Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Morphological changes induced in human red cells by rabbit univalent antibody

In an attempt to study size and shape variations induced in red cells by fixation of relatively large amounts of antibody protein in the absence of haemagglutination, microscopic analysis has been performed on human O red cells incubated with rabbit univalent anti-human red cell antibodies. These were obtained by recombining half-molecules of anti-red cell γG-immunoglobulin with half-molecules of non-specific γG-immunoglobulin.

Changes in cell morphology accounted essentially for a diameter increase during incubation at 37° in the presence of the recombined fraction. These changes were a progressive variation towards the sphere and then the disk shape. The amount of univalent material effective in determining such variation was estimated to be near to 45,000 molecules/cell, i.e. about twice the amount of bivalent molecules per cell giving 50 per cent agglutination.

The possibility that the mechanism of action of either the bivalent γG-immunoglobulin antibody or the recombined univalent molecule might be the same in respect to cytotoxicity is discussed.

     

Distinct subpopulations of thymus-dependent lymphocytes: Tracing of the differentiation pathway of T cells by use of preparatively electrophoretically separated mouse lymphocytes

Thymus-dependent cells from thymus and peripheral lymphoid organs were preparatively separated by means of free flow electrophoresis into various subpopulations which were defined in terms of θ (theta) antigen content, negative surface charge, graft-versus-host (GvH) reactivity, hydrocortisone sensitivity, cell volume and morphological details. Most thymocytes in the cortex have a low negative surface charge, high θ antigen content, are hydrocortisone-sensitive and immuno-incompetent. On the basis of electronic cell sizing this group consists of a large population of 90 μm³ cells (T₁) and a small population of 175 μm³ cells (T₂), the latter being less hydrocortisone-sensitive than the former.

A minority of thymocytes resides in and around the medulla and has high negative surface charge, a medium θ antigen content, is hydrocortisone-resistant and reveals low GvH reactivity. These cells are medium sized (125 μm³), electrophoretically bimodal (T₃ had a medium and T₄ a high negative surface charge) and on the basis of morphological criteria are metabolically more active than the thymocytes of low negative surface charge.

In the peripheral lymphoid organs, all thymus-dependent cells show high negative surface charge and have the lowest observed θ antigen content and the highest observed GvH reactivity. These cells fall into two populations of which one is 125 μm³ with lower negative surface charge and the other is 90 μm³ with a somewhat higher negative surface charge. These 125 μm³ cells (T₄), which morphologically resemble the 125 μm³ thymocytes, are less GvH-reactive than the 90 μm³ (T₅) cells, which seem to be resting cells.

On the basis of these data, a possible sequence of steps in the maturation of T cells was constructed as follows: in the cortex of the thymus T₁ thymocytes are transformed into T₂ and these develop into T₃ and T₄ thymocytes which have higher negative surface charge, lower θ antigen content and are in an advanced stage of maturity. After further loss of θ antigen these cells, which are in the medulla, emigrate into the periphery and are finally transformed into highly immunocompetent T₅ cells possessing the highest observed negative surface charge.

     

Haemorrhagic reactions elicited at sites of passive cutaneous anaphylaxis by the intravenous injection of aggregated γ-globulin

In guinea-pigs injected intradermally with a small amount of antibody and challenged 2 hours later, by the intravenous route, with a mixture of homologous antigen and aggregated γ-globulin, haemorrhagic reactions of the Arthus type develop at the sites of intradermal sensitization. This effect was obtained with γ-globulins of different species (human, rabbit and horse) by using different techniques for aggregation (heat, mercaptoethanol—urea and bis-diazobenzidine) and was always correlated with the ability of the aggregated globulin to fix complement.

Fluorescein labelled aggregates of γ-globulin were detectable in the wall of vessels at sensitized sites.

In experiments performed with guinea-pig antibodies, the localizing effect was observed only with γ₁, whereas the γ₂, Arthus-producing fraction proved completely ineffective.

Histamine and histamine liberators are not sufficient for eliciting the effect obtained with sensitizing antibody plus homologous antigen. It is postulated, therefore, that other effects occurring at the site of specific sensitization may also be responsible for the phenomenon.

