Adult Still's disease with nephrotic syndrome at presentation.
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Adult↗
Biomedical subjects
A Jassim
Publications and source records attributed to A Jassim.
Characterization of a 50-kDa component of epithelial basement membranes using GDA-J/F3 monoclonal antibody.
Using the monoclonal antibody GDA-J/F3, a 50-kDa noncollagenous component of human skin basement membrane zone was identified. Immunofluorescence stainings of normal human skin with the GDA-J/F3 antibody showed a linear fluorescence decorating the basement membrane zone. With immunoelectron microscopy, the epitope was localized to the insertion points of the anchoring fibrils into the lamina densa. The antigen is distinct from collagen VII, from the main structural protein of the anchoring fibrils, and from several other structural molecules of the basement membrane zone, because the GDA-J/F3 antibody did not react with purified basement membrane components in vitro. In serum-free cultures, the antigen was synthesized and secreted by normal and transformed human keratinocytes and to a lesser extent by normal human skin fibroblasts. Immunoprecipitation of radiolabeled epithelial cell-conditioned medium with the GDA-J/F3 antibody yielded two polypeptides that migrated on SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 46 and 50 kDa under nonreducing conditions. Using reducing gels, only the 50-kDa polypeptide was observed. The antigen was resistant to digestion with bacterial collagenase but sensitive to trypsin and pepsin. It also bound to heparin and DEAE cellulose at low ionic strength and alkaline pH. These findings indicate that the GDA-J/F3 antigen is a small globular disulphide-bonded protein with a potential to interact with basement membrane proteoglycans. Integration of the GDA-J/F3 antigen into the histoarchitecture of the dermo-epidermal junction is dependent on the presence of collagen VII, because the GDA-J/F3 epitope was missing in several patients with a genetic blistering disorder of the skin, epidermolysis bullosa dystrophica, who lacked collagen VII and anchoring fibrils.
Antibodies, Monoclonal↗
The use of GDA-J/F3 monoclonal antibody for the detection of dead spermatozoa (necrosperm) during semen analysis.
The distinction between immotile necrosperm (dead spermatozoa) and those with immotility due to other causes is of the utmost clinical importance. The supravital dyes currently used for the identification of necrosperm are not highly reliable or accurate. In this study, GDA-J/F3 monoclonal antibody (MoAb) which reacts with the fibrous sheath (FS) was used as a specific probe for the detection of necrosperm using indirect immunofluorescence (IIF). Previously, several lines of evidence indicated the reaction of the antibody with necrosperm. This was confirmed in the current study where GDA-J/F3 MoAb failed to react with viable swim-up separated spermatozoa; such cells were only stained following sperm demembranation with 1% Triton X-100. Furthermore, by using immunogold electron microscopy of a normozoospermic sperm sample, all the spermatozoa which reacted with GDA-J/F3 MoAb showed damaged cytoplasmic membranes. Following these initial studies, sperm samples were obtained from 42 men attending infertility clinics and assessed by conventional semen analysis and GDA-J/F3 MoAb screening using IIF. The results showed a wide variation in sperm immotility and GDA-J/F3 reactivity; the ranges were 19-99 and 0-50% respectively. This novel immunological approach provides a simple and specific method of necrosperm enumeration for the investigation of male infertility.
Antibodies, Monoclonal↗
AJ-FS9 monoclonal antibody detects masked antigens within the human sperm tail fibrous sheath.
AJ-FS9 is one of a new series of monoclonal antibodies (mAbs) raised by immunizing mice with isolated human sperm tail fibrous sheath (FS). Using indirect immunofluorescence (IIF) of human spermatozoa dried onto slides, the AJ-FS9 mAb reacted with the principal piece of occasional spermatozoa. Following their enzymatic treatment with trypsin, dispase or collagenase, but not sulphatase, all the spermatozoa were stained at their principal piece. The ultrastructural localization of the antigens to the FS was established by immunogold electron microscopy, which showed the distribution of gold particles on the FS outer surface of spermatozoa sequentially treated with 1% Triton and dispase; spermatozoa demembranated by Triton alone showed no reaction. For biochemical characterization, spermatozoa were lysed with 1% Triton, and the sperm pellet was run through a reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotted and immunostained with AJ-FS9. The results showed the reaction of the antibody with three protein bands with molecular masses ranging between 46 and 56 kDa. IIF screening of human testicular cryostat sections with AJ-FS9 mAb showed its reactivity with occasional sperm tails; but following their dispase treatment, all spermatozoa were stained. The restricted staining of the assembled FS of maturing sperm tails indicated the late appearance of the antigens during spermatogenesis. The antibody did not react with sperm cell precursors or other cell types within/without the seminiferous tubules. Untreated and dispase-treated frozen sections of skin, oesophagus, tongue, liver, kidney, stomach, ileum or their blood vessels showed no reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
The target antigen for GDA-J/F3 monoclonal antibody in the human sperm tail fibrous sheath is a non-collagenous asialo-glycoprotein: implications and significance.
