A simple bactericidal antibody test for sero-diagnosis of typhoid fever

Serum samples were collected from 24 confirmed cases of typhoid fever, 15 clinically suspected cases and 23 normal healthy controls. The convalescent sera were obtained in 13 of the 24 confirmed typhoid cases. In all, 13 paired sera, 11 acute phase only, 15 clinically suspected and 23 normal serum samples were tested for eliciting bactericidal antibodies to Salmonella typhi. In addition, the Widal test was also performed for comparison. All the 24 acute phase sera as well as 13 convalescent sera were found to be positive by bactericidal antibody test (titre 1:80 or above). Of 15 clinically suspected cases, 5 were positive whereas one of the 23 normal controls sera gave a false positive reaction. In contrast, the Widal test could detect only one of the 24 cases in the acute stage, but all 13 cases showed antibodies at a diagnostic titre level during the convalescent stage. None of the 15 clinically suspected cases or 23 normal controls were positive by the Widal test. The feasibility of using a bactericidal antibody test in sero-diagnosis of typhoid fever is discussed.     

Simultaneous detection of Salmonella typhi antigen and antibody in serum by counter-immunoelectrophoresis for an early and rapid diagnosis of typhoid fever

Serum samples were collected from 26 proved cases and 15 clinically suspected cases of typhoid fever and from 23 normal controls. The convalescent phase serum samples were obtained in 18 of the 26 proved cases of typhoid fever. Salmonella typhi antigen and antibody were detected simultaneously in the serum samples by counter-immunoelectrophoresis (CIE). The conventional Widal test was also performed to elicit S. typhi H and O agglutinins. Out of the 26 cases proved by culture examination, 25 were detected during early stages of the disease (24 positive for S. typhi antigen and 1 for antibody) by CIE. Out of 15 clinically suspected cases of typhoid fever, 4 were positive for S. typhi antigen and none positive for antibody. All the 18 convalescent phase serum samples were positive for antibody, but were negative for S. typhi antigen. None of the 23 normal controls gave any false positive reaction for either antigen or antibody. In contrast, the Widal test could detect only 1 out of 26 cases in the acute stage, and all the 18 cases where convalescent serum samples were obtained were positive for antibodies at diagnostic titres. None of the 15 clinically suspected cases and 23 normal controls were positive by the Widal test. The feasibility of using simultaneous S. typhi antigen and antibody detection by CIE in diagnosis of typhoid fever is discussed.     

A rapid immunoperoxidase assay for determination of IgG antibodies to measles virus

A new indirect peroxidase antibody to membrane antigen (IPAMA) technique for the detection of IgG specific antibodies against measles virus is described. The technique utilizes as antigen measles-infected Vero cells dried on glass slides and stored at −70°C. Sera of 50 healthy medical students and laboratory workers and 24 sera of measles, encephalitis, and subacute sclerosing panencephalitis (SSPE) patients were checked by IPAMA and the results have been compared with the results obtained by the hemaglutination-inhibition (HI) test. There is good agreement between the results of both techniques as to the presence or absence of antibody in 48 out of the 50 tested. The advantages of the techniques are discussed.     

A simplified procedure for the preparation of antibodies to serum fibronectin

In the present paper we describe in detail a simple peocedure for the preparation of monosopecific antisera to human and mouse serum fibronectin.A similar procedure could also be used to prepare antibodies to fibronectin from other species. The procedure, based on the recently reported affinity of fibronectin for gelatin, essentially consists of two steps. (1) Immunization of rabbits with fibronectin purified from serum by affinity chromatography using gelatin coupled to CNBr-activated Sepharose 4B. (2) Absorption of the antiserum obtained by an immunoabsorbent prepared using fibronectin-free serum proteins that remained after absorbing serum with gelatin-Sepharose.The antisera obtained were monospecific, as determined by immunoelectrophoresis and did not show any difference with respect to antisera prepared by different procedures.     

Development of a simple passive haemagglutination-inhibition assay for Listeria monocytogenes lipoteichoic acid

A simple assay for listeria lipoteichoic acid was devised. It is based on the property of this substance to attach spontaneously to erythrocytes. Thus coated rabbit erythrocytes were agglutinated by a rabbit antiserum raised towards dead listeria. Inhibition of this passive agglutination was used as a measure of listeria lipoteichoic acid. Cross-reactivity of lipoteichoic acid from Lactobacillus fermenti in the assay and inhibition by simple sugars suggest that the assay is specific for terminal galactose.     