     

Shigella-: Rabbit Gamma-Globulin Allotypes as Genetic Markers for the Source of Antibody Produced in Recipients of* • Incubated Lymph Node Cells*

Rabbits homozygous for each of an allelic pair of allotypes of γ globulin (A4 and A5) were used as donors and recipients of transferred antigen-incubated lymph node cells, the cells of donors of one allotype being in each case transferred to recipients of the other. When agglutinins to Shigella (the source of the antigen with which the lymph node cells were incubated) appeared in the sera of the recipient animals, the allotype of the agglutinin was determined by adsorbing it to Shigella on a glass slide, then treating the preparations with fluorescein-conjugated rabbit anti-A5-γ-globulin and anti-A4-γ-globulin antisera, respectively. In each case the reactions of the recipients' sera were positive for γ globulin of the donors' allotype but not of their own. Positive reactions were given only by sera above a certain range of agglutinin titre.

After sufficient decline of the agglutinin level in the sera of the recipients, these animals were actively immunized with Shigella. The agglutinins that now appeared in these rabbits gave positive reactions with the fluorescent antibody against their own allotype.

These data indicated that in moderately X-irradiated rabbits given antigen-incubated rabbit lymph node cells, the antibody that subsequently appears in the recipient's serum has been synthesized by the donor's cells.

These experiments also illustrate the use of allotypes as genetic markers in lymph node cell transfer experiments.

     

Temperature changes produced by the injection of catecholamines and 5-hydroxytryptamine into the cerebral ventricles of the conscious mouse

1. Changes in temperature were determined following injection of noradrenaline, adrenaline, isoprenaline, dopamine and 5-hydroxytryptamine (5-HT) into the cerebral ventricles of the conscious mouse.

2. Noradrenaline (1-20 μg) and dopamine (10-160 μg) caused falls in body temperature. Adrenaline (1-20 μg) caused a slight and transient rise in body temperature followed by a fall. Isoprenaline (5-20 μg) caused a rise in body temperature, hypothermia only occurring after very high doses (200 μg) of this catecholamine.

3. α- and β-adrenergic blocking agents, phentolamine (> 2 μg) and propranolol (> 5 μg) respectively, caused falls in body temperature when injected into the cerebral ventricles of the mouse.

4. Specific drug antagonism studies were limited owing to the intrinsic effects of the α- and β-adrenergic blocking agents. However, some evidence was obtained to indicate that noradrenaline mediated its effects through a central α-type adrenergic receptor.

5. 5-HT (10-160 μg) caused a fall in body temperature. The action of this indoleamine and the catecholamines in regard to thermoregulatory function is discussed.

     

Quantitative estimation of 17alpha-hydroxypregn-4-ene-3,20-dione (17alphaOH-progesterone) in adrenal venous blood and adrenal glands

1. A method is described for the accurate estimation of small quantities of 17α-hydroxypregn-4-ene-3,20-dione (17αOH-progesterone) in blood and adrenal glands.

2. The dog adrenal was found to secrete 17αOH-progesterone under conditions of operative stress at a rate of 5-10 μg/g tissue/hr. This is similar to the secretion rate of aldosterone.

3. The secretion of 17αOH-progesterone was decreased after hypophysectomy and increased in response to adrenocorticotrophic hormone (ACTH).

4. The adrenal gland of the dog, the monkey and the guinea-pig was found to contain between 0·6 and 6 μg of 17αOH-progesterone/g tissue. The concentration was usually somewhat smaller than that of pregnenolone or progesterone and between 1 and 20% of the concentrations of the glucocorticoids.

5. A comparison of adrenal steroid contents and secretion rates under conditions of stress indicates a rate of synthesis in the order of 30-40 n-moles of 17αOH-progesterone/g adrenal tissue/min.

     

Preparation of anti-β1C antisera and their use in fluorescent antibody techniques

Antisera against components of guinea-pig complement were raised in rabbits by:

(1) Using as antigen a suspension of killed Proteus species bacteria which had been allowed to combine with their homologous antiserum in the presence of guinea-pig complement in optimum proportions.

(2) By injection of the β_1C component of guinea-pig complement adsorbed to zymosan particles. The antisera raised by the two methods contained antibodies mainly against the β_1C component of complement.

When coupled with FITC both antisera were found useful in detecting sites of complement fixation. The fluorescence anti-complement technique was found four times more sensitive than the indirect method for detecting antigen—antibody reactions in the presence of diminishing concentrations of antigen. It was only twice as sensitive for detecting antigen—antibody reactions in the presence of diminishing concentrations of antibody. The comparisons were based upon both visual assessment and photometric measurement.

