Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Design and performance of a compact scanning transmission X-ray microscope at the Photon Factory.

We present a new compact instrument designed for scanning transmission X-ray microscopy. It has piezo-driven linear stages, making it small and light. Optical components from the virtual source point to the detector are located on a single optical table, resulting in a portable instrument that can be operated at a general-purpose spectroscopy beamline without requiring any major reconstruction. Careful consideration has been given to solving the vibration problem common to high-resolution microscopy, so as not to affect the spatial resolution determined by the Fresnel zone plate. Results on bacteriogenic iron oxides, single particle aerosols, and rare-earth permanent magnets are presented as examples of its performance under diverse applications.

Treatment of pressure ulcers in patients with declining renal function using arginine, glutamine and ß-hydroxy-ß-methylbutyrate.

The aim of this study is to examine the efficacy on healing pressure ulcers (PU) of using a supplement combination containing arginine, glutamine and ß-hydroxy-ß-methylbutyrate, which was given to two elderly patients with renal dysfunction. The PU was surgically opened, decompressed and treated by drugs. A half quantity of the defined dose of the supplement combination, with an enteral nutrition product, was administered to the patients twice a day. This combination improved the PUs, with no effect on renal function. This novel finding may provide a nutritional rationale of arginine, glutamine and ß-hydroxy-ß-methylbutyrate for PUs associated with renal dysfunction.

Structural basis for the interaction of human ß-defensin 6 and its putative chemokine receptor CCR2 and breast cancer microvesicles.

Human ß-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved ß-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and ß2-ß3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure-function relation of the hBD6-CCR2 interaction, which is a promising target for the design of novel anticancer agents.

SU-D-213CD-03: Live Video-Guided Volumetric Tracking of Respiration Motion.

PURPOSE: To introduce video surface imaging guidance in synchronization with 4D cone-beam CT (CBCT) scans, and in combination with respiration- gated or target-tracked dose delivery to treat mobile tumors, without collaterally damaging nearby critical structures. METHODS: The approach uses the concept that the integral of balanced forces over the moving surfaces is directly proportional to the lung volume changes. The respiratory motions, representing the lung volume variations, were measured with the dynamic volume under the moving surfaces of the thorax and abdomen. Sequential surface images on several patients and volunteers were acquired for the feasibility study. Respiratory motions were repeatedly measured on volunteers undertaking a quiet (normal) or a forced (deep) breath. The dynamic volume under the moving surfaces were robustly fitted with a linear trend and a trigonometric wave function that was compared with the fitted curves for target moving trajectories derived from forty 4D-CBCT scans. RESULTS: A large chest wall superior-outward movement was the unique characteristic of a forced breath that had doubled the volume variations and elongated the respiration period from quiet breath of ∼4 seconds to >6 seconds. Under a quiet breath, target motion trajectories could be easily described by single sine functions that were consistent with dynamic surface volume modeling except for having different motion amplitudes. The accuracy in synchronization of the real-time surface motion with respiration motion was within the measurement uncertainty of ∼2 mm. CONCLUSIONS: The analytical results with a hypothetical single sine platform allow us to accurately predict internal target motion with use of real-time video images. Synchronization of dynamic volume with respiratory motion appears applicable for association of 4D medical imaging with 4D videoimaging.

SU-E-T-648: Comparison of VMAT Vs Arc Treatment Plans for Patients Undergoing SBRT of Early-Stage Non-Small Cell Lung Cancer (NSCLC).