A study of the rat passive cutaneous anaphylaxis (PCA) reaction for the assay of mouse IgE antibody

The rat PCA reaction for assay of mouse IgE antibody was investigated. PCA reactivity was examined in rats with different characteristics in terms of strain, sex, age and rearing conditions. Sprague-Dawley (S-D), Fischer and Wistar strain rats were almost equally appropriate for the assay. The male rat was recommended in spite of the comparable PCA reactivity to the female since the male showed less non-specific bluing. PCA reactivity of the germ-free rat was comparable to that of the conventional animal. PCA reactivity appeared at 3 weeks of age, increased up to 11–13 weeks and remained constant until 40 weeks of age. PCA titers assayed in the rostral, caudal, lateral or central side of the back were comparable. The PCA reaction was poor when elicited with a 15 min sensitization period, became progressively stronger to reach a plateau at 4 h and remained constant until 72 h. Heating conditions required to demonstrate thermolability of IgE antibodies were studied. Incubation of undiluted antiserum at 56°C for 2 h abolished the reaginic activity. Reaginic activity of an antiserum diluted in saline was relatively resistant to heat, but diluted reaginic antibody was equally sensitive to heat in the presence of normal mouse serum (NMS) or HGG in the dileunt. NMS must be coexistent with reaginic antibody on heating to confer thermolability since preheated NMS used for dilution had no effect on the PCA titer of an antiserum.     

Topographical representation in rabbit cerebellar flocculus for various afferent inputs from the brainstem investigated by means of retrograde axonal transport of horseradish peroxidase.

A small amount of horseradish peroxidase (HRP) was injected into several small areas of the flocculus and adjacent ventral paraflocculus of albino rabbits. Labeled cells were surveyed through the inferior olive, vestibular nuclear complex, nucleus reticularis tegmenti pontis, pontine nucleus and abducens nucleus. Distribution of labeled neurons in these structures varied depending on the location of injection sites, indicating the existence of a fine topographical organization for afferent inputs in the flocculus and adjacent ventral paraflocculus.     

A sensitive micromethod for measuring human reverse haemolytic plaque-forming cells

A micromethod is described for detecting human immunoglobulin-secreting cells (ISC) using a reverse haemolytic plaque assay (RHPA). Plaques are developed in monolayers of indicator erythrocytes on the botton of the wells in 96-well, flat-bottomed platic microtitre trays. The technique detects more ISC than the conventional RHPA in agarose and this increased sensitivity is not due to the formation of artifact plaques. Together with its relative simplicity and low cost, the sensitivity of the microwell method makes it suitable for use in large scale studies of human B lymphocyte function.     

Biosynthetic incorporation of [75Se]selenomethionine: a new method for labelling lymphocyte membrane antigens

A novel approach for radiolabelling lymphocyte membrane antigens is described. This technique is based on the use of the gamma-emitting amino acid analogue [75Se]selenomethionine. Human HLA-A, B, C and DR heavy and light chains and mouse Ia antigens were efficiently labelled by this technique and were precipitated with monoclonal antibodies. Approximately the same radioactivity was incorporated into the HLA-A, B, C chains whether [75Se]selenomethionine, [35S]methionine or [3H]leucine were used as precursors. Easily detectable as a gamma-emitter, [75Se]selenomethionine thus constitutes a useful biosynthetic label of lymphocyte surface antigens. The same method was used to label immunoglobulins produced by hybridomas and to determine the nature of the secreted light chains.     

Ontogeny of macrophage function II. Increase of A-cell activity and decrease of phagocytic activity of peritoneal macrophages during ontogenetic development of immune responsiveness in mice

A parallelism was found between ontogenesis of murine immune responsiveness in anti-sheep erythrocyte antibody responses and that of immune-participating accessory cell (A-cell) activity of thioglycollate-stimulated peritoneal macrophages. Phagocytic activity, however, did not develop in parallel with A-cell activity. Thus, phagocytic activity was highest in neonatal mice and tended to decrease until about 2 weeks of age when A-cell activity and immune responsiveness tended to increase toward adult levels.     

