Nanoparticle analysis of cancer cells by light transmission spectroscopy.

We have measured the optical properties of cancer and normal whole cells and lysates using light transmission spectroscopy (LTS). LTS provides both the optical extinction coefficient in the wavelength range from 220 to 1100nm and (by spectral inversion using a Mie model) the particle distribution density in the size range from 1 to 3000nm. Our current work involves whole cells and lysates of cultured human oral cells in liquid suspension. We found systematic differences in the optical extinction between cancer and normal whole cells and lysates, which translate to different particle size distributions (PSDs) for these materials. Specifically, we found that cancer cells have distinctly lower concentrations of nanoparticles with diameters less than 100nm and have higher concentrations of particles with diameters from 100 to 1000nm-results that hold for both whole cells and lysates. We also found a power-law dependence of particle density with diameter over several orders of magnitude.

DNA-based species detection capabilities using laser transmission spectroscopy.

Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel (Dreissena bugensis) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications.

High-resolution spectroscopy with a femtosecond laser frequency comb.

The output of a mode-locked femtosecond laser is used for precision single-photon spectroscopy of 133Cs in an atomic beam. By changing the laser's repetition rate, the cesium D1 (6s 2S(1/2)-->6p 2P(1/2)) and D2 (6s 2S(1/2)-->6p 2P(3/2)) transitions are detected and the optical frequencies are measured with accuracy similar to that obtained with a cw laser. Control of the femtosecond laser repetition rate by use of the atomic fluorescence is also implemented, thus realizing a simple cesium optical clock.

Testing the stability of fundamental constants with the 199Hg+ single-ion optical clock.

Over a two-year duration, we have compared the frequency of the 199Hg+ 5d(10)6s (2)S(1/2)(F=0)<-->5d(9)6s(2) (2)D(5/2)(F=2) electric-quadrupole transition at 282 nm with the frequency of the ground-state hyperfine splitting in neutral 133Cs. These measurements show that any fractional time variation of the ratio nu(Cs)/nu(Hg) between the two frequencies is smaller than +/-7 x 10(-15) yr(-1) (1sigma uncertainty). According to recent atomic structure calculations, this sets an upper limit to a possible fractional time variation of g(Cs)(m(e)/m(p))alpha(6.0) at the same level.

rIL-2-stimulated splenocytes reactivate the NO2-producing ability of macrophages infected by Leishmania donovani.

Infection with L. donovani down-regulates immunity and parasite clearance by macrophages. Treatment with IL-2-stimulated-splenocytes activate parasiticidal action in vitro in peritoneal macrophages of C57BL/6 (Lsh(s)) mice and also was effective in stimulating infected macrophages to produce NO2-.

Synergism of IL-2-stimulated splenocytes and Pentostam enhances the killing of Leishmania donovani in vitro.

Control of infections by the obligate intramacrophage protozoan parasite Leishmania donovani is traditionally done with pentavalent antimonial drugs. It was of interest to determine if immunotherapy with IL-2-stimulated splenocytes would enhance drug action in an in vitro model system. It is confirmed that nontoxic doses of Pentostam decrease infection in a dose-dependent manner in vitro in terms of both the number of amastigotes/100 macrophages and the % of infected macrophages; the curative effect was most apparent when the drug was used on the 7th day of the infection, when the parasite was in its proliferative phase. It is also confirmed that recombinant IL-2-stimulated splenocytes induced infected macrophages to reduce significantly their parasite burden, especially when the infection was treated in the nonproliferative phase of the parasite, also in a dose-dependent manner. Leishmanicidal action in the infected macrophages was induced by cytokine(s) released from the lymphokine-stimulated cells. Immunotherapy in the nonproliferative phase, combined with drug treatment in the proliferative phase, reduced the infection to levels significantly below those produced by either treatment alone. Immunotherapy with IL-2-stimulated splenocytes in combination with Pentostam is, therefore, an excellent candidate treatment for the effective reduction of experimental infections by L. donovani.

IL-2-stimulated splenocytes reduce infections by Leishmania donovani in vivo.

The traditional treatment of infections produced by the obligate intramacrophage protozoan Leishmania donovani involves the use of antimonial drugs. Because these drugs may have toxic side effects (and are sometimes ineffective), the potential efficacy of alternative therapy with lymphokine-stimulated leucocytes was assessed. Macrophage-depleted C57BL/6 splenocytes from mice inoculated 2 wk earlier with L. donovani were stimulated in vitro with the interleukin-2-containing supernatant of MLA 144 cells and transferred intravenously into syngeneic infected mice. Compared to infected mice that had received unstimulated normal or infected spleen cells, animals treated with lymphokine-stimulated splenocytes had significantly reduced parasite loads. Efficacy was further enhanced significantly by supplementary intraperitoneal injections of the MLA 144 supernatant; the effector function of the stimulated splenocytes was dose dependent. The rescue of animals infected by L. donovani from parasite-induced down-regulation of immunity could be an important part of a strategy for the effective treatment of kala azar; lymphokine-stimulated cells are potential candidate agents to restore curative immune responses of experimental visceral leishmaniasis.

