Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells.

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

Hilotherapy for the management of perioperative pain and swelling in facial surgery: a systematic review and meta-analysis.

Hilotherapy is the application of cold compression at a regulated temperature through a face mask. Studies that have evaluated its efficacy have focused on postoperative oedema, pain, and the patient's comfort. However, there is no clear consensus in favour of its use, so we have made a systematic review and meta-analysis to evaluate relevant published reports. We searched PubMed, EMBASE, MEDLINE, the Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials to identify studies. Sixty-one records were screened, six of which met the inclusion criteria and four of which were suitable for meta-analysis. All data suitable for meta-analysis were derived from studies of elective and traumatic facial skeletal surgery. Hilotherapy was associated with significant reductions in facial pain on postoperative day 2 (p<0.00001), and facial oedema on days 2 (p=0.0004) and 3 (p=0.02). Patients reported more comfort and satisfaction with hilotherapy than with cold compression (p<0.00001). The effect of hilotherapy on ecchymosis and formation of haematomas remains uncertain. Well-designed, randomised, controlled trials of its use after aesthetic facial surgery are required.

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress.

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis.

Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737.

Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.

Facing the World: audit of activity 2002-2010.

BACKGROUND: Craniofacial anomalies, although uncommon, can have considerable effects on the individual, their family and society.(1-4) They carry with them a large morbidity and require a highly specialized, multidisciplinary approach to treatment.(5) Facing the World (FTW), was founded in 2002, to offer facial reconstructive surgery to children with complex, craniofacial anomalies with no prospect of local treatment, from developing countries anywhere in the world. METHODS: We present an 8-year audit of the cases treated by FTW, where children are brought from their own countries to the UK for treatment. Patient selection takes place prior to their arrival in the UK by a multidisciplinary team. Specifically the condition has to be correctable to a degree that justifies the risks involved with the surgery, and the disruption to the child and their family. RESULTS: Since inception, FTW has evaluated more than 300 cases and provided treatment in the UK for over 24 cases from 18 different countries. We present our range of cases and complications. We discuss our complication rate of 28% and mortality rate of 4% (1 case). CONCLUSIONS: Key to the sustainability of FTW is the development of local healthcare infrastructure within the developing countries to facilitate eventual local management of the more straightforward cases and follow up of these patients by well-trained medical staff. By establishing these programs, FTW aims to not only change these children's lives but to raise awareness, and help to expand the global craniofacial network whereby in the future, satellite partners will be present to help manage these conditions locally. LEVEL OF EVIDENCE: III.

Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.

Time-lapse video microscopy allows analysis of the interaction between individual CTLs and adherent peptide-pulsed targets, from contact, to lymphocyte detachment, APC rounding, phosphatidylserine exposure and finally loss of plasma membrane integrity characteristic of end-stage apoptosis. Using in vitro-stimulated effectors specific for the ovalbumin K(b)OVA(257) (OT-I) and influenza A virus D(b)NP(366) and D(b)PA(224) epitopes, no significant correlation was found between the duration of CTL contact and the time to phosphatidylserine exposure or loss of membrane integrity. Furthermore, there were minimal indications that transgenic T cells specific for the K(b)OVA(257) epitope (TCR) diversity had any effect. However, when the analysis was repeated with D(b)NP(366) and D(b)PA(224)-specific CTLs recovered directly from the lungs of mice with influenza pneumonia, the lower avidity D(b)NP(366)-specific set was found to elute much more quickly. Shorter contact time may allow individual CTLs to lyse more targets, suggesting that lower TCR/epitope avidity may be more beneficial than higher epitope avidity for cell-mediated immunity.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity.

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance.

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

Nasal tip necrosis--an unusual presentation of rheumatoid vasculitis.

We describe an unusual case of cutaneous necrosis of the nasal tip presenting to a facial reconstructive surgeon. The patient had developed this painless necrosis over a period of about 10 days. Her past medical history included rheumatoid arthritis. She described an exacerbation of her arthritic symptoms in the weeks preceding the development of the nasal tip necrosis. Her rheumatoid arthritis had been managed with corticosteroid and immunosuppressive therapy for more than 3 years. She had not previously experienced extra-articular manifestations (EAMs). A biopsy was taken and histological analysis identified a lymphocytic vasculitis. She was referred to her rheumatologist, and surgical management of her necrotic nasal tip commenced.

Complications of 278 consecutive abdominoplasties.

