A phase II study of temsirolimus added to low-dose weekly carboplatin and paclitaxel for patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).

Background: Activating events along the PI3K/mTOR pathway are common in head and neck squamous cell carcinomas (HNSCC), and preclinical studies suggest additive or synergistic effects when combining mTORC1 inhibitors with carboplatin and paclitaxel chemotherapy. Patients and methods: In this single-institution phase II study, the combination of temsirolimus 25 mg, carboplatin AUC 1.5, and paclitaxel 80 mg/m2 administered on days 1 and 8 of a 21-day cycle was evaluated in 36 patients with recurrent and/or metastatic (R/M) HNSCC. The primary end point was objective response rate after two cycles of treatment. Secondary end points include the safety and tolerability profile and overall survival. Correlative studies with exome mutational analysis were performed in pre-treatment biopsy samples from 21 patients. Results: Fifteen (41.7%) patients had an objective response, which were all partial responses, and 19 (52.3%) patients had stable disease as best response. The two patients who were designated as 'non-responders' were removed from study prior to two cycles of treatment, but are included in the efficacy and safety analyses. The median duration on study was 5.3 months and the median progression-free survival and overall survival were 5.9 months (95% confidence interval, 4.8-7.1) and 12.8 months (95% confidence interval, 9.8-15.8), respectively. The most common grade 3 and 4 adverse events were hematologic toxicities. Three (3.8%) patients developed neutropenic fever on study. Three of four patients with PIK3CA mutations experienced tumor regressions, and responses were also seen in patients with other genetic alterations in the PI3K/mTOR pathway. Conclusion: The combination of temsirolimus with low-dose weekly carboplatin and paclitaxel appears to have meaningful clinical efficacy in the treatment of R/M HNSCC. This regimen has a relatively high response rate compared to other treatments evaluated in R/M HNSCC, and potential associations with genetic alterations in the PI3K/mTOR pathway should be further explored.

Development of metronidazole-resistant lines of Blastocystis sp.

Metronidazole (MTR) is frequently used for the treatment of Blastocystis infections, but with variable effectiveness, and often with treatment failures as a possible result of drug resistance. We have developed two Blastocystis MTR-resistant (MTR(R)) subtype 4 WR1 lines (WR1-M4 and WR1-M5), with variable susceptibility to a panel of anti-protozoal agents including various 5-nitroimidazoles, nitazoxanide and furazolidone. WR1-M4 and WR1-M5 were developed and assessed over an 18-month period and displayed persistent MTR resistance, being more than 2.5-fold less susceptible to MTR than the parent isolate. The MTR(R) lines grew with a similar g time to WR1, but were morphologically less consistent with a mixture of size. All Blastocystis isolates and the MTR(R) lines were most susceptible to the 5-nitroimidazole drug ronidazole. WR1-M5 was apparently cross-resistant to satranidazole and furazolidone, and WR1-M4 was cross-resistant to nitazoxanide. These MTR(R) lines now provide a valuable tool for the continued assessment of the efficacy and mechanism of action of new and established drugs against a range of Blastocystis sp. subtypes, in order to identify a universally effective drug and to facilitate understanding of the mechanisms of drug action and resistance in Blastocystis.

Sequence map of the 2 Mb Giardia lamblia assemblage A chromosome.

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.org) has identified 4 scaffolds (CH991771, CH991780, CH991782, and CH991767) belonging to the 2 Mb, Assemblage A, chromosome. Because of the density of probe sequences, we have been able to predict the orientation of the scaffolds and have identified erroneous inclusions in scaffold CH991767. Exclusion of erroneously included sequences resulted in a 1.96 Mb chromosome sequence. This study brings together experimental data and the GiardiaDB data to compile the sequence of a whole chromosome.

Orally administered Giardia duodenalis extracts enhance an antigen-specific antibody response.

We have identified novel adjuvant activity in specific cytosol fractions from trophozoites of Giardia isolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281-284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments when Giardia extract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent assay to be comparable to those generated by the "gold standard," mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.

Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal.

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha-AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein.

A line of FVB (H-2q) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development of inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive ("ignorant") to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups of young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2q background of these mice, incorporation of a Db allele into the genome allowed comparison of Db-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization.

An event-related potential investigation of posthypnotic recognition amnesia.

Forty-two individuals selected for high hypnotizability or for low hypnotizability were taught lists of words during hypnosis and assessed for recognition following hypnosis using event-related potential (ERP) procedures, both before and after the cue to reverse amnesia. A subgroup of low-hypnotizable participants were asked to simulate hypnotic behavior. All participants had larger late positive component (LPC) amplitudes to learned than to unlearned words, regardless of whether amnesia was reported. The highly hypnotizable participants who reported recognition amnesia, however, had significant changes in attention-related (P1 and N1) and recognition-related (N400 and LPC) ERP component amplitudes as a function of whether amnesia was reported. These data suggest that posthypnotic amnesia may involve alterations in the processes of attention, selection, and accessibility.

Immunological responses in human papillomavirus 16 E6/E7-transgenic mice to E7 protein correlate with the presence of skin disease.

The human papillomavirus (HPV) oncogenes, E6 and E7, are believed to contribute to the development of cervical cancers in women infected with certain HPV genotypes, most notably HPV-16 and HPV-18. Given their expression in tumor tissue, E6 and E7 have been implicated as potential tumor-specific antigens. We have examined an HPV-16 E6- and E7-transgenic mouse lineage for immune responses to these viral oncoproteins. Mice in this lineage express the HPV-16 E6 and E7 genes in their skin and eyes, and on aging, these mice frequently develop squamous cell carcinomas and lenticular tumors. Young transgenic mice, which had measurable E7 protein in the eye but not in the skin, were immunologically naive to E7 protein. They mounted an immune response to E7 on immunization comparable to that of nontransgenic controls, suggesting a lack of immune tolerance to this protein. Older line 19 mice, which are susceptible to skin disease associated with transcription of the E6 and E7 open reading frames, had measurable E7 protein in their skin. These older transgenic mice spontaneously developed antibody responses to endogenous E7 protein, particularly in association with skin disease. Also detected in older mice were delayed-type hypersensitivity responses to E7. These finding parallel the humoral immune response to E7 protein in patients with HPV-associated cervical cancer and suggest that line 19 mice may provide a model for studying the immunobiology of HPV-associated cancers.

Human papillomavirus (HPV) type 18 E7 protein is a short-lived steroid-inducible phosphoprotein in HPV-transformed cell lines.

We used a capture ELISA to quantify the E7 protein of human papillomavirus type 18 (HPV-18). In HeLa cells, which express low levels of immunoreactive E7 protein (iE7), iE7 had a mean half-life of 13.5 min. In HPV-18 E7 recombinant baculovirus (E7rec BV)-infected Sf21 cells, which express higher levels of E7, the half-life of iE7 was much longer (90 min and > 24 h, with two different E7rec BVs). For two transformed human cervical cell lines expressing HPV-18 E7, exposure of the cells to hydrocortisone resulted in a twofold increase in steady-state levels of the E7 protein: no similar effect was observed with progesterone, oestrogen or testosterone. The half-life of iE7 was unaltered by hydrocortisone or progesterone exposure. An immunoassay which distinguished Ser33-phosphorylated E7 from E7 not phosphorylated at this residue (Ser33dephospho-E7), showed that in HeLa and Sf21 cells the majority of E7 was phosphorylated: the half-life of both species of E7 was similar in HeLa cells, but the half-life of Ser33dephospho-E7 was much shorter (90 min) in Sf21 cells than that of Ser33phospho-E7 (> 24 h). A HeLa-fibroblast fusion cell line with tumorigenic potential (CGL-1) had a similar ratio of dephospho-E7 to total E7 (0.06), as a similar fusion cell line (CGL-4) with no tumorigenic potential (0.03). We conclude that E7 is a labile phosphoprotein, and that the expression and steady-state level of the E7 protein in eukaryotic cells may be influenced by the hormonal environment of the cells.

