Determination of human leukocyte interferon by a microfluorimetric immunologic method]

The possibility of quantitative determination of human leucocyte interferon using FITC-labeled antiinterferon antibodies was studied. A highly specific antiinterferon immunoglobulin was obtained as a result of longterm immunization of a donkey with human leucocyte interferon followed by fractionation and immuno-absorbtion of immune plasma. This immunoglobulin was labeled with FITC and used for human leucocyte interferon assay in direct and indirect reactions of fluorescence immunoinhibition. The titres of different human leucocyte interferon preparations in this immunoassay were comparable with the titres of the same preparations detected by interferon inhibition of viral cytopathic effect.     

Production of antibodies against the porcine luteinizing hormone by two immunization methods.

Two methods of rabbit immunization were applied: multisite intradermal injections of small doses and intramuscular injections of large hormone doses for obtaining antibodies against the porcine luteinizing hormone (LH). A high titre, specificity towards LH and affinity were obtained in rabbits immunized by both methods. In most animals the highest titre was noted 10 weeks after the first immunization.     

Raising Antibodies to Staphylococcal Enterotoxins A and B

A method is described for raising specific antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) by intravenous inoculation of rabbits with small doses of enterotoxin diluted in sterile physiological saline. A course of six injections over a period of two weeks was given on four occasions. After the third course, 3/5 rabbits given SEA had a titre ± 1/50 and 4/6 given SEB had a titre of 1/50. Titres were not appreciably enhanced by the fourth series of injections. Only minor non-specific reactions which would require no absorption were found at the end of the series. It is believed that the findings in this study would be reproducible and that the method is likely to be suitable for raising antisera to other enterotoxins. Reference is also made to preliminary experiments in which latex sensitized with SEA or SEB was inoculated intravenously into rabbits.     

Infection of small ruminants with Ehrlichia spp. in Sicily

In 1991, the experimental infection of a goat with pooled blood from goats that were positive for anti-Ehrlichia canis, E. risticii, E. equi and E. phagocytophila antibodies was monitored (physical examination, cell blood count, microscopical examination of blood smears, serology) for 180 days. The infection produced a clinical condition characterized by intermittent fever, anaemia and leukopenia with neutropenia during the first 40 days. Recurrent leukocytosis with lymphocytosis was noticed afterwards. A permanent high-level thrombocytosis appeared after the 18th day. During the first week, cytoplasmic basophilic inclusion bodies were seen in smears of peripheral venous blood stained with May-Grunwald-Giemsa, first in mononuclear cells and then in neutrophils (in max 3% of circulating leukocytes). Seroconversion occurred during the 2nd week and the highest antibody titre (IFAT) was registered vs E. equi (10,240) at the 19th day, vs E. canis (320) at the 24th and vs E. risticii (80) at the 30th day. At the end of the observation period the infected goat was still positive for E. equi (titre 160) and E. canis (titre 10) only. The preinoculation serum of the infected goat was reactive with E. phagocytophila antigen (serum was tested for IF antibodies to E. phagocytophila at 1:200 dilution only, because of the limited quantities of antigen available), but the qualitative evaluation of fluorescence showed an increase from the 7th day, maximum intensity between the 14th and the 40th day and passed to negative from the 74th day. Although it was based on microscopy and serology only and not carried out in a SPF goat, the above experiment gave evidence of the existence of species of the E. phagocytophila genogroup in Italy for the first time.     

Haemagglutination-inhibiting and neutralizing antibodies to type 3 and 5 rhinoviruses in human sera and nasal washings.

Presence of antibodies to RV 3 and RV 5 was tested by HIT and NT in 60 human sera. Antibodies to RV 3 were detected in 23 sera by HIT in a titre range of 1:4--1:64 and in 19 sera by NT in a titre range of 1:4--1:256. Antibodies to RV 5 were detected in 31 sera by HIT in titres of 1:4--1:268 and 27 sera by NT in the same titre range. In a group of 22 persons with unequivocal serum antibodies nasal secretory antibodies were found in 11 subjects in titres of 1:4--1:32. In a group of 16 persons without detectable serum antibodies, presence of secretory antibodies (titre 1:4) was only found in four cases.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3162 = 4.8 % ) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand.
Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1 %) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida.
Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3/62 = 4.8%) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand. Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1%) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida. Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

Production of human leukocyte interferon for clinical use under immunoelectrophoretic control.

