Propagation characteristics of Airy beams: dependence upon spatial coherence and wavelength.

We generate a broadband "white light" Airy beam and characterize the dependence of the beam properties on wavelength. Experimental results are presented showing that the beam's deflection coefficient and its characteristic length are wavelength dependent. In contrast the aperture coefficient is not wavelength dependent. However, this coefficient depends on the spatial coherence of the beam. We model this behaviour theoretically by extending the Gaussian-Schell model to describe the effect of spatial coherence on the propagation of Airy beams. The experimental results are compared to the model and good agreement is observed.

Induction of glutamine synthetase in embryonic retina: its dependence on cell interactions.

A relation between enzyme induction in embryonic cells and cellular organization is indicated by the finding that the levels of glutamine synthetase induced by hydrocortisone in the embryonic neural retina in vitro are dependent on the associations between the retina cells. Intact retina tissue, aggregates of dissociated cells, and cells in monolayer culture showed a decreasing response, in this order, to glutamine synthetase induction. With time of culture, the enzyme activity continued to rise in the intact retina and in cell aggregates, but activity declined in monolayer cultures even though the inducer was continuously present. Dispersed cells cultured in monolayer without the inducer showed after 24 hours a loss of inducibility which could not be reversed by reaggregating such modified cells but could be prevented by maintaining the freshly dispersed cells at a low temperature.

Optical micromanipulation using supercontinuum Laguerre-Gaussian and Gaussian beams.

We characterize a single beam supercontinuum "white light" trap and determine the trap stiffness in the transverse trapping plane. We realize a holographic white light trapping system using a spatial light modulator, and explore the generation of a dual beam trap and characterize its performance. We also demonstrate optical trapping and rotation of particles using a supercontinuum vortex beam. It is shown that orbital angular momentum can be transferred to spheres trapped in a supercontinuum vortex. Quantified rotation rates are demonstrated.

Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.

Cationic lipids show promise as vectors for transfer of CFTR cDNA to airway epithelia of patients with cystic fibrosis (CF). However, previous studies have not compared the effect of DNA-lipid to DNA alone. Recently, we developed a formulation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) that generated greater gene transfer in mouse lung than previously described DNA-lipid vectors. Therefore, we tested the hypothesis that DNA-lipid complexes were more effective than DNA alone at transferring CFTR cDNA to airway epithelia in vivo. We administered complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study. Electrophysiologic measurements showed that DNA-lipid complexes partially corrected the Cl- transport defect. Importantly, the pCF1-CFTR plasmid alone was at least as effective as complexes of DNA with lipid. Measurements of vector-specific CFTR transcripts also showed gene transfer with both DNA-lipid and DNA alone. These results indicate that nonviral vectors can transfer CFTR cDNA to airway epithelia and at least partially restore the Cl- transport defect characteristic of CF. However, improvements in the overall efficacy of gene transfer are required to develop a treatment for CF.

The binding of carcinoembryonic antigen by antibody and its fragments.

In order to assess the potency of antigenic fragments of carcinoembryonic antigen (CEA) in the radioimmune assay; it is necessary to know whether the high affinity of goat anti-CEA antibody (which makes possible the detection of as little as 10--11M CEA) is due to bivalent binding of the CEA molecule. Immunoglobulin G and the F(ab')2 and Fab fragments derived from it were prepared from an anti-CEA serum and tested for their abioity to bind CEA. Equivalent concentrations of binding sites of the bivalent F(ab)2 and univalent Fab fragments of anti-CEA were identical to the immunoglobulin G fraction in the standard inhibition curve. Fragments of CEA obtained by trypsin digestion produced equivalent inhigition curves when tested with either immunoglobulin G, F(ab')2, or Fab". This, increased avidity due to bivalent binding to a single antigen molecule cannot be invoked to explain the sensitivity observed in the CEA assay. This high sensitivity implicates the protein rather than the carbohydrate as an important part of the antigenic determinant(s) of CEA.

Interactions between chondroitin sulfate and concanavalin A.

