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Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.
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Adyuvantes Inmunológicos , Flagelina , Vacunas Antirrábicas , Rabia , Vacunas de Productos Inactivados , Vacunas Antirrábicas/inmunología , Vacunas Antirrábicas/administración & dosificación , Animales , Flagelina/inmunología , Ratones , Rabia/prevención & control , Rabia/inmunología , Vacunas de Productos Inactivados/inmunología , Perros , Virus de la Rabia/inmunología , Salmonella typhimurium/inmunología , Femenino , GatosRESUMEN
The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (ÐÐ-1413, ÐÐ-342, ÐBl-29, ÐBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions.
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The phenomenon of tritium decay catastrophe is introduced. A technical example is provided regarding the radioligand [methoxy-3H] levosulpiride whose long-term behavior is consistent with the concept of tritium decay catastrophe.
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In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme-1 min-1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.
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Active Alpha-1 antitrypsin (AAT) circulates in blood in two isoforms in dynamic equilibrium: 1) native AAT, which binds irreversibly to neutrophil elastase, and 2) thiol-modified AAT, which binds reversibly to neutrophil elastase, either of which can be inactivated by oxidation, cleavage, covalent binding, and bound antibodies. Anti-AAT antibodies used for detecting AAT in plasma bind to active and inactive protein, thereby overestimating the concentration of functional protein. Active AAT can be quantitated by measuring its inhibition of active-site titrated elastase. A method for measuring active AAT in the presence of competing proteinase inhibitors in plasma has been developed and is described herein.
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Elastasa de Leucocito , Plasma , Anticuerpos , Inhibidores de Proteasas , Compuestos de SulfhidriloRESUMEN
Iris pallida Lam., also known as Sweetie Iris, is a perennial ornamental and medicinal plant that produces a wide range of secondary metabolites. The Sweetie Iris was recently reported to have high allelopathic properties with the potential to be explored in sustainable weed management. This study aimed to identify and evaluate the contributions of compounds involved in the inhibitory effects of the rhizome of Sweetie Iris. High-performance liquid chromatography (HPLC) analysis was used to determine the content of ß-ionone in the rhizome of Sweetie Iris. The phytotoxicity of ß-ionone was evaluated on lettuce (Lactuca sativa L.) and other test plants. The content of ß-ionone in the crude extract of Sweetie Iris rhizome was found to be 20.0 mg g-1 by HPLC analysis. The phytotoxicity bioassay showed that ß-ionone had strong inhibitory activity on the growth of lettuce (Lactuca sativa L.) and the other test plants, including Taraxacum officinale, Stellaria media, Eleusine indica, Amaranthus hybridus, Vicia villosa, and Brassica napus. At a concentration of 23.0 µg mL-1, ß-ionone inhibited the growth of all test plant species treated. Therefore, ß-ionone is an active compound among the other allelopathic substances contained in the rhizome of Sweetie Iris.
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Helicobacter pylori HpfutC, a glycosyltransferase (GT) 11 family glycoprotein, has great potential for industrial 2'-fucosyllactose (2'-FL) production. However, its limited catalytic activity, low expression, and poor thermostability hinder practical applications. Herein, a semi-rationally designed site-saturation mutation was applied to engineer the catalytic activity and thermostability of HpfutC. The 6 single point mutants (K102T, R105C, D115S, Y251F, A255G and K282E) and 6 combined mutants (V1, V2, V3, V4, V5, and V6) with enhanced enzyme activity were obtained by mutant library screening and ordered recombination mutation. The optimal mutant V6, with an optimum temperature of 40 °C, was not a metal-dependent enzyme, yet the reaction was facilitated by Mn2+. Compared to wild-type HpfutC, mutant V6 exhibited a 2.3-fold increase in specific activity and a 2.18-fold increase in half-life at 40 °C, respectively. Kinetic parameters indicated that the Km values of mutant V6 were 34.5 % (lactose) and 25.0 % (GDP-L-fucose) lower than those of the wild enzyme, whereas the kcat/Km values were 1.20 and 1.25-fold higher than those of the wild enzyme. Further, 3D-structure analysis revealed that the highly rigid structure, formation of new hydrogen bonds, increased hydrophobic residues and redistribution of electrostatic charges on the surface may be responsible for the elevated enzyme activity and thermostability. The strategy adopted in this study is of great significance to the solution of the technical bottleneck of HpfutC and the industrial application of 2'-FL.
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Helicobacter pylori , Helicobacter pylori/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Mutación , Temperatura , Estabilidad de EnzimasRESUMEN
Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-ß-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (ß-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-ß-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.