The case notes of 278 consecutive patients who underwent abdominoplasty, during a five-year period, in one institution under the care of four surgeons were reviewed. Patient details, early and late complications and revision procedures were noted. Seventy-five percent of patients had a 'full' abdominoplasty with undermining to costal cartilage and repositioning of the umbilicus and 23% had 'mini abdominoplasties', 2% were revision operations. Eighteen percent of patients suffered from early complications the most common of which were seroma (5%), haematoma (3%), infection (3%), skin or fat necrosis (2.5%) and delayed healing (2%). Twenty-five percent of patients had late complications which were often relatively minor. These included 'dog ears' (12%), localised fatty excess (10%) and unsatisfactory scars (8%). Twenty-four percent of patients underwent revision surgery. Most commonly further liposuction (12%), dog ear revision (10%) and scar revision (5%). Analysis failed to reveal significant risk factors. Despite an apparently high complication and revision rate the subjective impression is of a satisfied patient cohort.

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

The management of midline transcranial nasal dermoid sinus cysts.

The most common congenital midline nasal masses are nasal dermoid sinus cysts (NDSC) [Hughes GB, Sharpino G, Hunt W, Tucker HM. Management of the congenital midline nasal mass--a review. Head Neck Surg 1980;2:222-33.]. Their clinical importance hinges on their potential to communicate with the central nervous system. Preoperative diagnosis of an intracranial extension allows for referral to a craniofacial team with the appropriate skills and experience for a transcranial approach. All patients with a NDSC require imaging with high resolution multiplanar MRI scans and complimentary fine cut CT scan to reveal the anatomical extent of the tract and its relationship to the anterior cranial fossa. A single-stage craniofacial approach to resection of midline NDSC extending to the anterior cranial base is effective with minimal morbidity [Yavuzer R, Bier U, Jackson IT. Be careful: it might be a nasal dermoid cyst. Plast Reconstr Surg 1999;103:2082-3; Denoyelle F, Ducroz V, Roger G, Garabedian EN. Nasal dermoid sinus cysts in children. Laryngoscope 1997;107:795-800; Rohrich RJ, Lowe JB, Schwartz MR. The role of open rhinoplasty in the management of nasal dermoid cysts. Plast Reconstr Surg 1999;104:2163-70; Rahbar R, Shah P, Mulliken JB, et al. The presentation and management of nasal dermoid-a 30-year experience. Arch Otolaryngol Head Neck Surg 2003;129:464-71; Posnick JC, Bortoluzzi P, Armstrong DC, Drake JM. Intracranial nasal dermoid sinus cysts: computed tomographic scan findings and surgical results. Plast Reconstr Surg 1994;93:745-54 [discussion 755-56]; Bartlett SP, Lin KY, Grossman R, Kratowitz J. The surgical management of orbitofacial dermoids in the pediatric patient. Plast Reconstr Surg 1993;91:1208-15.]. The cyst and tract are accessed through a combination of a nasal and transcranial approach. This allows visualisation and dissection of the tract with only a small incision on the nasal dorsum to include the cutaneous punctum when present. Transnasal endoscopic techniques have been advocated where the dermoid is located within the nasal cavity and there is little or no cutaneous involvement [Weiss DD, Robson CD, Mulliken JB. Transnasal endoscopic excision of midline nasal dermoid from the anterior cranial base. Plast Reconstr Surg 1998;101:2119-23.]. We present a review of five cases referred to our unit between 1999 and 2004 with a diagnosis of a midline nasal dermoid sinus cyst and radiological evidence of intracranial communication. All cases had a communication with the anterior cranial fossa diagnosed preoperatively and were treated surgically with a craniofacial approach. An intracranial extension was identified at operation in each case and this was confirmed on histopathology. The only significant complication resulted from an early postoperative infection, requiring re-operation. There were no recurrences and acceptable aesthetic outcomes have been observed in all cases.

Demographics and macroeconomic effects in aesthetic surgery in the UK.

Media interest in aesthetic surgery is substantial and suggestions of demographic changes such as reductions in age or an increase in the number of male patients are common. In spite of this, there is no peer reviewed literature reporting demographics of a contemporary large patient cohort or of the effect of macroeconomic indicators on aesthetic surgery in the UK. In this study, computer records 13006 patients presenting between 1998 and the first quarter of 2003 at a significant aesthetic surgery centre were analysed for procedures undergone, patient age and sex. Male to female ratios for each procedure were calculated and a comparison was made between unit activity and macroeconomic indicators. The results showed that there has been no significant demographic change in the procedures studied with patient age and male to female ratio remaining constant throughout the period studied for each procedure. Comparison with macroeconomic indicators suggested increasing demand for aesthetic surgery in spite of a global recession. In conclusion, media reports of large scale demographic shifts in aesthetic surgery patients are exaggerated. The stability of unit activity in spite of falling national economic indicators suggested that some units in the UK might be relatively immune to economic vagaries. The implications for training are discussed.