The metastatic site does not influence PD-L1 expression in advanced non-small cell lung carcinoma.

INTRODUCTION: PD-L1 expression by immunohistochemistry (IHC) testing with Tumor Proportion Score (TPS) ≥50% and ≥1% is required to be eligible for first- and second-line Pembrolizumab treatment for metastatic non-small cell lung cancer (NSCLC) respectively. Stage IV NSCLC often presents with metastasis to multiple distant sites which are easily accessible for biopsy. Knowing whether PD-L1 IHC TPS can be indifferently measured from different metastatic site is therefore an important clinical question. In this study, we evaluated PD-L1 expression in NSCLC from varied distant metastatic sites. METHODS: A total of 580 NSCLC specimens of distant metastases were retrieved for study, including 35 paired samples from two different metastatic sites. The metastatic sites included brain, bone, remote lymph nodes, serous membranes (pleura, pericardium and peritoneum), extra-thoracic solid organs and skin/soft tissues. The samples were cytology cell blocks, small biopsies or surgical resections. IHC was performed using Dako PD-L1 IHC 22C3 pharmDx. A total of 100 viable tumor cells was required for adequacy. TPS ≥ 50% and 1-49% were defined as high and low PD-L1 expression respectively. RESULTS: PD-L1 TPS scores were not significantly different across a range of distant metastatic sites nor between metastases in paired samples. CONCLUSION: Our results suggest that the PD-L1 TPS scoring is similar across different metastatic sites and any site biopsied will yield necessary information for guiding clinical management.

Curative pelvic exenteration for recurrent cervical carcinoma in the era of concurrent chemotherapy and radiation therapy. A systematic review.

OBJECTIVE: Pelvic exenteration requires complete resection of the tumor with negative margins to be considered a curative surgery. The purpose of this review is to assess the optimal preoperative evaluation and surgical approach in patients with recurrent cervical cancer to increase the chances of achieving a curative surgery with decreased morbidity and mortality in the era of concurrent chemoradiotherapy. METHODS: Review of English publications pertaining to cervical cancer within the last 25 years were included using PubMed and Cochrane Library searches. RESULTS: Modern imaging (MRI and PET-CT) does not accurately identify local extension of microscopic disease and is inadequate for preoperative planning of extent of resection. Today, only half of pelvic exenteration procedures obtain uninvolved surgical margins. CONCLUSION: Clear margins are required for curative pelvic exenterations, but are poorly predictable by pre-operative assessment. More extensive surgery, i.e. the infra-elevator exenteration with vulvectomy, is a logical surgical choice to increase the rate of clear margins and to improve patient survival following surgery for recurrent cervical carcinoma.

Copeptin and arginine vasopressin at high altitude: relationship to plasma osmolality and perceived exertion.

PURPOSE: A diuresis is a key part of acclimatisation to high altitude (HA). Arginine vasopressin (AVP) is a hormone involved in salt and water balance and may potentially have a role in the development of altitude illness. ProAVP (copeptin) is more stable than AVP and is assayed by a straightforward, automated method. We investigated the relationship of AVP to copeptin and the copeptin response to exercise and altitude illness in a large cohort during a field study at HA. METHODS: 48 subjects took part in a 10-day trek at HA. Venous blood samples were taken at 3,833, 4,450 and 5,129 m post-trek (exercise) and the following day at rest. Daily recordings of symptoms of altitude illness, oxygen saturations and perceived exertion were carried out. RESULTS: AVP and copeptin levels increased with exercise and correlated closely (ρ 0.621 p < 0.001), this was strongest in the stressed state when AVP secretion was highest, at 5,129 m post-exercise (ρ 0.834 p < 0.001). On two-way ANOVA, both altitude (F = 3.5; p = 0.015) and exercise (F = 10.2; p = 0.002) influenced copeptin levels (interaction F = 2.2; p = 0.08). AVP levels were influenced by exercise (F = 14.4; p = 0.0002) but not altitude (F = 2.0; p = 0.12) with no overall group interactions (F = 1.92.6; p = 0.06). There was no association between copeptin or arginine vasopressin and altitude illness. Copeptin correlated with the Borg RPE score and was significantly higher in the group with a Borg score ≥15 (7.9 vs. 3.7 p < 0.001). CONCLUSION: We have shown that arginine vasopressin and copeptin levels correlate and are suppressed below 5,129 m. Furthermore, we have demonstrated that exertion, rather than altitude illness or increasing osmolality, is the stimulus for increases in copeptin.

Ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation-induced migration in melanoma cells.

BACKGROUND: There is growing evidence that inflammation may exacerbate cancer metastasis and several clinical studies show that taking nonsteroidal anti-inflammatory drugs appears to reduce metastases. OBJECTIVES: The aims of this study were: (i) to examine the effects of ibuprofen on the major proinflammatory cytokine tumour necrosis factor (TNF)-alpha induction of migration of C8161 and HBL human melanoma cells; (ii) to develop ibuprofen-releasing hydrogels (Pluronics F127) for future topical use in reducing metastatic spread of primary melanoma; and (iii) to examine whether the actions of ibuprofen might be explained by induction of apoptosis. METHODS: Melanoma cells were exposed to 300 U mL(-1) TNF-alpha for a 24-h period prior to making a scratch wound to which ibuprofen or ibuprofen-loaded hydrogels were then added. The effects of relevant concentrations of ibuprofen on cell viability and apoptosis were examined. RESULTS: Ibuprofen at 10(-3) mol L(-1) significantly reduced TNF-alpha-stimulated migration of both cell types to that of nonstimulated cells (P < 0.001). TNF-alpha-unstimulated cell migration was not significantly affected. Cells responded similarly to SS and SR forms of ibuprofen. Cells treated with ibuprofen sodium salt-loaded hydrogels showed a significant reduction in migration when compared with unloaded hydrogels. Ibuprofen induced apoptosis in HBL cells but had no effect on C8161 melanoma cells apoptosis at concentrations that reduced migration. CONCLUSIONS: These results demonstrate that TNF-alpha upregulated malignant melanoma migration in vitro and that this could be reduced by ibuprofen both in solution and delivered from a hydrogel. These effects of ibuprofen cannot be attributed simply to induction of apoptosis.

Clinical pharmacokinetics of 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine in human immunodeficiency virus-infected patients.

The pharmacokinetics and bioavailability of 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosi ne (cyclic HPMPC) were examined at four doses in 22 patients with human immunodeficiency virus infection. Two groups of six patients received a single dose of cyclic HPMPC at 1.5 or 3.0 mg/kg of body weight by each of the oral and intravenous routes in a random order with a 2-week washout period between administrations. Additional patients received single intravenous doses of cyclic HPMPC at 5.0 mg/kg (n = 6) or 7.5 mg/kg (n = 4). Serial serum and urine samples were collected at intervals over 24 h after dosing. The concentrations of cyclic HPMPC and cidofovir in serum and urine samples were determined by validated reverse-phase ion-pairing high-performance liquid chromatography methods with derivatization and fluorescence detection. After intravenous administration of cyclic HPMPC, concentrations of cyclic HPMPC declined in a biexponential manner, with a mean +/- standard deviation half-life of 1.09 +/- 0.12 h (n = 22). The pharmacokinetics of cyclic HPMPC were independent of dose over the dose range of 1.5 to 7.5 mg/kg. The total clearance of cyclic HPMPC from serum and the volume of distribution of intravenous cyclic HPMPC were 198 +/- 39.6 ml/h/kg and 338 +/- 65.1 ml/kg, respectively (n = 22). The renal clearance of cyclic HPMPC (132 +/- 27.3 ml/h/kg; n = 22) exceeded the creatinine clearance (86.2 +/- 16.3 ml/h/kg), indicating active tubular secretion. The cyclic HPMPC excreted in urine in 24 h accounted for 71.3% +/- 16.0% of the administered dose. Cidofovir was formed from cyclic HPMPC in vivo with a time to the maximum concentration in serum of 1.64 +/- 0.23 h (n = 22). Cidofovir levels declined in an apparent monoexponential manner, with a mean terminal half-life of 3.98 +/- 1.26 h (n = 22). The cidofovir excreted in urine in 24 h accounted for 9.40% +/- 2.33% of the administered cyclic HPMPC dose. Exposure to cidofovir after intravenous administration of cyclic HPMPC was dose proportional and was 14.9% of that from an equivalent dose of cidofovir. The present study suggests that intravenous cyclic HPMPC also has a lower potential for nephrotoxicity in humans compared to that of intravenous cidofovir. The oral bioavailabilities of cyclic HPMPC were 1.76% +/- 1.48% and 3.10% +/- 1.16% with the administration of doses of 1.5 and 3.0 mg/kg, respectively (n = 6 per dose). The maximum concentrations of cyclic HPMPC in serum were 0.036 +/- 0.021 and 0.082 +/- 0.038 microgram/ml after the oral administration of doses of 1.5 and 3.0 mg/kg, respectively. Cidofovir reached quantifiable levels in the serum of only one patient for each of the 1.5- and 3.0-mg/kg oral cyclic HPMPC doses.

