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Biomedical subjects

J Castillo-Olivares

Publications and source records attributed to J Castillo-Olivares.

10 recordsLinked to original sources
PubMed2003

Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

Animals↗
PubMed2001

Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (G(L)): induction of virus-neutralizing antibody and assessment of protection in ponies.

An Escherichia coli-expressed recombinant protein (6hisG(L)ecto) comprising the entire ectodomain (aa 18-122) of equine arteritis virus (EAV) glycoprotein G(L), the immunodominant viral antigen, induced higher neutralizing antibody titres than other G(L)-derived polypeptides when compared in an immunization study in ponies. The potential of the recombinant G(L) ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 microg of protein. All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1-2 weeks after the administration of a booster on week 5 (VNAb titres of 1.8-3.1), 13 (VNAb titres of 1.4-2.9) or 53 (VNAb titres of 1.2-2.3). Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb. Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx. The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion. These results provide the first evidence that a sub-unit vaccine protects horses against EAV. The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines.

Amino Acid Sequence↗
PubMed2000

Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (G(L)) of equine arteritis virus.

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.

Animals↗
PubMed1984

Time-related efficacy of liver cell isografts in fulminant hepatic failure.

We and others have reported that dispersed liver cells transplanted into the spleen parenchyma of syngeneic rats remained functional and viable for a long time. This report describes our results with hepatocellular transplantation as a therapeutic method in a model of fulminant hepatic failure (FHF) in the rat. 60 male Sprague-Dawley rats weighing 200-250 g were used. The FHF was reached through an Eck's fistula with 2/3 hepatectomy at the same time. This model produced lethal hepatic failure in a highly reproducible manner. Liver cells were isolated by the collagenase method. 40 X 10(6) hepatocytes suspended in Hanks' balanced salt solution were transplanted into the spleen parenchyma 24 hr before (group 1), at the same time as (group 2), and 24 hr after (group 3) FHF was achieved. Additional sham-operated animals (groups 4 and 5) and a control group (group 6) were used. The hepatocellular transplantation markedly increased the survival of the animals with induced FHF to 80% (group 1) and 60% (group 2)--but not in group 3 (20%),--compared with 10% in the control group. This study shows that dispersed liver cells transplanted into the spleen can provide sufficient support to allow animals with lethal hepatic failure to survive and recover. Nevertheless the efficacy of transplantation is a time-related phenomenon with the FHF induction.

Animals↗
PubMed1980

Human pancreatic polypeptide secretion in conditions of exogenous and endogenous hyperglycaemia.

The effects of exogenous and endogenous hyperglycaemia on human pancreatic polypeptide secretion have been studied. In normal subjects elevation of plasma glucose concentration by glucose infusion both depressed the basal levels of circulating human pancreatic polypeptide (by 40-50%) and consistently reduced the human pancreatic polypeptide response to the ingestion of a portion-rich meal (areas above pre-meal value: 19.5 +/- 4.1 (mean +/- SEM) vs. 9.6 +/- 2.1, p < 0.01) as well as to caerulein infusion (areas above pre-caerulein value: 8.8 +/- 2.2 vs. 4.6 +/- 1.4, P < 0.01). In diabetic subjects treated with sulphonylureas or diet (fasting plasma glucose: 166 +/- 11 mg/dl, n = 24), human pancreatic polypeptide secretion evoked by food was similar to that of 24 healthy individuals (areas above basal value: 46.6 +/- 9.9 and 33.6 +/- 3.6, respectively). In insulin dependent diabetics (fasting plasma glucose: 231 +/- 19 mg/dl, n = 21) the human pancreatic polypeptide response to the meal (area above basal value: 78.2 +/- 13.7) was significantly greater than that of the controls as well as that of the noninsulin-dependent group (P < 0.05). Since the administration of pancreatic polypeptide to man has been shown to decrease pancreatic exocrine output, postprandial human pancreatic polypeptide hypersection may contribute to the decreased exocrin function of the pancreas often found in insulin-dependent diabetics.

Adolescent↗