Occupational stress amongst care staff working in nursing homes: an empirical investigation.

A questionnaire survey of care staff in nursing homes examined staff stress. Staff completed questionnaires covering Type A behaviour, job satisfaction, psychological well-being, relaxation behaviour, coping skills and demographic details. A specific measure of nursing home stress was developed following a pilot study. From a total sample of 375, 112 (30%) responses were obtained. On the stress questionnaire the major stressors were found to be 'unsatisfactory wages', 'shortage of essential resources', 'not enough staff per shift', 'feeling undervalued by management', 'lifting heavy patients' and 'working with colleagues who are happy to let others do the work'. Factor analysis of stress questionnaire responses identified five major stress groupings. These were, 'differing expectations about patient care', 'management factors', 'lack of support from other staff', 'feeling inadequately trained to deal with job demands' and 'home-work conflicts'. Examination of stress outcomes showed that many staff were under pressure, with high levels of smoking and alcohol intake. Given the increasing importance of nursing home care for the elderly the present study is timely. The implications of the findings for further research, and for the training of staff in nursing homes are considered.

Effects of antipsychotic drugs on latent inhibition: sensitivity and specificity of an animal behavioral model of clinical drug action.

Latent inhibition (LI) of a conditioned emotional response (CER) has been proposed as a quantitative measure of selective attention. We have assessed the parallels of the pharmacology of LI in rats with the clinical pharmacology of schizophrenia. Drug and vehicle treated rats were divided into groups and preexposed 20 times to cage illumination as a CS, or not preexposed. All groups were conditioned with 2 CS-footshock pairings. The following day CER, as measured by interruption of drinking in response to CS presentation, was recorded. LI was observed as a decreased CER in preexposed relative to non-preexposed animals. LI was enhanced by haloperidol 0.3 mg/kg after 7 or 14 daily treatments, but not after a single acute dose. Haloperidol doses of 0.3 and 0.03 mg/kg enhanced LI, while doses of 0.003 and 3.0 mg/kg had no effect. Haloperidol enhancement of LI was unaffected by the coadministration of the anticholinergic agent trihexyphenidyl. Enhancement of LI is exhibited by the antipsychotic drugs fluphenazine, chlorpromazine, thiothixene, thioridazine, mesoridazine, and metoclopramide but not clozapine. The non-antipsychotic drugs pentobarbital, imipramine, chlordiazepoxide, trihexyphenidyl, and promethazine failed to enhance LI. LI exhibits striking parallels to the clinical pharmacology of schizophrenia.

An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.

ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.

Protein and DNA evidence for two demes of Blastocystis hominis from humans.

Analysis of 10 stocks of Blastocystis hominis isolated from human stools revealed two discrete groups of organisms. Proteins of the two groups were immunologically distinct and hybridization with random probes generated from the DNA of one stock showed that the DNA content of the two groups was different. Further studies are required to determine whether these should be classified as discrete species or whether these groups are epidemiologically significant.

The in-vitro activity of drugs against Blastocystis hominis.

The development of an assay to measure the sensitivity of drugs against Blastocystis hominis using the incorporation of 3H-hypoxanthine is described. The activity of 42 compounds have been measured. Four of the 5-nitroimidazoles tested (satranidazole, S75 0400 A, flunidazole and ronidazole) were found to be more active than metronidazole, the drug commonly used to treat infections caused by B. hominis in humans. Other potentially useful compounds include emetine, furazolidone and quinacrine. Ketoconazole and iodoquinol reported to have therapeutic activity in infections caused by this parasite were found to be significantly less active than metronidazole.

Chromosomes of Blastocystis hominis.

Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to greater than 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.

Endocytosis in cultures of Blastocystis hominis.

