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Biomedical subjects

J P Martin

Publications and source records attributed to J P Martin.

At least 19 recordsLinked to original sources

Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus.

Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton-mediated process occurred. The productive infection of SEC may contribute to the progression of the infection.

Animals

Development of a sedation policy for upper GI endoscopy based on an audit of patients' perception of the procedure.

OBJECTIVE: To assess patients' perception of upper GI endoscopy, both with and without sedation, and based on this information to propose a sedation policy for the procedure. METHODS: A prospective audit of the experience of patients undergoing upper GI endoscopy both with and without sedation. RESULTS: Of the 500 patients in the study, 237 had endoscopy with sedation and 263 with local anaesthetic throat spray. Sedated patients found the procedure more tolerable, but most unsedated patients felt the extra discomfort was more than compensated for by other advantages. 87% of those patients sedated and 70% of those not sedated would choose to have the procedure repeated in the same way. CONCLUSION: Upper GI endoscopy using local anaesthetic throat spray alone is well tolerated by patients. We have developed a policy on sedation which we believe could result in many more patients electively choosing to have the procedure without sedation, resulting in potential savings on cost and manpower.

Administration, Topical

[Unilateral adrenal hematoma in adults. Contribution of MRI in follow-up].

We describe a case of unilateral and spontaneous adrenal hematoma. Initial and follow up MRI examinations showed serial modifications typical of hematoma and allowed to exclude a preexisting non hyperfunctioning tumor. MRI findings modified the therapeutic attitude, a voiding surgery.

Adrenal Gland Diseases

Frequency and prognostic evaluation of 3p21-22 allelic losses in non-small-cell lung cancer.

Previous loss of heterozygosity (LOH) studies of chromosome 3p loci have displayed a 60% deletion frequency in non-small-cell lung cancers (NSCLC), as opposed to small-cell lung cancers, in which the 3p deletion is consistently found. However, the high stromal-cell admixture found in NSCLC and the use of the Southern-blot method lead to under-evaluation of this frequency. In this study, we used a very precise microdissection technique followed by PCR amplification of 6 3p21-22 polymorphic genomic sequences to analyze LOH in 86 NSCLC and in normal adjacent tissue. We found the sensitivity of the microdissection-PCR-based LOH technique higher than the sensitivity of the Southern-blot technique: 87% of the squamous-cell carcinomas and 84% of the large-cell undifferentiated carcinomas showed a clear LOH for a 3p21-22 locus. All doubly informative cases but 4 showed concordant deletion at all 3p21-22 loci. The analysis of 3p microsatellite sequences displayed only 2 cases of genomic instability, one of them also displaying features of tumoral heterogeneity as regards the instability genotype. Four carcinomas in situ adjacent to these NSCLC showed the same allelic profile as the invasive tumors. The only prognostic factors in this study were the disease stage and histology. The 3p21-22 deletion was not related to the stage of the disease and did not appear to be a significant prognostic factor of survival. 3p21 loss appears, so far, to be the most frequent and the earliest genetic alteration described in NSCLC, but does not seem to carry significant prognostic information in invasive tumors.

Alleles

Involvement of the three inter-alpha-trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family.

Partial cDNAs coding for each of the three human inter-alpha-trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3-related ITI form and a low-amount H1-related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.

Alpha-Globulins

The human inter-alpha-trypsin inhibitor genes respond differently to interleukin-6 in HepG2 cells.

The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL-6, suggesting that IL-6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL-6-treated cells confirmed these data. The nuclear run-on assay supports the regulatory effect of IL-6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL-6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre-alpha-trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL-6.

Alpha-Globulins

Gene expression and protein distribution of inter-alpha-trypsin inhibitor in three human hepatoma cell lines.

In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno-precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously.

Alpha-Globulins

T cell immunodeficiency involved in pathogenicity of attenuated MHV3 mutants.

Viral pathogenicity results when there is an imbalance between viral replication and the host's immune defenses. The immune system plays and important role in the outcome of an acute disease induced by the mouse hepatitis virus type 3 (MHV3). Of use in the study of the role of viral properties involved in its pathogenicity is the attenuated escape mutants. We reported that two MHV3 escape mutants were attenuated in their ability to deplete T cell subpopulations in the spleen in BALB/c mice according to inoculation route and time postinfection. The highly attenuated CL12 mutant cannot induce depletion in T cells following intraperitoneal (i.p.) or intranasal (i.n.) inoculations, at three days postinfection (p.i.). The less attenuated 51.6 mutant, however, maintained the ability to deplete T cells following i.p. inoculation, as described for the pathogenic MHV3. In contrast, no depletion of T cells following i.n. inoculation was induced with this mutant. The use of such mutants enables us to dissect the role of each compartment of the immune system.

