FITC-labeled Monoclonal Antibody Research Articles

Progression of HIV-1 infection

We present data on the distribution of human immunodeficiency virus (HIV-1) proviral DNA in different subsets of peripheral blood mononuclear cells (PBMCs) over an observation period of eight months. Eleven patients with well documented HIV-1 infection were studied. The PBMCs were obtained at two intervals and purified by fluorescence-activated cell sorting (FACS) after staining with FITC-labelled monoclonal antibodies. Varying numbers of FACS-sorted CD4+ cells, CD8+ cells and peripheral monocytes were assayed for HIV-1 proviral DNA (env and gag region) by PCR. Samples from patients at CDC stages II or III had to contain 10(3)-10(4) cells in order to allow detection of proviral HIV-1 DNA. At CDC stage IV, however, HIV-1 DNA was detected in as few as 100 CD4+ T-lymphocytes. In contrast, in peripheral monocytes HIV-1 DNA was not regularly found. CD8+ cells did not harbor detectable amounts of proviral DNA. During an observation period of eight months, the rate of infected CD4+ T-lymphocytes increased significantly in three patients while staying constant in the remaining eight patients. This increase of the infection rate was paralleled by clinical progression in one patient and by a decrease of the absolute number of CD4+ cells in another patient. The percentage of CD4+ cells harboring the viral genome increases in the course of the disease. These results may help to explain the decrease in CD4+ T-lymphocyte counts during HIV-1 infection.

130 INHIBITION OF ADHESION OF S-FIHBRIATED E.COLI TO BUCCAL EPITAECIAL CELLS ST HUMAN MILK FAT GLOBULE MEMBRANE MUCINS: A NEW ASPECT OF PROTECTIVE FUNCTION Of THE NON-IMHUNOGLOSULIN FRACTION

In search of factors of human milk that inhibit invasion of pathogenic bacteria we have analyzed the effect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated E.Coli to human buccal epithelial cells. S-fimbriae are a common feature of E.Coli strains causing sepsis and meningitis in the newborn period and are bound to epthelia via sialyl-(α 2-3) or sialyl-(α 2-6) galactoside structures. Whole milk fat globules (MFG) could be agglutinated by the above mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin and α1acid glykoprotein. In addition, pretreatment of MFG with Vibrio cholerae neuraminidase markedly reduced bacteria - induced agglutinations indicating the involvement of neuraminic acid containing glykoproteins. Lipid droplets of infant formula or arteficial lipid emulsions (IntralipidR), in contrast, could not be agglutinated. MFG were present in stools of breast fed newborn babies as shown by staining with a FITC-labelled monoclonal antibody directed against HMFGM and could be agglutinated by bacteria. To further characterize relevant HMFGM components they were separated by get chromatography. Of the fractions obtained mucins whose quality was confirmed by Western blot and monosaccharide analysis showed the most pronounced inhibitory effect on adhesion of S-fimbriated E.Coli to human buccal epithelial cells. Our data suggest that MFG besides their nutritive function provide protection against bacterial infections by inhibition of bacterial adhesion over the length of the entire intestine. Supported by Deutsche Forschungsgemeinschaft Schr 381/3-1

Culture confirmation and typing of herpes simplex virus isolates from clinical specimens with new monoclonal antibody typing reagents

FITC-labelled monoclonal antibody typing reagents for herpes simplex virus (HSV) (PathoDx, Diagnostic Products Corp., Los Angeles, California) were compared to another commercially available FITC-labelled monoclonal antibody typing reagent for culture confirmation and typing of HSV isolates from clinical specimens. A total of 344 clinical specimens were evaluated for culture confirmation and typing by the two systems. There were 296 HSV-positive cultures identified by cytopathic effect (CPE). The PathoDx typing reagents identified 143 isolates as HSV-1, and 151 isolates as HSV-2, with two isolates staining with both the type 1 and type 2 reagent. The PathoDx typing reagents had a 100% concordance with the other set of typing reagents.

