Open, randomized, multi-center phase II study comparing efficacy and tolerability of Erlotinib vs. Carboplatin/Vinorelbin in elderly patients (>70 years of age) with untreated non-small cell lung cancer.

BACKGROUND: Targeting the epidermal-growth-factor-receptor (EGFR) in non-small cell lung cancer (NSCLC) is an established treatment option with less toxicity compared to conventional chemotherapy. This study was undertaken to determine whether Erlotinib is non-inferior compared to chemotherapy as a first-line therapy in unselected elderly patients. MATERIALS AND METHODS: Patients ≥ 70 years with untreated, metastatic NSCLC were randomized to Erlotinib (E), 150 mg/day or Carboplatin (AUC5) plus Vinorelbine (25mg/m(2) on days 1 and 8) every three weeks (CV). Primary endpoint was progression-free survival (PFS). After progression, crossover was strongly recommended. Secondary endpoints were duration of response, 1-year survival, overall survival (OS), response rate (RR), quality of life (FACT-L), assessment of comorbidities by simplified comorbidity score (SCS) and Charlsons' comorbidity score, safety and assessment of molecular markers. RESULTS: Between June 2006 and August 2008 284 pts were randomized to E (144) and CV (140). PFS was significantly inferior with E (median PFS 2.4 versus 4.6 months [HR 1.6, 75% CI 1.22-2.09, p: 0.0005]) as well as RR (7.8% v 28.3%, p: 0.0001). No significant difference in OS appeared (median E: 7.3 months versus CV: 8.4 months, HR: 1.24 [75% CI 0.9-1.71]). In never smokers PFS (median PFS: 3.7 v 4.3 m, E v CV, HR 0.72, 75% CI 0.35-1.48) and OS (median: 16.5 versus 17 months, HR 0.99 [75% CI 0.38-2.57]) were comparable. More skin toxicity and diarrhea was seen with E compared to more myelotoxicity, neurotoxicity and constipation with CV. Less severe adverse events were observed with E (81 v 102, E v CV). CONCLUSION: CV had an increased efficacy compared with E in an unselected population of elderly patients with advanced NSCLC.

[Treatment of small-cell lung cancer]. / Therapie des kleinzelligen Lungenkarzinoms.

Treatment of small cell lung cancer (SCLC) is based on the stage of disease. While combination of chemo- and radiotherapy preferably as concomitant chemoradiotherapy represents standard treatment in patients with locally advanced tumors (UICC stage I-III), patients with metastatic disease (stage IV) should be treated with an established platinum based chemotherapy regimen. After chemotherapy and in case of an achieved tumor response treatment should be completed by an adjuvant radiation of the brain in patients with adequate performance status. In patients with a very early stage of disease without involvement of lymph node metastasis a surgical approach in combination with an adjuvant chemotherapy can be discussed. In patients with relapsed tumors second line therapies like the topoisomerase I inhibitor Topotecan have proven efficacy. Up to now neither molecular targeted therapies nor cytotoxic or immunological maintenance strategies have provided any benefit to patients with SCLC.

Mice deficient in the interferon type I receptor have reduced REM sleep and altered hypothalamic hypocretin, prolactin and 2',5'-oligoadenylate synthetase expression.

We report that mice with a targeted null mutation in the interferon type I receptor (IFN-RI), which cannot respond to such IFNs as IFNalpha and IFNbeta, have a 30% reduction in time spent in spontaneous rapid eye movement sleep (REMS) as a consequence of a reduced number of REMS episodes. Time spent in nonrapid eye movement sleep (NREMS) was essentially unaltered in IFN-RI knockouts (KOs) compared to 129 SvEv controls. Body temperature and locomotor activity were similar in both strains of mice. Hypothalamic expression of mRNAs for molecules previously linked to sleep-wake regulation and an IFN-inducible antiviral gene, 2',5'-oligoadenylate synthetase 1a (OAS), were determined by real-time reverse-transcriptase polymerase chain reaction (RT2-PCR). The level of hypocretin A mRNA was elevated in IFN-RI KO mice compared to 129 SvEv mice, while prolactin mRNA and OAS mRNA levels were suppressed. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) mRNA levels were unchanged relative to controls. Serum prolactin levels were similar in both strains. Results are consistent with the hypothesis that increased hypocretin and reduced prolactin in the hypothalamus of IFN-RI KO mice are responsible for their reduced REMS. In addition, the reduced OAS expression may result in modulation of prolactin receptor signaling and thus contribute to suppression of REMS.