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 microgram/ml) reacted only within a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 microgram/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low Ph and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Estradiol induces an accumulation of free heparan sulfate glycosaminoglycan chains in uterine epithelium.

Blastocyst implantation in the mouse uterus is triggered by an estrogen surge that induces a number of characteristic changes in biosynthetic activity of the endometrial stroma and epithelium, including a reduction in the thickness of the epithelial plasma membrane coat, immediately preceding attachment of the blastocyst. A brief exposure of the uterine luminal surface to 1% Nonidet-40 (NP-40) nonionic detergent extracted this surface coat along with membrane-associated and soluble [35S]sulfate-labeled proteoglycans specifically from the epithelium. Extraction from the underlying stroma appeared to have been prevented by the epithelial basal lamina, which was minimally affected by 1% NP-40, but was extracted by subsequent treatment with 1% NP-40 in 1 M KCl. About 70-80% of the [35S]sulfate in proteoglycans extracted from the uteri of ovariectomized mice with NP-40 was associated with heparan sulfate proteoglycans that eluted from Sepharose CL-4B in a peak at 0.55 Kd. All remaining radioactivity could be accounted for by dermatan sulfate/chondroitin sulfate proteoglycans. Specifically, in the NP-40 extract of uteri from ovariectomized mature mice and intact immature mice, but not in the culture medium or residual tissue, estradiol induced a pronounced increase in the relative amount of radioactivity in a 0.8 Kd peak, which appeared to contain only free heparan sulfate glycosaminoglycan chains. The same relative increase was seen in the NP-40 extract of uteri from intact 3.5-day pregnant mice. We conclude that estradiol induces cellular changes that result in an increased accumulation of epithelial heparan sulfate proteoglycan degradation product immediately preceding normal attachment of the blastocyst.

C-reactive protein concentration in children: relationship to adiposity and other cardiovascular risk factors.

Whether or not C-reactive protein (CRP) predicts heart disease in adults because it is a marker of damage or atherosclerosis is difficult to assess. In children, there is no confounding with coronary disease or active smoking. We measured CRP in 699 children aged 10-11 years. CRP levels were 47% higher in girls than boys, and rose with age by 15%/year. CRP levels were 270% (95% CI, 155-439%) higher in the top fifth than the bottom fifth of Ponderal index (weight/height(3)). After adjustment, CRP levels remained 104% (95% CI, 23-236%) higher in the 56 children of South Asian origin. CRP was unrelated to: birth weight, height, social class, Helicobacter pylori infection or passive smoke exposure. CRP was correlated with several cardiovascular risk factors, but only fibrinogen (r = 0.33, P = 0.0001), HDL-cholesterol (r = -0.13, P = 0.0006), heart rate (r = 0.12, P = 0.002) and systolic blood pressure (r = 0.08, P = 0.02) remained statistically significant after adjustment. We conclude that adiposity is the major determinant of CRP levels in children while physical fitness has a small independent effect. The strong relationships with fibrinogen and HDL-cholesterol suggest a role for inflammation throughout life in the development of atherosclerosis and cardiovascular disease. Longitudinal studies are needed to determine whether these associations reflect long term elevations of these risk factors in some individuals, or short term fluctuations in different individuals.

Effect of constant light on DMBA mammary tumorigenesis in rats.

A study of light, and mammary tumorigenesis was conducted in rats. One-hundred female Sprague-Dawley rats were divided by weight into two groups. One group was exposed to constant light (LL) from 26 days of age, and the second group was exposed to 8 h light and 16 h dark per day (LD). Both groups received an 8 mg dose of a chemical carcinogen, dimethylben-zanthracene (DMBA) at 52 days of age. At 13 weeks post-DMBA, there were significantly fewer mammary tumors in the LL group compared with the LD group. Constant light was clearly demonstrated to have a profound effect on mammary tissue development. Although virgin, the majority of the LL rats (29/50) had gross evidence of lactation at 141 days of age. None of the LD rats (0/50) showed evidence of milk production. These results suggest that constant light not only substantially accelerated mammary gland development, but pushed development of the tissue past the stage normally observed in virgin animals (to the lactation stage).