Effect of salmon calcitonin on the infiltration of T lymphocytes in a synovium-like membrane of a rat inflammatory model

The effect of salmon calcitonin (SCT) on the number of T lymphocytes accumulating in air pouch lining membranes produced on the back of Wistar rats was studied. SCT was given from day 0 through day seven following the air cavitation by daily injections of 2.5-20 micrograms/kg directly into the air cavity. Compared with saline controls, treatment with SCT caused a clear reduction in percentage of T lymphocytes, T helper cells and T non-helper subset in the synovial-like air pouch lining as revealed by FITC-labeled monoclonal antibodies and quantified by flow cytophotometric measurements. This effect may be due to a reduction in local vascular permeability caused by SCT.

ＬＡＫ活性およびＩｎｔｅｒｌｅｕｋｉｎ２添加培養後の単核球表面抗原の変化に対するＣｏｒｔｉｃｏｓｔｅｒｏｉｄの影響 ｉｎ ｖｉｔｒｏにおける検討

In the treatment of human cancer, the efficacy of the adoptive transfer of lymphokine-activated killer (LAK) cells combined with interleukin 2 (IL2) has been emphasized. However, administration of large doses of IL2 may cause significant adverse effects, such as "capillary leak syndrome". Moreover, the administration of corticosteroid may reduce the toxicity of IL2. However the efficacy of adoptive immunotherapy is abrogated by the administration of corticosteroid. In this context, the effect of corticosteroid on LAK activity and the change of membrane antigen phenotype on PBMC (peripheral blood mononuclear cells) was studied in vitro by using methylprednisolone (M-PSL). LAK cells were induced from PBMC, which had been separated from the peripheral blood of normal donors, and incubated with IL2 for 4 days. LAK activity was measured in a 4 h chromium release assay against K-562 cell and Daudi cell. The suppression of LAK activity by M-PSL depended on both concentration and working period of M-PSL. This inhibition was recognized even at rather low densities, such as 0.5 nmol/ml M-PSL. And the proliferation of PBMC after IL2 incubation, which was measured by incorporation of [3H]TdR, was reduced dose-dependently by M-PSL. In contrast, pretreatment within 24 hours of PBMC with M-PSL resulted in no effect on LAK activity. Furthermore M-PSL had no effect when added directly to a 4 h chromium release assay. The analysis of membrane antigen phenotype on PBMC was performed by the direct immunofluorescence method using FITC-labeled monoclonal antibodies. The proportions of T cells and NK cells, which were justified as precursors of LAK cells, increased after IL2 incubation both with and without M-PSL. Therefore no effect of M-PSL was confirmed in those circumstances. The ratio of HLA-DR (+) cells increased after IL2 incubation without M-PSL, while M-PSL reduced expression of this antigen. In contrast, IL2 receptor (CD25)(+) cells, markers of T cell and NK cell activation, significantly increased after IL2 incubation with or without M-PSL. These results suggest that the inhibitory effect of M-PSL on LAK activation was caused not by preventing a triggering process of activation of precursor cells, but by a possible inhibition of proliferation, though other effects of corticosteroid remain to be elucidated. Also, it is emphasized that caution should be exercised in the administration of corticosteroid in adoptive immunotherapy because of the inhibitory effect induced by M-PSL.

An analysis of staining methods for the detection ofCryptosporidiumspp. oocysts in water-related samples

The ability of five staining techniques, originally developed for the rapid identification of Cryptosporidium spp. oocysts in faecal samples, to detect oocysts in water and water-related samples was assessed. All the stains used (modified Ziehl Neelsen, auramine-phenol (Lempert), Wright-Giemsa, safranin-methylene blue and FITC-labelled monoclonal antibody) stained oocysts after storage in water for 2 months at 4 degrees C (71-89% of control values). Storage of oocysts below 0 degrees C greatly reduced the staining ability of auramine-phenol. With the exception of oocysts stored in raw and final waters, the histochemical stains proved less useful in detecting oocysts than the monoclonal antibody. Organisms of similar size and shape took up these stains, causing confusion in interpretation. Cold Ziehl Neelsen and the FITC-labelled monoclonal antibody were best at identifying oocysts from a waterborne outbreak. Screening with a fluorescent antibody, followed by confirmation with cold Ziehl Neelsen, where possible, are the currently recommended procedures for the detection of oocysts in water-related samples.

