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Biomedical subjects

T L Lin

Publications and source records attributed to T L Lin.

At least 55 records · Page 3Linked to original sources

Evidence of reproductive failure and lack of perinatal transmission of Bartonella henselae in experimentally infected cats.

Five female specific pathogen-free (SPF) cats inoculated intradermally with B. henselae and bacteremic for 4 weeks, and one cat inoculated with 0.9% NaCl, were bred with uninfected SPF male cats. The uninfected female became pregnant with one breeding, while three infected cats became pregnant 1-12 weeks later, after repeated breedings. Two infected females either did not become pregnant or maintain pregnancies despite repeated breedings. Infected cats produced anti-B. henselae IgM and IgG antibodies. Fetuses and kittens of infected cats were not infected and did not produce anti-B. henselae antibodies. Male cats bred with infected females did not become infected or seroconvert. Maternal anti-B. henselae IgG antibodies detected in sera of kittens 2 weeks post-partum were no longer detectable 10 weeks post-partum. These findings suggest that B. henselae causes reproductive failure in female cats, but is not transmitted transplacentally, in colostrum or milk, or venereally. Infected cats immunosuppressed with methylprednisolone acetate after their kittens were weaned had no detectable bacteria in tissues, suggesting that they were no longer infected.

Animals↗

Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection.

The current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin V-FITC labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes.

Animals↗

The commissioning of a flexible low-cost multipurpose X-ray beamline at SRRC.

A low-cost multipurpose X-ray beamline has been commissioned at the bending magnet B15 of the Synchrotron Radiation Research Center (SRRC). The beamline is constructed in such a way to accommodate the various needs of small research consortia, from universities or research institutes, whose requirements of a beamline facility are quite diverse while under limited funding support. Flexibility is the special feature of this beamline. It is capable of performing quick test measurements without a prolonged reviewing process. Switching between different techniques, such as white-beam irradiation, EXAFS and X-ray scattering, can be achieved within 1 h. Novel experiments, such as energy-dispersive small-angle scattering experiments and energy-dispersive reflectivity measurements, can also be performed.

Journal Article↗

A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus.

Infectious bursal disease virus (IBDV), a member of the birnaviridae family, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5' termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA polymerase of IBDV. A proteinase K digestion based approach and acid-guanidium-phenol-chloroform (AGPC) RNA extraction method were used to extract dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and purity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates with the presence of a genome-linked protein in IBDV. Although dsRNA obtained by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pairs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of longer fragments (1958 bp) of IBDV cDNA by PCR.

Animals↗

Research notes: ELISPOT assay for detection of antibody secreting cells to infectious bursal disease virus in chickens.

The ELISPOT assay was used to enumerate antibody secreting cells (ASC) in spleens of chickens infected with infectious bursal disease virus (IBDV). In the first experiment, chickens were orally challenged with IBDV. Spleens were collected 14 d later and used for ELISPOT assay. The assay detected 16 to 28 IgG ASC per 10(6) splenocytes and 3 to 6 IgM ASC per 10(6) splenocytes. In the second experiment, chickens were vaccinated against IBDV and orally challenged with IBDV 14 d after vaccination. Spleens were collected 7 d postchallenge. The ELISPOT assay detected 18 to 128 IgG ASC per 10(6) splenocytes and 4 to 6 IgM ASC per 10(6) splenocytes. The results indicated that the ELISPOT assay can be used to measure isotype-specific antibody responses to IBDV or other avian pathogens.

Animals↗

Inhibition of nitric oxide synthase expression by PPM-18, a novel anti-inflammatory agent, in vitro and in vivo.

