Evaluation of the efficacy and safety of the combination of gemcitabine and nedaplatin for elderly patients with advanced non-small-cell lung cancer.

OBJECTIVE: The aim of the present study was to retrospectively assess the safety and efficacy of the combination of gemcitabine and nedaplatin in elderly patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Patients ≥75 years with previously untreated NSCLC who underwent chemotherapy consisting of gemcitabine (800 mg/m(2) on days 1 and 8) and nedaplatin (80 mg/m(2) on day 1) every 3 weeks were retrospectively analyzed. RESULTS: Of the 35 patients, 28 were men and 7 were women, with a mean age of 78 years (range 75-87); 10 patients had stage IIIB disease and 25 patients had stage IV disease. The overall response rate was 45.7% (95% confidence interval 28.8-63.4). The median survival time was 14 months (range 3-44). Grade 3-4 toxicities included neutropenia in 74.3%, thrombocytopenia in 48.6%, anemia in 34.3%, hepatic dysfunction in 11.4%, and infection in 2.9%. There were no treatment-related deaths. There were no differences in response rate and survival between patients aged 75-79 years and patients ≥80 years, although grade 3-4 thrombocytopenia and anemia were significantly more frequent in patients ≥80 years. CONCLUSION: Our results suggest that the combination of gemcitabine and nedaplatin is effective and well tolerated for selected elderly patients with advanced NSCLC.

Stimulating effect of MDP and its adjuvant-active analogues on guinea pig fibroblasts for the production of thymocyte-activating factor.

Synthetic muramyl dipeptide (MDP) could stimulate skin fibroblasts of the guinea pig to produce thymocyte-activating factor, which augments the proliferative response of thymocytes to phytohemagglutinin (PHA). Adjuvant-active analogues of MDP also stimulated fibroblasts to produce the factor, whereas adjuvant-inactive analogues failed to do so. Thus a marked parallelism was found between adjuvant activity of these compounds and the stimulating effect on fibroblasts. Thymocyte-activating factor derived from MDP-stimulated fibroblasts was found in the fraction of mol wt 30,000-60,000 by gel filtration on a Sephacryl S-200 column. Furthermore, this factor did not exhibit T cell growth factor activity.

Vaccination of experimental monkeys against Plasmodium falciparum: a possible safe adjuvant.

Owl monkeys (Aotus trivirgatus griseimembra) were effectively immunized against a human malaria parasite, Plasmodium falciparum. Two injections of antigen, primarily mature segmenters with fully developed merozoites, mixed with adjuvant (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine and liposomes) were administered intramuscularly at a 4-week interval. Approximately 2 weeks after the second vaccination, the monkeys were challenged with the homologous strain of P. falciparum. All immunized monkeys survived the challenge. The substitution of Freund's complete adjuvant is an encouraging step toward the development of an effective and safe vaccine for human malaria.

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma.

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

Toll-like receptors, NOD1, and NOD2 in oral epithelial cells.

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Conformational studies of synthetic lipid A analogues and partial structures by infrared spectroscopy.

Synthetic lipid A analogues and partial structures were analyzed and compared with natural hexaacyl lipid A from E. coli applying Fourier transform infrared spectroscopy. The investigations comprised (i) the measurement of the beta <=> alpha phase transition of the acyl chains via monitoring of the symmetric stretching vibration of the methylene groups, (ii) an estimation of the supramolecular aggregate structures evaluating vibrations from the interface like ester carbonyl and applying theoretical calculations (iii) a determination of the inter- and intramolecular conformations monitoring functional groups from the interface and the diglucosamine backbone (ester carbonyl, phosphate). The phase transition temperature Tc was found to be nearly a linear function of the number of acyl chains for most bisphosphoryl compounds indicating comparable packing density, whereas the deviating behaviour of some samples indicated a higher packing density. From the determination of the supramolecular aggregate structures (cubic, HII) of natural hexaacyl lipid A by X-ray small-angle diffraction, the existence of the same aggregate structures also for the synthetic hexaacyl lipid A was deduced from the nearly identical thermotropism of the ester carbonyl band. From this, a good approximation of the supramolecular structures of all synthetic samples was possible on the basis of the theory of Israelachvili. The analysis of the main phosphate band, together with that of the Tc data and former colorimetric results, allowed the establishment of a model of the intermolecular conformations of neighbouring lipid A/LPS molecules. The biological relevance of the findings is discussed in terms of the strongly varying biological activity (between high and no activity) of the samples.

