Phase I, first-in-human study of MSC-1 (AZD0171), a humanized anti-leukemia inhibitory factor monoclonal antibody, for advanced solid tumors.

BACKGROUND: Activation of leukemia inhibitory factor (LIF) is linked to an immunosuppressive tumor microenvironment (TME), with a strong association between LIF expression and tumor-associated macrophages (TAMs). MSC-1 (AZD0171) is a humanized monoclonal antibody that binds with high affinity to LIF, promoting antitumor inflammation through TAM modulation and cancer stem cell inhibition, slowing tumor growth. In this phase I, first-in-human, open-label, dose-escalation study, MSC-1 monotherapy was assessed in patients with advanced, unresectable solid tumors. MATERIALS AND METHODS: Using accelerated-titration dose escalation followed by a 3 + 3 design, MSC-1 doses of 75-1500 mg were administered intravenously every 3 weeks (Q3W) until progression or unmanageable toxicity. Additional patients were enrolled in selected cohorts to further evaluate safety, pharmacokinetics (PK), and pharmacodynamics after escalation to the next dose had been approved. The primary objective was characterizing safety and determining the recommended phase II dose (RP2D). Evaluating antitumor activity and progression-free survival (PFS) by RECIST v1.1, PK and immunogenicity were secondary objectives. Exploratory objectives included pharmacodynamic effects on circulating LIF and TME immune markers. RESULTS: Forty-one patients received treatment. MSC-1 monotherapy was safe and well tolerated at all doses, with no dose-limiting toxicities. The maximum tolerated dose was not reached and the RP2D was determined to be 1500 mg Q3W. Almost half of the patients had treatment-related adverse events (TRAEs), with no apparent trends across doses; no patients withdrew due to TRAEs. There were no objective responses; 23.7% had stable disease for ≥2 consecutive tumor assessments. Median PFS was 5.9 weeks; 23.7% had PFS >16 weeks. On-treatment changes in circulating LIF and TME signal transducers and activators of transcription 3 signaling, M1:M2 macrophage populations, and CD8+ T-cell infiltration were consistent with the hypothesized mechanism of action. CONCLUSIONS: MSC-1 was very well tolerated across doses, with prolonged PFS in some patients. Biomarker and preclinical data suggest potential synergy with checkpoint inhibitors.

Analysis of the acute postoperative pain experience following oral surgery: identification of 'unaffected', 'disabled' and 'depressed, anxious and disabled' patient clusters.

BACKGROUND: Pain is defined as both a sensory and an emotional experience. Acute postoperative tooth extraction pain is assessed and treated as a physiological (sensory) pain while chronic pain is a biopsychosocial problem. The purpose of this study was to assess whether psychological and social changes occur in the acute pain state. METHODS: A biopsychosocial pain questionnaire was completed by 438 subjects (165 males, 273 females) with acute postoperative pain at 24 hours following the surgical extraction of teeth and compared with 273 subjects (78 males, 195 females) with chronic orofacial pain. Statistical methods used a k-means cluster analysis. RESULTS: Three clusters were identified in the acute pain group: 'unaffected', 'disabled' and 'depressed, anxious and disabled'. Psychosocial effects showed 24.8 per cent feeling 'distress/suffering' and 15.1 per cent 'sad and depressed'. Females reported higher pain intensity and more distress, depression and inadequate medication for pain relief (p < 0.001). Distress and depression were associated with higher pain intensity. The developed questionnaire had tested reliability (test-retest r = 0.89) and estimated validity. CONCLUSION: Cluster analysis showed constituent groups with a range of psychosocial effects in acute postoperative dental extraction pain and is associated with an increase in pain intensity.

Adolescent girls and their babies: achieving optimal birthweight. Gestational weight gain and pregnancy outcome in terms of gestation at delivery and infant birth weight: a comparison between adolescents under 16 and adult women.