Animals

Data quality assurance, monitoring, and reporting.

In conclusion, the quality assurance and monitoring program is an integral and continuing part of study operations. A system must be devised and implemented by the coordinating center investigators, with the endorsement of the study leadership and support of the field site and resource center personnel. Proactive mechanisms for promoting high-quality data acquisition and reporting must be implemented. Data quality monitoring must address the entire process by which the data are gathered, transmitted, stored, and analyzed. Data quality should be monitored continually, with summary reports prepared and distributed to the study leadership. Appropriate training and certification enhance data quality, and site visits allow data collection and storage processes to be observed directly. The quality assurance and monitoring system must be documented. It should be flexible enough so that new means of quality assurance or monitoring can be added when necessary during the course of the study. At the completion of the study, quality monitoring results should be summarized in a final report regarding the level of quality achieved by the study investigators and personnel. Finally, for a quality assurance and monitoring program to be successful, the coordinating center investigators and personnel must provide prompt feedback and suggestions for corrective action whenever a data quality problem is discovered. This need can be met only when the coordinating center staff understand data quality goals and are up to date with all phases of data management and reporting. Delays in initiating any stage of data management and quality monitoring may result in uncorrectable data problems. Thus, knowledgeable and efficient coordinating center personnel are essential to achieving good data quality studywide.

Clinical Trials as Topic

Pretreatment with a blocking monoclonal antibody to P-selectin accelerates pharmacological thrombolysis in a primate model of arterial thrombosis.

It has been established that the fibrin content of a developing thrombus can be dramatically reduced with the use of the GA6 monoclonal antibody, which is directed against P-selectin (CD62p). This effect is probably related to diminished tissue factor activity on monocytes in the presence of P-selectin antagonism. Therefore, we hypothesized that an occlusive arterial thrombus formed in the presence of a P-selectin monoclonal antibody would be more susceptible to lysis with standard thrombolytic therapy. To test this hypothesis, 22 male cynomolgus monkeys were anesthetized and instrumented for induction of thrombosis of a femoral artery. Endothelial injury was induced by passing a 150-microA anodal current through a small electrode that was placed in the femoral artery. Blood flow through the artery was continuously monitored using an ultrasonic transit-time flowmeter. The GA6 monoclonal antibody (1 mg/kg) or control, isotype matched mouse IgG1 (P23 or P7) was administered i.v. 1 hr before electrolytic endothelial injury. In the P23 group (n = 11), an occlusive thrombus formed in 52.1 +/- 8.5 min, and in the GA6 group (n = 11), an occlusive thrombus formed in an average time of 52.0 +/- 8.1 min. After formation of an occlusive thrombus, the current was terminated and intravenous heparin (100 U/kg + 50 U/kg/hr) was administered to prevent clot extension.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Evidence of feline immunodeficiency virus replication in cultured Kupffer cells.

OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection.

Animals

Mouse hepatitis virus type 3 infection provokes a decrease in the number of sinusoidal endothelial cell fenestrae both in vivo and in vitro.

Fenestrations of hepatic endothelial cells play an active role as a sieving barrier allowing extensive exchange between the blood and liver parenchyma. Alteration of these structures may be induced in the course of various pathological events and provoke important perturbations of liver function. We demonstrate here that sinusoidal endothelial cells are permissive for mouse hepatitis virus 3 (MHV3) in vivo and in vitro and that this infection leads to a striking decrease in the number of fenestrae. The disappearance of these structures observed under scanning electron microscopy or in cryofracture preparations in vivo and in vitro cannot be reversed by the action of cytochalasin B on the microfilament network. The decrease in the porosity seems to be related directly to the productive infection of the endothelial cells, because it was not observed in A/J mice resistant to the virus and in susceptible BALB/c mice immunized with a thermosensitive mutant in which no viral replication occurs. In conclusion, a viral infection of liver endothelial cells may cause extensive loss of the fenestrations and thus lead to important functional pertubations.

Animals

Tandem orientation of the inter-alpha-trypsin inhibitor heavy chain H1 and H3 genes.