Reduction in antiviral activity of human interferon-gamma in acidic media with reference to structural change.

The fluorescence intensity of a unique tryptophan 36 in human interferon-gamma was drastically decreased below pH 4 with a concomitant decrease of antiviral activity. The region of residues 32-42 of human interferon-gamma was found by calculation to have a low hydrophobicity together with a high helical hydrophobic moment, and the net electric charge of this region having an amphiphilic helical structure changed significantly near pH 4. These results suggest that the region of residues 32-42 plays an important role in exhibiting antiviral activity.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia.

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations 'RA with severe dysplasia (RASD)' and 'RA with minimal dysplasia (RAminiD)'. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger-Huet anomalies of mature neutrophils > or =3% and/or micromegakaryocytes (mMgk) of megakaryocytes > or =10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P=0.06; RASD vs RAEB, P=0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

New system for assessing the prognosis of refractory anemia patients.

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies >/=3% were classified as high risk (12 patients); of patients without pseudo-Pelger-Huët anomalies >/=3%, those with intermediate/poor karyotype according to IPSS, Hb /=10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype.

Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy.

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1(+)), while 35.8% showed homozygous deletions of GSTT1 (GSTT1(-)). The GSTT1(-) group had a worse prognosis than the GSTT1(+) group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1(-) was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1(-) group than the GSTT1(+) group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Lower expression of activating transcription factors 3 and 4 correlates with shorter progression-free survival in multiple myeloma patients receiving bortezomib plus dexamethasone therapy.

Bortezomib (BTZ), a proteasome inhibitor, is widely used in the treatment of multiple myeloma (MM), but a fraction of patients respond poorly to this agent. To identify factors predicting the duration of progression-free survival (PFS) of MM patients on BTZ treatment, the expression of proteasome and endoplasmic reticulum (ER) stress-related genes was quantified in primary samples from patients receiving a combination of BTZ and dexamethasone (BD). Fifty-six MM patients were stratified into a group with PFS<6 months (n=33) and a second group with PFS⩾6 months (n=23). Of the 15 genes analyzed, the expression of activating transcription factor 3 (ATF3) and ATF4 was significantly lower in patients with shorter PFS (P=0.0157 and P=0.0085, respectively). Chromatin immunoprecipitation analysis showed that these ATFs bind each other and transactivate genes encoding the pro-apoptotic transcription factors, CHOP and Noxa, which promote ER stress-associated apoptosis. When either ATF3 or ATF4 expression was silenced, MM cells partially lost sensitivity to BTZ treatment. This was accompanied by lower levels of Noxa, CHOP and DR5. Thus low basal expression of ATF3 and ATF4 may attenuate BTZ-induced apoptosis. Hence, ATF3 and ATF4 could potentially be used as biomarkers to predict efficacy of BD therapy in patients with MM.

Chemical structure and biological activity of a lipid A component from Helicobacter pylori strain 206.

The chemical structure of a lipid A, which was obtained as a minor component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta-(1 -6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid, (R)-3- hydroxyhexadecanoic acid, and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2-, 3- and 2'-positions, respectively. Compared with the other major lipid A from the same strain, which was previously reported [Suda Y, Ogawa T, Kashihara W et al. Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide. J Biochem 1997; 121: 1129--1133], the structure was very similar with one exception. An (R)-3-hydroxyhexadecanoic acid was present at the 3-position of the novel lipid A component. The structure is apparently identical to one of the proposals by Moran et al. [Moran AP, Lindner B, Walsh EJ. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol 1997; 179: 6453--6463], who concluded the same structure as the so-called major lipid A from the H. pylori strain NCTC 11637 but without isolating a homogeneous component. The endotoxic properties and pro-inflammatory cytokine-inducing activities of this novel tetra-acyl type lipid A were lower than those of previously reported major tri-acyl type lipid A.

Preclinical increase in activity of muscle microsomal trypsin-like protease in murine muscular dystrophy, C57BL/10-mdx.

Preclinical alterations of protease activities in skeletal muscles from 10-day-old dystrophic mouse, C57BL/10-mdx, were examined by using 10 fluorogenic peptide substrates. Among the activities tested, only Boc-Val-Pro-Arg-MCA-hydrolyzing enzyme of the muscle microsomes showed an about 6-fold higher level of activity in mdx mouse. The increase in activity was not observed in tissues other than skeletal muscle. The enzyme had a pH optimum between 8.5 and 11.0, and was inhibited with DFP and variety of trypsin inhibitors. The enzymatic activity transiently increased at 1-2 weeks of age, the preclinical or very early stage of the disease. These results imply that the increased level of a trypsin-like protease possibly present in muscle microsomes may be closely related to the manifestation of muscular dystrophy.