Human leukocyte interferon (HLIF) can be contaminated by several antigens which may include also potentially pathogenic agents. For this reason, gel filtration as a separation method was included into the routine procedure of preparation and purification of HLIF for clinical use. Since production of HLIF requires the cultivation of leukocytes in the presence of serum (or albumin), serum proteins represent then the majority of proteins contaminating IF preparations. Measuring the total protein content, as a control of various antigens present in HLIF preparations during purification, becomes ineffective because, essentially, it indicates only the decrease of the amount of proteins derived from cultivation medium. For a better visualization of dissociation of different antigens from molecules with IF activity during the purification procedure, the method of quantitative immunoelectrophoresis was applied utilizing a sheep antiinterferon serum in assay. Our results indicate that this method represents a valuable control test.     

A Survey of Rubella Antibodies in Health Personnel and a Report of Vaccine Trial

Five hundred and seventy female health science personnel between the ages of 18 and 25 years were examined for antibodies to the rubella virus by the hemagglutination inhibition technique. Approximately 90% of the subjects had a titre of 1:20 or higher. The geometric mean titre of the positive sera was 1:251. Sixty-four of the 77 persons with an antibody of 1:20 or less volunteered to take the vaccine and were examined six weeks later for the development of antibodies. The conversion rate for the 51 persons who were negative at the dilution of 1:20 at the outset was 94%; the rate of antibody increment for the 13 persons who were positive at 1:20 at the outset was 77%. Among the 51 persons who developed antibodies, the geometric mean titre was 1:57 and among the other 13 it was 1:49. Although the trial was conducted in adult females, the number of side effects from the vaccine was remarkably scanty and insignificant. This trial would seem to emphasize the importance of avoiding the use of rubella vaccine in women of child-bearing age without first excluding pregnancy with meticulous care and using active and controllable contraceptive methods for the two months following vaccination.     

The immune response of brown trout (Salmo trutta L.) to injection with soluble antigens.

The immune response of brown trout (Salmo trutta) to horse serum and keyhole limpet haemocyanin was studied. Intraperitoneal and intramuscular injections were used, with and without adjuvant, in 209 fish. Complement-fixing antibodies (CFA) and precipitins were produced to both antigens. CFA were detected after 8 days to haemocyanin and after 13 days to horse serum. Maximum CFA titres to a single intraperitoneal injection of horse serum or haemocyanin were reached at 44 and 43-46 days respectively. Precipitins to a single injection of haemocyanin given intraperitoneally were detected after 19 days using gel diffusion. Similarly using the intramuscular route they were detected after 22 days. However, using counter-current electrophoresis, precipitins were detected after 8 days by the intraperitoneal route and after 9 days by the intramuscular. Precipitins to horse serum given intraperitoneally were demonstrated after 22 days by both gel diffusion and counter-current electrophoresis. Fish given 2 intraperitoneal injections of haemocyanin in adjuvant reached maximum CFA titres after 55 days; a 3rd injection on day 56 did not produce a marked increase in titre. Fish given intramuscular injections of haemocyanin in adjuvant showed maximum CFA titres at day 43. After a 3rd injection on day 56, maximum CFA titres were reached between days 92 and 106. Intramuscular injections gave significantly higher titres than those given by the intraperitoneal route. Some fish which showed no precipitins by gel diffusion were positive by counter-current electrophoresis. Precipitating antibodies to haemocyanin migrating in the beta2-gamma1 region were detected by immuno-electrophoresis.     

Subacute sclerosing panencephalitis--the continuing threat

Clinical, epidemiological and laboratory findings of four patients with subacute sclerosing panencephalitis (SSPE), diagnosed in Croatia in 2002, were examined. Patient age at disease onset ranged from 5-11 years. All patients were vaccinated regularly with MMR-vaccine. Two patients had a history of measles infection at the age of six and seven months, respectively. In the other two patients, the disease started immediately after the varicella infection. Complement fixing antibody titre to the measles virus (MV) ranged from 1:1024 to 1:65536 in serum, and from 1:16 to 1:128 in cerebrospinal fluid (CSF). In CSF, no antibodies to varicella-zoster virus were found. Brain tissue samples were obtained at autopsy from two patients. In one patient, electron microscopy demonstrated intranuclear viral inclusions (MV nucleocapsids). MV antigen was detected in brain imprints using IFA in both of them. Viral RNA was found in brain tissue samples only, while plasma, serum and CSF were negative. Nucleotide sequence analysis showed that the viruses detected in brain tissue belong to the wild-type MV D6 genotype.     