Effects of 50- or 60-hertz, 100 microT magnetic field exposure in the DMBA mammary cancer model in Sprague-Dawley rats: possible explanations for different results from two laboratories.

In line with the possible relationship between electric power and breast cancer risk and the underlying melatonin hypothesis, 50-Hz magnetic field (MF) exposure at microtesla flux densities for either 13 or 27 weeks significantly increased the development and growth of mammary tumors in a series of experiments from Löscher's group in Germany. Löscher's group used the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in Sprague-Dawley rats. The finding could not be replicated when a similar experimental protocol was used in a study conducted by Battelle in the United States. In the present paper, investigators from the two groups discuss differences between their studies that might explain the apparent discrepancies between the results. These differences include the use of different substrains of Sprague-Dawley rats (the U.S. rats were more susceptible to DMBA than the European rats), different sources for diet and DMBA, differences in environmental conditions, and differences in MF exposure metrics. Furthermore, the effects of MF exposure reported by Löscher's group, albeit significant, were weak. We also discuss the general problem of replicating such weak effects.

Combined use of neostigmine and ocular motility measurements in the diagnosis of myasthenia gravis.

Forty-four patients with diplopia caused by myasthenia gravis, ocular myopathies, and ocular motor nerve palsies underwent complete orthoptic evaluations before and after intramuscular injection of neostigmine (Prostigmin) methylsulfate. A test score combining the results of binocular and uniocular measurements was developed that allowed correct correct classification of 70% (31) of patients studied.

Storage and transport of cultures for Herpes simplex virus, type 2.

Tryptic soy broth and brain-heart infusion broth maintained herpes simplex virus within +/- 1 serial dilution of the original titer in all of 51 samples held for one to three days at 4 C. In contrast, holding temperatures of -20 C or 22 C resulted in losses of titer of 10(2) or more. Holding periods as long as five days prior to inoculation did not delay the appearance of the cytopathic effect following inoculation. Unmodified tryptic soy broth or brain-heart infusion broth and holding periods as long as three days at 4 C prior to arrival in the laboratory give as satisfactory results as do traditional viral culture media and prompt inoculation of the specimen.

Hemochromatosis heterozygotes may constitute a radiation-sensitive subpopulation.

A primary mechanism of radiation-induced DNA damage is by generation of free radicals. Chronically increased oxidative stress from elevated levels of iron in the body may increase radiation sensitivity by decreasing cellular oxygen radical scavenging capability. Hemochromatosis heterozygotes have elevated body iron. Low-level radiation sensitization by iron may be particularly pertinent for risk of breast cancer. Since 10% of the population appears to be heterozygous for the hemochromatosis gene, a radiosensitizing effect would have pervasive implications.

Micronuclei induced by radon and its progeny in deep-lung fibroblasts of rats in vivo and in vitro.

Genotoxic damage induced by radon and its progeny was investigated using the micronucleus assay in deep-lung fibroblasts to compare the response induced in vitro with that induced from inhalation of radon and its progeny in vivo. Male Wistar rats were exposed to 0, 115, 213 and 323 working-level months (WLM) of radon and its progeny by inhalation. After sacrifice, the cells were isolated and grown in culture, and the frequency of micronuclei was determined. A linear increase in the frequency of micronuclei was measured as a function of exposure [micronuclei/1000 binucleated cells = (29 +/- 9) + (0.47 +/- 0.04) WLM]. To compare exposure in WLM to dose in mGy, and to study how cell proliferation influences the way inhalation of radon and its progeny induces micronuclei, lung fibroblasts were isolated and exposed in vitro to graded doses from radon and its progeny after either 16 or 96 h in tissue culture. Cell cycle stage at the time of exposure was determined using flow cytometry. Primary lung fibroblasts exposed as either nondividing or dividing cells showed dose-dependent increases in micronuclei [micronuclei/1000 binucleated cells = (33 +/- 40) + (593 +/- 68)D and micronuclei/1000 binucleated cells = (27 +/- 69) + (757 +/- 88)D, respectively, where D is dose in Gy]. Results showed no significant influence (P = 0.20) of cell proliferation at the time of exposure on the frequency of micronuclei induced by radon and its progeny. Comparing dose-response relationships for nondividing cells to the exposure response for cells exposed by inhalation of radon and its progeny, it was estimated that a 1-WLM exposure in vivo caused the same amount of cytogenetic damage as produced by 0.79 mGy in vitro. In vivo/in vitro research using the micronucleus assay in lung fibroblasts serves as a powerful tool to estimate effective dose to cells in the respiratory tract after inhalation of radon and its progeny. Such studies form the basis for understanding the relationship between exposure, dose and biological damage.