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Esterasas , Proteínas Fúngicas , Lignina , Nitrofenoles , Especificidad por Sustrato , Esterasas/metabolismo , Esterasas/genética , Esterasas/química , Nitrofenoles/metabolismo , Lignina/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hidrólisis , Colorimetría/métodos , Pruebas de Enzimas/métodosRESUMEN
A systematic study of the distribution of the Naturally Occurring Radioactivity in stone dust and crushed stone, has been carried out with an objective of establishing reliable baseline data on the radiation level and hence to evaluate hazard indices approach and the production of radioactive heat (RHP) due to radiation exposure to the workers and to the inhabitants of the studied area. Twenty-six samples have been collected from different locations in the State of Rio de Janeiro (Brazil). To calculate the specific activity, gamma ray spectrometry and a detector of High Purity Germanium (HPGe; Canberra, 30% relative efficiency) was used. The activity concentration of 238U, 226Ra 232Th and 40K ranged from 29.3 ± 18.6 to 206.8 ± 21.5 Bq kg-1, 30.3 ± 1.0 to 134.3 ± 1.8, 27.9 ± 0.7 to 86.2 ± 0.9 Bq kg-1, and 734.9 ± 35.1 to 1204.8 ± 53.5 Bq kg-1, respectively. The mean values of the Iex, Iin, Iγ, Iα, AUI, IRP112Rn, IPA, IPI and IYu indices were 0.68 ± 0.09, 0.92 ± 0.12, 0.93 ± 0.12, 0.43 ± 0.07, 0.88 ± 0.20, 1.25 ± 0.21, 0.34 ± 0.05, 0.75 ± 0.10, 0.63 ± 0.08 and 34. ± 6.31, respectively. The average radioactive heat production (RHP) of 2.01 ± 0.28 µ Wm-3 was above the values found in the literature, which may contribute to the heat flow in the study area because the raw materials that make up the samples showed a high value of environmental radioactivity. The Brazilian Hazard Index to assess the radiological risk of crushed stone and stone dust was created and the average value was 0.74 ± 0.10, a value below 1, which means only moderate control over the use of these materials, with no indication of restriction to the its use.
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@#<b>Objective</b> To measure the specificactivity of natural and synthetic radionuclides in the environmental soil of Panjin, China and determine the content of radionuclides in the surface soil, and to conduct a scientific assessment of the radiation health risks of residents in this area. <b>Methods</b> Thirty-one surface soil samples were collected within the jurisdiction of Panjin, and a high-purity germanium detector was used for γ spectrum analysis to obtain the content of radionuclides and the current environmental radioactivity level. The two independent samples mean <i>t</i>-test was used to compare the specific activity data of radionuclides in soil samples between Panjin and Liaoning Province or China. <b>Results</b> The meanspecific activities of natural radionuclides <sup>238</sup>U, <sup>226</sup>Ra, <sup>232</sup>Th, <sup>40</sup>K, and synthetic radionuclide <sup>137</sup>Cs in the surface soil samples of Panjin were 18.7 Bq/kg, 19.6 Bq/kg, 23.5 Bq/kg, 604.6 Bq/kg, and 0.9 Bq/kg, respectively. <b>Conclusion</b> The specific activities of natural and synthetic radionuclides in the surface soil samples of Panjin Area at the background level, causing a very low health risk to the people in this area.
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Xylanase is a high-profile glycoside hydrolase with applications in brewing, feed, pharmacy and bioenergy industries, but most of xylanases are in active below 30 ℃. In order to obtain low temperature active xylanase, a xylanase gene, XYN11A, was cloned from Penicillium sp. L1 and expressed in Pichia pastoris GS115. After purification and enzyme assay, optimal pH and temperature were determined to be 3.5 to 4.0 and 55 ℃. This enzyme was stable at acid and neutral condition (pH 1.0 to 7.0) or under the treatment of 40 ℃ for 1 hour. This xylanase displayed strong resistance to all tested ions and chemicals. Noteworthily, XYN11A maintained a higher activity of 6 700 U/mg than a lot of GH11 xylanase, and demonstrated higher activity (24% to 58%) at lower temperature from 20 to 40 ℃. After beechwood xylan hydrolysis for 16 h, the hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose and barely of xylose, thus XYN11A could be used for the production of prebiotic xylooligosaccharide. Possessing the features of acidophilic, highly active at lower temperature and oligosaccharide production, XYN11A demonstrated great potential in food and feed industrials.