Artificial immunization of pigeons against Argas polonicus (Ixodoidea, Argasidae)

Two subcutaneous injections of salivary gland antigen (SGA) or larval homogenate (LH) at 2-week intervals induced a resistance in pigeons to Argas (Argas) polonicus Siuda, Hoogstraal, Clifford and Wassef larvae and induced anti-tick antibodies. The number of larvae rejected after LH immunization was significantly higher compared to SGA immunization but lower than the number of larvae rejected after two natural infestations at 2-week intervals. The antibody titre reached a peak on day 6 following the first inoculation of LH, and 11-13 days after SGA inoculation. The maximum antibody titre was recorded 6 days after a second challenge for both antigens. The highest antibody titre was reached after the first inoculation with LH but only after the second inoculation with SGA. The sera of pigeons immunized either with SGA or LH cross-reacted with the other antigen as demonstrated by ELISA. SDS-PAGE and immunoblot studies demonstrated several differences in the protein profiles of these antigens, the presence of 34 and 35 kdal proteins in SGA and their absence in LH.     

Vaccinia virus persistence in a child against the background of immune deficiency

A young girl, vaccinated against smallpox 6 years before suffered from a persistent vaccinia virus infection and a congenital skin disease, i.e. epidermolysis bullosa. The virus was isolated from skin lesions at the vaccination site and remote sites and repeatedly from the blood; it was not isolated from bone-marrow specimen, saliva, pharynx or urine. The titre of virus-neutralizing antibodies was low (1:10), immunoglobulins A, M and G were within age-related limits; antibodies against measles and tetanus were at protective levels, skin tests were positive. Staphylococcal antitoxin titre was extremely high. The child's mother, not vaccinated against smallpox, possessed vaccinia--virus-neutralizing antibodies at high titre (over 1:320). Examination of the child did not show any quantitative immune deficiency. Immune deviations were found in the lymphocyte blast-transformation reaction on stimulation with PHA and specific antigen, as well as in the nonspecific-suppression test. The possible genesis of the virus persistence and the role of the virus in the clinical course of the disease are discussed.     

Circulating Antibody Response in BCG Vaccination,Tuberculous Infection and Sarcoidosis

The bentonite flocculation test was used to differentiate antibodies formed by BCG vaccination from those produced by infections with virulent tubercle bacilli. Of 116 BCG-vaccinated nurses, only two (1.7%) showed antibody titres higher (1:128) than the threshold titre of 1:64 established for tuberculous patients.The bentonite test was also used to follow the course of infection in 54 patients with tuberculosis. A good correlation was found between the clinical course of the disease and O.T.-bentonite titres on repeated serological testing.Tuberculosis-like antibodies were demonstrated in sarcoidosis patients. These antibodies, however, are globulins (7S), in contrast to the macroglobulins (19S) found in tuberculous patients.     

Kinetics of antibody response by Dot-ELISA in rabbits immunized with adult Haemonchus contortus antigen

Whole adult soluble extract of Haemonchus contortus as an antigen along with Freund's complete adjuvant, was used to immunize rabbits. Antisera from immunized rabbits were collected at intervals of 30, 60, 90, 120, 150 and 180 days. For the detection and titration of anti-H. contortus antibodies in these sera, Dot-ELISA was developed. Sera collected 30 days post-immunization exhibited a titre of 1:5,000 in all the rabbits except one, where a titre of 20,000 was recorded. Later, all the rabbits attained the highest titre of 40,000 at different periods of post-immunizations, which were maintained 150-180 days. These high titre sera can be of immense use in the identification and characterization of immunodominant antigens of adult H. contortus.     

昆虫细胞中存在角蛋白纤维

本文采用细胞分级抽提结合整装细胞电镜制样技术,分别在两种昆虫细胞:斜纹夜蛾(SL)细胞;甜菜夜蛾(SE)细胞中显示了一个精细的中等纤维网络结构,纤维自胞核发出,排列错综复杂,其单丝清晰可见,直径约为8～10nm;间接免疫荧光染色结果表明角蛋白抗体在两种细胞中均能显示出清晰的荧光纤维网络,而且荧光纤维的分布有所不同;用角蛋白抗体对这两种细胞全蛋白进行免疫印迹实验,均可显示49KD,68KD的两个主要多肽条带,说明这两种昆虫细胞中等纤维的主要成分为角蛋白.     