1,25-dihydroxycholecalciferol stimulates ICAM-1 expression of human alveolar macrophages in healthy controls and patients with sarcoidosis.

Synthesis and release of 1,25-dihydroxycholecalciferol (1, 25-(OH)2D2) by alveolar macrophages (AM) have been shown to be increased in granulomatous lung disease. ICAM-1 plays a major part in leukocyte homing to sites of chronic inflammation, which is a crucial step during the inflammatory response. Whether 1,25-(OH)2D2 alters the ICAM-1 expression of AM in humans has not been studied. Bronchoalveolar lavage (BAL) was performed in 12 healthy volunteers, in 13 patients with sarcoidosis (active disease n = 8, inactive disease n = 5), and in 9 patients with chronic bronchitis. AM were incubated with different concentrations of 1,25-(OH)2D2 (10(-11) to 10(-6) M) with and without priming with interferon-gamma (IFN-gamma) and with and without preincubation with 10(-8) M dexamethasone. In addition, the metabolites of vitamin D, 24, 25-dihydroxycholecalciferol and 25-hydroxycholecalciferol, were used. The AM expression of ICAM-1 (cELISA) and the release of tumor necrosis factor-alpha (TNF-alpha) (bioassay) by AM were determined. In healthy volunteers the ICAM-1 expression on AM was significantly and dose-dependently increased by 1,25-(OH)2D2, but not by 24, 25-dihydroxycholecalciferol and 25-hydroxycholecalciferol. Priming with IFN-gamma resulted in an additive effect. Preincubation with dexamethasone inhibited ICAM-1 expression. Addition of 1,25-(OH)2D2 after inhibition by dexamethasone increased ICAM-1 expression significantly. TNF-alpha secretion of AM from healthy volunteers was significantly reduced by 1,25-(OH)2D2. In sarcoidosis patients ICAM-1 expression was significantly higher compared with healthy volunteers. Incubation with 1,25-(OH)2D2 resulted in a further significant increase of ICAM-1 expression. TNF-alpha secretion of AM was increased compared with healthy volunteers. 1,25-(OH)2D2 reduced TNF-alpha secretion; however, this difference was not significant. 1, 25-(OH)2D2 has an immunomodulating effect on human AM both in healthy volunteers and in sarcoidosis patients with enhanced expression of ICAM-1. It may serve as an autocrine mediator in inflammatory lung disease.

Host nuclear abnormalities and depletion of nuclear antigens induced in Trichinella spiralis-infected muscle cells by the anthelmintic mebendazole.

Infection by the parasitic nematode Trichinella spiralis induces cell cycle repositioning (chronic suspension in apparent G2/M) and genetic reprogramming in differentiated mammalian skeletal muscle cells. These changes occur in association with dramatic enlargement of infected host cell nuclei (as large as 17 microm in diameter) and nucleoli. Nuclear antigens (NA) that colocalize with host chromatin have been detected by antibodies to T. spiralis antigens, but the functions of these NA are unresolved. Mebendazole (MBZ) preferentially binds parasite versus host beta-tubulins, is implicated in inhibiting secretion in nematodes and induces cytoplasmic changes in muscle cells infected with T. spiralis. These infected cell changes might be indirect via MBZ inhibition of parasite secretions. This effect would have implications for host/parasite interactions and was evaluated here. MBZ treatment of chronically infected mice caused: (1) a significant deformation of host nuclei and diminution of nucleoli by 4 and 6 days of treatment (dot), respectively; (2) a reduction of nuclear lamins A/C in infected cell nuclei that was concomitant with nuclear deformation; and (3) significant reductions in total RNA, general protein and acid phosphatase activity levels. These changes were associated with the depletion of NA from host nuclei detected by 4 dot. However, DNA content of infected cell nuclei was not detectably reduced and muscle gene expression was not reactivated. The cellular changes documented are likely to account for previously described cytoplasmic alterations induced by MBZ. Concomitant depletion of NA from infected cell nuclei suggests a role of these products in regulating nuclear functions of host cells.

