Immunological analysis of human lymphocytes bearing different surface receptors]

Cell surface receptors on human lymphocytes being studied, essential differences were revealed in a relative content of E-, EA- and EAC-rosette-forming cells in peripheral blood and tonsils. In tonsils, part of lymphocytes carrying receptors for sheep erythrocytes have been shown to possess C'3-receptors for sheep erythrocytes. Apparently, C'-receptor,--being B-lymphocyte surface marker,--may also appear at definite stages of T-cell differentiation.     

Detection of anti-sperm antibodies on the surface of live spermatozoa by the method of flow cytometry

Detection of antisperm antibodies is an important step of the diagnosis of infertility. In present study we conducted comparative studies with the detection of antisperm antibodies in IgG, IgA and IgM classes on the surface of living spermatozoa by flow cytometry and with Mixed Agglutination Reaction and Sperm immobilization. Our data show that flow cytometry may be used to verify that immunoglobulins are on the surface of living spermatozoa, either retrieved directly from an ejaculate or following exposure to cervical mucus or serum and to determine their isotype, proportion of sperm which bound antibody and quantity of immunoglobulins bound to cell surface.     

One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.     

Genetic studies in Saudi Arabia: red cell enzyme, haemoglobin and serum protein polymorphisms.

Population genetic studies in Saudi Arabia are performed for EsD, GPT, AcP, ADA, AK, 6-PGD, PGM, C3, Tf, Hp, Gc, Pi, Bf, Hb, ABO-blood groups and Rh-factor, level of the third component of complement and immunoglobulins. The data are compared with reported frequencies in European and African populations.     

The effect of anti-Anisakis simplex antibody levels on C3 and C4 complement components in human sera

Previously, an in vitro effect was observed on the complement system not only of the excretory-secretory products but also of somatic antigens from L3 Anisakis simplex larvae. In the present work the effect of anti-A. simplex specific antibodies on C3 and C4 levels in human sera was investigated. Up to 309 samples of sera were tested to determine levels of C3 and C4 and anti-A. simplex antibodies, including immunoglobulins IgG, IgM, IgA and IgE. Significant differences were observed between levels of C3 and C4 and all immunoglobulins except for IgE. In the case of immunoglobulins, the probability that an anti-A. simplex positive subject has a C3 deficiency was 3.8 times higher than a subject without specific antibodies. In conclusion, an association between elevated levels of anti-A. simplex antibodies and C3 and C4 deficiency was demonstrated.     

Separation of precursor T cells and Ig-secreting B cells from the large lymphocytic cells of human tonsil.

Large lymphocytic cells of human tonsils concentrating in the lighter fractions of a BSA gradient contained both precursors of E rosette-forming cells and lymphocytes carrying μ and γ determinants. These two subpopulations of cells could be separated by retaining the Ig-carrying cells on an anti-Fab column. Cells which did not bind to the column matured within 3–5 days into E rosette-forming cells. The Ig-carrying cells were eluted by medium supplemented with immunoglobulins and continued to secrete γ determinants in vitro. Both types of cells were also distinguished by different profiles of spontaneous [³H]thymidine incorporation, the persisting capacity for division of the E-rosette-maturing cells suggesting that they represent an earlier stage of lymphocyte maturation (precursor T cells).     

Establishment of leukemia cell lines, BALM-6, BALM-7 and BALM-8 with B-cell characteristics from a patient with acute lymphoblastic leukemia.

Three leukemia cell lines, BALM-6, -7 and -8 were established from the bone marrow of a patient with acute lymphoblastic leukemia. Based on immunophenotypic and the cytogenetic analysis and on the expression of immunoglobulins on both the cell surface and in the cytoplasm, BALM-6, BALM-7 and BALM-8 are clonal leukemic cell populations of B cell origin.     

Nature of Hamster Complement-fixing Antibody to Adenovirus 12 Tumor Antigen

The presence of complement-fixing (CF) antibody reactive with T antigen and with viral C antigen in hamsters bearing adenovirus 12-induced tumors has been confirmed. Antibody activity in serum obtained at a time when the host was bearing large tumors was found to be associated exclusively with 7S immunoglobulins. Two populations of 7S immunoglobulins showing CF reactivity were distinguished by electrical charge, as determined by diethylaminoethyl cellulose chromatography and immunoelectrophoresis. No antibodies of the 19S IgM type were detected in the serum of hamsters bearing large tumors.     