Ganciclovirが有効であった腎移植後のCytomegalovirus Nephropathyの一症例

A 32-year-old man who had received a kidney transplant from a living related donor, contracted cytomegalovirus (CMV) pneumonitis in the 8th month. He was treated with human interferon-beta and cured of the pneumonitis. After that, his serum creatinine value increased gradually. Renal biopsy revealed the cells with intranuclear inclusion bodies in the renal tubulus and the cells were positive for CMV antigens by direct immunofluorescence test using FITC-labeled mouse monoclonal antibody against an early antigen. He was hospitalized with persistent CMV viruria and treated with ganciclovir. Ganciclovir was administered daily in doses of 3 mg per kg per day for 32 days by intravenous drip infusion and thereafter the same dose was given 3 times weekly for 8 weeks. His urine was positive for CMV before the ganciclovir treatment and became negative on the 31st day after the treatment. The anti-CMV effect of ganciclovir was evidenced by gradual decrease in titer (PFU) of infectious CMV in the urine samples. His serum creatinine value decreased from 3.2 mg/dl to 2.8 mg/dl, and no adverse effect was noticed. Thus, ganciclovir is considered to be efficacious against CMV infections in kidney transplant recipients.

Pluripotent hemopoietic stem cells give rise to osteoclasts in vitro: effects of rGM-CSF

Studies involving bone marrow transplantation of osteopetrotic rodents have provided evidence for the lineage of the ostcoclast. Recent investigations have demonstrated that pluripotent hemopoietic stem cells (PHSC) isolated from the bone marrow of normal animals cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia osteopetrotic rats. A criticism of these findings is that the microenvironment of the osteopetrotic bone and the bone marrow compartment may be unique in its ability to induce the differentiation of these stem cells into osteoclasts. To test this hypothesis, PHSC were co-cultured with fetal metatarsal bones from normal animals. PHSC were isolated from normal bone marrow using FITC-labelled monoclonal antibodies directed against rat Thy 1.1 and fluorescence-activated cell sorting. The PHSC or whole mononuclear bone marrow were co-cultured with 20-day fetal rat metatarsal rudiments. In some cultures, recombinant mouse granulocyte-macrophage colony-stimulating factor (rGM-CSF) (250 U per culture) was added in addition to the PHSC. After 7 days the fetal bones were prepared for light and electron microscopy and the number of osteoclasts generated in vitro was determined. The PHSC isolate generated as many osteoclasts as the whole mononuclear bone marrow. The addition of rGM-CSF did not enhance the generation of osteoclasts in either control bones or in bones cultured with PHSC. These results are equivalent to those reported in the osteopetrotic transplant system.

The effects of transplantation of granulocyte-macrophage progenitors on bone resorption in osteopetrotic rats.

Osteopetrosis in the ia (incisors absent) rat is the result of reduced bone resorption due to abnormal osteoclasts. This mutant and others have been used to determine the precursor(s) to osteoclasts. Hemopoietic stem cells, isolated from bone marrow of normal littermates, cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. These studies were conducted to define further the precursor to the osteoclast by evaluating the effects of the transplantation of granulocyte-macrophage progenitors on bone resorption in the ia rat. Granulocyte-colony forming cells (G-CFC), granulocyte-macrophage-colony forming cells (GM-CFC), and macrophage-colony forming cells (M-CFC) were isolated from normal bone marrow using an FITC-labeled monoclonal antibody directed against rat Thy-1.1 and fluorescence-activated cell sorting. The isolates were evaluated in soft agar culture; granulocyte isolates generated 71% G-CFC of all colonies formed and were enhanced 27 times over unfractionated cells. Mixed isolates generated 57% GM-CFC of all colonies formed and were 15 times enhanced, while macrophage isolates were 75% M-CFC with an enhancement factor of 18. The isolated populations were injected into 3-week-old ia recipients and evaluated for the ability of these cellular isolates to correct the bone resorption defect by measuring the size of the tibial marrow cavity and by identifying morphologically normal osteoclasts. In addition, isolated populations of cells were labeled with FITC and injected into ia donors to determine if labeled osteoclasts developed.(ABSTRACT TRUNCATED AT 250 WORDS)

A bone marrow fraction enriched for granulocyte-macrophage progenitors gives rise to osteoclasts in vitro

