Search PubMed⌕ Search

Biomedical subjects

T H The

Publications and source records attributed to T H The.

At least 73 records · Page 4Linked to original sources
PubMed1995

The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection.

A small polypeptide from pUL57 of human cytomegalovirus was identified as a major target for the immunoglobulin M antibody response. This antigen seems to be superior to antigenic fragments from pp150 and p52 in the identification of sera from acutely infected patients. It may therefore represent an essential antigen for recombinant immunoglobulin M antibody tests for human cytomegalovirus.

Acute Disease↗
PubMed1995

Comparative immunoblot analysis with ten different, partially overlapping recombinant fusion proteins derived from five different cytomegalovirus proteins.

Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with an apparent molecular weight of 38 kD, ppUL99 (MW of 28 kD), the basic phosphoprotein ppUL32 (MW of 150 kD), the lower matrix protein ppUL83 (65 kD), and the DNA binding protein ppUL44 (MW of 52 kD). Some fragments overlapped and responses to these recombinant proteins were compared. Antibody responses appeared to be predominately directed at fusion proteins of ppUL32, the basic phosphoprotein and at ppUL44, the major DNA binding protein. Some differences were found in the responses to these partly overlapping fusion proteins. In addition, an IgM response mainly present in primary infections to fusion proteins XP1 and G2, again representing ppUL32 and ppUL44, was found.

Antibodies, Viral↗
PubMed1995

The cytomegalovirus antigenemia assay: a plea for standardization.

Its high diagnostic accuracy, rapidity, quantitative nature and technical simplicity have made the cytomegalovirus (CMV) antigenaemia assay one of the cornerstone methods for diagnosis and management of active CMV infection in immunocompromised patients. Many technical variations have been introduced in an effort to optimize the assay. Now, standardization of the assay and quality control are becoming of increasing importance. In this review we first discuss the nature and origin of the CMV antigens in distinct blood cells during active CMV infection. Further, some of the most important technical variations of the assay are considered and a proposal for standardization is made to indicate how quality control might be achieved. Acceptance of these proposals by the international community would be an important step forward, e.g. towards multicentre antigenaemia-directed intervention studies.

Antigens, Viral↗
PubMed1994

Quantification of low frequency white blood cells expressing human cytomegalovirus antigen by image cytometry.

Human cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromised patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen-positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen-positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen-positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen-positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen-positive cells than patients with a HCMV infection who remained nonviremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques.

Antigens, Viral↗
PubMed1994

Epitope mapping of SCLC-cluster-2 MAbs and generation of antibodies directed against new EGP-2 epitopes.

Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it difficult to generate and select antibodies against other potentially useful EGP-2 epitopes. Using PCR, we have generated mutant EGP-2 cDNA (delta EGP-2) from which the coding sequences for a putative immunodominant 6-kDa intra-chain loop structure has been removed. delta EGP-2 transfected COS-7 cells reacted with MM104, an antibody detecting a linear epitope on EGP-2, but were not recognized by any cluster-2 MAb. To generate new anti-EGP-2 antibodies we constructed another mutant EGP-2 protein (delta EGP-2) from which additional domains, irrelevant for antibody generation (signal peptide, trans-membrane and cytoplasmic domains), were removed. delta EGP-2 was introduced in a prokaryotic expression system that adds a polyhistidine affinity tag to the delta EGP-2 N-terminus, making possible one-step purification by immobilized metal-ion-affinity chromatography (IMAC). Western blot analysis showed that sera derived from mice immunized with purified delta EGP-2 had high-titer antibodies to reduced EGP-2 samples. We conclude that the availability of prokaryotic and eukaryotic EGP-2-expression constructs might facilitate the selection of new anti-EGP-2 MAbs otherwise difficult to obtain.

Amino Acid Sequence↗
PubMed1994

Detection of a putative 30-kDa ligand of the cluster-2 antigen.

The cluster-2 antigen, also called EGP-2, is a 38-kDa transmembrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. Monoclonal antibodies (MAbs) directed against EGP-2 have been extensively studied as anti-tumor agents, yet the function of the antigen is not known. In the present study we used a biotinylated recombinant soluble derivative of the EGP-2 (sEGPbio) as a probe to detect a possible EGP-2 ligand, using various carcinoma cell lines as a substrate. The recombinant soluble EGP-2 was expressed in the Autographa californica nuclear polyhedrosis virus (baculovirus) expression system. The sEGP-2, to which we engineered a poly-histidine affinity tag, was purified from infected Spodoptera frugiperda insect cells using immobilized metal-ion-affinity chromatography (IMAC). In Western blot analysis the sEGPbio probe bound to a 30-kDa protein band in 2 out of 5 of the assessed carcinoma cell lines, suggesting that this band may be an EGP-2 ligand. Interestingly, binding only occurred when, prior to SDS-PAGE, cell lysates had been subjected to a reducing agent (2-mercapto-ethanol). The physiological significance of this phenomenon and nature of the detected 30-kDa protein band remains to be determined.

