Risk of Cervical Abnormalities for Women With Multiple Sclerosis Treated With Moderate-Efficacy and High-Efficacy Disease-Modifying Therapies.

BACKGROUND AND OBJECTIVES: The impact of immunomodulatory therapies on the risk of cervical pre-cancer and invasive cancer development is important for the health and safety of women with multiple sclerosis (wwMS). We investigate the risk of cervical abnormalities in wwMS treated with disease-modifying therapies (DMTs). METHODS: This is a multicenter cohort study with data collected from 1998 to 2019 in Victoria, Australia. Data linkage was performed using matching records from the MSBase Registry, the National Human Papillomavirus (HPV) Vaccination Program Register, and the Victorian Cervical Cytology Register. The primary outcome was the detection of any type of cervical abnormality as determined by cytology or histology. Survival methods were used to assess the time to cervical abnormality detection on cervical screening tests (CSTs). Crude and adjusted Cox proportional hazards models were used to determine time to and magnitude of association of DMTs with the risk of cervical abnormality. In a sensitivity analysis, we constructed standardized survival curves averaged over the same set of covariates to determine the commensurate population-average (marginal) causal effects. RESULTS: We included 248 wwMS. The incidence of abnormal CSTs was lower (p < 0.001) for women not exposed to moderate-high-efficacy therapy (10.2 per 1,000 patient-years [95% confidence interval (CI) 5.5-14.9]), compared with those exposed (36.6 per 1,000 patient-years [95% CI 21.7-51.6]). Exposure to higher efficacy treatment was associated with a 3.79-fold increased hazard (95% CI 2.02-7.08, p < 0.001) of developing a cervical abnormality relative to those not exposed. When adjusted for vaccination status, smoking, hormonal contraceptive use, and socioeconomic status, the risk remained elevated at 3.79 (95% CI 1.99-7.21, p < 0.001). Marginal hazard ratios declined over time, ranging from 3.90 (95% CI 2.09-7.27) at 20 years of age to 2.06 (95% CI 1.14-3.73) at 70 years of age. DISCUSSION: A greater than three-and-a-half-fold increased risk of cervical abnormalities was found after exposure to moderate-high-efficacy DMTs. This risk persisted despite adjusting for HPV vaccination status, hormonal contraception use, smoking, and socioeconomic status. If confirmed in future studies, we would advocate for wwMS exposed to moderate-high-efficacy DMTs to be treated in line with immune-deficient paradigm in cervical screening and HPV vaccination programs. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that highly active MS therapy compared with less active therapy increases the risk of developing cervical abnormalities among women with MS.

The Prevalence of Domestic Violence in the Lives of Female Heterosexual Partners of Sex Addicts.

This study provides evidence that rates of domestic violence (DV) run considerably higher in the lives of heterosexual women who identify as partners of sex addicts (PSAs) than in the general population. Data collected from 558 survey participants, from a variety of high-income nations, revealed that 92.1% had ever experienced any form of DV perpetrated by their partner and 57.7% had experienced physical and/or sexual intimate partner violence with their partner. The study also tests several hypotheses about sex addiction behaviors and PSA intimate partner violence (IPV), to help those working with these populations understand what factors may be contributing to, or mitigating, these women's experiences of violence.

Applying Principles of Evolutionary Biology to Plastic Surgery at an Organizational Level Predicts an Extinction Event.

As an organized profession, plastic surgery struggles delivering a clear message regarding scope of practice to patients given the diversity of procedures performed. Whereas granting licensure to practice medicine resides with governmental bodies, certification rests with organizations. However, certification is not required to practice plastic surgery. Since plastic surgery operationalizes techniques rather than working within a defined body organ, competition for patients is intense. Mapping territorial interactions between healthcare providers while parsing taxonomy elucidates individual, community, organizational, and governmental levels, creating various selection pressures. Applying evolutionary biology as a framework predicts the termination of plastic surgery over time as a unique specialty. An entirely new domain, Restorative Healthcare, is proposed which circumvents an extinction outcome.

