Possible importance of antibiotics and naltrexone in neurodegenerative disease.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
Biomedical subjects
Y P Agrawal
Publications and source records attributed to Y P Agrawal.
Low dose naltrexone therapy in multiple sclerosis.
The use of low doses of naltrexone for the treatment of multiple sclerosis (MS) enjoys a worldwide following amongst MS patients. There is overwhelming anecdotal evidence, that in low doses naltrexone not only prevents relapses in MS but also reduces the progression of the disease. It is proposed that naltrexone acts by reducing apoptosis of oligodendrocytes. It does this by reducing inducible nitric oxide synthase activity. This results in a decrease in the formation of peroxynitrites, which in turn prevent the inhibition of the glutamate transporters. Thus, the excitatory neurotoxicity of glutamate on neuronal cells and oligodendrocytes via activation of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid class of glutamate receptor is prevented. It is crucial that the medical community respond to patient needs and investigate this drug in a clinical trial.
Apoptosis↗
The vWF content of factor VIII concentrates.
Explore the source record for details and available documents.
Factor VIII↗
Complement and anti-alpha-galactosyl natural antibody-mediated inactivation of murine retrovirus occurs in adult serum but not in umbilical cord serum.
Many retroviral vectors for hematopoietic cell and other clinical gene therapy are derived from murine packaging cell lines. The exposure of these retroviruses and packaging cell lines to adult human serum (AS) inactivates them by complement and anti-alpha-galactosyl natural antibody-mediated mechanisms. We show that virus stability and infection efficiency of CRIP/BAG, a murine packaging cell line derived amphotropic retrovirus vector is reduced > 95% following a 30-min incubation in AS. This inactivation is prevented by replacing AS with umbilical cord serum (CS), wherein full retroviral transduction efficiency is maintained after 30 min of incubation. The loss of retrovirus transduction efficiency in AS was smaller upon blockage of anti-alpha-galactosyl antibodies with galactose alpha 1-3-galactose. Serum levels of CH 100, as well as C1q complement which inactivates retroviruses by an antibody-independent mechanism were similar in AS and CS. The high stability of CRIP/BAG retrovirus vector in CS is likely due to its lower levels of maternally derived anti-alpha-galactosyl antibodies. These results have implications for in vivo gene transfer in adults and also in newborns since neonates do not produce natural antibodies during the initial months of life.
Adult↗
Interaction of vesicular stomatitis virus-G pseudotyped retrovirus with CD34+ and CD34+ CD38- hematopoietic progenitor cells.
Retroviral vectors have had limited success in mediating gene transfer to hematopoietic stem cells, particularly in primates, due in part to low or absent expression of the amphotropic receptor (RAM-1). We have been interested in determining whether retrovirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) would allow more efficient gene delivery to hematopoietic stem cells as the VSV-G receptors appear to be ubiquitously present phospholipids. However, we previously found that completion of retroviral vector reverse transcription does not occur in CD34+ CD38- hematopoietic stem cells that were exposed to VSV-G pseudotyped retrovirus. To determine at which stage the block to infection of CD34+ CD38- cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped viral particles could bind to CD34+ CD38- cells. Virus binding to CD34+ cells was saturable at 4 degrees C but nonsaturable at 37 degrees C, up to a multiplicity of infection of 1080. This suggests that surface levels of phospholipid receptors available for viral binding are limiting on CD34+ cells. Cytokine stimulation increased virus binding to CD34+ cells. However, no increase in the level of surface phosphatidylserine (PS), a strong candidate for the VSV-G receptor, was seen as detected by the PS-specific reagent, annexin V. This suggests that another molecule is serving as the VSV-G receptor on CD34+ cells. Here, we show that once virus binding to cytokine-stimulated CD34+ CD38- cells has occurred, virus fusion proceeds efficiently as determined by octadecyl rhodamine (R18) fusion assays. Taken together with our previous observation that reverse transcription does not occur in CD34+ CD38- cells, we suggest that there are intracellular mechanisms leading to blockage of complete reverse transcription of the retrovirus in CD34+ CD38- cells. This has important implications for retrovirus-mediated gene transfer to quiescent stem cells.
ADP-ribosyl Cyclase↗
Seminal fluid antisperm antibodies measured by direct flow cytometry do not correlate with those measured by indirect flow cytometry, the indirect immunobead test, and the indirect mixed antiglobulin reaction.
