Analysis of thymic epithelial cell proliferation in vitro by combining bromodeoxyuridine and keratin labeling in an immunofluorescence assay

A simple method of analyzing thymic epithelial cell (TEC) proliferation has been developed by combining bromodeoxyuridine (BrDU) and keratin labeling in an immunofluorescence assay. The first reagent specifically visualizes the cells entering the S phase of the cell cycle, whereas the second immunostaining reveals which of the proliferating BrDU-positive cells actually belong to the epithelial lineage. This method, besides being rapid and free of radioactivity, appears to be reliable in view of the minor variations in the percentages of BrDU+ TEC observed in several distinct experiments. Thus, BrDU/keratin immunolabeling appears to represent a useful tool for the analysis of in vitro TEC proliferation.     

Formation of ectopic neuromuscular junctions in adult rats

The formation of ectopic neuromuscular junctions between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle was examined in adult rats. Formation of new junctions was induced by denervating the soleus muscle by cutting the tibial nerve. Junctional transmission began 3 days after denervation when denervation was done 3 weeks after transplantation of the foreign nerve, and progressively later when denervation was done 8, 16 and 24 weeks after transplantation. The rate at which the transmission, once started, acquired mature characteristics was approximately the same in each case. Initially, spontaneous m. e.p. p.s were infrequent, long lasting with a skewed amplitude distribution. The e. p.p. s evoked by stimulation of the foreign nerve were then commonly below threshold for eliciting action potentials and were occasionally no larger than the size of the spontaneous m. e.p. p.s. M.e. p.p. characteristics became normal in the 2nd week after transmission had started. Fully effective evoked transmission with every innervated fibre responding with overshooting action potential occurred 1–3 months after onset of transmission.     

Multiomics uncovers developing immunological lineages in human

The development of the human immune system during embryonic and fetal life has historically been difficult to research due to limited access to human tissue. Experimental animal models have been widely used to study development but cellular and molecular programmes may not be conserved across species. The advent of multiomic single-cell technologies and an increase in human developmental tissue biobank resources have facilitated single-cell multiomic studies focused on human immune development. A critical question in the near future is "How do we best reconcile scientific findings across multiple omic modalities, developmental time, and organismic space?" In this review, we discuss the application of single-cell multiomic technologies to unravel the major cellular lineages in the prenatal human immune system. We also identify key areas where the combined power of multiomics technologies can be leveraged to address specific immunological gaps in our current knowledge and explore new research horizons in human development.     

IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG.

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.     

The CA 125 tumour-associated antigen: a review of the literature

CA 125 is an antigenic determinant on a high-molecular-weight glycoprotein recognized by a monoclonal antibody which was raised using an ovarian cancer cell line as an immunogen. During the last 5 years the studies reviewed in this paper have provided information concerning the nature, distribution and clinical significance of CA 125. The CA 125 determinant is expressed by epithelial ovarian tumours and various other pathological and normal tissues of Müllerian origin. The function of the glycoprotein expressing CA 125 remains unclear but the distribution of the antigen suggests that it may have a physiological role. The highest serum levels of CA 125 are found in ovarian cancer patients, but elevation of serum CA 125 may also be associated with other malignancies and benign and physiological states, including pregnancy, endometriosis and menstruation. Despite limitations of sensitivity and specificity serum CA 125 estimation is of clinical value in the pre-operative diagnosis and monitoring of ovarian malignancy and may be a prognostic indicator for this disease. The role of CA 125 in screening for early-stage ovarian cancer is currently under investigation. Recent reports suggest that serum CA 125 measurement may also be of value as a prognostic indicator in endometrial cancer and as a reflection of disease status in advanced endometriosis.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Natural materno-fetal transfer of antibodies to PspA and to PsaA

PspA and PsaA are Streptococcus pneumoniae surface proteins and potential pneumococcal vaccine antigens. The aim of this study was to characterize the transplacental transfer of antibodies to PspA and to PsaA. Paired mother and cord blood sera were obtained at delivery from 28 women. Concentrations of antibodies against PspA, PsaA, tetanus toxoid (vaccine-induced antibodies) and P6-outer membrane protein (OMP) of nontypeable Haemophilus influenzae were determined by ELISA. Antibodies to PspA of the IgG, IgG1 and IgG2 antibodies were also determined. The geometric mean percentage (GM%) of the paired infant:mother antibody were calculated. Results: The GM% of the infant:mother antibody concentrations against PspA, PsaA and P6-OMP antibodies were 64.7% (3.3 micro g/ml in infants vs. 5.1 micro g/ml in mothers), 50.4% (6.8 micro g/ml vs. 13.5 micro g/ml) and 66.7% (5.6 micro g/ml vs. 8.4 micro g/ml), respectively; the GM% of antibodies against tetanus toxoid was 104.5% (4.6 micro g/ml vs. 4.4 micro g/ml). Transplacental transfer of IgG1 was more efficient than that of IgG2 (approximately 120%vs. 65%). A transplacental transfer of antibodies to PspA and to PsaA exist. Moreover, these data suggest an active placental transfer of IgG1 antibodies to PspA since the concentration of these antibodies were consistently higher in cord sera than in the mother's sera.     

Monoclonal antibody to desmosomal glycoprotein 1--a new epithelial marker for diagnostic pathology

Desmosomes are intercellular adhesive junctions that occur in almost all epithelia and should therefore be useful as epithelial markers in tumour diagnosis. Here, we describe a monoclonal antibody, 32-2B, to a major desmosomal glycoprotein (dgl) which reacts with human tissues in paraffin sections. This antibody was tested for its ability to stain epithelia and tumours. It reacted with all epithelia tested and with every specimen of a wide range of carcinomas. It also stained meningiomas, another desmosome-containing tumour. It did not stain other types of tumours including lymphomas, melanomas, and various sarcomas, or normal tissues which lack desmosomes. These characteristics demonstrate that 32-2B is a reliable epithelial marker that may have a useful role in diagnostic histopathology.