Phase 1/2a clinical trial of gene-corrected autologous cell therapy for recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) patients have mutations in the COL7A1 gene and thus lack functional type VII collagen (C7) protein; they have marked skin fragility and blistering. This single-center phase 1/2a open-label study evaluated the long-term efficacy, safety, and patient-reported outcomes in RDEB patients treated with gene-corrected autologous cell therapy.METHODSAutologous keratinocytes were isolated from participant skin biopsies. Epidermal sheets were prepared from cells transduced with a retrovirus carrying the full-length human COL7A1 gene. These gene-corrected autologous epidermal sheets measured 5 × 7 cm (35 cm2) and were transplanted onto 6 wound sites in each of 7 adult participants (n = 42 sites total) from 2013 to 2017. Participants were followed for 2 to 5 years.RESULTSNo participants experienced any serious related adverse events. Wound healing of 50% or greater by Investigator Global Assessment was present in 95% (36 of 38) of treated wounds versus 0% (0 of 6) of untreated control wounds at 6 months (P < 0.0001). At year 1, 68% (26 of 38) of treated wounds had 50% or greater healing compared with 17% (1 of 6) of control wounds (P = 0.025). At year 2, 71% (27 of 38) of treated wounds had 50% or greater healing compared with 17% (1 of 6) of control wounds (P = 0.019).CONCLUSIONC7 expression persisted up to 2 years after treatment in 2 participants. Treated wounds with 50% or greater healing demonstrated improvement in patient-reported pain, itch, and wound durability. This study provides additional data to support the clinically meaningful benefit of treating chronic RDEB wounds with ex vivo, C7 gene-corrected autologous cell therapy. This approach was safe and promoted wound healing that was associated with improved patient-reported outcomes.TRIAL REGISTRATIONClinicaltrials.gov identifier: NCT01263379.FUNDINGEpidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH R01 AR055914, Office of Research and Development at the Palo Alto Veteran's Affairs Medical Center, and the Dermatology Foundation.

Cortisol response to operative stress with anesthesia in healthy children.

BACKGROUND: Supraphysiological "stress dosing" is generally given to adrenally insufficient patients undergoing operative procedures and/or general anesthesia. However, the normal responses of cortisol to surgery are poorly documented, especially in small children. Recent studies in adults suggest that massive glucocorticoid dosing is not needed, especially in minimally invasive surgery. OBJECTIVE: We sought to characterize the normal cortisol secretion rate in healthy children undergoing minimally and moderately invasive urological procedures. DESIGN AND SETTING: This was a prospective observational study conducted at a tertiary referral center. PATIENTS: Thirty healthy children, ages 5 months to 6 years, were studied undergoing elective urological procedures. METHODS: Procedures were performed by a single surgeon; anesthesia was by a standard protocol. Sera were obtained at 5 points: iv catheter placement, intubation, 50% completion of surgery, anesthesia reversal, and 1 hour postoperative. Cortisol and cortisone were quantitated by liquid chromatography-tandem mass spectrometry. RESULTS: Group mean cortisol values ranged from 4.21 to 5.71 µg/dL across the 5 time points; none of these mean values differed significantly (P < .05). There were no differences according to age, time of procedure, caudal anesthesia, and moderate vs minimally invasive procedures; 3 patients had higher values. There was a modest diminution in cortisone across the 5 time points. CONCLUSIONS: Minimal and moderately invasive urological procedures do not result in a cortisol stress response in healthy children. Peak cortisol levels were seen 1 hour postoperatively. These data suggest that current guidelines for stress dosing in adrenally insufficient patients substantially exceed physiological requirements during minimally invasive procedures.

Efficient induction of spawning of northern leopard frogs (Lithobates pipiens) during and outside the natural breeding season.

BACKGROUND: Amphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals. METHODS: The method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions. RESULTS: We tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly(10), D-Ala(6), Pro-NHEt(9)-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5-6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully. CONCLUSION: We provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

Avotermin for scar improvement following scar revision surgery: a randomized, double-blind, within-patient, placebo-controlled, phase II clinical trial.

BACKGROUND: Skin scarring is associated with psychosocial distress and has a negative effect on quality of life. The transforming growth factor (TGF)-ß family of cytokines plays a key role in scarring. TGF-ß3 improves scar appearance in a range of mammalian species. This study was performed to assess the efficacy of intradermal avotermin (TGF-ß3) for the improvement of scar appearance following scar revision surgery. METHODS: Sixty patients (35 men and 25 women; age, 19 to 78 years; 53 Caucasians; scar length, 5 to 21 cm) received intradermal avotermin (200 ng/100 µl/linear cm wound margin) and placebo to outer wound segments immediately after, and again 24 hours after, complete (group 1) or staged (group 2) scar revision surgery. A within-patient design was chosen to control for interindividual factors that affect scarring. The primary efficacy variable was a total scar score derived from a visual analogue scale, scored by a lay panel from standardized photographs from months 1 through 7 following treatment. RESULTS: : Primary endpoint data from the combined surgical groups showed that avotermin significantly improved scar appearance compared with placebo (total scar score difference, 21.93 mm; p = 0.04). Profilometry showed a greater reduction in scar surface area from baseline with avotermin treatment compared with placebo, significant in group 2 at months 7 and 12 (difference, 41.99 mm and 25.85 mm, respectively; p = 0.03 for both comparisons). Histologic analysis from group 2 showed that, compared with placebo treatment, collagen organization in avotermin-treated scars more closely resembled normal skin in 14 of 19 cases. Avotermin was well tolerated. CONCLUSION: Avotermin administration following scar revision surgery is well tolerated and significantly improves scar appearance compared with placebo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I.(Figure is included in full-text article.).