We studied the effect of PPM-18, a chemically synthesized naphthoquinone derivative and also an anti-inflammatory agent, on the lipopolysaccharide (LPS)-activated inducible NO synthase (iNOS) expression in rat alveolar macrophages. Pretreatment of macrophages with PPM-18 (0.1-10 microM) significantly inhibited nitrite production, iNOS protein expression and iNOS mRNA accumulation. PPM-18 did not directly affect the enzymic activities of iNOS and other constitutive NOS forms. The LPS-induced increase in nuclear transcription factor kappaB (NF-kappaB) p65 and p50 in nucleus was suppressed by PPM-18 (10 microM). Moreover electrophoretic mobility-shift assays demonstrated that PPM-18 inhibited DNA binding to NF-kappaB induced by LPS in whole cells but not when added in the nuclear extract, suggesting that PPM-18 did not interfere directly with the binding of NF-kappaB to DNA and that some events had to be processed before NF-kappaB could bind DNA. Examination of NF-kappaB showed that PPM-18 stabilized the NF-kappaB inhibitor, IkappaBalpha, by preventing its degradation from NF-kappaB. Therefore the stabilization of IkappaBalpha might have contributed to the inhibition of NF-kappaB activation. These results also indicate strongly that NF-kappaB is involved in the production of NO on stimulation by LPS. PPM-18 significantly decreased the production of tumour necrosis factor alpha in response to LPS. PPM-18 protects mice against LPS-induced lethal toxicity. These results also indicate that PPM-18 is a potent inhibitor of iNOS expression by blocking the binding of NF-kappaB to promoter and exerts a beneficial effect in the mouse model of sepsis.

Animals↗

Quantitative competitive polymerase chain reaction for detection and quantification of infectious bursal disease virus cDNA and RNA.

A polymerase chain reaction (PCR)-based method to measure complementary DNA (cDNA) and RNA levels of infectious bursal disease virus (IBDV) was developed. Quantification was achieved by quantitative competitive PCR (QC-PCR) amplification. A competitor, a deletion mutant of the wild type IBDV cDNA, was 10-fold serially diluted and co-amplified with IBDV cDNA after being reversely transcribed from the viral RNA. After agarose gel electrophoresis, staining, and densitometric scanning, the bands on the digitized images were analyzed and quantified by computer-assisted image analysis. Complementary DNA of standard, as well as variant strains, of serotype 1 IBDV was detected and quantified using the same QC-PCR procedures. The assay could measure IBDV cDNA levels ranging from 1 microgram to 45 fg and RNA levels ranging from 9 micrograms to 45 fg. The results indicated that QC-PCR is sensitive, easy to perform, and suitable for routine quantitation of IBDV cDNA or RNA levels.

Animals↗

Experimental infection of young specific pathogen-free cats with Bartonella henselae.

Eighteen 12-week-old specific pathogen-free cats, blood culture- and serum antibody-negative for Bartonella henselae, were randomly allocated to groups and were intravenously inoculated with 10(10) (group 1), 10(8) (group 2), or 10(6) (group 3) B. henselae or with saline (group 4) or were not inoculated (group 5). Cats were humanely killed at 2, 4, 8, 16, and 32 weeks after inoculation. All B. henselae-inoculated cats were bacteremic by 2 weeks after infection. Bacteremia persisted until 32 weeks after infection in 1 cat. Cats in groups 1 and 2 had fever (>39.7 degrees C) and partial anorexia by 2 weeks after infection that lasted 2-7 days. All infected cats had Bartonella-specific IgM and IgG serum antibodies and lymphocyte blastogenic responses. Histopathologic lesions were observed in multiple organs of infected cats through 8 weeks after infection. Cats were readily infected with B. henselae by intravenous inoculation, developed histopathologic lesions that apparently resolved, and developed B and T lymphocyte responses to infection.

Animals↗

Temperature effect on the sensitivity of ELISA, PA and WB to detect anti-HIV-1 antibody and infectivity of HIV-1.