A study of the function of the electron-dense pits in the vacuolar and granular forms of Blastocystis hominis was undertaken. Immuno-electron microscopy using anti-clathrin antibody and colloidal gold demonstrated clathrin to be associated with all forms of the pits and some cytoplasmic vesicles. Cationized ferritin traced the pathway of endocytosis from the surface of the coated pits through internalization via electron-dense coated vesicles and uncoated vesicles and tubules in the cytoplasm. The cationized ferritin particles accumulated in the central vacuole, suggesting a metabolic or storage role for this structure. Differences in the accumulation of cationized ferritin particles were noted between vacuolar and granular forms. The hydrolytic enzyme acid phosphatase was not detected within the central vacuole suggesting that this structure does not act as a lysosome.

Respiratory epithelial permeability after cigarette smoke exposure in guinea pigs.

The purpose of this study was to determine the pathology of cigarette smoke-increased permeability at the bronchioalveolar junction of the guinea pig. After exposure to either smoke or room air, guinea pigs were anesthetized and fluorescein isothiocyanate-dextran (FITC-D, mol wt 10,000) was aerosolized into their lungs. Blood samples taken through a carotid arterial cannula were analyzed by gel chromatography and spectrofluorometry for the presence of FITC-D. The results confirmed that, after smoke exposure, increased amounts of intact FITC-D molecules with a reported Einstein-Stokes radius of 22.2 A crossed the respiratory epithelium into the vascular space. Transmission electron-microscopic studies showed that the FITC-D diffused across damaged type I pneumocyte membranes and cytoplasm to reach the basal lamina and entered the alveolar capillaries through endothelial tight junctions. Damage to the alveolar epithelium was more frequent for the smoke-exposed animals than the room air-exposed animals (P less than 0.05). We conclude that smoke exposure damages type I cells and that inhaled FITC-D crosses the epithelial barrier at damaged type I cells of the bronchioloalveolar junctions.

Ultrastructural variation of Blastocystis hominis stocks in culture.

An ultrastructural study of 10 different Blastocystis hominis stocks was undertaken. Three distinct morphological forms, vacuolar, granular and amoeboid, were distinguished. Numerous variations in the organelles and general cell structure were observed between stocks. B. hominis displayed considerable size variation in the vacuolar forms, ranging from 4 to 63 micron. Thickness and density of the surface coat varied between different stocks. Beneath the surface coat the bilaminar cell membrane displayed electron-dense pits. The nature and quantity of the vacuolar contents varied, and in the granular form four morphologically different inclusions were seen. The organelles which showed the greatest variation between stocks were the mitochondria, varying in shape, electron-density, type of cristae and presence of inclusions. There was minimal variation between stocks with regard to endoplasmic reticulum, Golgi complex and nuclei. Budding of material between the cytoplasm and central vacuole was observed in some stocks. Indications of phagocytic behaviour of B. hominis were seen in the amoeboid form and in the vacuolar form of one stock.

The use of the internal perimeter to compare airway size and to calculate smooth muscle shortening.

Previous studies from our laboratory suggest that the internal airway perimeter (Pi) defined by the folded epithelial surface remains constant as the airways narrow. To test this hypothesis, we treated adjacent slices of resected lung lobes with either theophylline or carbachol and determined the dimensions of the airways in these lung slices. Transverse sections of contracted (n = 58) and relaxed (n = 55) airways were used to measure the Pi defined by the epithelial surface, lumen area (Ai), external perimeter (Pe) defined by the outer edge of the smooth muscle layer, and the external area (Ae). Wall area (WA = Ae - Ai) was calculated. The frequency distribution of internal perimeters was not significantly different for the contracted and relaxed airways, and when the square root of wall area was plotted against Pi, the regression lines for the contracted and relaxed airways were almost identical. The "relaxed" external perimeter was calculated Per = square root Pi2 + (4 pi WA), and the percentage of muscle shortening (PMS) was determined: PMS = [(Per - Pe)/Per] x 100. We conclude that Pi and WA are constant in airways whether the smooth muscle is relaxed or contracted and that Pi can be used as a marker of airway size and, under controlled conditions, can be used to calculate the smooth muscle shortening present in a given airway.