Coupled antisera raised by the first method gave brighter specific fluorescence than antisera raised by the second method when used in the highest concentration which did not give non-specific staining.

The usefulness for detection of viral antigens of sera raised by both methods is discussed.

     

The passive transfer of delayed hypersensitivity in the guinea-pig: II. The ability of passively transferred antibody to cause local inflammation and retention of antigen and the role of these phenomena in the passive transfer of delayed hypersensitivity

It is known that serum increases the passive transfer of delayed hypersensitivity to certain antigens, such as bovine γ-globulin, in the guinea-pig. This synergic effect of serum in the passive transfer of delayed hypersensitivity to bovine γ-globulin was partly, but not completely, produced by serum to haemocyanin when a mixture of bovine γ-globulin and haemocyanin was used for skin testing. It was concluded that part of the synergic action of serum was due to a local inflammatory reaction.

The ability of serum to cause local retention of antigen in the skin was studied using antigens labelled with radioactive iodine. Immune serum favoured the local retention of antigen. The passive transfer of antiserum to bovine γ-globulin, egg albumin and haemocyanin specifically increased the retention of antigen two- to twelve-fold. This ability of serum to cause the local retention of antigen at the site of intradermal injection was present in serum taken 3 weeks after immunization with bovine γ-globulin in Freund's complete adjuvant but absent in serum taken at 1 week. Antiserum also altered the distribution of antigen at the skin site. Autoradiography showed that it increased the area over which an appreciable concentration of antigen occurred.

Active immunization with bovine γ-globulin had a slight effect on the total amount of antigen retained in the skin after intradermal injection. It had a greater effect on the distribution of antigen. In control guinea-pigs 87 per cent of the total amount of bovine γ-globulin retained at 20 hours was found within a radius of 6.5 mm of the centre of injection. In contrast in guinea-pigs immunized with bovine γ-globulin in Freund's complete adjuvant 43 per cent was found beyond this radius. A similar change in the distribution of human serum albumin was seen in guinea-pigs immunized with bovine γ-globulin when the albumin was mixed with bovine γ-globulin. This indicated that factors other than the formation of immune precipitates were sometimes responsible for the local retention of antigen. The total amount of purified protein derivative of tuberculin (PPD) retained and its distribution in the skin was uninfluenced by immunization.

It was concluded that the synergic effect of immune serum on the passive transfer of delayed hypersensitivity was due in part to some aspect of the inflammation caused by antibody antigen reaction and in part to the local retention of antigen caused by antibody.

     

Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels

Gamma globulin complexes were demonstrable in certain joint fluids from patients with rheumatoid arthritis by analytic and density gradient centrifugation. They form a continuum of high molecular weight components ranging from 7S to 30S and were dissociable primarily to 7S γG globulin. The larger complexes were also detectable by precipitation reactions with C1q and with γM rheumatoid factor. This permitted the isolation and partial characterization of the complexes. Non-immunoglobulin constituents were not detectable. Evidence was obtained that 7S γG globulin rheumatoid factors represented an important constituent of the complexes.

A relationship was encountered between the amount of γ globulin complex present in the joint fluids and diminution in total haemolytic complement activity. All fluids with abundant γ globulin complexes contained markedly lowered complement levels. A decrease in levels of C1q and β_1A was found to correlate with the amount of γG globulin complexes. Although patients who had diminished complement levels in their joint fluid have serum γM rheumatoid factor, its titre does not correlate well with the extent of complement depression.

Joint fluids with abundant γ globulin complexes manifested an anticomplementary effect. This activity was most apparent at 37°C when fresh rheumatoid serum was used as a source of complement. Evidence indicating the participation of γM rheumatoid factor in this anticomplementary effect was obtained. All fluids with significant anticomplementary activity formed precipitin bands with C1q on agarose plates. γM rheumatoid factor was not necessary for this reaction.

     

The attenuation of rod signals by backgrounds

1. The paper which precedes this investigated the nerve interaction between two flashes, λ at centre (Fig. 1a) and ϕ on the surround region (but not on the centre). The size of the inhibitory nerve signal V generated by ϕ is given by V = ϕ(ϕ + σ), where σ is the semi-saturation constant.

2. A former paper (Alpern & Rushton, 1967) had shown that when the flash ϕ falls upon a steady background θ, V suffers attenuation in the G-box (Fig. 1b) down to the fraction θ_D/(θ_D + θ) where θ_D is the eigengrau or receptor noise. Thus, in general, the nerve signal N is given by [Formula: see text].