BACKGROUND: This study is designed to resolve the problem of whether temperature or freeze/thaw cycle will have any impact on the sensitivity for detection of anti-HIV-1 antibody by particle agglutination (PA), enzyme-linked immunosorbent assay (ELISA) and western blotting (WB). To reduce potential risk for laboratory personnel exposed to HIV-infection, it will be useful to determine the temperature effect on HIV infectivity. METHODS: Testing sera were incubated at different temperatures or treated with several cycles of freeze and thaw. PA, ELISA and WB were used to detect anti-HIV-antibodies, whereas syncytia formation assay and polymerase chain reaction (PCR) were applied to detect HIV-infection. RESULTS: The data showed that certain temperature points (no treatment, 25 degrees C for 1 hr, 2 hrs and 4 hrs, 37 degrees C for 30 minutes and 60 minutes, 56 degrees C for 30 minutes and 60 minutes, 65 degrees C for 15 minutes and 30 minutes) had no impact on the testing results of ELISA, PA and WB in detection of anti-HIV-1 antibody. In addition, testing results of 50 normal human serum samples which had been heated to 56 degrees C for 30 minutes were still negative by ELISA and PA. Only the samples incubated at 65 degrees C for 60 minutes had slight differences in results. Freeze and thaw treatments of the serum did not alter anti-HIV testing results, either. Treatments of supernatant of HTLV-IIIB culture at 56 degrees C for 30 minutes and 60 minutes, 65 degrees C for 15 minutes and 30 minutes could eliminate the syncytia formation caused by HIV-infection. Further analysis of the samples by PCR was able to detect HIV-specific sequences in all the treatments. CONCLUSIONS: Anti-HIV antibody is quite stable in serum, even when it is pre-heated to 56 degrees C for 30 minutes. Freeze and thaw treatment of serum samples up to seven cycles did not change the results, either. In addition, to minimize the potential risk of laboratory personnel exposed to HIV infection, pre-treatment of serum samples with heat at 56 degrees C for 30 minutes or 60 minutes can reduce HIV infectivity. However, laboratories still must emphasize the importance of universal precautions rather than heat-inactivation of serum to prevent occupational transmission of HIV.

Agglutination Tests↗

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Animals↗

Passive immunization of channel catfish (Ictalurus punctatus) against the ciliated protozoan parasite Ichthyophthirius multifiliis by use of murine monoclonal antibodies.

Fish acquire immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection. The immune response includes the elaboration of humoral antibodies against a class of abundant surface membrane proteins referred to as immobilization antigens (i-antigens). Antibodies against these proteins immobilize the parasite in vitro, suggesting a potential role for the i-antigens in protective immunity. To test this hypothesis, passive immunization experiments were carried out with naive channel catfish, Ictalurus punctatus, using immobilizing murine monoclonal antibodies (MAbs). Fish were completely protected against lethal challenge following intraperitoneal injection of 20 to 200 micrograms of MAb. Although fish succumbed to infection at lower doses, palliative effects were observed with as little as 2 micrograms of antibody. In experiments in which animals were challenged at various times following inoculation, an inverse relationship between parasite load and serum immobilizing activity was seen. Of seven MAbs which conferred protection, all were immunoglobulin G class antibodies. The only immobilizing MAb that failed to protect was an immunoglobulin M antibody that was absent from surface mucosa as determined by enzyme-linked immunosorbent assay. The implications of these findings for the development of a vaccine against I. multifiliis and immunity against surface pathogens of fish are discussed.

Animals↗

Detection of IgM antibody to Japanese encephalitis virus infection by enzyme-linked immunosorbent assay (ELISA).

BACKGROUND: Japanese encephalitis (JE) is an important infectious disease in Taiwan, with reported cases observed all the year around. Laboratory tests for JE consist mainly of hemagglutination inhibition (HI) test and neutralization test (NT). Commercialized enzyme-linked immunosorbent assay (ELISA) kits for detection of JE-IgM are still not available. Therefore an attempt has been made to develop a sensitive and rapid ELISA for detection of the IgM antibody to JE to serve as an indication of recent Japanese encephalitis virus infection. METHODS: Both positive and negative JE serum specimens, confirmed by HI test, were checked for IgM antibody to JE by ELISA. The optimum concentration of biotin-IgG and avidin horse-radish peroxidase conjugate used in MAC-ELISA were 1/2000 and 1/ 15000, respectively. RESULTS: From 1987 to 1989, 118 paired serum specimens of HI-confirmed JE patients were tested for JE-IgM by ELISA. The positive rate of JE-IgM was 65.7% (25/38), 73.9% (17/23), 93.5% (29/31) and 88.8% (8/9) for the consecutive first to fourth weeks after onset of the disease. The JE-IgM antibody of 17 serum specimens collected from the 5th to the 10th week after onset of the disease were 100% detected. In addition, among the 13 HI-confirmed JE cases occurring in 1994, 84.6% of the acute phase serum specimens demonstrated the JE-IgM antibody. CONCLUSIONS: About 65.7% of the JE-IgM of the acute serum specimens collected within one week after onset of the disease were detected. The JE-IgM positive rate elevated as the days from disease onset increased. Therefore the appearance of JE-IgM could be used as an indication of recent JEV infection to serve as a rapid laboratory diagnostic tool.