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Objective:To investigate guidance levels of radionuclide for food and drinking water.Methods: According to the relevant standards and technical documents issued by international organizations, the computing methods of guidance levels of radionuclide for food and drinking water under the existing exposure situation and emergency exposure situation were analyzed and researched.Results: The calculated guidance levels of 17 radionuclides in drinking water and 20 radionuclides in food in existing exposure situation were provided, and the guidance levels of 24 radionuclides in food and drinking water in emergency exposure situation also were proposed. It was found that the relevant standards in China were lag and the standard of guidance levels of radionuclides activity concentrations for drinking water were deficient.Conclusion: It is suggested that the standards of guidance levels of radionuclide for food and drinking water in China should be formulated or revised by referring to international standard that issued from international organization and taking into account the specific conditions in China so as to preferably protect public health of our country.
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Objective To investigate radioactivity levels in the main agriculture products around a uranium mine in Northern Guangxi.Methods The agriculture products and soil samples were collected and analyzed by using HPGe gamma ray spectrometer.Results The specific activity of 226Ra in radish (including leaf),radish leaves and radish,collected in one place,were 45.0,66.7 and 32.3 Bq/kg,respectively.Those of 226Ra and 23SU in the radish soil collected in the same place were 19 672 and 85 917 Bq/kg,respectively.The transfer coefficients of soil-to-radish and soil-to-leaves were 1.61 × 10-3 and 3.40 × 10-3,consistent with those reported in relevant literature.Radioactivity levels in agricultural products in another survey was in consistence with those in the national survey for food products.Radioactivity levels in soil elsewhere near the radish site was consistent with the results of the national soil radioactivive background survey.Conclusions The soil in this place has been contaminated by the nearby uranium mine.It is important to investigate this place further and take the necessary measures.
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Aim Toanalyzethekeyqualityandestablishmeth-ods for essential quality control of human recombinant ASPP2 adenovirus.Methods TheviralstructuralgeneofE2Bandtar-get gene of ASPP2 were identified by PCR;The number of virus particles was measured by UV-SDS methods;Infectious titer was determined by TCID50 assay;Target protein of ASPP2 was ob-served by Western blot assay;The biological effects of recombi-nant adenovirus on liver cancer cells were evaluated by MTT as-say;A549 cells were used to check replication of the competent adenovirus(RCA)by the observation of the cytopathic effect. Results PCRanalysisofE2BandASPP2wasinconsistent with theoretical values;Particle numbers of virus were 5. 6 × 1012 VP/mL,infectious titer was 2 ×1011 IU/mL and specific activity was 3. 5%;ASPP2 protein expression could be detected when cells were infected with virus for 24 h;Growth inhibition of liver cancer cells could be found by adding recombinant ASPP 2 adenovirus;The level of RCA was less than 1 RCA/3. 0 ×1010 VP,in line with the standards of China Food and Drug Adminis-tration(SFDA).Conclusion Thequalitycontrolmethodswere established aiming at key characters of human recombinant AS-PP2 adenovirus,which may provide foundations for its quality standard and future applications.
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Objective To separate and screen components with fibrinolytic enzyme activity from twelve Chinese herbal medicines. Methods The components were extracted with water and precipitated with salt, and they were tested by fibrinolytic protein plates method. The active components with fibrinolytic activity were separated and screened which were compared with urokinase. Results Eleven of the twelve extracts showed fibrinolytic activity, while Trichosanthes kirilowii got the biggest fibrinolytic zone after 36 hours, followed by Alisma plantago-aquatica and Leonurus japonicus, and the Radix Astagali got the smallest one. According to the concentration of the protein, the area of the fibrinolytic zone and the specific activity of the components, the extract from Angelica sinensis exhibited the best specific activity at level of 48.46U/mg. Conclusion The extracts from Chinese herbal medicines except Semen Persicae exhibit fibrinolytic enzyme activity which can dissolve the fibrin in different degrees.