OBJECTIVE: To compare the direct flow cytometric (FCM) measurement of sperm-bound antibodies with the indirect FCM measurement of unbound antisperm antibodies in seminal plasma of the same ejaculates. To compare the FCM measurements with the indirect mixed antiglobulin reaction (MAR) and the indirect immunobead test (IBT) performed on the same seminal plasmas. SETTING: University hospital-based infertility service. PATIENTS: Eleven infertile men with a positive immunoglobulin (Ig) G direct MAR test and antibody positivity on the direct FCM measurements. Four control patients with a negative IgG-direct MAR test. MAIN OUTCOME MEASURES: The proportion of spermatozoa positive for IgG and IgA antibodies as measured by various tests. RESULTS: The direct and indirect FCM measurements for sperm-bound antibodies from seminal plasma did not correlate with each other. In general, IgG antibodies were not detectable by indirect FCM whereas IgA were detected in lower proportions than by direct FCM measurements. Weak correlation was observed between the indirect FCM, indirect MAR, and indirect IBT measurements. CONCLUSION: Nearly all of the IgG and some of the IgA antisperm antibodies in seminal fluid bind to spermatozoa. Thus, indirect tests to measure antisperm antibodies from seminal plasma are likely to miss the presence of antisperm IgG antibodies while detecting some cases of IgA antibodies.
Flow Cytometry↗
A placebo-controlled flow cytometric study of the effect of low-dose prednisolone treatment on sperm-bound antibody levels.
In this placebo-controlled study the effect of short term, low-dose prednisolone treatment on sperm-bound antibody levels was investigated using an objective flow cytometric (FCM) measurement. Twenty infertile men, positive for antisperm antibodies by the mixed antiglobulin reaction (MAR) test, were treated with either 20 mg/day prednisolone (n = 10) or placebo (n = 10) for 3 weeks. The two groups were similar with respect to sperm count, motility and antibody levels. The proportion of spermatozoa positive for IgG and IgA antibodies and the antibody load, expressed as the number of IgG and IgA antibody molecules bound per spermatozoa were measured by FCM. The study showed that, overall, the antibody levels measured before and after treatment with low-dose prednisolone or with placebo were not statistically different. However, in the prednisolone group a marked decrease in the proportion of spermatozoa positive for IgG and IgA antibodies was observed in two of the patients. An examination of the FCM data of these two patients revealed that, in their ejaculates, only the spermatozoa with low to moderate amounts of sperm-bound antibodies had responded to prednisolone treatment, while spermatozoa with high levels of sperm antibodies had remained resistant to prednisolone. These results point to the existence of an as-yet-undefined subset of prednisolone-responsive, antibody--positive spermatozoa.
Antibodies↗
Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells.
As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34+ cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34+ cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34+ cells into cycle were stem cell factor (SCF) plus one of the following: interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF). The maximum numbers of cells in S+G2M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34+ cells remained quiescent in the first G0/G1 phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34+CD38- subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-G pseudotyped virus containing the neomycin phospho-transferase gene (neo), we exposed CD34+ cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34+ cells and the CD34+CD38- subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT-neoDNA was detectable in CD34+ cells that have been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34+ cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the 34+CD38- subset showed that RT-reoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34+ cells but not in CD34+CD38- cells. These results suggest during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34+CD38-cells in addition to the lack of mitotic activity.
ADP-ribosyl Cyclase↗
Flow cytometric quantitation of C-myc and P53 proteins in bovine papillomavirus type 1-transformed primary mouse fibroblasts.
Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast cell lines were analyzed via flow cytometry (FCM) for expression of p53 and c-myc proteins along with their DNA content. In comparison to the nontransformed control cell line, significantly elevated levels of both the p53 and the c-myc protein were present in some but not all of the transformed cell lines. Quantitation of p53 and c-myc proteins in cell lines containing BPV-1 DNA revealed that the tumorigenic cell lines expressed higher levels of both the p53 (P = 0.0034; Mann-Whitney U test) and the c-myc protein (P = 0.0039; Mann-Whitney U test) as compared to the nontumorigenic cell lines. On average, at least 9,000-10,000 p53 or c-myc protein molecules per cell were detected in the transformed tumorigenic cell lines. These results show that quantitative FCM can be reliably used to detect very low levels (3,000 molecules per cell) of specific protein, and FCM is a useful tool to study the virus-induced changes in the levels of nuclear proteins within a cell population and in tumorigenesis.