Cytoplasmic islet-cell antibodies (ICA) and cytoplasmic complement- fixing antibodies (CF-ICA) in patients with newly detected and short-lasting diabetes mellitus type 1

The study aimed at assessing ICA and CF-ICA in the serum of patients with newly diagnosed and short-lasting diabetes mellitus type 1. Sixty patients with newly diagnosed diabetes type 1 (39 patients) and short-lasting diabetes of the same type (21 patients) aged between 2 and 34 years were classified. Anti-islet antibodies were detected with indirect immunoflourescence in specimens of fresh, frozen human pancreast in the tested group ICA were found in 53% of cases. At the time of diagnosis, ICA were found in 76% of children and in 14% of adult patients whereas respective data for diabetes mellitus lasting up to 2 years were 40% and 64%. Complement-fixing islet cytoplasmatic antibodies were found only in patients with ICA (47% of such cases). These antibodies were found in children with newly diagnosed diabetes mellitus (36%). In case of adults CF-ICA were detected in 7% of newly diagnosed diabetes mellitus cases and in 45% of cases with the disease lasting for 2 years. Titres of ICA ranged from 1:1 to 1:128 whereas titres CF-ICA from 1:1 to 1:8. No correlation between ICA titre and CF-ICA titre was noted.     

Detection of desmin-containing intermediate filaments in cultured muscle and nonmuscle cells by immunoelectron microscopy

Antibodies raised against chicken gizzard smooth muscle desmin were shown to be specific by immunofluorescence cytochemistry and immunoautoradiography after two-dimensional polyacrylamide gel electrophoresis. Embryonic chick heart cell cultures (permeabilized with Triton X-100) and enucleated adult chicken erythrocyte ghosts (Granger, B. L., E. A. Rapasky, and E. Lazarides, 1982, J. Cell Biol. 92:299-312) were then used for immunoelectronmicroscopic localization of desmin. As expected, all intermediate filaments (IF) of the cardiac myocytes were labeled heavily and uniformly with the desmin antibodies. No periodicity or helicity was detectable along the labeled IF. Of interest was the intermittent but clear labeling of the IF of the nonmuscle, fibroblastic cells in the identical cultures. These antibodies did not bind vimentin from embryonic chick heart homogenates; furthermore, they did not label IF of avian erythrocytes known to contain vimentin but not desmin. We conclude that IF of cardiac fibroblastic cells contain low, but significant, concentrations of desmin and that this protein probably forms a copolymer with vimentin in these cells.     

Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts

Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.     

Persistence of influenza serum antibodies in humans following immunization with a bivalent A/Victoria and A/New Jersey vaccine.

The persistence of serum antibodies 1 year after immunization with a bivalent vaccine containing recombinant viruses that were antigenically identical with A/Victoria/3/75 (H3N2) and A/New Jersey/8/76 (Hsw1N1) viruses was measured in 128 persons aged 18 to 65 years. Serum samples were tested with the hemagglutination inhibition assay against the two vaccine antigens and against A/Texas/1/77 (H3N2) and A/USSR/90/77 (H1N1) viruses. Prior to vaccination 56% and 79% of the participants had been found to be seronegative to A/Victoria and A/New Jersey antigens respectively; the geometric mean antibody titres were low (1:5 to 1:11) except in persons aged 51 to 65 years, whose mean titre of antibody to the A/New Jersey antigen was 1:23, and persons aged 26 to 35 years, whose mean titre of antibody to the A/USSR antigen was 1:25. By 3 weeks after vaccination 85% of the seronegative persons had a fourfold or greater rise in titres of antibodies to the viruses in the vaccine, and 70% had a fourfold increase in titre of antibody to the A/Texas antigen. Of the persons aged 26 to 35 years (seronegative and seropositive) 68% had a fourfold or greater increase in titre of antibody to the A/USSR antigen. There was no change in the mean titres of 19 unvaccinated control subjects during the observation period. At 6 and 12 months after vaccination the titres of antibodies to the A/Victoria and A/New Jersey antigens had declined moderately in all age groups from those observed 3 weeks after vaccination. The rate of decline was similar for the various antibodies except that to the A/USSR antigen in persons 26 to 35 years of age, in whom the decline was much slower.