Amino Acid Sequence↗
PubMed1994

Natural killer cell responses in renal transplant patients with cytomegalovirus infection.

Natural killer (NK) cell function in relation to immunophenotypical signs of NK activation was studied prospectively in 15 renal transplant patients with cytomegalovirus (CMV) infection. NK activity (expressed as the percentage of K562 lysis) before onset of CMV infection reached a median of 6% (range 2-18%), comparable to values observed in noninfected controls [7% (range 7-21%), P < .1]. During CMV infection, NK activity rose to a maximum of 25% (6-60%) (P < .001 vs. controls). Maximal values exceeded the upper level of controls in nine of 15 patients. NK activity was correlated to the number of CD56+HLADR+ cells in the peripheral blood (r = .57, P < .001). These data suggest that NK cells play a role in the recovery from CMV infection in a substantial number of patients and that immunophenotypical analysis of NK activation provides a surrogate marker of NK cell function.

Antigens, CD↗
PubMed1994

Poor induction of interleukin-2 receptor expression on CD8bright+ cells in whole blood cell cultures with CD3 mAb. Implications for immunotherapy with CD3 mAb.

To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed. However, in our clinic, such a treatment did not induce additional activation of T cells. To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant. Cultures using isolated blood mononuclear cells were used as a control. When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures. However, when stimulation was performed with CD3 mAb, CD8bright+ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor beta (IL-2R beta). This is in contrast to the situation found in MNC cultures where all CD8bright+ cells expressed CD25 or IL-2R beta to a high extent at the end of culture. When rIL-2 or recombinant interferon gamma (rIFN gamma) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright+ cells were induced to express CD25 or IL-2R beta. These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures. In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright+ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy. The presented results suggest that in vivo simultaneous administration of rIFN gamma or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8+ T cells than CD3 mAb only.

Antibodies, Monoclonal↗
PubMed1994

Phase I study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2.

In a phase I trial the toxicity and immunomodulatory effects of combined treatment with intravenous (i.v.) bispecific monoclonal antibody BIS-1 and subcutaneous (s.c.) interleukin 2 (IL-2) was studied in renal cell cancer patients. BIS-1 combines a specificity against CD3 on T lymphocytes with a specificity against a 40 kDa pancarcinoma-associated antigen, EGP-2. Patients received BIS-1 F(ab')2 fragments intravenously at doses of 1, 3 and 5 micrograms kg-1 body weight during a concomitantly given standard s.c. IL-2 treatment. For each dose, four patients were treated with a 2 h BIS-1 infusion in the second and fourth week of IL-2 therapy. Acute BIS-1 F(ab')2-related toxicity with symptoms of chills, peripheral vasoconstriction and temporary dyspnoea was observed in 2/4 and 5/5 patients at the 3 and 5 micrograms kg-1 dose level respectively. The maximum tolerated dose (MTD) of BIS-1 F(ab')2 was 5 micrograms kg-1. Elevated plasma levels of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were detected at the MTD. Flow cytometric analysis showed a dose-dependent binding of BIS-1 F(ab')2 to circulating T lymphocytes. Peripheral blood mononuclear cells (PBMCs), isolated after treatment with 3 and 5 micrograms kg-1 BIS-1, showed increased specific cytolytic capacity against EGP-2+ tumour cells as tested in an ex vivo performed assay. Maximal killing capacity of the PBMCs, as assessed by adding excess BIS-1 to the assay, was shown to be decreased after BIS-1 infusion at 5 micrograms kg-1 BIS-1 F(ab')2. A BIS-1 F(ab')2 dose-dependent disappearance of circulating mononuclear cells from the peripheral blood was observed. Within the circulating CD3+ CD8+ lymphocyte population. LFA-1 alpha-bright and HLA-DR+ T-cell numbers decreased preferentially. It is concluded that i.v. BIS-1 F(ab')2, when combined with s.c. IL-2, has a MTD of 5 micrograms kg-1. The treatment endows the T lymphocytes with a specific anti-EGP-2-directed cytotoxic potential.

Adjuvants, Immunologic↗
PubMed1994

Presence of human cytomegalovirus (HCMV) immediate early mRNA but not ppUL83 (lower matrix protein pp65) mRNA in polymorphonuclear and mononuclear leukocytes during active HCMV infection.