PURPOSE: To evaluate the dosimetric implications of using VMAT (Volume Modulated Arc Therapy) treatment planning techniques compared to traditional Arc therapy methods for patients undergoing SBRT (Stereotactic Body Radiation Therapy) for early-stage Non-Small Cell Lung Cancer (NSCLC). METHODS: Ten NSCLC cancer patients are planned with both VMAT and Arc techniques. The SBRT treatment plans comparison was quantified by several Dose-Volume Histogram (DVH) indicators including mean, maximum and minimum doses for GTV, ITV, PTV, OAR (Organs At Risk), and V95 (volume receiving at least 95% of the prescribed dose) for PTV. RESULTS: On average VMAT plans require for treatment delivery 16.6 ± 20.2 % more monitor units (MU) than the traditional Arc plans. The average PTV minimum, maximum and mean doses as a percentage of prescribed dose are 94.5 ± 3.9 %, 114.1 ± 3.3 % and 106.6 ± 1.6 % for VMAT vs 91.6 ± 4.4 %, 119.5 ± 5.3 % and 109.5 ± 2.5 % for the Arc technique. The V95 PTV coverage for VMAT plans range from 99.4 % to 100 % with a mean of 99.7 %, compared with a range of 96.8 % to 100 % with a mean of 99 % for the Arc plans. The maximum dose received by the lungs, spinal cord and chest wall show on average significant increases for Arc plans as opposed to VMAT plans (5.7 ± 6 % increase for lungs, 4.4 ± 9.2 % for cord and 2.4 ± 6.3 % for chest wall). The average mean doses and minimum doses for the OAR are similar for both techniques. CONCLUSIONS: The comparison of VMAT vs Arc plans for SBRT of NSCLC patients is subject to many variables, including GTV and PTV volume sizes, shape and their proximity relative to the OAR.

Genotoxic activity of Eucalyptus globulus essential oil in Aspergillus nidulans diploid cells.

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), alpha-pinene (8.9), beta-pinene (1.5), globulol (6.9), alpha-eudesmol (1.12), spathulenol (1.42), gamma-cadinene (1.45), trans-beta-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 microL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.

Evolutionary relationship between defensins in the Poaceae family strengthened by the characterization of new sugarcane defensins.

Plant defensins are small (45-54 amino acids), highly basic, cysteine-rich peptides structurally related to defensins of other organisms, including insects and mammals. Small putative proteins (MW < 10 kDa) containing eight cysteines were screened based on the sugarcane expressed sequence tag (EST) database. We selected ORFs that exhibited 25-100% similarity in primary sequence with other defensins in the NCBI database and that contained eight cysteines. This similarity is sufficient for folding prediction, but not enough for biological activity inference. Six putative defensins (Sd1-6) were selected, and activity assays showed that recombinant Sd1, Sd3 and Sd5 are active against fungi, but not against bacteria. Structural characterization, based on circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy showed that the structures of these Sds were compatible with alpha/beta proteins, a feature expected for plant defensins. Phylogenetic analysis revealed that sugarcane defensins could clearly be grouped within defensins from Poaceae family and Andropogoneae tribe. Our work demonstrates that defensins show strong conservation in the Poaceae family and may indicate that the same conservation occurs in other families. We suggest that evolutionary relationships within plant families can be used as a procedure to predict and annotate new defensins in genomes and group them in evolutionary classes to help in the investigation of their biological function.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Genotoxicity (mitotic recombination) of the cancer chemotherapeutic agents cisplatin and cytosine arabinoside in Aspergillus nidulans.

Cisplatin (cis-diamminedichloroplatinum, cis-DDP) and cytosine arabinoside (ara-C) are anticancer drugs used in the treatment of human cancer. The two chemotherapeutic drugs were tested in current research for their recombinogenic potential in diploid cells of Aspergillus nidulans. Non-cytotoxic concentrations of ara-C (0.4 and 0.8 microM) and cis-DDP (1.5, 3.0 and 6.0 microM) were strong recombinagens in A. nidulans UT448//A757 diploid strain, which induced homozygosis of recessive genetic markers, previously present in heterozygous condition. Drugs significantly increased homozygosity index (HI) values for five nutritional genetic markers when compared with those determined in the absence of anticancer drugs. Since mitotic recombination is a mechanism leading to malignant growth through loss of heterozygosity at tumor-suppressor loci, ara-C and cis-DDP may be characterized as secondary promoters of malignant neoplasia in diagnosed cancer patients, after chemotherapy treatment.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Implications of protein conformational diversity for binding and development of new biological active compounds.