Animals↗
Flow cytometric method to measure urea-resistant alkaline phosphatase from blood neutrophils for use in the prenatal screening of Down's syndrome.
The histochemical measurement of urea-resistant alkaline phosphatase from maternal blood neutrophils is known to have a high detection rate for the prenatal detection of Down's syndrome pregnancies. However, because the histochemical method is laborious and subjective to use, it has not gained widespread acceptance in prenatal screening programmes. We present a simple and objective method for the measurement of urea-resistant alkaline phosphatase by flow cytometry. The method should allow the design of larger studies aimed at evaluating the role of neutrophil urea-resistant alkaline phosphatase in the prenatal screening for Down's syndrome.
Alkaline Phosphatase↗
Comparison of flow cytometric measurement of seminal antisperm antibodies with the mixed antiglobulin reaction and the serum tray agglutination test.
OBJECTIVE: To compare the mixed antiglobulin reaction (MAR) and the serum tray agglutination test (TAT) with the flow cytometric (FCM) measurement of sperm antibodies. To evaluate by FCM the correlation in sperm antibody parameters between paired semen ejaculates obtained 2 to 4 weeks apart and the effect of washing on sperm antibody levels. SETTING: University-based infertility clinic. PATIENTS: The TAT and FCM were done on 81 patients with a positive immunoglobulin (Ig)G-MAR test. The correlation of sperm antibody parameters between paired semen samples was done in 16 men. A few samples were used to study the effect of washing on sperm antibody parameters. MAIN OUTCOME MEASURES: The proportion of spermatozoa positive for IgG and IgA antibodies as well as the number of antibody molecules per spermatozoa measured by FCM. RESULTS: In general, the degree of MAR positivity but not that of the TAT correlated with the sperm antibody load. With reference to FCM the MAR test had an 1% false positive rate, whereas the serum TAT had a 25% false negative rate. Correlation between FCM measurements of paired semen samples was good. Washing the spermatozoa once removed the loosely bound antibodies; further washes were not useful. CONCLUSION: Flow cytometry is a reliable technique that correlates with the MAR test. In patients with a strongly positive MAR, FCM is useful to identify patients with a low sperm antibody load. It is speculated that patients with a low sperm antibody load may respond better to IVF treatment.
Agglutination Tests↗
A flow cytometric study on the effect of low dose cyclic prednisolone treatment on sperm-bound antibody levels.
Using flow cytometry the changes in the proportion of spermatozoa positive for antisperm antibody, and for levels of sperm-bound immunoglobulin (Ig)G and IgA antibody and some other semen parameters were monitored during low dose cyclic prednisolone treatment to 11 male patients. Patients were given prednisolone 20 mg/day for the first 10 days of the partner's menstrual cycle and then 5 mg on days 11 and 12. The treatment was repeated for three cycles and the sperm parameters measured after the first cycle, third cycle and in four cases also 1 and 3 months after cessation of treatment. A clear reduction of sperm-bound IgG antibody levels was seen in 3/11 (27%) patients, while only IgA was reduced in 2/11 (18%) patients. Only minor changes in sperm-bound antibody levels were shown in 63% (7/11) of patients, and in one patient the levels of sperm antibody even increased. Thus, corticosteroid treatment did not reduce sperm-bound antibodies in the majority of cases. In those cases in which the levels of sperm antibody declined, the decrease in sperm-bound antibodies was noticeable even after one cycle of prednisolone treatment.
Autoantibodies↗
Comparison of poly- and monoclonal antibodies for determination of B-cell clonal excess in an routine clinical laboratory.
Flow cytometry (FCM) has gained wide use in the determination of clonality in B-cell lymphoproliferative diseases and many methodological variations exist. We have compared the suitability of a) dual fluorochrome (FITC/PE)-labelled monoclonal antibodies, b) single fluorochrome (FITC)-labelled monoclonal antibodies and c) F(ab')2 fragments of FITC-labelled polyclonal antibodies for flow cytometric determination of clonality using commercially available software and a short sample preparation protocol. The FCM method was validated by analysis of immunoglobulin heavy chain and light chain gene rearrangements. We recommend the use of FITC-labelled monoclonals to obtain three parameters, the kappa/lambda ratio, D and D/S(n) values (Kolmogorov-Smirnov statistics) instead of the commonly used kappa/lambda ratio and D values only. This allows the use of a rapid sample preparation protocol to blood and bone marrow aspirates without sacrificing sensitivity or specificity obtained by the usual FCM method.