3. This formula had only been established for a moderate range of values. In this paper we use extreme values to explore the limits of its validity. We find the equation to be true over the entire intensity range where N is measurable.

4. Six different types of experiment have been performed to test various features of the equation. For instance, if log N is plotted against log ϕ for various fixed values of θ, the curve is always the same with simply a vertical shift. And the shift is equal to log(1 + θ/θ_D) for all values both of θ and of ϕ.

5. The most interesting curve is the plot of log ϕ against log θ for fixed N. This is similar to the Weber—Fechner increment threshold but the criterion is not that ϕ be strong enough to be detected, but strong enough to generate an N signal just sufficient to inhibit some fixed λ flash. These curves (below the onset of saturation) are all the same except for vertical separation, and prove that the condition for flash detection is that a fixed signal, N₀, is generated of size 10^-5 of the maximum signal obtainable (i.e. with ϕ large and θ zero).

6. With strong backgrounds the curves of (5) above exhibit a marked saturation of the Aguilar & Stiles' type (1954). The family of curves each with a fixed N value shows a remarkable symmetry (Fig. 8) which in fact follows from the equation in (2) above. It has nothing to do with bleached pigment, but follows from the equation in (1) above. V there cannot exceed unity, thus when scaled by the G-box below the criterion level, further increase in ϕ will not bring improvement.

     

Antibody production in mice: I. The analysis of immunological memory

The development of immunological memory was analysed by the use of `in vivo culture technique'. The lymphoid cells from primed mice were transferred into heavily irradiated recipients and the size of memory cell population in primed donor mice was estimated quantitatively by the magnitude of secondary responsiveness.

The production of memory cells was detected about 1 week after the primary administration of antigen, and increased gradually up to approximately 6 weeks. The development of the memory cells carrying information for the synthesis of γG₁-antibodies (γG₁-memory cells) was initiated at the early stage of priming and followed by that of the γG₂-memory cells. Although the ratio of population of γG₂- to γG₁-memory cells changed with lapse of time in primed mice, the original ratio of each population in donor, at any stage after priming, was apparently maintained when cultured in recipients for at least 4 weeks. This indicates that the conversion from γG₁- to γG₂-memory cells does not occur.

From these results, it was suggested that the γG₁- and γG₂-memory cells develop independently in primed mice under the continuous stimulation of primarily administered antigen.

     

Passive sensitization of skin and lung by guinea-pig immunoglobulins

Guinea-pig γ₁- and γ₂-globulins have been purified by preparative electrophoresis followed by chromatography. No γ₁-globulin was detectable in purified γ₂-globulin, but purified γ₁-globulin always contained fast γ₂-globulin. Normal guinea-pig serum contained much less γ₁-globulin than immune serum. Antisera prepared against normal guinea-pig serum did not contain useful amounts of antibody specific for γ₁-globulin.

Guinea-pig lung tissue was sensitized by very low concentrations of guinea-pig γ₁-globulin (of the order of 6×10^-10 molar) but γ₂-globulin antibodies were almost inactive. No evidence was found that the trace of activity in γ₂-globulin was not due to very slight contamination with γ₁-globulin antibodies.

The finding that γ₁-globulin antibodies are far more potent than γ₂-globulin antibodies in sensitizing skin has been confirmed, but several lines of evidence suggest that γ₂-globulin antibodies may also have weak activity. Thus quantitative passive cutaneous anaphylaxis (PCA) tests showed that whenever the γ₂-globulin fraction contained antibody it appeared far more potent relative to γ₁-globulin than when the same proteins were tested on lung tissue. The PCA activity of moderate amounts of purified γ₂-globulin antibodies disappeared faster than the skin sensitization produced by small amounts of γ₁-globulin antibodies, and the γ₂-globulin preparations did not contain enough γ₁-globulin impurity to account for their PCA activity. No inhibition of skin responses was observed with the largest doses of antigen tested.

The most plausible explanation of these results is that, under the conditions of our experiments, γ₂-globulin antibody had weak PCA activity. Objections to this hypothesis are discussed. The PCA activity of γ₂-globulin antibody probably involves a mechanism different from that of the sensitization produced by the highly potent γ₁-globulin antibody.