During an active human cytomegalovirus (HCMV) infection, leukocytes, harbouring the HCMV lower matrix protein pp65 (ppUL83) are present in the peripheral blood and can be detected with the HCMV antigenaemia assay. In the present study, it was investigated whether the presence of pp65 in these cells was due to transcription of the virus genome or might be the result of uptake of this viral protein. Peripheral blood leukocytes of transplant recipients and AIDS patients with an active HCMV infection were investigated for the presence of HCMV immediate early (IE) antigen and pp65 using well characterized monoclonal antibodies, and for the presence of the corresponding mRNAs using non-radioactive in situ hybridization. Both mononuclear and polymorphonuclear cells were found to contain IE antigen and pp65. However, only mRNAs encoding IE antigen were found in these cells, whereas mRNAs encoding pp65 were not detected. In contrast, both IE antigen and pp65, as well as their corresponding mRNAs, were detected in the circulating late-stage HCMV-infected endothelial cells that were also present in the leukocyte fractions. These findings demonstrate that a restricted viral gene expression (transcription of IE genes) does occur in mononuclear and polymorphonuclear leukocytes. However, the abundant presence of the early antigen pp65 without detectable presence of the corresponding mRNA in these cells strongly indicates uptake of this protein by the phagocytic leukocytes, rather than de novo synthesis.

Antigens, Viral↗
PubMed1994

Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens.

DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of pp150 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of pp150 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.

Antibodies, Viral↗
PubMed1994

CMV infection after organ transplantation: immunopathological and clinical aspects.

Cytomegalovirus (CMV), a member of the Herpes virus family, is a seemingly harmless infectious agent for healthy individuals. However, it is one of the most important opportunistic pathogens in immunosuppressed patients, particularly in organ transplant recipients. Due to its monocytotropic character, CMV exhibits an intimate relationship with the host immune system, and is therefore of great interest to immunologists. In this review, a summary is given of recent developments in our understanding of the interaction between CMV and the host immune system and guidelines are provided for the clinical management of this infection.

Cytomegalovirus Infections↗
PubMed1993

Antibody responses to human cytomegalovirus-specific polypeptides studied by immunoblotting in relation to viral load during cytomegalovirus infection.

The humoral immune response to individual proteins of human cytomegalovirus (CMV) was studied by immunoblotting. CMV polypeptides present in an extract of CMV-infected fibroblasts in a late stage of infection were recognized by sera of healthy seropositive individuals and transplant recipients who suffered from a primary or secondary CMV infection. The results showed that the sera reacted with a maximum number of 18 polypeptides ranging in molecular weight from 28-235 kDa. In 60% or more of the healthy seropositives, polypeptides were recognized with an apparent molecular weight of 150, 98, 94, 58, 50, 44, 38, and 32 kDa. In the patient group, the most immunogenic polypeptides were those with an apparent molecular weight of 150, 104, 94, 66, 50, 38, and 32 kDa. A correlation was found between the antibody levels in sera from the healthy seropositives and the number of recognized polypeptides but no such relationship was seen in the transplant recipients. However, sera of patients with a high virus load during their secondary CMV infection, as detected by the CMV-antigenemia test, appeared to react less frequently and less intensely with the polypeptides than those with low viremia. A high number of antigen-positive leukocytes in the CMV-antigenemia test was related to a low frequency of polypeptides with molecular weights of 85, 76, 66, 44, 38, and 32 kDa recognized before transplantation.

Adult↗
PubMed1993

Selective MR imaging of labeled human peripheral blood mononuclear cells by liposome mediated incorporation of dextran-magnetite particles.

Human peripheral blood mononuclear cells (PBMCs) were incubated with large unilamellar vesicles (LUV) containing encapsulated dextran-magnetite particles (DMP). This resulted in an efficient incorporation of DMP. Electron microscopy revealed the presence of DMP in cells mainly in phagosomes and secondary lysosomes. DMP-labeled PBMCs showed a strong increase of the transverse relaxation rate (up to 16.6 s-1 for 5 x 10(7) cells/ml) and, accordingly, a great loss of signal intensity in MR imaging. The fraction of DMP containing PBMCs could be enriched by magnetic cell separation. The major population of the DMP containing cells proved to be monocytes. When PBMCs depleted of monocytes were used for labeling, DMP uptake was observed also in the peripheral blood lymphocytes. The labeling of PBMCs presented here may be used in future studies of selective MR imaging of in vivo cell migration in a variety of immunologically compromised tissue states, e.g., tumors, transplantations, and abscesses.

Contrast Media↗