appear to be any association of antioxidants with adverse effects for women, but data for these outcomes were limited.

Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain of cMyb to CBP KIX.

Association rates for interactions between folded proteins have been investigated extensively, allowing the development of computational and theoretical prediction methods. Less is known about association rates for complexes where one or more partner is initially disordered, despite much speculation about how they may compare to those for folded proteins. We have attached a fluorophore to the N-terminus of the 25 amino acid cMyb peptide used previously in NMR and equilibrium studies (termed FITC-cMyb), and used this to monitor the kinetics of its interaction with the KIX protein. We have investigated the ionic strength and temperature dependence of the kinetics, and conclude that the association process is extremely fast, apparently exceeding the rates predicted by formulations applicable to interactions between pairs of folded proteins. This is despite the fact that not all collisions result in complex formation (there is an observable activation energy for the association process). We propose that this is partially a result of the disordered nature of the FITC-cMyb peptide itself.

A mechanistic model for amorphous protein aggregation of immunoglobulin-like domains.

Protein aggregation is associated with many debilitating diseases including Alzheimer's, Parkinson's, and light-chain amyloidosis (AL). Additionally, such aggregation is a major problem in an industrial setting where antibody therapeutics often require high local concentrations of protein domains to be stable for substantial periods of time. However, despite a plethora of research in this field, dating back over 50 years, there is still no consensus on the mechanistic basis for protein aggregation. Here we use experimental data to derive a mechanistic model that well describes the aggregation of Titin I27, an immunoglobulin-like domain. Importantly, we find that models that are suitable for nucleated fibril formation do not fit our aggregation data. Instead, we show that aggregation proceeds via the addition of activated dimers, and that the rate of aggregation is dependent on the surface area of the aggregate. Moreover, we suggest that the "lag time" seen in these studies is not the time needed for a nucleation event to occur, but rather it is the time taken for the concentration of activated dimers to cross a particular solubility limit. These findings are reminiscent of the Finke-Watzky aggregation mechanism, originally based on nanocluster formation and suggest that amorphous aggregation processes may require mechanistic schemes that are substantially different from those of linear fibril formation.

Take home lessons from studies of related proteins.

The 'Fold Approach' involves a detailed analysis of the folding of several topologically, structurally and/or evolutionarily related proteins. Such studies can reveal determinants of the folding mechanism beyond the gross topology, and can dissect the residues required for folding from those required for stability or function. While this approach has not yet matured to the point where we can predict the native conformation of any polypeptide chain in silico, it has been able to highlight, amongst others, the specific residues that are responsible for nucleation, pathway malleability, kinetic intermediates, chain knotting, internal friction and Paracelsus switches. Some of the most interesting discoveries have resulted from the attempt to explain differences between homologues.

Summary of in vitro genetic toxicology assay results: expected and unexpected effects of recent study design modifications.

Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure-activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53-competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)-compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non-GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL-positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay.

Robotic surgery for benign gynaecological disease.