Immunotherapy with IL-2-stimulated splenocytes reduces in vitro the level of Leishmania donovani infection in peritoneal macrophages.

A study was done in vitro to determine if IL-2-stimulated lymphocytes (LAK cells) would activate infected macrophages to reduce their burden of Leishmania donovani. Macrophage-depleted splenocytes from normal or infected C57BL/6 (H-2b; LshS) mice, stimulated in vitro by the IL-2-containing supernatant of the MLA 144 cell line or by rIL-2, significantly reduced the number of syngeneic resting peritoneal macrophages infected by L. donovani; LAK cells from infected animals were significantly more effective in reducing the numbers of infected cells. Supernatants of MLA 144-stimulated spleen cells and rIL-2-stimulated splenocytes isolated in Millipore chambers also induced a significant reduction of the infection in vitro. Anti-Thy 1.2 eliminated the ability of the supernatant of MLA 144 to induce an activating function in C57BL/6 splenocytes; monoclonal anti-IL-2 abolished the ability of rIL-2 and of the MLA 144 supernatant to stimulate the splenocytes. Infected resting peritoneal macrophages of C57L (H-2b; LshR) mice were more responsive to activation than those of the C57BL/6 animals, irrespective of the phenotype of the stimulating LAK cells. Lymphokine stimulation reverses the immunological anergy induced in T lymphocytes by Leishmania donovani; IL-2-stimulated LAK splenocytes are highly effective in reducing the intensity of experimental visceral leishmaniasis in vitro in peritoneal macrophages.

Time course and intensity of infection in vitro in the resident peritoneal macrophages of resistant and susceptible mice exposed to different doses of Leishmania donovani promastigotes.

A study was made of the proliferation dynamics in vitro of Leishmania donovani amastigotes in the resting peritoneal macrophages of C57BL/6 (Lshs) and C57L/J (Lshr) mice. Monolayers were inoculated with 5, 50 or 500 promastigotes per macrophage and the number of infected cells and the number of parasites per cell were determined 1, 3, 5, 7 and 14 days following inoculation. Results indicate that, irrespective of the phenotype of the donor mouse and of the inoculum, only 50-65% of the cells became infected initially. Expansion of the infection proceeded more rapidly in monolayers of Lshs cells and may have involved the "recruitment" of non-susceptible macrophages, perhaps by the action of a soluble factor. Also irrespective of the inoculum and phenotype, only 3-6 amastigotes were present in each macrophage initially, suggesting a limited number of ligands for the attachment of the parasite to the cell. Amastigotes did not proliferate for 3-4 days and then divided actively until day 7, when more parasites were present in the macrophages of the susceptible phenotype. Differential expansion of the infection and the proliferation of amastigotes in vitro suggest that resting peritoneal macrophages may, indeed, express the Lsh gene.

Exposure to Toxoplasma gondii and cat ownership in Nova Scotia.

In this survey, 998 children and adolescents between 7 months and 17 years of age who attended a hospital diagnostic center in the city of Halifax, Nova Scotia, for routine evaluation were tested for Toxoplasma gondii antibody. The 5.2% prevalence rate of antibody for children living in the outlying rural areas was significantly higher than the 1.1% rate among the urban children (P = .0006). Seroprevalence increased with age for both rural and urban children. Cat ownership was associated with antibodies to Toxoplasma among rural children but not urban children. Rural children who lived in a house with more than one cat were two times more likely to be infected than children who had one cat and three times more likely to be infected than children with no cats. The geometric mean titer was also significantly higher among the rural children with more than one cat, 1:152, than rural children with one or no cats, 1:63 (P = .02). In light of these findings for children and adolescents, the association of Toxoplasma infection with cat ownership needs to be thoroughly evaluated among pregnant women in rural areas.

Prevalence of antibody to Toxoplasma gondii in the moose (Alces alces americana Clinton) of Nova Scotia, Canada.

The prevalence of antibodies to Toxoplasma gondii was investigated in the sera of 125 moose taken by hunters in 5 countries of Nova Scotia. Nineteen of these sera (15%) were positive by the indirect passive hemagglutination test, with titers above 1:64. This study adds further evidence to the prevalence of antibodies to T. gondii in the wildlife, extending this evidence to eastern Canada. The possibility that humans may acquire toxoplasmosis by ingesting undercooked infected meat from game animals is supported by the results of this investigation; the implications of these findings to public health are obvious.

Infectivity and route of penetration in rats after oral and intraperitoneal inoculations of bloodstream and in vitro-cultured metacyclic forms of Trypanosoma lewisi.