Importance of low serum vitamin B12 and red cell folate concentrations in elderly hospital inpatients.

To determine the functional importance of the low B12 and red cell folate concentrations repeatedly observed in the elderly 200 consecutive patients admitted to a geriatric unit were studied. Forty six of the patients had low serum concentrations of B12 (15), red cell folate (26), or both (five). Serum B12 and red cell folate concentrations correlated with mean cell volume, and serum B12 correlated with the neutrophil lobe count. Bone marrow deoxyuridine suppression was abnormal in 35% of the patients with low vitamin concentrations, but 55% of those with abnormal deoxyuridine suppression had morphologically normal bone marrow, and 73% had a normal mean cell volume. In patients with low vitamin values the deoxyuridine suppressed value correlated with the haemoglobin concentration and neutrophil lobe count. Thus synthesis of thymidylate was impaired by vitamin B12 or folate deficiency in at least 8% of newly admitted elderly patients, many of whom had normal blood counts despite the biochemical disturbance affecting haemopoiesis. A nutritionally depleted diet may have been responsible for many of the low vitamin values.

Immunophenotypic characterization of owl monkey peripheral blood mononuclear cells.

A two-phase study was initiated to delineate the peripheral blood lymphocyte populations present in owl monkeys and to correlate those populations with immune response and parasitism during malaria infection. The goal of phase I of the study was to elucidate a monoclonal antibody panel that could be used to characterize peripheral blood mononuclear cell (PBMC) populations with flow cytometric techniques. Forty-two monoclonal antibodies (reported to be reactive with human and macaque lymphocyte antigens) were screened for activity to owl monkey PBMC. Eleven monoclonals were found to react: anti-H42A (MHC Class II DP-like); anti-TH14B (MHC Class II DR-like); and anti-TH81A5 (MHC Class II DQ-like); anti-H58A (MHC Class I); anti-DH59B (granulocyte and monocyte); anti-B1 (B cell); anti-T4 (CD4); anti-Leu3a (CD4); anti-Leu11a (CD16); anti-60.3 (CD18); and anti-OKM1 (NK and monocyte). In a preliminary retrospective study correlating antibody titers, parasitemia values, and MHC Class I and Class II marker profiles on PBMC to test antigens used in malaria vaccine trials, a significant negative association was observed between cells bearing MHC Class II molecules and the other elements of the comparison. In summary, an appropriate panel of monoclonal antibodies has been identified for characterizing PBMC in owl monkeys, and preliminary studies indicate a possible association between clinical outcome and expressed phenotypic PBMC markers.

A simple automated colorimetric method for determination of N-acetyl- beta-D-glucosaminidase.

An automated method for the determination of N-acetyl-beta-D-glucosaminidase in serum or plasma using p-nitrophenol (pNP) N-acetyl-beta-D-glucosamine as substrate and a Pye Unicam AURA system is described. Normal samples had activities of 853 +/- 146 (SD) nmol pNP liberated/ml/h, with intra-assay coefficient of variation 1.2% and inter-assay coefficient of variation 1.6%. Inhibition of enzyme activity by heparin in plasma samples can be reversed by the addition of calcium chloride to the buffer.

Sonoporation of cultured cells in the rotating tube exposure system.

Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex, a gas-body- based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0-3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.