The oligosaccharides in a recombinant hepatitis B virus surface antigen (HBsAg) carrying the pre-S2 region derived from yeast.

The N- and O-linked oligosaccharides in a yeast-derived HBsAg M protein (pre-S2 + S) were analyzed. Two major structures of the N-linked oligosaccharides bound to residue Asn4 were determined to be high-mannose type oligosaccharides, Man7GlcNAc2 and Man8GlcNAc2, by two-dimensional sugar mapping of the corresponding pyridylamino oligosaccharides. Peptide mapping of the M protein, sequence analysis of the glycopeptides after beta-elimination under reducing conditions and sugar-composition analysis revealed that the O-linked oligosaccharides were composed solely of mannose and bound to residue Ser5, Thr6, Thr7, Ser27, Ser28, Ser29 and Thr31 in the pre-S2 region.

Elevated promotion of prostacyclin production by synthetic lipid A analogs in aged human endothelial cells in culture.

We examined the effects of E. coli lipopolysaccharide (LPS) and synthetic analogs of lipid A, a bioactive moiety of LPS, on the prostacyclin (PGI2) production by young and old human endothelial cells in vitro. PGI2 production by endothelial cells has been shown to decrease during in vitro cellular senescence as well as in vivo. LPS and all the analogs tested in this study did not stimulate PGI2 production by young endothelial cells more than twofold. However, LPS and the majority of the lipid A analogs examined stimulated the PGI2 production by old cells more than twofold (approximately two- to sixfold). These results indicate that the responses to certain stimuli sometimes differ markedly between young and old cells, and this should be carefully considered when evaluating the biological effects of various compounds. Furthermore, these results suggest that certain synthetic lipid A analogs can be used as drugs to prevent some age-related vascular diseases.

Radiotherapy for carcinoma of the uterine cervix by remotely controlled afterloading intracavitary system with high-dose-rate.

Between January 1978 and June 1985 seventy-nine patients with carcinoma of the uterine cervix were treated by a remotely controlled afterloading system (RALS) with high-dose-rate irradiation following external beam therapy. It was given once a week with a dose per fraction of 6 Gy to point A, 3 to 6 times, using applicators equipped with 1 Ci 60Co bead in each ovoid and two 1 Ci 60Co beads in a tandem 1 cm apart. Five-year survival rates were 85, 68, and 51 percent in Stages I, II, and III, respectively. Recurrences were noted in 12 patients (20%) with a minimum follow-up of 2 years: 4 patients in the parametrium, 2 in the central area, 5 in both the parametrium and central areas, and 5 in the para-aortic lymph node. Complications were noted in 13 patients (22%): temporary rectal bleeding in 7, stenosis of the sigmoid in 2, recto-vaginal fistula in 1, ileo-sigmoidal fistula in 1, and perforation of the intestine in 2 patients. The treatment results of the high-dose-rate schedule for RALS appear to be comparable to those of conventional low-dose-rate systems reported by others. Therefore a fraction dose of 6 Gy to point A once a week by RALS should be considered to be practical, economical, and acceptable to patients.

Age factor relevant to the development of radiation pneumonitis in radiotherapy of lung cancer.

The significance of age factor for the development of radiation pneumonitis is evaluated in 62 patients with lung cancer between 1977 and 1985. The younger group consists of those less than 70 years old and the elderly group of those 70 years old or more. Radiation doses ranged from 1.5 to 2 Gy, 3 to 5 times per week, therefore the delivered doses were converted to nominal single doses (rets dose). Severe radiation pneumonitis was more often observed in the elderly than in the younger regardless of radiation field size and chemotherapy (n.s.). The onset of radiation pneumonitis occurred earlier in a field size of 90 sq cm or more than in that of less than 90 sq cm in both age groups; there was no significant difference between the two age groups in each field size. The pneumonitis was more frequently noted with increasing rets dose in both age groups (n.s.) regardless of field size and chemotherapy. It is concluded that there is no significant difference in the development of radiation pneumonitis between the younger group and the elderly group, but the pneumonitis is inclined to be more severe in the latter.