Treatment-induced changes of lymphocyte subsets in patients with adenoid cystic carcinoma of the head and neck.

PURPOSE: Adenoid cystic carcinoma (ACC) of the head and neck is a rare and highly malignant tumor, characterized by perineural growth and early distant metastases. The composition of immune cells in the peripheral blood and the tumor microenvironment is critical to tumor growth and control. However, little is known about the frequency and function of the relevant immune cell subsets in this entity. METHODS: In ACC patients (n = 11) and matched healthy donors (n = 11), the frequency of peripheral blood T and B cells was measured by flow cytometry at different treatment stages of disease (24 samples). Cells were further characterized by their expression of CCR7, PD-1, CD39 and CD73. Tumor-infiltrating lymphocytes (TIL) were analyzed by immunohistochemistry for ten patients and for three patients by flow cytometry. RESULTS: CD4+ T cells had significantly lower frequency after radiotherapy (RT). All other cell frequencies, including Treg, were stable through course of the disease. In B cells, CD73 was reduced after RT. CCR7 expression on T and B cells in patients with relapse/metastases (R/M) differed significantly from patients with active disease. PD-1 remained stable. Treg were more present in TIL compared to peripheral blood. CONCLUSION: Composition of lymphocyte subgroups behaves similar to squamous cell carcinoma in the head and neck, except for Treg, which remained stable. Nevertheless, the CD4+/Treg ratio was lower after RT, which could stand for an immunosuppressive effect in these patients. Therefore, it could be beneficial treating ACC with combined RT and immunomodulatory drugs.

HER2 expression and markers of phosphoinositide-3-kinase pathway activation define a favorable subgroup of metastatic pulmonary adenocarcinomas.

OBJECTIVES: Pulmonary adenocarcinomas (ADC) can be sub-grouped based on dominant oncogenic drivers. EGFR mutations define an entity of metastatic ADC with favorable prognosis and high susceptibility to EGFR tyrosine kinase inhibition. In contrast, the clinical impact of additional ERBB family members in ADC is less defined. To this end we prospectively studied HER2 expression, gene amplification, and mutation in relation to outcome of patients with advanced or metastatic ADC. MATERIALS AND METHODS: Diagnostic tumor biopsies from 193 sequential patients with stage III/IV ADC were prospectively studied for HER2 expression by immunohistochemistry (IHC). Cases with IHC scores 2+ or 3+ were analyzed by HER2 chromogenic in situ hybridization (CISH), and sequencing of HER2 exons 20 and 23. Additional prospectively determined biomarkers included PTEN, cMET, pAKT, and pERK expression, KRAS, EGFR, BRAF and PIK3CA mutations, and ALK fluorescence ISH (FISH). RESULTS AND CONCLUSION: HER2-IHC was feasible in 176 (91.2%) cases. Of 53 (30%) cases with IHC scores 2+/3+, 45 (85%) could be studied by CISH and 34 (64%) by sequencing. The lower number of HER2-mutational analyses resulted from exhaustion of tumor tissue and DNA following mutational analysis of KRAS, EGFR, BRAF and PIK3CA. HER2 amplification was detected in 4 cases (2.3%), while no mutation was found. HER2 expression correlated with expression of pAKT and cMET. Expression of HER2 and pAKT was associated with favorable overall survival in stage IV disease. HER2-expressing ADC more frequently harbored KRAS mutations, while HER2 expression was absent in all 4 cases with BRAF mutation. HER2-IHC was not predictive of HER2 gene amplification or mutation, which both were rare events in prospectively studied patients with advanced or metastatic ADC. Expression of HER2 and pAKT define a population of patients with stage IV ADC with a distinct disease course, who could benefit from specifically tailored pharmacotherapies.

Rehabilitation after primary total hip replacement. Comparison between Italian and international protocols.

AIM: In intensive rehabilitation departments (Code 56) there is a high case-load of patients with recent total hip replacement (THR). Whereas there has been a progressive standardisation and perfecting of prosthetic materials and surgical techniques, time-frames and modalities of rehabilitation programmes are still very variable. Following the Ministerial Guidelines, issued in 1998 by the Italian National Health System, and the introduction of Accreditation Requirements, methods must become more uniform and there must be increased scientific rigour in treatment so as to reduce variability and the subjective nature of the service provided. Ana-lysis of the working methods of large Rehabilitation Centres may help to focus on the problems more clearly and stimulate any improvements that may be required. The study analyses and compares rehabilitation protocols for hip replacement patients adopted in Italian and international rehabilitation centres. METHODS: Thirty-four post-THR rehabilitation protocols were analysed; 14 Italian plus 20 international. RESULTS: The analysis revealed that some factors are unanimously considered important and are therefore codified: 1) posture and positioning; 2) prevention of deep vein thrombosis; 3) rapid return to mobility; 4) education of patients to joint care. CONCLUSIONS: Weight-bearing on the operated limb is not yet a standardised aspect and thus the physiatrist's attention should be focused on this for a return to walking that is safe (for patients and for implanted prosthesis) correct (from the biomechanical and kinematic standpoints) and that plays a normal role in performing activities of daily living.