Studies involving bone marrow transplantation of osteopetrotic rodents have provided evidence for the lineage of the osteoclast. Recent investigations have demonstrated that isolates of bone marrow containing an enriched population of granulocyte-colony-forming cells (G-CFC) or granulocyte macrophage-colony-forming cells (GM-CFC) from normal animals cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia osteopetrotic rats. Macrophage-colony-forming cell isolates were ineffective in this transplant system. A criticism of these findings is that the microenvironment of the osteopetrotic bone and the bone marrow compartment may be unique in their ability to induce the differentiation of these stem cells into osteoclasts. To test this hypothesis, G-CFC, GM-CFC, and M-CFC were co-cultured with fetal metatarsal bones form normal animals. The CFC were isolated from normal bone marrow using FITC-labeled monoclonal antibodies directed against rat Thy 1.1 and fluorescence-activated cell sorting. The isolates were evaluated in soft agar culture; granulocyte isolates generated 74% G-CFC of all colonies formed and were enhanced 30 times over unfractionated cells. Mixed isolates generated 60% GM-CFC of the colonies formed and were 12 times enhanced, while macrophage isolates were 77% M-CFC with an enhancement factor of 28. The various CFC isolates or whole mononuclear bone marrow were co-cultured with 20-day fetal rat metatarsal rudiments for 7 days and then prepared for light and electron microscopy. The number of osteoclasts generated in vitro by each isolate was determined. The G-CFC and GM-CFC isolates generated significantly more osteoclasts than found in the control cultures ( p < .01), but the M-CFC isolates were ineffective in giving rise to osteoclasts. These results are equivalent to those reported in the osteopetrotic transplant system, and support the idea that the osteoclast specific cell lineage diverges from the macrophage lineage prior to the M-CFC stage.

蛍光標識モノクローナル抗体 (Microtrak Herpes) による単純ヘルペスウイルス感染症の診断

蛍光標識モノクローナル抗体 (Microtrak Herpes) による単純ヘルペスウイルス感染症の診断

Enhancement of Ia-like antigen expression by interferon-gamma in polymorphonuclear leukocytes.

The effects of interferons (IFNs) on Ia-like antigen expression in human peripheral blood polymorphonuclear leukocytes (PMNs) were examined in vitro using flow cytometry and FITC-labelled monoclonal antibody specific for the Ia-like antigen OKIa1. Recombinant human IFN-gamma markedly enhanced the expression of Ia-like antigen on PMNs and increased the number of Ia-positive PMNs. In contrast, natural human IFN-alpha and-beta had no effect on the expression when compared on the basis of antiviral activity.

Detection of Chlamydia trachomatis by using FITC-labeled monoclonal antibodies (MicroTrak method)

The usefulness of FITC-labeled monoclonal antibodies (Micro TrakTM Culture Confirmation Testand Direct Specimen Test) for the detection of Chlamydia trachomatis from clinical specimens wasstudied. For examination of the specificity of Micro TrakTM method 15 reference strains (serovar A-Kand L1-L3) of C. trachomatis and one of Chlamydia psittaci were used. The reagents of Micro TrakTMmethod reacted strongly to all 15 strains of C. trachomatis but not to a strain of C. psittaci at all. A total of1034 clinical cases were tested for Micro TrakTM method. Culture Confirmation Test brought on thesame results for detection of chlamydial inclusions as Giemsa or iodine staining after cell culture ofspecimens. Number of inclusions detected from specimens by Culture Confirmation Test tended to morethan those by Giemsa or iodine staining method, and the inclusions stained with Micro TrakTM methodwere easier to be found out. Therefore, it would be concluded that Micro TrakTM Culture Confirmation Test is superior in detection of chlamydial inclusions to Giemsa or iodine staining which are now ususallyutilized. In Direct Specimen Test negative-coincidence ratio (number of negative-Direct Specimen Testcases in negative-culture cases/number of negative-culture cases) was 89.0%. This would show thatreactions of Micro TrakTM method in direct smears were almost specific to C. trachomatis organisms. Onthe other hand, positive-coincidence ratio (number of positive-Direct Specimen Test cases in positiveculturecases/number of positive-culture cases) was 69.7%. It is suggested that positive ratio isunequivocally influenced by the method for collection of specimens and for making direct smears, andthe experience of observation with fluorescence microscopy. Therefore, the carefulachievement andskillfulness of suitable method for detection of chlamydial organisms must bring better results. It mightbe suggested that Micro TrakTM Direct Specimen Test is a really rapid and easy-to-perform method fordetection of C. trachomatis and to be performed as a substitute method for cell culture method in clinicallaboratories.