Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1.

To correlate specific local immune responses with protection from corneal scarring, we examined immune cell infiltrates in the cornea after ocular challenge of vaccinated mice with herpes simplex virus type 1 (HSV-1). This is the first report to examine corneal infiltrates following ocular challenge of a vaccinated mouse rather than following infection of a naive mouse. Mice were vaccinated systemically with vaccines that following ocular challenge with HSV-1 resulted in (i) complete protection against corneal disease (KOS, an avirulent strain of HSV-1); (ii) partial protection, resulting in moderate corneal disease (baculovirus-expressed HSV-1 glycoprotein E [gE]); and (iii) no protection, resulting in severe corneal disease (mock vaccine). Infiltration into the cornea of CD4+ T cells, CD8+ T cells, macrophages, and cells containing various lymphokines was monitored on days 0, 1, 3, 7, and 10 postchallenge by immunocytochemistry of corneal sections. Prior to ocular challenge, no eye disease or corneal infiltrates were detected in any mice. KOS-vaccinated mice developed high HSV-1 neutralizing antibody titers (> 1:640) in serum. After ocular challenge, they were completely protected against death, developed no corneal disease, and had no detectable virus in their tear films at any time examined. In response to the ocular challenge, these mice developed high local levels of infiltrating CD4+ T cells and cells containing interleukin-2 (IL-2), IL-4, IL-6, or tumor necrosis factor alpha (TNF-alpha). In contrast, only low levels of infiltrating CD8+ T cells were found, and gamma interferon (IFN-gamma)-containing cells were not present until day 10. gE-vaccinated mice developed neutralizing antibody titers in serum almost as high as those of the KOS-vaccinated mice (> 1:320). After ocular challenge, they were also completely protected against death. However, the gE-vaccinated mice developed low levels of corneal disease and virus was detected in one-third of their eyes. Compared with KOS-vaccinated mice, the gE-vaccinated mice had a similar pattern of IFN-gamma, but a delay in the appearance of CD4+ T cells, CD8+ T cells, and IL-4-, IL-6-, and TNF-alpha-containing cells. In sharp contrast to those of the KOS-vaccinated mice, no cells containing IL-2 were detected in the eyes of gE-vaccinated mice at any time. Mock-vaccinated mice developed no detectable neutralizing antibody titer and were not protected from lethal HSV-1 challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of baculovirus-expressed herpes simplex virus type 1 glycoprotein K.

The DNA region encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) was inserted into a baculovirus transfer vector, and recombinant viruses expressing gK were isolated. Four gK-related recombinant baculovirus-expressed peptides of 29, 35, 38, and 40 kDa were detected with polyclonal antibody to gK. The 35-, 38-, and 40-kDa species were susceptible to tunicamycin treatment, suggesting that they were glycosylated. The 38- and 40-kDa species corresponded to partially glycosylated precursor gK (pgK) and mature gK, respectively. The 29-kDa peptide probably represented a cleaved, unglycosylated peptide. The 35-kDa peptide probably represented a cleaved, glycosylated peptide that may be a precursor to pgK. Indirect immunofluorescence with polyclonal antibody to gK peptides indicated that the recombinant baculovirus-expressed gK was abundant on the surface of the insect cells in which it was expressed. Mice vaccinated with the baculovirus-expressed gK produced very low levels (< 1:10) of HSV-1 neutralizing antibody. Nonetheless, these mice were partially protected from lethal challenge with HSV-1 (75% survival). This protection was significant (P = 0.02). Despite some protection against death, gK-vaccinated mice showed no protection against the establishment of latency. Surprisingly, gK-vaccinated mice that were challenged ocularly with a stromal disease-producing strain of HSV-1 had significantly higher levels of ocular disease (herpes stromal keratitis) than did mock-vaccinated mice. In summary, this is the first report to show that vaccination with HSV-1 gK can provide protection against lethal HSV-1 challenge and that vaccination with an HSV-1 glycoprotein can significantly increase the severity of HSV-1-induced ocular disease.     