Oxidized welwitindolinones from terrestrial fischerella spp

3-Hydroxy-N-methylwelwitindolinone C isonitrile (3), 3-hydroxy-N-methylwelwitindolinone C isothiocyanate (4), and the novel cyclic ether N-methylwelwitindolinone D isonitrile (6) are three new alkaloids from two terrestrial Fischerella spp. belonging to the Stigonemataceae. Photooxidation of N-methylwelwitindolinone C isonitrile (1) leads to isonitriles 3 and 6. Isonitrile 3 is readily hydrated to 3-hydroxy-N-methylwelwitindolinone C formamide (5), an artifact produced during the isolation procedure.

Pre-evaluation and system optimization of the Elecsys thyroid electrochemiluminescence immunoassays.

We present the results of a pre-evaluation of the thyroid function test free thyroxine, free triiodothyronine and third generation TSH using the Elecsys electrochemiluminescence immunoassay system. A collaborative field study between the development center of the manufacturer and a clinical chemistry laboratory addressed the reliability and comparability of the new Elecsys assays to established methods under clinical laboratory conditions using samples from routine in vitro thyroid testing. Preliminary (reference) formulations of the reagents and several electrochemiluminescent pilot models were used for assay measurements, either in the company's research center or in the clinical setting. The new thyroid assays were compared with the respective Enzymun-Test assays, performed on the ES300 automated immunoassay analyzer. A WHO standard was used for standardization of TSH, whereas an equilibrium dialysis method was applied for free triiodothyronine. The free thyroxine assay was standardized against the Enzymun-Test free thyroxine assay, which had previously been calibrated against equilibrium dialysis. The aim of this field study was to support the optimization of the technology used for Elecsys in an early stage of development and thereby prepare the ground for the adaptation of the immunoassays to the final Elecsys 2010 random access analyzer. A subsequent multicenter evaluation demonstrated that the requirements of routine thyroid testing in terms of reliability were fulfilled by the system.

Isolation of a nitrile-containing indole alkaloid from the terrestrial blue-green alga hapalosiphon delicatulus

Ambiguine G nitrile is a new indole alkaloid from the terrestrial blue-green alga Hapalosiphon delicatulus (UH isolate IC-13-1). It is the first nitrile to be found in the Stigonemataceae.

Elecsys CEA, PSA and AFP. Clinical results of a multicentre evaluation.

Three tumormarker assays, Elecsys CEA, PSA and AFP, have been evaluated in an international multicentre study to characterize their clinical performance and to verify the comparability with the corresponding tests of the Enzymun-Test product line and other methods. For each of the markers results were obtained from four laboratories. On the basis of 314 and 199 specimens respectively, (preliminary) reference ranges could be established for CEA and PSA. For the prostate marker, the age dependence of the antigen level could be clearly confirmed. Mean concentrations range between 0.51 ng/ml (< 40 years) and 3.57 ng/ml (> 70 years). Referring to CEA, 95th percentiles of 4.31 ng/ml and 2.69 ng/ml were elaborated for smokers and nonsmokers. In general, good to excellent correlations (r > 0.98) were found between the Elecsys and Enzymun-Tests. Regarding the systematic comparability of both systems, most of the slopes derived from the individual method comparison studies are within the +/- 10% range of the respective standardization results. The specific distribution pattern of the individual tumormarker values elaborated with sample material of known clinical background, reflects the well established categorization of different benign and malignant diseases according to their characteristic marker levels. Of utmost importance, however, is the excellent comparability of the Elecsys assays with the corresponding Enzymun-Tests and the FDA approved AIA 1200 tests from TOSOH in follow-up studies. Almost superimposable concentration curves guarantee that identical diagnostic information is derived from all three methods. Especially for PSA, a series of measurements on sera of prostatectomized patients proved the usability and clinical value of the test also for this particular indication. For either one of the Elecsys tests, the feasibility of using plasma as sample material was verified.

A sensitive enzyme immunoassay for pregnancy-associated plasma protein A (PAPP-A): a possible first trimester method of screening for Down syndrome and other trisomies.

Pregnancy-associated plasma protein A (PAPP-A) is a large glycoprotein produced mainly by the trophoblast during pregnancy and released into the maternal circulation. Its biological function is unknown. In the second trimester i.e. when Down syndrome (DS) screening is routinely performed, the level of maternal serum PAPP-A was found to be within the normal range in pregnancies affected by fetal trisomy 21. However, PAPP-A was shown to be a potent marker for DS before 14 weeks of gestation. Only radioimmunoassays (RIAs) based on labelled antigen competition reached the required sensitivity for early pregnancy PAPP-A determinations; but they have a very short shelf life due to inherent tracer half-life and, in the case of PAPP-A, instability of the labelled antigen after three weeks. We describe a convenient and novel enzyme immunoassay (ELISA) with high sensitivity and a long shelf life.