Enzymes and chemicals were used to analyse the biochemical structure of the antigenic epitope recognized by GDA-J/F3 monoclonal antibody (MoAb) in the human sperm tail fibrous sheath. Treatment of sperm dried onto slides with trypsin or dispase enzymes abolished their immunofluorescence staining with GDA-J/F3 MoAb, thus indicating the proteinaceous nature of the antigen. The proteolytic cleavage of GDA-J/F3 protein by trypsin, which also caused sperm decapitation, indicated the presence of peptide bonds involving the carboxyl groups of the basic amino acids, arginine and/or lysine. The epitope was also glycosylated as demonstrated by its sensitivity to sodium metaperiodate treatment which was dose-dependent. The GDA-J/F3 antigenic epitope lacked sialic acid since pre-treatment of spermatozoa with sialidase enzyme (neuraminidase) had no effect on their reactivity with the antibody. The lack of collagenous domains in the GDA-J/F3 antigen was demonstrated by the failure of collagenase to abrogate sperm immunostaining with the MoAb. Furthermore, type VII collagen of the skin basement membrane (BM) was previously thought of as a potential target antigen for GDA-J/F3 MoAb. This was ruled out since several monoclonal and polyclonal antibodies failed to detect the antigen in the spermatozoa using immunofluorescence and Western blotting. These data, therefore, show that the target antigen for GDA-J/F3 MoAb is a non-collagenous asialo-glycoprotein, and by inference provide the first evidence for the glycosylation of the sheath proteins as another step of post-translational modification occurring during sperm tail development.
Antibodies, Monoclonal↗
AJ-FS1 monoclonal antibody detects a novel group of non-glycosylated antigens within the human sperm tail fibrous sheath.
AJ-FS1 is one of a new series of monoclonal antibodies (MoAbs) raised by immunizing mice with isolated human sperm tail fibrous sheath. Using indirect immunofluorescence (IIF), the AJ-FS1 MoAb did not react with the surface antigens of viable sperm, but did stain the flagellar principal piece of sperm dried onto slides or those demembranated with 1% Triton X-100. The specificity of the antibody for the fibrous sheath was confirmed by immunogold electron microscopy which showed the distribution of gold particles on the outer fibrous sheath surface, and its reaction with the Western blots of purified fibrous sheath preparations where multiple protein bands with mol. wt ranging between 97 and 28 kDa were identified. The same peptides were also detected in urea-dithiothreitol (DTT) fraction of sequentially extracted spermatozoa but not in Triton or Triton--DTT sperm lysates. The failure of sodium metaperiodate to abrogate the antibody reaction in both Western blotting and IIF indicated the non-glycosylated nature of the antigens. IIF screening of human testicular cryostat sections with AJ-FS1 MoAb showed its reactivity with the assembled fibrous sheath of maturing sperm tails only; thus indicating the late appearance of the antigens during spermatogenesis. The antibody did not react with skin, oesophagus, tongue, liver, kidney, placenta, uterus, cervix or their blood vessels. The significance of these results is discussed together with the importance of AJ-FS1 MoAb as a specific probe for the characterization of the fibrous sheath antigens in both normal and abnormal flagella.
Antibodies, Monoclonal↗
AJ-p90: a novel protein of the perinuclear theca in human sperm subacrosome.