Interaction of Chlamydia pneumoniae and human alveolar macrophages: infection and inflammatory response.

The obligate intracellular pathogen Chlamydia pneumoniae is associated with chronic respiratory, atherosclerotic, and rheumatic disease. The alveolar macrophage (AM) is a potential target cell for the pathogen and may contribute to respiratory immunopathology. We therefore investigated in vitro the interaction between chlamydiae and macrophages with cocultures of C. pneumoniae and AM from 12 healthy volunteers. Inflammatory responses were evaluated through lucigenin-amplified chemiluminescence; secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin 8 (IL-8); and expression of intercellular adhesion molecule-1 (ICAM-1) and human leukocyte antigen-DR (HLA-DR). C. pneumoniae readily induced productive infection in the AM. Inclusions containing replicating pathogens could be maintained for up to 120 h. Morphologically similar infection patterns were seen ex vivo in AM collected from six patients with known C. pneumoniae pneumonia. AM responded to the infection with a marked, dose-dependent release of reactive oxygen species, TNF-alpha, IL-1beta, and IL-8. ICAM-1 expression remained unchanged, but HLA-DR was significantly upregulated. Our data indicate that the release of antimicrobial mediators cannot prevent chlamydial infection and replication in AM, but may be involved in amplification of the local inflammatory response in C. pneumoniae pneumonia.

Role of interleukin-8 in community-acquired pneumonia: relation to microbial load and pulmonary function.

In pneumonia local phagocyte activation is crucial for clearing of pathogenic microorganisms. In this context alveolar macrophage interleukin-8 secretion, phagocyte oxidative response and concentrations of lavage proteins were quantified, including interleukin-8, in 31 patients with pneumonia, 13 age matched patients with peripheral lung consolidation and six healthy volunteers; these findings were related to the impairment of gas exchange and the bacterial load in the alveolar space. Increased interleukin-8 levels were found in bronchoalveolar lavage fluid (BALF) and in alveolar macrophage supernatants from patients with pneumonia (214 ng/10(5) AM +/- 121 vs 71 ng/10(5) AM +/- 35 and 66 ng/10(5) AM +/- 30, p < 0.05). Interleukin-8 release from alveolar macrophages correlated with the upregulated spontaneous luminol enhanced oxidative response of pulmonary phagocytes but not with the neutrophil count in BALF. In pneumonia patients a significant difference was found between patients with 10(4) or more colony forming units (CFU)/ml BALF of one pathogen and patients with less CFU or nonspecific microbiological results (261 ng/10(5) AM +/- 89 vs 179 ng/10(5) AM +/- 81 and 7.5 ng/ml BALF +/- 17 vs 0.44 ng/ml BALF +/- 1, p < 0.05). Further, a negative correlation between interleukin-8 release of alveolar macrophages and the arterial pO2 at the time of BALF could be demonstrated (r = -0.47, p < 0.05). The results demonstrate local cellular activation in community-acquired pneumonia, which is related to the bacterial load in the alveolar space and to impairment of gas exchange. This is consistent with the hypothesis that pulmonary phagocytes play a central role in the pathogenesis of bacterial pneumonia, contributing not only to bacterial clearing but also to local tissue damage.

A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes.

Two mAb, C6B6 and 7D10, each significantly reduced infection of mice by Cryptosporidium parvum and reacted with a 23-kDa glycoprotein (p23) of geographically disperse C. parvum isolates. The antibodies were used to identify plaques in a cDNA library prepared from C. parvum sporozoite mRNA. cDNA insert sequences from positive plaques were determined and used to isolate additional clones encoding p23 coding sequences. A consensus open reading frame of 333 base pairs, encoding 111 amino acids, was identified in this collection of cDNAs. The predicted amino acid sequence contained one N-glycosylation site, but lacked hydrophobic membrane spanning regions. Epitope mapping revealed that mAb 7D10 defines the linear epitope QDKPAD which occurs twice in the C terminal region of the peptide encoded by the ORF. This same C terminal peptide region contains a non-linear epitope bound by mAb C6B6. Serum from mice immunized with synthetic C terminal peptide reacted with sporozoite p23. The occurrence of neutralization-sensitive epitopes encoded by defined regions of the C. parvum genome suggests that recombinant proteins or synthetic peptides containing these epitopes may prove useful for inducing immune responses that diminish infection.