Free amino Acid content and metabolic activities of setting and aborting soybean ovaries

Fruits of soybean (Glycine max [L.] Merr.) that are destined to abscise shortly after anthesis grow more slowly than fruits that will be retained. In this work, amino acid composition, protein metabolism, and nucleic acid metabolism were studied in setting and abscising soybean ovaries from anthesis to 6 days after anthesis to provide additional evidence of chemical processes associated with abscission. Principal free amino acids were asparagine, aspartic acid, glutamic acid, serine, and glutamine. Percent aspartate and glutamate declined as the ovaries grew, with aspartate declining more in abscising and glutamate more in setting ovaries. Percent glutamate was positively correlated to percent abscission throughout the period. Proline, serine, and leucine were positively correlated to abscission from 0 to 2 days after anthesis, whereas significant negative correlations were observed at these ages for ethanolamine and arginine. ⁷⁵Se fed as selenate and ¹⁴C fed as sucrose, glycine, and alanine were readily incorporated into soluble and insoluble proteins in a 24-hour in vitro incubation. Radioactivity of total proteins, expressed on a perovary basis, was negatively correlated with percent abscission and positively correlated with ovary weight. [¹⁴C]Glutamine and serine followed the opposite pattern, with greater protein labeling in abscising than in setting ovaries. When data were expressed as disintegrations per minute per milligram ovary fresh weight, protein labeling from alanine was seen to be significantly greater in abscising ovaries at anthesis and throughout the sampling period. Nucleic acid labeling from uridine was highly correlated to ovary weight; labeling from thymidine was greater in setting than abscising ovaries at anthesis and in abscising ovaries at later stages of development. It is concluded that abscising ovaries can continue amino acid metabolism almost up to the date of separation from the raceme, and that the involvement of alanine, glutamine, aspartate, glutamate, and other amino acids in soybean flower abortion deserves further study.     

Biochemical characterization of soybean ovary growth from anthesis to abscission of aborting ovaries

Soybean (Glycine max [L.] Merr.) ovary growth was measured from anthesis to 6 days after anthesis (DAA) to establish a timetable of biochemical events that might be useful in identifying processes that initiate abscission. Two procedures were developed to provide samples with either high or low percent pod set for `IX93-100,' a semideterminate line having long racemes. Characteristics measured were fresh and dry weight, soluble and insoluble protein, soluble carbohydrate, starch, RNA, and DNA. Setting ovaries grew more rapidly than abscising ovaries. Since there was a daily increase in ovary weight in both groups, all measured characteristics showed daily increases when expressed on perovary basis. Statistically significant differences between groups were detected between 2 and 5 DAA for most characteristics. When chemical composition was expressed on concentration basis, starch level was significantly higher in setting ovaries at 5 and 6 DAA. Regression analysis showed that these deviations between setting and abscising samples started between anthesis and 1 DAA. We conclude that processes leading to eventual shedding of fertilized ovaries (called flower abortion in soybeans) commence soon after anthesis of the shed flower, and that setting and abscising ovaries do not differ in protein, soluble carbohydrate, starch, or nucleic acid content when abscission processes begin.     

Role of Opioid Receptors on Food Choice and Macronutrient Selection in Meat-Type Chick

The present study was designed to examine the role of opioid receptors on food choice and macronutrient selection in neonatal chicks. In this study, 13 experiments designed, experiments 1–3 for effect of specific opioid receptors on appetite and experiments 4–13 on effect of opioid receptors on food choice and macronutrient selection in meat-type chick. In experiment 1, chicken intracerebroventricular (ICV) injected with 125, 250 and 500 pmol of DAMGO (µ-opioid receptor agonist). Experiment 2 was conducted to investigate the effect of DPDPE (δ-opioid receptor agonist) at doses of 20, 40 and 80 nmol. In experiment 3 ICV injection of the U-50488H (κ-opioid receptor agonist, of 10, 20 and 40 nmol) was done. In experiment 4, birds injected with saline and different diets: standard diet without fat, diet containing nutrient energy 20 % higher than standard, diet containing nutrient energy 20 % lower than standard and standard diet containing fat were offered to them to investigate desire of chicken to diets. Experiments 5–7 were similar to experiment 4, except, birds ICV injected with 125, 250 and 500 pmol of DAMGO. In experiments 8–10 chicken received ICV injection of DPDPE (20, 40 and 80 nmol). The experiments 11–13 was similar to previous experiments which birds injected with different doses of U-50488H (10, 20 and 40 nmol), respectively. Then the cumulative food intake measured until 180-min post injection. According to the results, ICV injection of DAMGO diminished food intake while DPDPE and U-50488H increased appetite (P < 0.05). Despite anorexigenic effect, ICV injection of DAMGO increased birds desire to eat fat containing standard diet compared to the standard diet without fat (P < 0.05). These findings suggest endogenous opioids governing preferences for fat rich foods.     