Mild hyperoxia limits hTR levels, telomerase activity, and telomere length maintenance in hTERT-transduced bone marrow endothelial cells.

Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O(2)). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%-6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O(2) increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O(2), elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis.

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

The disruptive physician: righteous Maverick or dangerous Pariah?

BACKGROUND: "Disruptive physician" is a term appearing more frequently in many hospital bylaws. It has significant negative implications that can lead to loss of privileges for plastic surgeons. METHODS: Exploring the various definitions of disruptive physician reveals palpable differences between those of the Joint Commission and the American Medical Association. These discrepancies expose plastic surgeons to potential harm when actively addressing quality issues in the hospital environment. RESULTS: The disruptive label can be inappropriately leveraged by hospital administrators against plastic surgeons who confront quality issues. Moreover, the term disruptive is open to subjective interpretation. Challenging the disruptive label in court reveals only that the justice system is concerned that the actual process leading to the disruptive charge is followed appropriately as outlined within the organizational bylaws; the courts are not interested in the actual quality issues and generally will not second-guess the judgment of peer review panels or hospital administrators. CONCLUSIONS: Plastic surgeons would benefit from familiarizing themselves with these issues. Hospitals should be required to use root cause analysis when dealing with quality issues raised by members of the medical staff. Furthermore, findings from root cause analysis should be privileged from legal discovery in all jurisdictions to permit honest exploration of quality issues. When a conflict does arise, consideration of mediation should be given to resolve disputes.

Ethical issues for the plastic surgeon in a tumultuous health care system: dissecting the anatomy of a decision.

BACKGROUND: Society anticipates that plastic surgeons will make ethical decisions that are solely in the best interest of their patients. However, a variety of competing factors exert an influence on all decision-making processes. METHODS: Multiple competing factors that commonly influence decision-making by plastic surgeons, on both conscious and subconscious levels, are identified. By exploring the ramifications of these factors, a more ethical outcome can be achieved. RESULTS: Some of these competing interests that can sidetrack ethical decision-making include personal finances (e.g., ownership of surgical centers, selection of procedures, pricing); outside regulations (e.g., Emergency Medical Treatment and Active Labor Act of 1986 and care of the uninsured); and professional duty (e.g., informed consent, discussion of error). CONCLUSIONS: Plastic surgeons who are aware of the competing interests that influence their decision-making processes stand a greater chance of achieving ethical outcomes. Nevertheless, with the growing volume of nonreimbursed care and expectations of perfect outcomes, achieving uniformly ethical decisions without burdensome self-sacrifice is difficult at best.

Junctional epidermolysis bullosa associated with hypoplastic enamel and pervasive failure of tooth eruption: Oral rehabilitation with use of an overdenture.

This report describes a previously unreported case of generalized hypoplastic enamel and failure of eruption of the permanent maxillary teeth and only partial eruption of the permanent mandibular teeth in an 18-year-old male diagnosed with junctional epidermolysis bullosa. Similar anomalies were reported to have affected the deciduous dentition. Beginning at 4 years of age, oral rehabilitation has been conservatively managed with the fabrication of various maxillary complete overdentures. The use of this prosthesis has provided an economical, nonsurgical treatment option when oral soft tissue permits and with relative ease of construction.

Pulmonary hypertension in neurofibromatosis.

Two cases of severe pulmonary arterial hypertension in patients with neurofibromatosis are reported. The published research is reviewed. In conclusion, it is suggested that the association between these conditions be recognized in the classification of pulmonary hypertension.

Hypertonic saline treatment of cystic fibrosis.