Intercellular adhesion molecule-1 (ICAM-1) is upregulated on alveolar macrophages from AIDS patients.

In acquired immune deficiency syndrome (AIDS) patients, alveolar macrophages (AMs) have an increased ability to serve as accessory cells during the generation of an immune response. In addition to soluble mediators, like cytokines, molecules of the major histocompatibility complex (MHC) class II and adhesion molecules, like intercellular adhesion molecule-1 (ICAM-1), play a major role in the regulation of these cellular interactions. Using an enzyme-linked immunosorbent assay (ELISA) technique and immunocytochemical staining, we investigated ICAM-1 and human leucocyte antigen-DR (HLA-DR) expression on AMs from 20 AIDS (HIV+) patients in context with other parameters of macrophage activation, such as tumour necrosis factor-alpha (TNF-alpha) secretion and the release of superoxide anion, comparing the results to a group of healthy volunteers. In addition, we quantified soluble ICAM-1 in the bronchoalveolar lavage fluid (BALF) using a commercially available kit. We found a nearly twofold increase in ICAM-1 expression (0.81 +/- 0.30 (SD) versus 0.42 +/- 0.12 ELISA units (EU) (mean +/- SD)), whilst the number of HLA-DR+ AMs was slightly decreased in AIDS-patients (80 +/- 5 versus 89 +/- 3%). Furthermore, soluble ICAM-1 in the BALF of these patients was significantly increased (41.9 +/- 26.1 versus 19.1 +/- 5.1 ng.ml-1). ICAM-1 levels on AMs in the patient group correlated strongly with the sodium fluoride triggered release of superoxide anion (O2-) but not with the spontaneous secretion of TNF-alpha by AMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of mononuclear cell alveolitis in pulmonary sarcoidosis.

BACKGROUND: Alveolitis in pulmonary sarcoidosis is characterised by an accumulation of highly activated macrophages and CD4+ lymphocytes in the alveolar compartment. The role of intercellular adhesion molecule 1 (ICAM-1) expression on alveolar cells has been studied in this context. METHODS: Using a sandwich ELISA technique, ICAM-1 expression on alveolar macrophages from 17 consecutive untreated patients with pulmonary sarcoidosis and six healthy normal volunteers was quantified. In addition, parameters of macrophage activation (tumour necrosis factor alpha (TNF alpha) and superoxide anion release) were evaluated. RESULTS: Significantly elevated expression could be demonstrated on alveolar macrophages from patients with pulmonary sarcoidosis compared with healthy controls (mean (SD) 0.74 (0.24) ELISA units (EU) v 0.46 (0.12) EU). On subdividing the patients into those with active and those with inactive disease, only the former showed increased ICAM-1 levels on alveolar macrophages (0.82 (0.27) EU) compared with control alveolar macrophages. No differences were detected in serum levels of soluble ICAM-1 between patients and controls. ICAM-1 expression on alveolar macrophages from patients with sarcoidosis correlated with the spontaneous release of TNF alpha but not with the release of the superoxide anion by the activated macrophages. There was no correlation with the percentage of lymphocytes or the absolute number of CD4+ cells in bronchoalveolar lavage fluid. CONCLUSIONS: Increased ICAM-1 surface expression on alveolar macrophages reflects disease activity in the pulmonary compartment. Considering the significance of adhesion molecules during antigen presentation and lymphocyte activation, ICAM-1 expression on alveolar macrophages may have an important role in the immune process of pulmonary sarcoidosis.

Differential expression of interleukin-6, intracellular adhesion molecule 1, and major histocompatibility complex class II molecules in renal carcinoma cells stimulated with S fimbriae of uropathogenic Escherichia coli.