Changes in contents of chlorophyll,proteins, and saccharides in leaves during plant development of determinate,semideterminate, and indeterminate lines of soybean

Major differences betwean the determinate (dt₁Dt₂,dt₁dt₂), semideterminate (Dt₁Dt₂), and indeterminate (Dt₁dt₂) near-isogenic lines of glycine max (L.) Merr, mainly appeared after R1 (reproductive) stage. Increases in specific leaf matter (SLM) between Rl and R6 stages showed that determinate lines have higher SLM than semideterminate or indeterminate lines. Soluble protein and starch also accumulated more rapidly in determinate lines. Insoluble protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), reducing saccharides, chlorophyll, and soluble saccharides contents of leaf from determinate lines resembled those of semideterminate and indeterminate lines.     

A new analytical formula for neutron capture gamma dose calculations in double-bend mazes in radiation therapy

BackgroundPhotoneutrons are produced in radiation therapy with high energy photons. Also, capture gamma rays are the byproduct of neutrons interactions with wall material of radiotherapy rooms.AimIn the current study an analytical formula was proposed for capture gamma dose calculations in double bend mazes in radiation therapy rooms.Materials and methodsA total of 40 different layouts with double-bend mazes and a 18 MeV photon beam of Varian 2100 Clinac were simulated using MCNPX Monte Carlo (MC) code. Neutron capture gamma ray dose equivalent was calculated by the MC method along the maze and at the maze entrance door of all the simulated rooms. Then, all MC resulted data were fitted to an empirical formula for capture gamma dose calculations. Wu–McGinley analytical formula for capture gamma dose equivalent at the maze entrance door in single-bend mazes was also used for comparison purposes.ResultsFor capture gamma dose equivalents at the maze entrance door, the difference of 2–11% was seen between MC and the derived equation, while the difference of 36–87% was found between MC and the Wu–McGinley methods.ConclusionOur results showed that the derived formula results were consistent with the MC results for all of 40 different geometries. However, as a new formula, further evaluations are required to validate its use in practical situations. Finally, its application is recommend for capture gamma dose calculations in double-bend mazes to improve shielding calculations.     

Water‐miscible ionic liquids as novel effectors for the firefly luciferase reaction

Ionic liquids (IL) are used as a new class of solvents for various reactions. Especially using IL in biocatalysis in an aqueous milieu has attracted considerable attention because enzymes show remarkable differences in their catalytic features in IL‐containing reaction media. Firefly luciferase is widely used in many analytical techniques, because light production of firefly luciferase is one of the most sensitive analytical measures in the ultrasensitive detection of adenosine‐5′‐triphosphate, e.g. for measuring microbial contamination and monitoring gene expression, as well as for monitoring tumor growth and metastasis in whole animals. Firefly luciferase is an unstable enzyme and its inactivation can lead to low sensitivity in the above‐mentioned assays. The present study addresses the comparative influence of six different water‐immiscible IL, the 3‐methylimidazolium derivatives [BMIM]Cl, [HMIM]Cl, [BMIM]Br, [EMIM]Br, [HMIM]Br, and [BMIM]BF₄, on the kinetic properties, structural stability, and function of firefly luciferase from Photinus pyralis using circular dichroism, fluorescence spectroscopy, and a bioluminescence assay. The incubation of luciferase with various IL showed that, with the exception of [BMIM]BF₄, the activity and stability of luciferase was considerably increased in the presence of IL, compared to luciferase in aqueous medium. Moreover, K_m for the substrate adenosine‐5′‐triphosphate in the presence of IL (except for [BMIM]BF₄) decreased while K_m for luciferin remained constant.     

Adaptive and innate transforming growth factor beta signaling impact herpes simplex virus 1 latency and reactivation

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor β (TGF-β) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-β signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-β receptor type II transgenic mouse lines. These mice have specific TGF-β signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-β signaling during acute and latent infections. Limiting innate cell but not T cell TGF-β signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-β signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-β signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-β signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.