Continuous ECG measurements of intracardiac activity from the surface of Langendorff-perfused guinea pig hearts.

The present paper describes a method by which it is possible to continuously detect early atrial and His-bundle activity from the surface of intact Langendorff-perfused guinea-pig hearts, by appropriate placement of two electrodes and the use of a custom designed instrumentation-amplifier. In some experiments the surface ECG recordings were compared with intracardiac ECG recordings. No difference in ECG durations could be observed between intracardiac and extracardiac measurements. In further experiments changes in ECG durations and heart rate were measured for 2 h. After 30 min equilibration time, no changes in heart rate and conduction time could be observed. In order to locate the best surface electrode positions to detect His-bundle activity, vector ECG recordings were taken at high gain. This vector ECG signal contained a His-loop which was split into a larger and a smaller part. The main and initial vector was directed to the left and the smaller to the right ventricle. The best recordings of the His-bundle activity could be observed when the electrodes were positioned as follows; one in a posterior position, near the valve plane and the other one in the opposite position near the initial part of the anterior interventricular artery and in the direction of the large His-loop. We conclude that the ECG surface recordings are a valuable tool for measuring impulse propagation through various segments of the cardiac conduction system in preparations of guinea-pig hearts, perfused by the Langendorff method.

Effects of low concentrations of antibiotics on Escherichia coli adhesion.

We have previously shown that subinhibitory concentrations of antibiotics may influence the adhesion of Escherichia coli SS142 to human epithelioid tissue culture cells. This report shows that these effects are not limited to E. coli SS142 or to our tissue culture system. Most of the 10 E. coli strains studied showed decreased adhesion to Intestine 407 tissue culture cells after growth in 25% of the minimum inhibitory concentration of streptomycin, tetracycline, trimethoprimsulfametrole, chloramphenicol, and clindamycin. Nalidixic acid at 25% of the minimum inhibitory concentration caused an increase of adhesion. The hemagglutinating activity of the five hemagglutinating strains and the adhesiveness of E. coli SS142 to human buccal cells were similarly affected by low concentrations of the above-mentioned antibiotics. We conclude that E. coli adhesion to human epithelioid tissue culture cells is a valid model of bacterial adhesion because of its high accuracy and reproducibility.

An assay for measuring specific adhesion of an Escherichia coli strain to tissue culture cells.

Escherichia coli SS142 has been found to adhere specifically to the human epithelioid tissue culture cell line Intestine 407, but not to other tissue culture cells. This paper describes an accurate, reproducible and objective method of assessing the rate of adhesion of radiolabelled bacteria to these cellular monolayers. Adhesion was found to be linear with time for 60 min and with bacterial concentrations up to 10(9) bacteria/ml. The binding appeared to be irreversible. Adhesion was not affected by changes in the composition of the medium, its pH or ionic strength, or by the assay temperature within physiological limits, but was diminished at very high ionic strength or low temperature. It increased with increasing cell density of the monolayers. Under appropriate conditions the assay could be used for comparative determinations of the rate of adhesion of different, or differently treated bacteria.

Adsorption and structural studies on activated carbons.

The porous structure of activated carbon is examined from the point of view of gas adsorption, and in relation to classical methods such as X-ray diffraction and electron microscopy. It is suggested that the different approaches to the problem of microporosity should provide complementary information, which can be useful for a better understanding of static and dynamic adsorption processes in activated carbons.

Chemical and physical properties of mycobacteriophage D29.

Mycobacteriophage D29 has a head of uniform size (average diameter 65 nm) and regular shape and a tail of variable length. The stability of the bacteriophage is optimal between pH 9 and 10. The virus contain double-stranded DNA and six structural polypeptides, three major and three minor. The molecular weights of these six polypeptides, as determined by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, are 150 000, 138 000, 13 000, 66 000 and 24 000. The virus contains no lipids as shown by (a) the lack of structural changes after inactivation of the bacteriophage with chloroform, (b) the absence of lipids containing [32P]phosphate or [35S]sulfate in labeled virus, and (c) the absence of an electron paramagnetic resonance spectrum in bacteriophage which had been incubated with a nitroxide-containing fatty acid.

Phase transitions in the membrane of a marine bacterium, Pseudomonas BAL-31.

An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL-31, host cell of the lipid-containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cis16:1 or trans16:1 showed one transition at 9.4 degrees C and 12.4 degrees C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL-31 had one transition at approximately 12 degrees C, on the other hand there was no clear cut phase transition using extracted lipids. Replication of bacteriophage PM2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in tran16:1. Other cases were not studied.

Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.

Practical, stereoselective synthesis of Cecropia juvenile hormone.

The high-yield, stereoselective conversion of geraniol (3,7-dimethyl-trans-2,6-octadien-1-ol) to the insect juvenile hormone, methyl 12,14-dihomojuvenate (methyl cis-10-epoxy-3,11-dimethyl-7-ethyl-trans, trans-2,6-tridecadienoate), has been performed by means of a nine-step route that avoids chromatography of intermediates.