A perinuclear theca protein of the human sperm subacrosome was detected using LH43 monoclonal antibody which was originally raised against human keratinocytes. Using indirect immunofluorescence, the antibody stained the acrosomal zone (AZ) of dried ejaculated spermatozoa but did not react with viable cells, thus suggesting that the antigen was intracellular. This was confirmed by immunogold electron microscopy which also revealed the ultrastructural localisation of the antigen to the subacrosomal fibrils. Throughout spermatogenesis the antigen was only detected on the AZ of developed testicular spermatozoa and its expression was continued during their epididymal passage. Biochemically, the protein was insoluble in Triton, and dithiothreitol (DTT) was required for its solubilisation. In Western blotting of sperm and keratinocyte lysates, the antibody detected similar 90-kDa protein doublets (AJ-p90). These biochemical features exclude the identity of AJ-p90 with various cyto- and karyo-skeletal antigens, including the intermediate filaments and microfilaments. AJ-p90 therefore represents a novel product of the subacrosomal perinuclear theca. The significance of these data is discussed together with the importance of the antibody for probing the perinuclear theca in normal and abnormal germ cells, including multinucleated spermatids which also showed reactivity with the antibody.
Acrosome↗
Antigenic determinants of human sperm tail fibrous sheath proteins.
Mouse anti-fibrous sheath antisera (MAFA) produced by immunizing mice with purified preparations of human sperm tail fibrous sheath (FS) reacted with the principal piece of less than 10% of freshly isolated spermatozoa which were immotile and probably dead. Following their demembranation by detergents or repeated freezing and thawing, all spermatozoa were stained. This was also demonstrated on spermatozoa dried onto slides, but the undiluted xenoantisera showed additional reactivity with the acrosomal zone (AZ). Using immunogold electron microscopy, the target antigens were ultrastructurally localized to the FS, and a few spermatozoa showed some reaction at the AZ subacrosomal perinuclear theca. Following titration of the antibodies, the anti-AZ-reaction became undetectable at a dilution of 1:20 while their reactivity with the principal piece continued to a 1:400-dilution. These results indicated that the xenoantisera probably contained an additional unrelated antibody component which reacted with the AZ. Western blotting and staining of purified FS with MAFA detected seven major protein bands with MW ranging between 25 kDa and 97.4 kDa. In human testes, the 1:50 diluted MAFA reacted with sperm tails only, indicating the late expression of the antigenic determinants during spermatogenesis. MAFA did not react with oesophagus, stomach, duodenum, ileum, nasal lining tissues, uterus, pericardium, pancreas, thyroid gland, or cultured fibroblasts. The xenoantisera did, however stain the skin epidermis and cultured keratinocytes which exhibited filamentous cytoplasmic staining although their target antigens could not be biochemically identified. These results indicate that the FS proteins express antigenic determinants which are not shared with other cytoskeletal elements within the sperm flagellum or a variety of somatic tissues.
Animals↗
Impaired antibody diffusion within the cytoplasmic membrane-fibrous sheath interspace of the human sperm tail.
Monoclonal and polyclonal antibodies specific for the fibrous sheath (FS) were used to assess their diffusion within the subcytoplasmic membrane space of the human sperm tail. Using indirect immunofluorescence (IIF) and immunogold electron microscopy (IEM), the staining of the entire FS with these antibodies was observed only when the spermatozoa were completely demembranated by detergents or dried onto slides. Partial damage to sperm cytoplasmic membranes by freezing and thawing resulted in segmental staining of the FS. The IEM of such spermatozoa with the antibodies revealed the distribution of gold particles on the FS in denuded areas only and adjacent segments which retained their cytoplasmic membrane were not stained. These results indicate that partial disruption of the sperm plasma membranes does not lead to free diffusion of antibodies within the cytoplasmic membrane-FS interspace. This property appears to be unique to sperm cells and is due to their specialized structural organization.
Antibodies↗
Isolation and biochemical characterization of the human sperm tail fibrous sheath.
The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.
Antibodies, Monoclonal↗
AJ-p97: a novel antigen of the human sperm tail fibrous sheath detected by a neurofilament monoclonal antibody.