Conservation of genetic linkage between heat shock protein 100 and glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei and Trypanosoma cruzi.

The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.

The properties and function of the glycosylphosphatidylinositol-phospholipase C in Trypanosoma brucei.

The purpose of this review is to consider recent results obtained concerning the properties and function of the glycosylphosphatidylinositol-phospholipase C (GPI-PLC) in Trypanosoma brucei. A mutagenesis study that provides evidence that the GPI-PLC is more closely related to bacterial PI-PLCs than previously realised is described. The variant specific glycoprotein (VSG), which dominates the surface of the mammalian stages of the trypanosome, is almost certainly the major substrate of the GPI-PLC. The hydrolysis of the GPI-anchor of the VSG under stress conditions and hypotonic lysis is well established. To investigate whether this hydrolysis of the GPI-anchor plays any role during the life cycle a GPI-PLC null mutant has been made. The phenotype indicates that the gene is non-essential, but its absence alters the course of infection in mice.

The epidermolytic (exfoliative) toxins of Staphylococcus aureus.

Two epidermolytic toxins, produced by different strains of Staphylococcus aureus, split human skin at a site in the upper epidermis. Clinical effects are most common in infants, but adults are susceptible. Epidermolysis may also be observed in the mouse, in vivo and in vitro, and in a few other mammals. Recent in vitro experiments have demonstrated an inhibition by chelators and point to metal-ion, possibly Ca2+, involvement. The epidermolysis effect is insensitive to a wide range of other metabolic inhibitors. The toxin amino acid sequences are similar to that of staphylococcal proteinase, and new experiments by chemical modification and site-directed mutagenesis have shown that toxicity depends on 'active serine' residues of a catalytic triad similar to that found in serine proteases. Furthermore the toxins possess esterolytic activity, also dependent on the 'active serine' sites. However, the toxins have low or undetectable activity towards a range of peptide or protein substrates. In histological and related studies, the toxins bound selectively to an intracellular skin protein, profilaggrin, but there was no evidence that the toxin can enter intact epidermal cells. Therefore, although the circumstantial evidence that the toxins act by proteolysis is convincing, a specific skin proteolytic substrate for the toxin has not been identified.

The esterolytic activity of epidermolytic toxins.

The two epidermolytic toxins were shown to have intrinsic N-t-butyloxycarbonyl-L-glutamic acid alpha-phenyl esterase activity. The activity was dependent on free toxin pKa values of 6.6 and 6.8 for ETA and ETB respectively. ETB incorporated 0.97 mol of radiolabelled di-isopropyl phosphorofluoridate/mol of protein with loss of esterolytic and epidermolytic activities. The correspondence of epidermolytic and esterolytic activities in ETA and ETB during thermal inactivation and reaction with di-isopropyl phosphorofluoridate, together with the inactivity of the mutant protein ETA S195G, demonstrates that the two activities are dependent on a single active serine residue in each protein.

The role of the serine protease active site in the mode of action of epidermolytic toxin of Staphylococcus aureus.

The sequences of the epidermolytic toxins and V8 serine proteinase share about 25% identity, including the catalytic triad at the proteinase active centre. Here we have altered the putative ETA active-site serine-195 to glycine by site-directed mutagenesis. No epidermolytic activity was detected when up to 100-fold greater amounts of the homogeneous mutant ETA were injected subcutaneously into neonatal mice showing that serine-195 is required for toxicogenesis.