The new generation of biologically active compounds developed during the 20(th) century relied on knowledge of enzymology and protein structure, and were based initially, on the understanding that protein-protein and small molecule-protein interactions occurred through a lock-and-key mechanism. Later, evidence suggested that this mechanism was usually followed by a conformational change, known as induced fit. Recent studies on protein dynamics, mainly by nuclear magnetic resonance (NMR) relaxation measurements, have shown that proteins are not structured in a unique conformation. Rather, they frequently have regions of conformational diversity. In the present review we will discuss a novel view of binding, put forward in by several research groups in the last 5 to 10 years. In the free state, protein regions displaying conformational diversity exhibit equilibria among pre-existing conformations. In the presence of a ligand, one of these conformations is stabilized, so that the ligand does not need to induce a new conformation. Upon ligand binding there is a population shift toward the bound conformational state. Conformational diversity of binding sites of several proteins has been measured and has important practical as well as thermodynamical consequences: binding sites can be mapped without prior knowledge of the ligand and also evolution of binding sites depends mostly on the free state, occurring at least partially independently of the ligand.

Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes.

The polymerase chain reaction showed high sensitivity for detecting Trypanosoma cruzi in the blood of mice, independent of clonal genotype (19, 20-T. cruzi I; 32, 39-T. cruzi II) or phase of the infection (acute or chronic).

[Ocular complications in patients infected with human immunodeficiency virus seen at the Acquired Immunodeficiency Syndrome Clinical Center].

PURPOSE: To examine the ocular complications in patients infected with human immunodeficiency virus(HIV) in Japan. METHODS: The medical records of 322 patients seen at the acquired immunodeficiency syndrome(AIDS) Clinical Center from July 1, 1997 through December 31, 1998 were reviewed, and the HIV-associated ocular complications were correlated with serum CD 4+ T-lymphocyte counts. RESULTS: Ocular complications were found in 51 patients: 35 cases with retinal microvasculopathy, 17 cases with cytomegalovirus retinitis(9 quiescent, 6 active, and 2 recurrent), and 1 case each with tuberculous uveitis, phthisis bulbi after necrotizing herpetic retinopathy, conjunctival Kaposi's sarcoma, papilledema, divergence palsy, hemianopia, and abducens palsy. Retinal microvasculopathy was present in patients with CD 4+ T-lymphocyte counts above 500/mm3, but was more common in patients with cell counts below 200/mm3. Among 6 patients with active cytomegalovirus retinitis, 5 patients had a CD 4+ T-lymphocyte count below 50/mm3 at the onset of retinitis, while one patient developed retinitis after the cell count increased to over 200/mm3 with highly active antiretroviral therapy. CONCLUSION: Cytomegalovirus retinopathy may occur in patients with a CD 4+ T-lymphocyte count of more than 200/mm3.

Treatment of malignant astrocytomas with repetitive resections: a longitudinal study.

BACKGROUND: The impact of repeated surgical resection on the survivorship of patients with malignant astrocytomas is an issue of some controversy in the medical literature. OBJECTIVES: To clarify this issue through a retrospective analysis of treatment outcomes in a brain tumor clinic. METHODS: The patient records from the Brain Tumor Clinic at Hahnemann University Hospital for the period 1988 to 2000 were reviewed. From these, 112 cases of glioblastoma multiforme and 50 cases of anaplastic astrocytoma were chosen for analysis. RESULTS: The group of patients with glioblastomas showed a median survival of 415 days. When analyzed as subgroups based on the number of surgical resections, the median survival was 393 days in the group with biopsy only, 380 days in the group with one surgical resection, and 548 days in the group with two or three resections. Using the Kaplan-Meier method to generate survival plots and the log rank test to compare groups, repeat debulking was found to be a significant predictor of survival (P = 0.173). The group of patients with anaplastic astrocytomas showed a median survival of 1,311 days. When analyzed by subgroups, the patients with biopsy only had a median survival of 544 days, those with one debulking 1,589 days and those with two or three debulkings 1,421 days. There was a trend toward increased survival with debulking and the log rank test again showed statistical significance (P = 0.1998). CONCLUSIONS: This study indicates that repeated surgical resections offer increased survival for both glioblastomas and anaplastic astrocytomas.

Toxic shock syndrome toxin-1 accelerated collagen-induced arthritis in mice.