Using indirect immunofluorescence (IIF), the RT97 anti-neurofilament monoclonal antibody (MoAb) detected an intracellular antigen in the principal piece of human ejaculated sperm tails. Its localisation to the tail fibrous sheath (FS) was confirmed by immunoelectron microscopy (IEM), which showed the binding of the gold particles to the outer FS surface. During spermatogenesis the antigen was first expressed on the spermatid FS, and its expression was continued on ejaculated mature sperm. In Western blotting of sperm lysates, the RT97 reacted with a 97 kDa protein (AJ-p97) which lacked disulphide bonds. This antigen was not detected on mouse or rat sperm tail FS, suggesting a sequence divergence of the AJ-p97 during evolution. The significance of these results and the relationship of AJ-p97 to neurofilaments are discussed, together with the use of the antibody as a probe for the structural dissection of the FS and for analysing the molecular events that take place during spermiogenesis, especially those involved in sperm tail morphogenesis.
Animals↗
Human sperm tail fibrous sheath undergoes phosphorylation during its development.
The phosphorylation of the human sperm tail fibrous sheath as a maturational step during its development is reported for the first time. This was demonstrated using GDA-J/F3 and RT97 monoclonal antibodies (MoAb) which recognize the fibrous sheath. In indirect immunofluorescence microscopy of frozen sections of human adult testes, the two antibodies reacted with the assembled fibrous sheath only, but the numbers of sperm tails stained with RT97 were consistently lower than those treated with GDA-J/F3. Furthermore, by using double indirect immunofluorescence, although the majority of spermatozoa were doubly stained with the two MoAbs, some GDA-J/F3-positive sperm tails were negative with RT97. In epididymal and ejaculated spermatozoa, the two antibodies stained all the tails. This indicated that the ontogenic appearance of the GDA-J/F3 epitope precedes that of RT97. In Western blotting and/or indirect immunofluorescence of spermatozoa, treatment of samples with alkaline phosphatase abolished the reactivity of RT97 while that of GDA-J/F3 MoAb was not affected. This finding indicated that the RT97 but not the GDA-J/F3 epitope was phosphorylated. Together, these results therefore reveal that during tail morphogenesis, the fibrous sheath undergoes phosphorylation as part of its structural maturation. Screening of sperm cell precursors recovered from oligozoospermic donors showed reaction of some abnormal germ cells with GDA-J/F3 MoAb but not with RT97, suggesting the possible failure of phosphorylation of the fibrous sheath protein in these cells. The significance of these findings is discussed together with the biological importance of phosphorylation to the fibrous sheath.
Alkaline Phosphatase↗
Detection of a novel basement membrane antigen by GDA-J/F3 anti-human sperm fibrous sheath monoclonal antibody.
Basement membrane zones (BMZ) of human epithelia were stained with GDA-J/F3 monoclonal antibody, which was originally raised against sperm cells. Using indirect immunofluorescence and immunoperoxidase techniques, the antibody reacted with the BMZ of stratified squamous epithelia (skin and its appendages, tongue, lip, oesophagus and cervix). It also stained the BMZ of trachea, nasal ciliated mucosa, some mammary ducts of lactating and resting breast, amnion and ureter but failed to react with that of stomach, ileum, colon, rectum, kidney, liver, fallopian tube, lung or their blood vessels. In testes, the antibody did not react with the BMZ of the seminiferous tubules although the sperm tails were stained. Split-skin immunofluorescence and immunoelectron microscopy localized GDA-J/F3 antigen to the inferior border of the lamina densa of the BMZ. In human foetuses, the epidermally associated antigen was detected at an estimated gestational age of 9 weeks, and in the amnion at 15 weeks. The antibody reacted with tissues from monkey but not from mouse, rat, cow or pig suggesting the late appearance of the antigen during evolution. Although the GDA-J/F3 was difficult to characterize biochemically, its tissue distribution, ontogeny and ultrastructural localization suggests that this antigen may be a type VII collagen-associated protein, whose expression is altered in recessive dystrophic epidermolysis bullosa. This disease could represent abnormalities in type VII collagen structure, assembly, transport or interaction with associated proteins.
Animals↗
GDA-J/F7 monoclonal antibody: a new marker for sperm cell precursors in human semen.
Germ cells isolated from semen of oligospermic human donors were found to react with GDA-J/F7 monoclonal antibody (MoAb). Their reactions with this antibody were demonstrated by using fluorescein activated cell sorter (FACS) analysis and indirect immunofluorescence (IIF) test. In the IIF test, the MoAb recognised an antigen on the surface of the sperm cell precursors (SpP) as well as on mature spermatozoa. The specificity of the antibody reaction with the SpP was further confirmed by immunoelectron microscopy. The MoAb did not react with peripheral blood lymphocytes or polymorphonuclear cells but did show cross-reactivity with monocytes. This antibody therefore provides the first marker for the SpP and could be used as a probe for their distinction from leucocytes. This could have clinical application in seminal analysis.