Postsynaptic inhibition underlying spike suppression of secondary vestibular neurons during quick phases of vestibular nystagmus

Synaptic mechanisms of spike suppression of vestibular neurons during quick phases of vestibular nystagmus were investigated by intracellular recording in the rostrolateral part of the cat medial vestibular nucleus. When repetitive spike discharges of vestibular neurons were abruptly suppressed at the quick phase, the membrane potential shifted steeply in the hyperpolarizing direction. After the commissural IPSP was inverted into depolarization by intracellular injection of Cl^? ions, the hyperpolarizing deflection of the membrane potential at the quick phase was also inverted into a depolarizing potential. The results indicate that an abrupt generation of IPSPs in vestibular neurons underlies the quick phase suppression of spike activity in these neurons.     

The influence of the gamma system on cross-correlated activity of Ia muscle spindles and its relation to information transmission.

In the decerebrate cat during static or slowly varying (less than 0.3 Hz) muscle stretches, the activity of muscle spindle (MS) pairs was poorly correlated and such correlation was not changed after ventral root sectioning. With slightly faster sinusoidal stretches (0.3-20 Hz) activity in pairs of MS was also poorly correlated. However, sectioning of the ventral roots produced preferential firing frequencies in the same muscle spindle pairs and increased their degree of correlation. The increase in correlation between MS activity detected after suppressing the gamma bias appeared to arise from extrinsic 60 Hz power line vibration in the range of micrometers. Nevertheless, activation of the gamma system could suppress such phase locking. When frequencies above 20 Hz were used, the gamma system could not decorrelate the MS pair activity since the two units became locked to their common input signal. It is suggested that decorrelation of MS activity by gamma influence may improve the fidelity of the information transmitted by the Ia MS ensemble by filtering distortion harmonics, as well as damping tremor oscillations in the stretch reflex loop.     

Interactions of porcine IgG and porcine lymphocytes with protein-A Sepharose

Interaction of Protein-A Sepharose with porcine IgG showed that the binding capacity for porcine IgG was slightly lower than for human IgG. A small proportion of antibody activity of porcine serum was not reactive with Protein-A Sepharose. The interaction occurred with Fc fragments obtained either by papaĭn or by trypsin splitting of IgG but not with Fc' fragments. However, Fab fragments obtained after papaĭn or trypsin hydrolysis as well as Fab'₂ and Fab' fragments obtained by pepsin cleavage were partly retained onto the Protein-A Sepharose column. No antigenic differences were observed between retained and non-retained Fab, Fab' and Fab'₂ fragments with specific anti-porcine light chain antiserum. Thus, the interaction between Protein-A from S. aureus and porcine IgG is mediated not only through the Fc fragment, but also partly through the Fab part of the molecule. The resolubilized heavy chains did not bind to Protein-A Sepharose, suggesting that structural requirements are necessary for recognition. A subpopulation of porcine lymphocytes could be adsorbed onto Protein-A Sepharose beads, a binding specifically inhibited in the presence of porcine IgG.     

Non-selective staining of neurones and glial cells by the fluorescent dye merocyanine 540 in tissue cultures of mouse cerebellum.

Both glial and neuronal cell populations are stained by the vital fluorescent dye merocyanine 540 in monolayer cultures and single cell suspensions of early postnatal mouse cerebellum.     

Transneuronal transport of tritiated fucose and proline in the visual pathways of the brushtailed possum, Trichosurus vulpecula

Tritiated fucose and proline after injection into one eye are transported transneuronally to the primary visual cortex in the brushtailed possum Trichosurus vulpecula. On the contralateral side in layer IV a continuous band of label extends throughout primary visual cortex. On the ipsilateral side in layer IV a lighter band of label extends through the binocular part of the visual cortex. Ocular dominance columns cannot be demonstrated in Trichosurus visual cortex using these methods.     

Cyclic GMP and cyclic AMP levels in median eminence, hypothalamus and pituitary gland of the rat after decapitation or microwave irradiation.