Quantitative characteristics of the T- and B-systems of immunity in healthy persons of different age groups]

The authors carried out a complex immunomorphological study of the peripheral blood lymphocytes in 160 healthy persons of different age groups. The tests of spontaneous and complementary rosette formation of lymphocyte blasttransformation with PHA were used, assessing it by the H3-thymidine incorporation, lymphocyte straining by Coon's direct and indirect methods for detection of surface immunoglobulin receptors; G, A, and M immunoglobulins blood serum content was studied by Mancini's method. Mean indices characterizing the T- and B-populations of lymphocytes in persons of different age were calculated on the basis of these studies.     

pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat

Rat and rabbit IgG immunoglobulins conjugated to horseradiah peroxidase as a histochemical marker bind at 0 degrees C to the luminal surface of absorptive cells in isolated segments of jejunum from 10-12-day old rats. Binding is observed at pH 6.0, near the normal luminal pH of the duodenum and jejunum at this age, but not at pH 7.4. Furthermore, no binding occurs when cells are exposed at pH 6.0 to either free peroxidase or peroxidase conjugated to chicken or sheep IgG immunoglobulins or bovine serum albumin. The sensitivity of binding to pH suggests a means whereby immunoglobulins which are selectively absorbed by the cells can be released efficiently at the abluminal surface.     

Localization by monoclonal antibodies of various surface antigens of hamster spermatozoa and the effect of antibody on fertilization in vitro

To determine the importance during fertilization of various plasma membrane components of the hamster spermatozoon, monoclonal antibodies were generated in the mouse against specific sperm surface antigens. BALB/C mice were immunized with washed hamster spermatozoa from the cauda epididymidis and immune splenocytes fused with myeloma cells (P3 X 63 Ag8). The sperm-specific immunoglobulins were detected in hybridoma cultures by a solid-phase assay (ELISA). Five monoclonal antibodies bound specifically to the surface of intact hamster spermatozoa, three immunoglobulins to restricted regions of the head and tail plasmalemma as detected by immunofluorescence. In two cases, the affinity of the membrane antigen was modified during passage through the epididymis. Monoclonal antibodies to the sperm head or to the head and tail inhibited fertilization in vitro by blocking sperm attachment to the zona pellucida and the oolemma.     

Localisation du site de l'infection urinaire chez l'enfant par la recherche de bactéries recouvertes d'anticorps

The demonstration by immuno-fluorescence of antibodies on the surface of urinary bacteria, a new method of determining the site of a urinary tract infection, was found to be as valuable in children as it is in adults. A clear correlation exists between a positive test result and renal parenchymal infection on one hand, and a negative result and lower urinary tract infection on the other. Moreover, immunoglobulins were still detectable in original positive urine samples that had been standing at 4degrees C for 7 weeks. The constant finding of IgA on bacteria suggests a particular synthesis for this class of immunoglobulin. A pathophysiologic role for complement would appear to be excluded by the facts that the serum concentrations of C3 were normal and that C3 was invariably absent from the bacterial surface.     

Early Events in the Pathogenesis of Foot-and-Mouth Disease in Pigs; Identification of Oropharyngeal Tonsils as Sites of Primary and Sustained Viral Replication

A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV) infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi) characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils. De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18–24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not co-localize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.     

Evidence of a true pharyngeal tonsil in birds: a novel lymphoid organ in Dromaius novaehollandiae and Struthio camelus (Palaeognathae)