Adolescent↗

Experimental infection of the red-backed vole (Clethrionomys gapperi) with Borrelia burgdorferi.

Red-backed voles (Clethrionomys gapperi) were live trapped in northern St. Louis County, Minnesota (USA), in late September and October 1988 and experimentally inoculated with Borrelia burgdorferi. Spirochetes were isolated from most animals 14 and 28 days following inoculation. Thus, red-backed voles exposed to B. burgdorferi were susceptible to infection and could be a reservoir host, along with chipmunks (Tamias striatus) and other small rodents, in areas where white-footed mouse (Peromyscus leucopus) populations are low. No evidence of clinical disease was noted in any infected voles.

Animals↗

Evaluation of a rapid microfiltration test for detection of HIV-1 antibody in serum.

BACKGROUND: Since the number of asymptomatic Human Immunodeficiency Virus Type 1 (HIV-1) infections has increased rapidly recently, to detect HIV-1 antibody rapidly becomes very critical when a quick mass survey is required or especially badly injured persons need blood transfusions immediately. This study evaluated sensitivity and specificity of a 10-minute qualitative assay (Single Used Diagnosis System; SUDS HIV-1 test) which is performed in a plastic cartridge using a modified enzyme immunoassay and to compare the results so obtained with more conventional HIV-1 antibody detection systems. METHODS: A total number of 248 human serum samples collected at several local hospitals and health stations were delivered to this Institute from June 1993 to April 1994 for confirmation were used to compare results obtained by either particle agglutination (PA), Western blot, or the SUDS HIV-1 tests. RESULTS: High sensitivity and specificity of SUDS were achieved using the conjugation of a specific gag antigen of HIV-1 and a synthetic HIV-1 envelop peptide. There was good correlation to one another among results from all the three tests. CONCLUSIONS: Data from those three tests employed in this study revealed no significant differences in sensitivity and specificity. The simplicity and rapidity of the SUDS make it more suitable for use than the others, especially when an emergency room screening test or a blood bank test is required where urgent situations need urgent answers.

Agglutination Tests↗

Mutagenicity of airborne particulates from combustion of electric cables in a waste metal retrieval area.

The disposal of massive quantities of synthetic materials has become a very serious environmental problem around the world. When synthetic polymers are burnt or smolder in air, the combustion products are extremely complex, often consisting of several hundred compounds. In Taiwan, a serious environmental problem was caused by the open air burning of discarded electric cords or cables, sheathed in polyvinyl chloride (PVC), in a special waste metal retrieval area. The resulting air pollution was especially severe. To determine mutagenicity, air samples were obtained from the area and the mutagenic compounds were purified by LH-20 column chromatography and semi-preparative HPLC. The active fractions purified at each step were monitored for their mutagenicity using S. typhimurium TA98. The major mutagenic fractions of the airborne particulate samples from the metal retrieval area were found to correspond to those of PVC smog generated from burning waste cables in a laboratory combustion chamber. Moreover, HPLC and fluorescence spectrometry showed 1,8-DNP and 1,6-DNP to be the major mutagenic compounds in the airborne particulate samples from the metal retrieval area.

Air Pollution↗

Acute toxoplasmosis in a gray fox (Urocyon cinereoargenteus).

Toxoplasmosis was diagnosed in a gray fox (Urocyon cinereoargenteus) found moribund in Lake Loundes Park, Mississippi. There was almost total necrosis of mesenteric lymph nodes, focal hemorrhage and necrosis in the brain. Lesions in all of these organs were associated with numerous Toxoplasma gondii tachyzoites. Tissue cysts were seen in the brain. Organisms reacted with anti-T. gondii serum in an avidin-biotin immunohistochemical test. There was no evidence of concurrent distemper or rabies virus infection. Hammondia heydorni-like schizonts and gamonts were seen in sections of intestinal epithelium.

Animals↗