OBJECTIVE: To review the literature concerning the use of hypertonic saline (HS) in patients with cystic fibrosis and explain the rationale for its use. DATA SOURCES: A MEDLINE search was conducted through February 2007. Search terms included hypertonic saline, mucociliary clearance, cystic fibrosis, and human DNASE 1 protein. Additional data were identified through subsequent bibliographic reviews. STUDY SELECTION AND DATA EXTRACTION: All articles in English identified from the data sources were evaluated. Pertinent studies using HS in patients with cystic fibrosis were included in the analysis. DATA SYNTHESIS: Cystic fibrosis is caused by a deficiency in the cystic fibrosis transmembrane regulator gene, resulting in reduced chloride secretion and excessive sodium absorption. The most significant changes are seen in the airway lumen of the lungs. HS has been shown to improve mucociliary clearance versus placebo. A short-term efficacy trial showed a modest and variable increase in forced expiratory volume in 1 second (FEV(1)) over a 2 week period (15.0 +/- 16.0% from baseline vs 2.8 +/- 13.1% with HS 6% and NaCl 0.9%, respectively; p = 0.004). A long-term efficacy trial of either HS 7% or NaCl 0.9% twice daily for 48 weeks has shown a modest sustained improvement in FEV(1) and a significantly increased exacerbation-free survival rate (76% vs 62% for HS 7% and NaCl 0.9%, respectively; p = 0.03). CONCLUSIONS: HS preceded by a bronchodilator is an inexpensive, safe, effective additional therapy in cystic fibrosis patients with stable lung function. Its use has been associated with a modest improvement in lung function and reduced frequency of pulmonary exacerbations.

Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon.

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.

The paradox of heparin-induced thrombocytopenia: future anticoagulation.

Heparin and low molecular weight heparin agents are frequently administered to postoperative orthopedic patients. In patients with a history of heparin-induced thrombocytopenia, alternative anticoagulant agents to heparin for prophylaxis and treatment must be considered. Unfortunately, the data is limited in this patient population. Upon transitioning from heparin-induced thrombocytopenia treatment with direct thrombin inhibitors, argatroban, or lepirudin, careful consideration must be taken to avoid further thrombotic complications.

Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

Binding of small molecules to an adaptive protein-protein interface.

Understanding binding properties at protein-protein interfaces has been limited to structural and mutational analyses of natural binding partners or small peptides identified by phage display. Here, we present a high-resolution analysis of a nonpeptidyl small molecule, previously discovered by medicinal chemistry [Tilley, J. W., et al. (1997) J. Am. Chem. Soc. 119, 7589-7590], which binds to the cytokine IL-2. The small molecule binds to the same site that binds the IL-2 alpha receptor and buries into a groove not seen in the free structure of IL-2. Comparison of the bound and several free structures shows this site to be composed of two subsites: one is rigid, and the other is highly adaptive. Thermodynamic data suggest the energy barriers between these conformations are low. The subsites were dissected by using a site-directed screening method called tethering, in which small fragments were captured by disulfide interchange with cysteines introduced into IL-2 around these subsites. X-ray structures with the tethered fragments show that the subsite-binding interactions are similar to those observed with the original small molecule. Moreover, the adaptive subsite tethered many more compounds than did the rigid one. Thus, the adaptive nature of a protein-protein interface provides sites for small molecules to bind and underscores the challenge of applying structure-based design strategies that cannot accurately predict a dynamic protein surface.

Blockage of heregulin expression inhibits tumorigenicity and metastasis of breast cancer.

The growth factor heregulin (HRG), expressed in about 30% of breast cancer tumors, activates the erbB-2 receptor via induction of heterodimeric complexes of erbB-2 with erbB-3 or erbB-4. HRG induces tumorigenicity and metastasis of breast cancer cells. Our investigation into whether HRG is a factor likely to promote tumor formation independently of erbB-2 overexpression concludes that blockage of HRG expression suppresses the aggressive phenotype of MDA-MB-231 breast cancer cells by inhibiting cell proliferation, preventing anchorage-independent growth, and suppressing the invasive potential of the cells in vitro. More importantly, we observed a marked reduction in tumor formation, tumor size, and a lack of metastasis in vivo. These studies were achieved by blocking HRG expression in MDA-MB-231 cells using an HRG antisense cDNA. In the search for the mechanism by which blockage of HRG reverts this aggressive phenotype, we discovered that the cells in which HRG is blocked exhibit a marked decrease in erbB activation and a significant reduction in MMP-9 activity, demonstrating a direct causal role in HRG induction of tumorigenicity. Our study is the first report and serves as a proof of the concept that HRG is a key promoter of breast cancer tumorigenicity and metastasis independently of erbB-2 overexpression and should be deemed a potential target in developing therapies for breast cancer.