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ObjectiveTo develop an ELISA method for quantitative determination of enterovirus 71 (EV71) antigen.The method can be applied to detect EV71 antigen contents and analyze the correlation between immunogenicity and immunoprotection.It also can be used for tissue culture infective dose( TCID50 ) assay.MethodsA double antibody sandwich ELISA method was developed for quantitative determination of EV71 antigen on the basis of the high-affinity neutralizing monoclonal antibodies ( K8G2 and Y8H2-HRP).This method was compared with microscopic observation for the detection of EV71 TCID50.The correlation was analyzed between the specific activity of EV71 antigen and the EV71 neutralizing antibody titer in immune serum.ResultsThe linear range of this method was 0.125-4.0 U/ml and the R2 value was 0.9911.The reagent did not react with other antigens except EV71 antigen.The recovery ratio of this method was 0.89-1.16.The coefficient of variation was less than 15%.The heat recovery rate was above 85% when the reagent was in 37℃ for 9 days.There was a good correlation in TCID50 of EV71 between this method and microscopic observation,r=0.990.The specific activity of EV71 antigen had positive correlation with the neutralization titer of immune serum in 21 EV71 strains,r=0.930.ConclusionThe quantitative ELISA method for EV71 antigen was developed,which could be used to detect EV71 antigen contents and analyze TCID50.The specific activity of EV71 antigen detected by the method could be used to evaluate the immunoprotection of the vaccine potency test.
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Objective To investigate the specific activity of 24Na per unit neutron fluence,AB/Φ,in blood produced for Chinese reference man irratiaded by 252Cf neutron source,and to analyze the effects of scattering neutrons from ground,wall,and ceiling in irradiation site on it.MethodsA 252Cf neutron source of 3 × 108 n/s and the anthropomorphic phantom were used for experiments.The phantom was made from 4 mm thick of outer covering by perspex and the liquid tissue-equivalent substitute in it.The data of phantom dimensions fit into Chinese reference man.The weight ratios of H,N,O and C in substitute equal from source to long axis of phantom were 1.1,2.1,3.1 and 4.1 m,respectively.Both the neutron source and the position of xiphisternum of the phantom were 1.6 m above the floor.ResultsThe average specific activity of 24Na per unit neutron fluence was related to the irradiation-distances,d,and its The AB/ΦM corresponds to that of phantom irradiated by plane-parallel beams,and the value is about more 3% than that by BOMAB phantom reported in literature.It has shown that floor-( wall-)scattered neutrons in irradiation site have significant contribution to the specific activity of 24Na ,but they contributed relatively little to the induced neutron doses.Consequently,using the specific activity of 24 Na for assessing accidental neutron doses received by an individual,the contribution of scattered neutrons in accident site will lead dose to be overestimated,and need to be correct.
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The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.
O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.
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PURPOSE: N-(3-[18F]Fluoroporpy)-2beta- carbomethoxy-3beta-(4-iodophenyl) nortropane ([18F]FP-CIT) has been shown to be very useful for imaging the dopamine transporter. However, synthesis of this radiotracer is some what troublesome. In this study, we used a new method for the preparation of [18F]FP-CIT to increase radiochemical yield and effective specific activity. MATERIALS AND METHODS: [18F]FP-CIT was prepared by N-alkylation of nor-beta-CIT (2 mg) with 3-bromo-l-[18F]fluoropropane in the presence of Et3N (5-6 drops of DMF/CH3CN, 140 degree C, 20 min). 3-Bromo-l-[18F]fluoropropane was synthesized from 5 microliter of 3-bromo-l-trifluoromethanesulfonyloxypropane (3-bromopropyl -l-triflate) and nBu4N18F at 80 degree C. The final compound was purified by reverse phase HPLC and formulated in 13% ethanol in saline. RESULTS: 3-Bromo-l-[18F]fluoropropane was obtained from 3-bromopropyl-l-triflate and nBu4N18F in 77-80% yield. N-Alkylation of nor-beta-CIT with 3-bromo-l-[18F]fluoropropane was carried out at 140 degree C using acetonitrile containing a small volume of DMF as the solvents. The overall yield of [18F]FP-CIT was 5-10% (decay-corrected) with a radiochemical purity higher than 99% and effective specific activity higher than the one reported in the literature based on their HPLC data. The final [18F]FP-CIT solution had the optimal pH (7.0) and it was pyrogen-free. CONCLUSION:: In this study, 3-bromopropyl-l-triflate was used as the precursor for the [18F]fluorination reaction and new conditions were developed for purification of [18F]FP-CIT by HPLC. We established this new method for the preparation of [18F]FP-CIT, which gave high effective specific activity and relatively good yield.
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Cromatografía Líquida de Alta Presión , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Dopamina , Etanol , Concentración de Iones de Hidrógeno , Tomografía de Emisión de Positrones , SolventesRESUMEN
TF was prepared from spleen cellsof both HSV-1-immunized mice(TF_(HSV)-1)and control group animals(TFn)4 weeks after immunization.The leukocyte adherence inhibitionindex(LAII)demonstrating the spe-cific activity of TF_(HSV)-1 in vitrowas found to be significantly higherthan that of control(P