Morphological changes of Trypanosoma lewisi blood trypomastigotes cultured in Schneider's Drosophila medium (SDM), supplemented or not with uric acid (SDM + UA), were compared to those that occurred in a control medium (M-199). No difference in trypanosome morphology and numbers was observed between SDM + UA and SDM cultures; there was little transformation into metacyclic stages in M-199. No difference was observed between the capacity of SDM- or SDM + UA-cultured metacyclic stages to infect rats. The infectivity of bloodstream forms was always higher than that of the SDM- or SDM + UA-cultured forms, whether inoculated orally or intraperitoneally. The oral inoculation of rats with tritium-labeled culture and bloodstream forms showed that the metatrypanosomes from the cultures remained longer in the salivary glands and tongue of the animal than the blood trypanosomes.

Importance of lymphokines in the control of multiplication and dispersion of Leishmania donovani within liver macrophages of resistant and susceptible mice.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. The immunosuppressant cyclosporin A (CsA), which inhibits the production of interleukin (IL)-1, IL-2, and interferon-gamma, increased infections 3-fold without affecting expression of the Lsh gene. The objective of this study was to determine how activation of macrophages by lymphokines affects the multiplication and propagation of the parasite within liver macrophages. Susceptible C57BL/6J and resistant C57L/J mice were treated with 200 mg/kg CsA and then infected intravenously with 10(7) amastigotes. Two weeks later macrophages were collected from the liver by perfusion, plated on coverslips, and incubated for 4, 24, and 48 hr. The percentage of infected macrophages and the number of amastigotes/100 cells were determined after staining the cells with Giemsa's stain. The number of infected macrophages and amastigotes per macrophage was significantly greater in animals of both strains that had been treated with CsA. This study demonstrated clearly that lymphokines or other soluble mediators produced by T cells act, in part, to control infection by L. donovani by minimizing both multiplication within macrophages and their dispersion.

Killing of Leishmania donovani by activated liver macrophages from resistant and susceptible strains of mice.

Leishmania donovani is an obligate intracellular protozoan parasite of macrophages; liver macrophages are, however, the only population of cells which express the resistant Lsh gene phenotype when these cells are infected in vitro. It was of interest to study in vitro the action of Con A-stimulated spleen cell lymphokines (LK) to protect or to cure liver macrophages from infection by L. donovani. Liver and peritoneal macrophages (PEC) from resistant (C57L/J) and susceptible (C57BL/6J) mice were infected in vitro with promastigotes before or after LK treatment; the percentage of infected macrophages was determined 4, 24, 48 and 72 h post-infection. Both macrophage populations were protected or cured by treatment with lymphokines; the cells of the resistant strain were protected or cured more effectively than those of the susceptible strain. The capacity for cure or for protection following LK treatment of liver and PEC macrophages was similar within each strain. Supernatants from the IL-2-produced MLA-144 cell line had no effect to protect or cure macrophages. This study indicates that the response of macrophages to the action of LK is also important in determining the susceptibility of mice to L. donovani; this model in vitro provides a good approximation of the response of macrophages to therapy.

The effect of cyclosporin A in murine visceral leishmaniasis.

Leishmania donovani is an obligate intracellular protozoan parasite of macrophages whose control is effected by cellular mechanism. In order to determine the role of T-helper cells and their soluble mediators in the control of the infection, the effects of cyclosporin A (CsA) were studied in strains of mice (C57L/J and C57BL/6J) known to be resistant and susceptible. Mice treated with CsA (200 mg/kg) exhibited as early as day 15 a significant increase in the level of their infection. The number of parasites was 3 times higher in CsA-treated mice of both strains when compared to untreated controls. The proportional difference in the level of infection between C57BL/6J and C57L/J mice was, however, not affected by treatment. Infection and CsA treatment, respectively, reduced or abolished the capacity of spleen cells to produce IL-2. Infection also abolished the production of IL-1 by macrophages. Both strains were, however, able to recover from the infection without functional T-helper lympho-cytes and/or the IL-1 IL-2 which had been inhibited by treatment with CsA.

432-nm source based on efficient second-harmonic generation of GaAlAs diode-laser radiation in a self-locking external resonant cavity.

Using a potassium niobate crystal in a modified self-locking power-buildup cavity, we have frequency doubled the 865-nm output from a GaAlAs laser diode. With 12.4 mW of input power we have obtained a unidirectional output of 0.215 mW at 432 nm. In contrast to previous diode doubling experiments, the output was both single frequency and circular Gaussian. With better optics, substantially higher conversion efficiencies should be possible using this technique.

Atomic beam collimation using a laser diode with a self-locking power-buildup cavity.

We have demonstrated a self-locking power-buildup cavity for laser diodes. This device requires only a few simple optical elements and can provide a standing wave containing as much as 1000 times the power emitted by the laser diode. With this device we have obtained an intense standing wave of tunable light that was used to collimate a cesium atomic beam. We have studied the power and frequency dependence of the beam collimation.