The production of interleukin-6, intracellular adhesion molecule 1, and major histocompatibility complex class II molecules by a renal carcinoma cell line (ACHN) in response to S fimbriae of uropathogenic Escherichia coli was studied. S fimbriae adhered to ACHN cells and stimulated the production of interleukin-6 and intercellular adhesion molecule 1 but did not affect major histocompatibility complex class II expression by renal carcinoma cells. Our data demonstrate that S fimbriae of E. coli display immunomodulating properties on kidney-derived epithelial cells.

Cleavage of tumor necrosis factor-alpha by Legionella exoprotease.

The role of the major secretory protein of Legionella pneumophila, a zinc protease, in Legionella infection is not known. Since an important step of the host reaction in Legionnaires' disease is the production of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages, we studied the interaction of Legionella protease and U-937 cells with respect to TNF-alpha. The Legionella protease was purified by fractionated precipitation, gel filtration and hydrophobic interaction chromatography. The purified enzyme was added to U-937 cells, a promyelocytic cell line. In the supernatants of PMA-treated U-937 cells we found low concentrations of TNF-alpha after incubation with protease. Therefore we pursued the hypothesis of direct enzymatic degradation of TNF-alpha by Legionella protease. Enzymatic cleavage of TNF-alpha was proven by SDS-PAGE, ELISA and TNF-alpha bioassay with L-929 cells. The degradation of TNF-alpha by the Legionella protease was shown in all three systems. Enzymatic degradation of TNF-alpha might be important for the pathogenesis of Legionnaires' disease.

Adherence to and cytotoxicity of Escherichia coli for eucaryotic cell lines quantified by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).

Adherence of Escherichia coli to human epithelial cells (HEp-2) was studied using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) which is cleaved by enzymes of eucaryotic or procaryotic cells to formazan. This method allows to quantify adherence of Escherichia coli to HEp-2 cells and offers the advantage of assaying a large number of eucaryotic cells without using specific antisera or radioactive material. Furthermore, toxic effects of isolated hemolysin cloned in Escherichia coli onto a renal tubular cell line (LLC-PK1) was investigated by this method, showing reduced cellular viability of tubular cells after an incubation period of 10 to 20 min. MTT is therefore considered to be useful to assay the adherence of Escherichia coli to eucaryotic cells and to quantify toxic effects in eucaryotic cells induced by bacterial virulence factors.

Estradiol induces proliferation of peroxisome-like microbodies and the production of 3-hydroxy fatty acid diesters, the female pheromones, in the uropygial glands of male and female mallards.

During the mating season the female mallards produce sex pheromones, diesters of 3-hydroxy fatty acids, in their uropygial glands. Subcellular fractionation by sucrose and Nycodenz density gradient centrifugations and electron microscopic examination of the fractions showed that diesters of 3-hydroxy acids and the enzymes that catalyze the formation and esterification of the 3-hydroxy fatty acids are located in the catalase-containing fractions, probably peroxisomes, whereas monoester synthesizing activities are located in the endoplasmic reticulum. Fatty acyl-CoA reductase that would provide fatty alcohol needed for the synthesis of monoester and diester waxes was found both in the peroxisomal and endoplasmic reticulum fraction. Upon daily intramuscular injection of estradiol into the females in the nonmating season, the short chain monoester waxes of the uropygial glands were replaced by long chain monoester waxes, and subsequently the monoester waxes were replaced by diester waxes. Injection of thyroxine with estradiol hastened the induction of the compositional changes including diester synthesis. Similar changes, including the synthesis of the female pheromones, were induced in the uropygial glands by the hormone treatment of males that do not normally produce diesters at any time during their life cycle. The structure and composition of the diesters induced by hormone treatment of both males and females were identical to those of the female pheromones produced during their mating season. Electron microscopic examination of diaminobenzidine-treated glands showed that peroxisomes proliferated in the gland of the females in the mating season and in the estradiol-treated males that produce the diesters.

Trichinella spp.: differential expression of acid phosphatase and myofibrillar proteins in infected muscle cells.