AIMS: Pregnancy amongst under 16s has been reported to result in worse outcomes for the baby, including low birthweight. This study aimed to find out whether the under 16s need to gain more weight during pregnancy to avoid this outcome. METHOD: A retrospective case control study of pregnancy outcomes in girls delivering before the age of 16 and women delivering aged 25-30. Data was collected from medical case notes, including maternal age, pregnancy weight gain and infant birth weight. RESULTS: Although weight gain amongst under 16s was similar to that in the control group, average birthweight of babies born to under 16s was less than in the older group. For both girls and older women greater weight gain in pregnancy did result in higher birth weights. DISCUSSION: As younger girls are still growing it may be necessary for them to achieve a greater pregnancy weight gain in order to achieve a satisfactory birth weight.

Evaluation of border sprays for managing the codling moth (Tortricidae: Lepidoptera) and the apple maggot (Tephritidae: Diptera) in Ontario apple orchards.

The efficacy of two insecticide control programs for managing the codling moth, Cydia pomonella (L.), and the apple maggot, Rhagoletis pomonella (Walsh), were compared in the Georgian Bay, London, Niagara, and Quinte apple production areas of Ontario during 1995, 1996, and 1997. In the border spray program, an initial cover spray of organophosphorus insecticide was applied to eradicate codling moths that may have colonized a test plot during the previous growing season. Subsequent sprays were applied only to a four-tree-wide zone (approximately 20 wide) around the perimeter of the plot to control immigrating codling moths or apple maggots. In the cover spray program, all sprays of organophosphorus insecticide were applied to the entire plot. Apple maggot injury was significantly greater in border spray program plots than in cover spray program plots only during 1995 in the London production area. There was no significant difference in codling moth injury between border spray and cover spray plots in the four production areas during the three-year study. The elimination of cover sprays from border spray plots during July and August may have left the apple crop more susceptible to damage by second generation larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris), in the London production area during 1995. There was a trend of increasing codling moth injury from 1995 to 1997 in two border spray plots, and apple maggot injury was detected in these plots during the third year of the study.

Studies of differential gene expression in clinically derived eosinophil populations.

BACKGROUND: Influx of eosinophils into the post-capillary bronchial epithelium and the subsequent release of inflammatory mediators is characteristic of the late phase of asthmatic attacks. The genes that serve to predispose the peripheral blood eosinophils of asthmatics to undergo this process are poorly defined. The aim of this report is to describe the differential gene expression of both the known pro-inflammatory genes 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) and novel cDNA sequences in eosinophils derived from clinical samples. METHODS: Novel cDNA sequences representing genes upregulated in peripheral blood eosinophils of asthmatic as compared with nonasthmatic patients were identified by differential display polymerase chain reaction (DDPCR). The differential expression of these sequences, in addition to known pro-inflammatory genes, were then studied by reverse dot blotting of amplified RNA generated from the eosinophils of nonasthmatic donors, asthmatic donors, asthmatic donors taking steroids, interleukin (IL) -3, IL-5, granulocyte-macrophage colony stimulating factor- (GM-CSF) treated eosinophils from asthmatic donors and the eosinophilic cell line AML14. RESULTS: Four unique DDPCR-generated 3'UTR DNA fragments were identified that showed differing patterns of expression between the eosinophil populations of interest. Expression of each of the novel clones was increased in the peripheral blood eosinophils of asthmatics and downregulated in those donors taking steroids. Expression of 5-lipoxygenase was not found to vary between the different eosinophil populations, whereas FLAP was induced by treatment with the cytokine cocktail in both primary eosinophils and the eosinophilic cell line AML14. CONCLUSION: The differential regulation of the novel cDNA sequences and FLAP in the range of eosinophil populations studied suggest that they may provide clinically relevant therapeutic targets. Moreover, the procedures used in these studies may provide a general approach to the study of differential gene expression in small numbers of cells such as those obtained from clinical samples.

Differential characteristics of human 15-lipoxygenase isozymes and a novel splice variant of 15S-lipoxygenase.

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells. In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401-429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete alpha-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates. Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo.

Radical resection for naso-lacrimal duct tumour.

Naso-lacrimal duct tumours are uncommon and present with epiphora and swelling. Since the naso-lacrimal duct is embedded in bone for the majority of its anatomical length, the late presentation of proptosis is due to orbital extension of the tumour. Radical surgical treatment is necessary to establish clear margins and facilitate reconstruction.