BACKGROUND: Robotic surgery is the latest innovation in the field of minimally invasive surgery. In the case of robotic surgery, instead of directly moving the instruments the surgeon uses a robotic system to control the instruments for surgical procedures. Robotic surgical systems have been used in various gynaecological surgeries for benign disease, such as hysterectomy (removal of the uterus), myomectomy (removal of uterine leiomyomas) and tubal reanastomosis (the reuniting of a divided tube). The mounting evidence demonstrates the feasibility and safety of robotic surgery in benign gynaecological disease. Robotic surgery is advertised as having promising advantages including more precise vision and procedures, improved ergonomics and shorter length of hospital stay. However, the main disadvantages of the robotic surgical system should not be overlooked, including the high cost of disposable instruments and retraining for both surgeons and nurses. OBJECTIVES: To assess the effectiveness and safety of robot-assisted surgery in the treatment of benign gynaecological disease. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group's Trial Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (Issue 2, 2011), MEDLINE and EMBASE up to November 2011 and citation lists of relevant publications. SELECTION CRITERIA: All randomised controlled trials (RCTs) comparing robotic surgery for benign gynaecological disease to laparoscopic or open surgical procedures. RCTs comparing different types of robotic assistants were also included. We contacted study authors for unpublished information, but failed in obtaining a response. DATA COLLECTION AND ANALYSIS: Two review authors independently screened studies for inclusion. The domains assessed for risk of bias were allocation concealment, blinding, incomplete outcome data and selective outcome reporting. Odds ratios (OR) were used for reporting dichotomous data with 95% confidence intervals (CI), whilst mean differences (MD) were determined for continuous data. Statistical heterogeneity was assessed using the I(2) statistic. We contacted the primary authors for missing data but failed in obtaining a response. MAIN RESULTS: Two trials involving 158 participants were included. Since one included trial was published in conference proceedings, limited usable data were available for further analysis. The only analysis in this trial showed comparable rates of conversions to open surgery between the robotic group and the laparoscopic group (OR 1.41, 95% CI 0.22 to 9.01; P = 0.72). One RCT showed longer operation time (MD 66.00, 95% CI 40.93 to 91.07; P < 0.00001), higher cost (MD 1936.00, 95% CI 445.69 to 3426.31; P = 0.01) in the robotic group compared with the laparoscopic group. Also, both studies reported that robotic and laparoscopic surgery seemed comparable regarding intraoperative outcome, complications, length of hospital stay and quality of life. AUTHORS' CONCLUSIONS: Currently, the limited evidence showed that robotic surgery did not benefit women with benign gynaecological disease in effectiveness or in safety. Further well-designed RCTs with complete reported data are required to confirm or refute this conclusion.

Pentoxifylline for endometriosis.

BACKGROUND: Endometriosis is a chronic, recurring condition that occurs during the reproductive years. It is characterized by endometrial tissue developing outside the uterine cavity. This endometrial tissue development is dependent on oestrogen produced primarily by the ovaries and, therefore, traditional management has focused on ovarian suppression. In this review we considered the role of modulation of the immune system as an alternative approach. This is an update of a Cochrane Review previously published in 2009 (Lu 2009). OBJECTIVES: To assess the effects of pentoxifylline, which has anti-inflammatory effects, in subfertile, premenopausal women for the management of endometriosis. SEARCH METHODS: For the first publication of this review we searched the following databases (from inception to December 2008) for trials: Cochrane Menstrual Disorders and Subfertility Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE, CINAHL, and PsycINFO. In addition, all reference lists of included trials were searched and experts in the field were contacted in an attempt to locate trials. This search was rerun to 23 November 2011, for this update. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing pentoxifylline with placebo or no treatment, medical treatment, or surgery in subfertile, premenopausal women were included. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials for inclusion, assessed trial risk of bias, and extracted data using data extraction forms. We contacted study authors for additional information and data. The domains assessed for risk of bias were sequence generation, allocation concealment, blinding, incomplete outcome data, and selective outcome reporting. Peto odds ratios (OR) were used for reporting dichotomous data with 95% confidence intervals (CI), whilst mean differences (MD) were expressed for continuous data. Statistical heterogeneity was assessed using the I(2) statistic. MAIN RESULTS: Four trials involving 334 participants were included. One RCT [n=34] showed pentoxifylline had no significant effect on reduction in pain (MD -1.60, 95% CI -3.32 to 0.12). There was no evidence of an increase in clinical pregnancy events in the pentoxifylline group compared with placebo (three RCTs [n=67] OR 1.54, 95% CI 0.89 to 266). One RCT studied recurrence of endometriosis [n=88] (OR 0.88,95% CI 0.27 to 2.84). No trials reported the effects of pentoxifylline on the odds of live birth rate per woman, improvement of endometriosis-related symptoms, or adverse events. AUTHORS' CONCLUSIONS: This review has been updated in 2011. The results of the original review published in 2009 remain unchanged. There is still not enough evidence to support the use of pentoxifylline in the management of premenopausal women with endometriosis in terms of subfertility and relief of pain outcomes.