The aim of this study was to explore the roles of toxic shock syndrome toxin-1 (TSST-1) in collagen-induced arthritis (CIA). DBA/1 mice were immunized with type II collagen (CII) and treated with TSST-1. Intraperitoneal and intravenous injections of TSST-1 aggravated CIA, enhancing its incidence and severity. CIA was accompanied by an increase in anti-CII IgG Ab levels. Intraperitoneal administration with TSST-1 enhanced IFN-gamma, TNF-alpha, and IL-4 production in DBA/1 mice. We discovered the mRNA expressions of IFN-gamma, IL-2, TNF-alpha, IL-1beta, and iNOS in spleen cells stimulated with TSST-1 in vitro. However, IL-12 and IL-4 mRNA expression were seen constitutively without stimulation. Only a little increase of IL-12 and IL-4 mRNA expression was seen at 2-3 h after treatment with TSST-1. Our experiments demonstrated that CIA was aggravated by the treatment with TSST-1, which may have induced various proinflammatory cytokines and the production of both Th1 and Th2 cytokines.

Genetic polymorphism of the MxA gene promoter and interferon responsiveness of hepatitis C patients: revisited by analyzing two SNP sites (-123 and -88) in vivo and in vitro.

We have previously reported a single nucleotide polymorphism (SNP) at nucleotide (nt) position -88 (G or T) within an interferon-stimulated response element-like sequence in the promoter region of the MxA gene, which correlated with responsiveness of hepatitis C patients to interferon. Upstream of it, we then identified another SNP (C or A at nt -123) and investigated whether this SNP also correlates with interferon responsiveness. The two SNPs showed a high linkage to each other: all the individuals having G at -88 had C at -123, and 73% of those having T at -88 had A at -123. As was expected from this observation, the SNP at -123 also exhibited a correlation with interferon responsiveness (C/C homozygotes were more frequent among nonresponders than among responders: 65% of 107 vs. 40% of 52, p = 0.0028). These in vivo data from patients were further supported by results from in vitro experiments. The MxA promoter sequence with A at -123 and T at -88 showed about 4-fold higher activity of upregulating the downstream reporter gene than that with C at -123 and G at -88, in a luciferase reporter assay.

Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi.

LuxO is the central regulator integrating the quorum sensing signals controlling autoinduction of luminescence in Vibrio harveyi. We have previously purified to homogeneity a new lux regulator, LuxT, that binds to the luxO promoter. Based on the sequence of the tryptic peptides of LuxT, degenerate oligonucleotides were designed for PCR of the genomic DNA. A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. The recombinant and native LuxT gave the same footprint binding between 117 and 149 bp upstream from the luxO initiation codon. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT was a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.

The role of Tc-99m HMPAO functional brain imaging in detection of cerebral radionecrosis.

BACKGROUND: Cerebral radionecrosis is a possible complication of brain radiation therapy in patients with primary or metastatic tumors. This retrospective study evaluated the role of Tc-99m hexamethyl propyleneamine oxime (HMPAO) scintigraphy in monitoring the effects of radiation on the brain. METHODS: Ninety-eight patients (41 female, 57 male) with a mean age of 51 years (range, 16-82 years) underwent 128 sets of single-photon emission computed tomography studies. TI-201 and Tc-99m HMPAO single-photon emission computed tomography studies were performed for tumor localization and evaluation of the effect of radiation on the cerebral cortex. Thirty concomitant neuropsychological tests and 96 anatomic imaging (computed tomography/magnetic resonance imaging) were performed. The average radiation dose was 52 Gy delivered as 1.8 to 2 Gy/fraction. Thirty-two patients received an average dose of 160 mCi of I-125 EGFr concomitantly. The average follow-up period was 34 months. Abnormalities away from the tumor site were interpreted as positive on HMPAO studies, neuropsychological testing, or anatomic imaging; otherwise, they were classified as negative. RESULTS: There were 10/45 (22%) and 75/83 (90%) abnormal HMPAO study results before and after radiation therapy. The HMPAO studies compared with neuropsychological testing showed 3/13 (23%) and 14/17 (82%) concordant abnormalities in addition to tumor site in patients pre- versus postradiation therapy. There was better concordance of HMPAO and anatomic imaging in 22/30 (76%) patients versus 24/67 (36%) patients before radiation therapy. HMPAO imaging after radiation therapy revealed significantly more perfusion abnormalities. There were significant differences between all of the aforementioned parameters. CONCLUSION Tc-99m HMPAO imaging is useful in the evaluation of the effects of radiation therapy on the brain and is highly concordant with neuropsychological testing. It is superior to anatomic studies in identifying radiation-induced changes.