Operational trial of ParaSight-F (dipstick) in the diagnosis of falciparum malaria at the primary health care level.

The rapid manual ParaSight-F test of Plasmodium falciparum malaria, an antigen capture test for detecting trophozoite-derived histidine rich protein-2 (PF HRP-2), is simple to perform and provides a definite diagnosis within 10 minutes. During an operational trial at health centers and mobile malaria units where microscopical diagnosis is not available and using defined symptom screening criteria, 3,361 subjects were tested yielding 618 positives (18.4%) for PF-HRP-2 by ParaSight-F. Microscopic examination of the same subjects by thick blood film examined 7 days later at a malaria clinic showed 578 falciparum, and 349 vivax and mixed infection (F+V) 41. The technology proved highly effective in detecting falciparum malaria at the peripheral levels where access to malaria laboratory services are difficult, thus allowing immediate administration of a complete course of treatment in the absence of a microscopic examination.

Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2) in human osteosarcoma cells.

The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 beta and TNF alpha in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were preincubated with inhibitors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1-30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2-0.4 microM). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released more PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.

Altered sensitivity of aspirin-acetylated prostaglandin G/H synthase-2 to inhibition by nonsteroidal anti-inflammatory drugs.

Aspirin (ASA) acetylates Ser516 of prostaglandin G/H synthase-2 (PGHS-2) resulting in a modified enzyme that converts arachidonic acid to 15(R)-hydroxy-eicosatetraeroic acid [15(R)-HETE]. ASA has pharmacological benefits that may not all be limited to inhibition of prostaglandin synthesis, and this study was initiated to further investigate the properties of ASA-acetylated PGHS-2 and of the mutation of Ser516 to methionine, which mimics ASA acetylation. Both the S516M mutant and ASA-acetylated form of PGHS-2 (ASA-PGHS-2) synthesize 15(R)-HETE and have apparent K(m) values for arachidonic acid within 10-fold of the apparent K(m) value for untreated PGHS-2. The time courses of turnover-dependent inactivation were similar for reactions catalyzed by PGHS-2 and ASA-PGHS-2, whereas the PGHS-2(S516M) showed a decrease in both the initial rate of 15-HETE production and rate of enzyme inactivation. The production of 15-HETE by modified PGHS-2 was sensitive to inhibition by most nonsteroidal anti-inflammatory drugs (NSAIDs), including selective PGHS-2 inhibitors. As observed for the cyclooxygenase activity of PGHS-2, the inhibition of 15-HETE production by indomethacin was time-dependent for both ASA-PGHS-2 and PGHS-2(S516M). However, two potent, structurally related NSAIDs, diclofenac and meclofenamic acid, do not inhibit either ASA-PGHS-2 or the PGHS-2(S516M) mutant. These results demonstrate that the sensitivity to inhibition by NSAIDs of the 15-HETE production by ASA-treated PGHS-2 is different than that of prostaglandin production by PGHS-2 and that Ser516 plays an important role in the interaction with fenamate inhibitors. The results also indicate that the conversion of arachidonic acid to 15-HETE by ASA-PGHS-2 is an efficient process providing a unique mechanism among NSAIDs that will not lead to arachidonic acid accumulation or shunting to other biosynthetic pathways.

A field trial of the ParaSight-F test for the diagnosis of Plasmodium falciparum infection.

The rapid manual ParaSight-F test for Plasmodium falciparum is an antigen capture test detecting trophozoite-derived histidine rich protein II, is simple and provides a definitive diagnosis within 10 min. Compared with 913 thick blood film examinations, the ParaSight-F test had 93.4% sensitivity and 98.2% specificity. Compared with 520 blood samples within the same study examined with the aid of the polymerase chain reaction, the ParaSight-F test had 91.6% sensitivity and 99.4% specificity. The ParaSight-F test could be a valuable diagnostic tool for falciparum malaria in any situation requiring rapid diagnosis in the absence of microscopical examination.

Arginine 120 of prostaglandin G/H synthase-1 is required for the inhibition by nonsteroidal anti-inflammatory drugs containing a carboxylic acid moiety.

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer.

Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.