Antibodies, Monoclonal↗
GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies.
GDA-J/F3 monoclonal antibody (MoAb) recognized an antigen in the fibrous sheath (FS) of human spermatozoa. This was based on: (i) intracellular localization of the antigen which was limited to the principal piece of the sperm tail; (ii) its absence from the cilia of trachea and nasal mucosa which lack FS; (iii) immunogold electron microscopy (IEM) which confirmed its ultrastructural localization to the FS. The antibody was used for the detection of abnormal germ cells in human semen. Nucleated cells other than spermatozoa (NCOS) obtained from oligozoospermic donors were screened with GDA-J/F3 MoAb using the indirect immunofluorescence test. The antibody which stains only the tails in normal cells, produced diffuse cytoplasmic immunofluorescence inside some spermatids. Using phase contrast microscopy, the tails in these spermatids were either present or undetectable. Electron microscopy studies of the NCOS showed the lack of the FS in those which had the tails (afibrous tail), or the presence of disorganized tails (tail dysgenesis) in the others. This antibody therefore provides a useful analytical tool for probing the FS as well as for the easy identification of certain abnormal germ cells in human semen.
Antibodies, Monoclonal↗
Analysis of HLA antigens on germ cells in human semen.
Nucleated cells other than sperm (NCOS) were isolated from human semen by centrifugation on a Ficoll density gradient. Using tissue-specific monoclonal antibodies (mAb) greater than 99% of the NCOS were found to be sperm cell precursors (SpP). These cells were tested for the expression of class I and II (DR, DP and DQ) HLA antigens by using specific mAb. The anti-HLA class I and II and anti-beta 2-microglobulin mAb reacted with less than 1% of the NCOS. This was demonstrated by indirect immunofluorescence microscopy and fluorescence-activated cell sorter analysis. These results were similar to those obtained from testing germ cells in frozen sections of normal adult testis using the same panel of mAb. In mixed lymphocyte-NCOS cultures, the SpP failed to stimulate allogeneic lymphocytes even when different concentrations of cells were used. These results indicate little or no expression of HLA class I and II including the HLA-D (T cell-defined) determinant on the SpP, a phenomenon which could be of biological importance.
Antibodies, Monoclonal↗
Immunological and morphological characterisation of nucleated cells other than sperm in semen of oligospermic donors.
Nucleated cells other than sperm (NCOS) were obtained from semen samples of oligospermic donors. The cells, after separation from sperm, were studied by immunological and morphological techniques including electron microscopy (EM). Using an indirect immunofluorescence (IIF) test, more than 95% of the NCOS reacted with xenoantisera specific for human sperm and germ cells but showed less than 5% reactivity with monoclonal antibodies (MoAbs) directed against surface markers of different leucocyte subsets or cytokeratin. This indicated that the majority of NCOS were germ cells in origin. These results were concordant to those obtained by EM which showed the presence of germ cells at various stages of differentiation. Our data therefore show that the application of immunological techniques could help and may replace laborious EM in the identification of the different cell types among NCOS. Also, the results emphasize that germ cells and not leucocytes appear to be the major cell type among NCOS, a concept which could have therapeutic and biological implications.
Adult↗
Molecular dissection of human testicular germ cell differentiation with monoclonal antibodies.
Monoclonal antibodies (MoAbs) were raised by immunising mice with nucleated cells other than sperm (NCOS) obtained from semen of an oligospermic donor. These MoAbs recognised different germ cell differentiation antigens (GDA) which appear at various stages of spermatogenesis and/or spermiogenesis. The GDA were found to be differentially expressed on spermatocytes, spermatids and on the various sub-regions of human spermatozoe. Some of the MoAbs also recognised changes in the antigenic structure of the sperm during their post-testicular maturation. Using solubilised sperm products, MoAb GDA-J/F1 reacted with a 92 kDa band in Western immunoblotting. The importance of these antibodies in the study of germ cell differentiation together with their potential clinical uses in the investigation of infertility, autoimmunity and immunological contraception are discussed.
Antibodies, Monoclonal↗