Antibodies↗
Reticulocyte analysis by flow cytometry using a modified gating procedure.
Explore the source record for details and available documents.
Blood Platelets↗
Use of quality control standards in clinical flow cytometry.
Flow cytometry is important in the clinical laboratory, especially in hematology and cancer diagnosis. With newer applications being continuously developed clinicians who will use the results and laboratory staff who produce them should be aware of the instrumental quality control procedures required. This will allow evaluation of the reliability as well as the comparison of results between different laboratories. While information about sample preparation as well as staining procedures is easily available, information about instrumental quality control procedures is less so. Various standards and controls are available commercially and their correct choice and usage requires an understanding of the methodology of instrumental quality control. Methods to evaluate the precision, sensitivity and accuracy of measurements are discussed. Most laboratories report immunophenotyping data as the percentage of antibody positive cells and make no attempt to quantitate the amount of antibody binding per cell. Methods for quantitating the number of bound antibody molecules per cell along with a simple technique to compare results between laboratories are discussed. These techniques should encourage laboratory staff and clinicians to standardise methodology and to exchange patient data between laboratories.
Flow Cytometry↗
Flow cytometry for measurement of antibodies to spermatozoa in subfertile men.
Explore the source record for details and available documents.
Antibodies↗
Gamma-glutamyl transpeptidase in rat epididymis: effects of castration, hemicastration and efferent duct ligation.
Gamma-Glutamyl transpeptidase (gamma-GT) was studied histochemically and biochemically in the rat epididymis after castration with or without testosterone treatment, or after hemicastration and ligation of the efferent ducts. There was a strong reaction to gamma-GT in the apical part of the epithelium in the caput epididymis, while in the corpus and cauda the reaction was confined mainly to the luminal contents. Castration caused a marked decline in epithelial gamma-GT activity within 10 days. Subsequent testosterone treatment (1 mg/day for 10 days) restored gamma-GT activity in the apical surface and lumen. After hemicastration of adult rats, and after hemicastration or ligation of the efferent ducts in immature 28-day-old rats, a small but significant (P less than 0.001) decrease was observed in gamma-GT activity in the epididymal caput compared to controls. The quantities of six other enzymes (beta-N-acetylglucosaminidase, beta-galactosidase, angiotensin-converting enzyme, alanyl amino-peptidase, dipeptidyl peptidase IV, acid phosphatase) also displayed significant changes after castration and restoration of activities by testosterone treatment. However, their distribution in the caput and cauda epididymis was more even than that of gamma-GT, and the changes after castration were less drastic. It is concluded that gamma-GT is a highly sensitive androgen-dependent secretory marker in the caput epididymis and may have an important function in sperm maturation.
Animals↗
Distribution of gamma-glutamyl transpeptidase in the mouse epididymis and its response to acivicin.
gamma-Glutamyl transpeptidase (gamma-GT), its substrate (GSH) and hydrolytic product (L-glutamic acid) were measured biochemically in mouse reproductive tissues. The epididymal caput and seminal vesicles showed the highest specific activities of gamma-GT, while GSH and L-glutamic acid were widely distributed in all tissues. Histochemically, gamma-GT displayed a strong apical and supranuclear reaction and a moderate basal activity in the ductuli efferents, a weak luminal reaction in the first, a moderate apical reaction in the second and a strong apical and supranuclear reaction in the third segment of the epididymal caput. In the epididymal corpus and cauda, the gamma-GT reaction was confined to the tubular lumina but an apical reaction was also present in the cauda. The daily administration of acivicin (12 mg/kg body weight), an irreversible inhibitor of gamma-GT, for 14 days resulted in a 60% suppression of the enzyme activity in the epididymal caput, while the gamma-GT inhibition in the kidney was greater than 95%. The treatment caused no change in the activity of alanyl aminopeptidase. Histochemically, the basal and supranuclear gamma-GT activities in the ductuli efferents and the third epididymal segment were suppressed, but the apical reactions were maintained. The in-vivo suppression of epididymal gamma-GT activity may have implications in the control of post-testicular sperm maturation.
Animals↗