Major alterations are induced in muscle cells infected by either Trichinella spiralis or Trichinella pseudospiralis. To investigate the response of muscle to these infections we have analyzed the expression of acid phosphatase (ACP, EC 3.1.3.2), adult skeletal muscle myosin heavy chain, and muscle tropomyosin proteins in infected mouse skeletal muscle cells. Using T. spiralis-infected cells, we provide strong evidence that the tartrate-sensitive ACP of these cells was synthesized by the infected cell and localized in lysosomes. Isoenzyme analysis indicated that the ACP activity was of host muscle cell origin and the specific activity of this ACP was 2.5 times greater than that in associated inflammatory cells. Increased ACP activity was also demonstrated in muscle cells infected by T. pseudospiralis. In synchronized muscle infections, increased ACP activity was detected at 5 days post-muscle infection for both parasites. ACP activity was further increased in infected muscle cells at later times tested. This increased infected cell ACP activity represents the earliest positive enzyme marker yet described indicating expression of the infected cell phenotype. In contrast, myofibrillar proteins were not detected in muscle cells chronically infected by T. spiralis but were detected in muscle cells infected by T. pseudospiralis. Decrease in myofibrillar protein levels was detected by 10 days post-muscle infection by T. spiralis. The data presented demonstrate significant differences and similarities in the phenotypes of muscle cells infected by these two parasites and establish criteria that could facilitate identification of parasite factors that may be involved in these phenomena.

Developmental changes in the expression of S-acyl fatty acid synthase thioesterase gene and lipid composition in the uropygial gland of mallard ducks (Anas platyrhynchos).

Developmental changes in the composition of the uropygial gland secretory lipids of the postembryonic mallard ducks (Anas platyrhynchos) were determined. During the first 3 weeks after hatching, the composition of the secretory lipids remained constant; the lipids consisted of long-chain wax esters composed of a complex mixture of n-, monomethyl, and dimethyl fatty acids esterified to n-C16 and n-C18 fatty alcohols. Afterward, as the ducks began to acquire adult feathers, short-chain wax esters composed of 2- and 4-monomethyl fatty acids began to appear with 2-methylhexanoyl and 4-methylhexanoyl as the major acyl components; esters of short-chain monomethyl fatty acids (less than or equal to C12) constituted 90% of the lipids when the ducks were 2 months old and had acquired adult plumage. The appearance of the short-chain acids in the acyl portion of the wax esters was accompanied by the appearance of S-acyl fatty acid synthase thioesterase, which can hydrolytically release short-chain acids from fatty acid synthase in the gland. Northern blot analysis showed that the gland-specific thioesterase gene transcripts began to appear in the gland only 3 weeks after hatching. The appearance of the transcripts and immunologically detectable thioesterase protein reached maximum levels 2 months after hatching, with the acquisition of the adult plumage. Thus, the developmental changes in lipid composition correlated with the changes in the level of expression of the thioesterase gene. Expression of other gland-specific genes has been previously found to begin just prior to hatching. The gland-specific thioesterase is the first case of delayed expression of a gland-specific gene.

Diesters of 3-hydroxy fatty acids produced by the uropygial glands of female mallards uniquely during the mating season.

The uropygial gland secretions produced by female mallards (Anas platyrhynchos) throughout the year were analyzed by thin-layer chromatography and combined gas-liquid chromatography and mass spectrometry. Most of the year, the secretion was composed of wax esters. With the beginning of the mating season in the middle of March, a polar component appeared which became the dominant and sole component of the secretion through April and May and as the mating season ended in June, wax esters became the sole component of the secretion. The polar components were identified to be diesters of n-C8, n-C10, and n-C12 3-hydroxy acids with n-C16 and n-C18 alcohols and n-C6 to C16 even chain acids. Immediately after the diester-producing period the female uropygial glands produced very long chain wax esters composed of fatty acids longer than C12. By the end of August, shorter chain wax esters composed of C6 and C12 acids became the dominant components of the secretion and this composition, previously considered characteristic of mallards, remained constant until March. The observed disappearance of the short chain waxes during the postnuptial period is similar to that in males. The dramatic changes in the composition of the uropygial glands similar to those observed in the female mallards during the mating season have not yet been observed in any other species.