Cyclic AMP and cyclic GMP content of the median eminence (ME), medial basal hypothalamus (MBH), posterior pituitary (PP) and anterior pituitary (AP) of inact male rats was measured by radioimmunoassay following decapitation or exposure to microwave irradiation (MW). Levels of both nucleotides in MW irradiated animals were higher in all three nervous tissues studied than in the AP. Following decapitation cyclic AMP levels increased strikingly in the MBH and ME, slightly in the AP, but remained unchanged in the PP. Cyclic GMP levels increased more markedly in the ME than in the MBH, slightly in the PP and did not change in the AP. The distinct synthesizing capability of the cyclic AMP and cyclic GMP generating systems in the ME, a region rich in neurohormones known to control anterior pituitary function, raises the possibility that these nucleotides are involved in the process of neurohormone secretion by the ME.     

Immunologic characterization of herpes simplex virus type 2 antigens ICP10 and ICSP11/12

Infected cell protein 10 (ICP10) or antigen 4 (Ag4) and infected cell-specific protein 11/12 (ICSP11/12) have been suggested as specific antigenic markers for cervical carcinoma. Experiments were designed to determine whether ICP10 and ICSP11/12 are distinct antigens and to determine the cellular localization of ICP10. Results indicate that an apparent 160 kdalton (kDa) protein analyzed by 8.5% polyacrylamide gels (= 144 kDa protein analyzed by 7.0% polyacrylamide gels) was detected in HSV-2-infected but not mock-infected extracts. This protein is an early virus-induced protein appearing 2-4 h after HSV-2 infection, and it was synthesized in the presence of successive blocks with cycloheximide and actinomycin D. These properties are characteristic for ICP10 (Ag4), thus establishing the identity of the 160 kDa/144 kDa protein as ICP10. Furthermore, Western blot analyses indicated that ICP10 and ICSP11/12 are distinct antigens recognized by antibodies in sera from immune rabbit or human cervical carcinoma patients. In addition, monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with ICP10. Antibodies in sera from rabbits immunized against ICP10 and monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with antigens on the plasma membrane surface of HSV-2-infected cells. Also, the reactivity of monoclonal antibodies with these antigens was blocked by the rabbit antibodies based on immunofluorescence analyses. These data provide evidence that ICP10 is antigenically distinct from ICSP11/12, and that ICP10 is present on the plasma membrane of HSV-2-infected cells. Also, our data confirm and extend the tentative identification of ICP10 with the HSV-2-induced ribonucleotide reductase recently suggested by Bacchetti et al. (J. Virol. 49, 591-593, 1984).     

Regional distribution of endorphin,met5-enkephalin and leu5-enkephalin in the pigeon brain

The distribution of β-endorphin and enkephalin in the pigeon forebrain by immunohistochemistry and radioimmunoassay is essentially analogous to mammals. Both endorphin- and enkephalin-reactive fibers have a similar periventricular distribution, but the enkephalin fibers are more extensive and are also found in the paleostriatum, limbic regions and brain stem, pituitary stalk and notably, penetrating the organum vasculosum hypothalami. There was poor correlation between endorphin and enkephalin regional contents by radioimmunoassay. In contrast, a highly significant correlation was observed between Met⁵-enkephalin and Leu⁵-enkephalin regional distribution. These data support the view that enkephalin neurons and endorphin neurons are independent central neuronal systems.     

Evidence for a GABAergic inhibitory nigrotectal pathway in the rat

The aim of the present study was to test the GABAergic nature of the inhibitory projection from substantia nigra, pars reticulata (SNr) to superior colliculus (SC) in the rat, through the use of extracellular recordings and microiontophoresis. The effect of SN stimulation on the spontaneous or glutamate-evoked firing of SC units was analyzed. Among 28 SC cells inhibited by SNr stimulation, 27 decreased their firing rate following iontophoretic application of either GABA or glycine. The effect of the iontophoretic administration of bicuculline on SNr-evoked inhibition was studied on 14 of these GABA- and glycine-sensitive neurons. Bicuculline reversibly blocked nigral inhibition on 12 neurons, with iontophoretic current which did not affect glycine depression.These results are consistent with the hypothesis that GABA is the inhibitory transmitter of the nigrotectal projection.     

Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells

A simple, objective, non-isotopic method for testing lymphocyte cytotoxicity is described. The evaluation of the test is based on photometric measurement of endogenous alkaline phosphatese activity of the residual adherent target cells.