The inter-alpha-trypsin inhibitor H1 (ITIH1) and inter-alpha-trypsin inhibitor H3 (ITIH3) genes have both previously been mapped to chromosomes 3 and 14 in the human and mouse, respectively. We now present evidence that these genes are physically linked. By using cDNA probes, a recombinant DNA phage has been isolated from a bacteriophage DNA library, which contains sequences flanking the 5' end of the ITIH3 gene and the 3' end of the ITIH1 gene. Restriction endonuclease mapping, PCR analysis and DNA sequence determination of the recombinant phage and comparison to genomic DNA revealed that the genes are in tandem, 2721 base pairs apart, with the ITIH1 gene to the 5' side of the ITIH3 gene. Their respective transcriptional units are thus on the same strand of DNA and most probably arose in evolution as the consequence of a duplication of a common ancestral gene.

Alpha-Globulins

Post-translational processing of the inter-alpha-trypsin inhibitor in the human hepatoma HepG2 cell line.

In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.

Alpha-Globulins

Detection by the polymerase chain reaction of two polymorphisms in exon 14 of the human inter-alpha-trypsin inhibitor heavy chain H1 gene.

Two polymorphisms were detected within exon 14 of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) gene. The polymorphisms are detected by digesting the same 202-bp polymerase reaction product with the PstI and Hph1 restriction endonucleases. These gene polymorphisms lead to the change of two amino acids in the mature protein. The polymorphisms can be used for the analysis of 3p21 deletion in human carcinomas as well as to develop a better understanding of the protein polymorphism already described.

Alpha-Globulins

The polymorphism of the plasma inter-alpha-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212.

We investigated the ITI protein polymorphism in linkage analysis, using DraI and SstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI*1 and ITI*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented by DraI 4.0 kb and DraI 2.4 + 1.6 kb, and by SstI 6.7 kb and SstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and by DraI 4.0/2.4 + 1.6 kb and SstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1-2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI*1 and ITI*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.

Alleles

Pathogenicity of neutralization escape mutants of mouse hepatitis virus: correlation with T- and B-cell depletions.

Viral pathogenicity is a result of an imbalance between viral replication and the host's immune defences. When the virus is lymphotropic, understanding the pathogenic process of the viral disease becomes complicated because virus/lymphocyte interactions can alter the cell's integrity and subsequently induce immunodeficiency. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3). The use of attenuated escape mutants provides a tool to study the role of viral properties involved in its pathogenicity. We selected MHV3 mutants by virtue of their resistance to neutralization by monoclonal antibodies (mAb), in order to study their pathogenic properties. We reported that two MHV3 escape mutants were attenuated in their pathogenic properties according to inoculation site and with regard to survival time and ability to deplete T- and B-cell subpopulations in the spleen, thymus and bone marrow of susceptible Balb/c mice. The highly attenuated CL12 mutant could not induce depletion in T or B cells following intraperitoneal (i.p.) or intranasal (i.n.) inoculations, at three days postinfection. The less attenuated 51.6 mutant, however, maintained the ability to deplete T and B cells following i.p. inoculation, as described with the pathogenic MHV3. In contrast, no depletion of T cells following i.n. inoculation was induced with this mutant, although B lineage cells decreased. The use of such mutants enabled us to examine the role of each compartment of the immune system, since the highly attenuated CL12 mutant induced no immunodeficiency, as defined by immune cell depletion, whereas the less attenuated 51.6 mutant maintained its ability to decrease only the B-cell compartment after i.n. inoculation. Results are discussed with regard to the virus/lymphocyte interactions during the pathogenic process.

Administration, Intranasal

Light microscopical detection of inter-alpha-trypsin inhibitor and its different mRNAs in cultured hepatoma Hep G2 cells using immunocytochemical and in situ hybridization techniques.

The Hep G2 hepatoma cell line synthesizes the inter-alpha-trypsin inhibitor (ITI). This protease inhibitor and the other proteins of this family include four polypeptides chains: three heavy chains (HC1, HC2, HC3) and one light chain (bikunin). In the present study, we have demonstrated by immunofluorescence that ITI is detected mainly in perinuclear cytoplasmic zones comparable to those of albumin or alpha-1-antitrypsin. The presence of the mRNAs of the four polypeptide chains in all Hep G2 cells of a non-synchronized culture have been demonstrated by in situ hybridization. An evaluation of the transcription of the four ITI genes through an analysis of markings brings to the fore a clearly much higher rate of mRNAs from the light chain than from the heavy chains. The mRNAs corresponding to the HC2 chains are more heavily represented than are those corresponding to the HC1 and HC3 chains. In Hep G2 cells in culture, a quantification of mRNAs based on the in situ hybridization technique shows that their relative quantities, in decreasing order, are those of L, HC2, HC3 and HC1.

Alpha-Globulins