ABSTRACT: BACKGROUND: Tonsils are secondary lymphoid organs located in the naso- and oropharynx of most mammalian species. Most tonsils are characterised by crypts surrounded by dense lymphoid tissue. However, tonsils without crypts have also been recognised. Gut-associated lymphoid tissue (GALT), although not well-organised and lacking tonsillar crypts, is abundant in the avian oropharynx and has been referred to as the "pharyngeal tonsil". In this context the pharyngeal folds present in the oropharynx of ratites have erroneously been named the pharyngeal tonsils. This study distinguishes between the different types and arrangements of lymphoid tissue in the pharyngeal region of D. novaehollandiae and S. camelus and demonstrates that both species possess a true pharyngeal tonsil which fits the classical definition of tonsils in mammals. RESULTS: The pharyngeal tonsil (Tonsilla pharyngea) of D. novaehollandiae was located on the dorsal free surface of the pharyngeal folds and covered by a small caudo-lateral extension of the folds whereas in S. camelus the tonsil was similarly located on the dorsal surface of the pharyngeal folds but was positioned retropharyngeally and encapsulated by loose connective tissue. The pharyngeal tonsil in both species was composed of lymph nodules, inter-nodular lymphoid tissue, mucus glands, crypts and intervening connective tissue septa. In S. camelus a shallow tonsillar sinus was present. Aggregated lymph nodules and inter-nodular lymphoid tissue was associated with the mucus glands on the ventral surface of the pharyngeal folds in both species and represented the Lymphonoduli pharyngeales. Similar lymphoid tissue, but more densely packed and situated directly below the epithelium, was present on the dorsal, free surface of the pharyngeal folds and represented a small, non-follicular tonsil. CONCLUSIONS: The follicular pharyngeal tonsils in D. novaehollandiae and S. camelus are distinct from the pharyngeal folds in these species and perfectly fit the classical mammalian definition of pharyngeal tonsils. The presence of a true pharyngeal tonsil differentiates these two ratite species from other known avian species where similar structures have not been described. The pharyngeal tonsils in these ratites may pose a suitable and easily accessible site for immune response surveillance as indicated by swelling and inflammation of the tonsillar tissue and pharyngeal folds. This would be facilitated by the fact that the heads of these commercially slaughtered ratites are discarded, thus sampling at these sites would not result in financial losses.     

Role of anaerobic streptococci in human pathology. Report I. Etiological significance of anaerobic streptococci in chronic tonsillitis]

The authors describe a method of isolation of anaerobic streptococci from the blood. A high seeding efficiency of anaerobic streptococci from the depth of the tonsils (75.1%) and from their surface (59.7%) and also from the blood (36%) pointed to the leading role of these microbes in the etiopathogenesis of chronic tonsillitis. In healthy individuals anaerobic streptococci are revealed (by culture method) on the surface of the tonsils but rarely (6%). Str. micros are the most incident in chronic tonsillitis (in 85.3% of cases-from the depth of the tonsils and in 77.5%-from the surface), whereas Str. anaerobius and Str. intermedius occur in individual cases only (8.4-7.5% and 6.3-15%, respectively). Pure culture of Str. micros was found in 36% of cases. Pathogenetic scheme of development of bacteriemia in chronic tonsillitis and rheumatism is presented. The isolated cultures were found to be resistant to the widely-used antibiotics.     

Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (C(initial E. coli O157)) can be derived from enrichment data according to the equation: C(initial E. coli O157) = C(initial coliforms) x C(final E. coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining C(final E. coli O157) in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms). Estimates of C(final coliforms) were determined from turbidity data. Initial E. coli O157 populations (C(initial E. coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of < 1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 10(3) to 10(8) cells ml(-1) enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.     

"Macromolecules to PDMS transfer" as a general route for PDMS biochips

"Macromolecules to PDMS transfer" technique relying on the direct entrapment of macromolecules spots during PDMS polymerisation is proposed as an alternative for the easy and simple PDMS surface modification. In the present work, the development of three different applications based on this procedure is presented as proof of the method potentialities. First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal anti-CRP antibodies was developed for sepsis diagnosis. The preserved integrity of the immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP in human sera (LOD=12.5 microg/L, detection ranging over two decades). Then, rheumatoid arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit immunoglobulins immobilisation allowed the detection of specific antibodies in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally, the "Macromolecules to PDMS transfer" procedure was used to easily and rapidly produce fibronectin-based cell culture arrays. The successful attachment of HeLa and BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin and vinculin.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.     

Immunochemical detection of Salmonella group B,D and E using an optical surface plasmon resonance biosensor

A surface plasmon resonance biosensor (Biacore) was used to detect Salmonella through antibodies reacting with Salmonella group A, B, D and E (Kauffmann-White typing). In the assay designed, anti-Salmonella antibodies immobilized to the biosensor surface were allowed to bind injected bacteria followed by a pulse with soluble anti-Salmonella immunoglobulins to intensify the signal. No significant interference was found for (mixtures of) 30 non-Salmonella serovars at 10(9) CFU ml(-1). A total of 53 Salmonella serovars were successfully detected at 1 x 10(7) CFU ml(-1), except those of groups C, G, L and P, as expected. The cut-off point was determined with an equicellular mixture of Salmonella enteritidis and Salmonella typhimurium at a final amount of 1.7 x 10(3) CFU per test portion. Although further work is needed to cover the detection of all relevant Salmonella serovars in food-producing animals and food products, this work demonstrates the merits of this alternative biosensor approach in terms of automation, sensitivity, specificity, simple handling and limited hands-on time.