     

Biological and physiocochemical properties of purified anti-DNP guinea-pig non-precipitating antibodies

Methods for isolation and purification of precipitating and non-precipitating guinea-pig antibodies are described.

The physicochemical properties of γ₁ and γ₂ non-precipitating antibodies are similar to γ₁ and γ₂ precipitating ones. Biological properties are also similar excepting the reverse Arthus reaction, which is positive with the precipitating and negative with the non-precipitating antibodies.

Bivalence of these antibodies was experimentally demonstrated. Precipitating antibodies K₀ do not differ greatly from those obtained with the corresponding non-precipitating ones. The incapacity to precipitates with the antigen may be a consequence of a steric impediment.

     

Studies on the Mechanism of Hypersensitivity Phenomena: V. Antigen-Antibody Interaction in the Guinea-Pig Small Intestine*†

A series of exploratory studies have been described in which an isometric apparatus has been applied to the study of the smooth muscle response to immune reactions.

Data are given for the dose-response relationships with several immune systems.

Evidence is provided which relates the length of the induction period to the quantity of antigen and magnitude of the smooth muscle response.

Segments of small intestine of some, but not all, guinea pigs react to the presence of rabbit anti-γ globulin. The differences that have been encountered may be due to genetic variation in guinea-pig γ globulins.

Previous findings have been confirmed that normal γ globulins may interfere with the sensitization of tissue or with the tissue response due to specific antibody.

In vitro sensitization of guinea-pig small intestine demonstrated that the smooth muscle response to one immune system may diminish a subsequent response of the same tissue to an unrelated antigen-antibody reaction.

     

Desensitization in vitro—The specific inhibition, by antigen, of the passive transfer of delayed hypersensitivity by peritoneal exudate cells

A preliminary experiment showed that the injection of bovine γ-globulin into guinea-pigs with delayed hypersensitivity to bovine γ-globulin reduced the 24-hour skin reactions to bovine γ-globulin and (to a lesser extent) PPD. The peritoneal exudate cells from the desensitized donors had a reduced ability to transfer delayed hypersensitivity to bovine γ-globulin but a normal ability to transfer delayed hypersensitivity to PPD.

Likewise, it was possible to diminish the passive transfer of delayed hypersensitivity to bovine γ-globulin by peritoneal exudate cells, by exposure of the cells to bovine γ-globulin in vitro. The recipients were tested immediately after cell transfer. This in vitro desensitization was specific, in that the transfer of delayed hypersensitivity to PPD was unaffected.

Exposure of cells in vitro to hypotonic conditions and antibody to guinea-pig γ-globulin did not prevent the passive transfer of delayed hypersensitivity.

     

Active cutaneous anaphylaxis in the guinea-pig: Immunological and inflammatory reactions

Active cutaneous anaphylaxis was studied in guinea-pigs sensitized by inhalation of egg albumen powder and producing high titre IgGγ1 and low titre IgE homocytotropic antibodies. On cutaneous antigen challenge an immediate increase in venular permeability occurred which declined rapidly over the next hour, normal permeability being restored by 2 hours. This was followed by a biphasic tissue eosinophilia with peaks at 3–6 hours and 14–16 hours. Tissue neutrophilia was also maximal at 14–16 hours with a preceding `shoulder' between 6 and 12 hours. Injection of antigen into non-sensitized animals produced only the early peak of leucocytosis.

Fluorescent protein tracing demonstrated that little antigen persisted in lesions older than 7 hours. Neutrophil phagocytosis of antigen was maximal between 1 and 3 hours; eosinophil phagocytosis of antigen was not observed. IgGγ1 and IgGγ2 were localized to granulocytes during the 3–6 hour peak of leucocytosis, but not in older lesions. IgGγ₁ was detected in eosinophils, but which granulocytes contained IgGγ2 antibodies remained uncertain. Binding of IgGγ1 antibodies to mast cells was not observed and C3 was not detected in any lesion. It is concluded that antigen induced the first peak of tissue leucocytosis; possible mediators of the second leucocytic peak include eosinophil chemotactic factor of anaphylaxis, mast cell products and leucoegresin.

     

Antigen-induced histamine release from peritoneal mast cells of mice producing reagin-like antibody

The time course of production in the mouse circulation, after different schemes of immunization, of reagin-like and γ₁ antibodies, as measured by the ability of the antisera to induce thermolabile long-latence and thermostable short-latence PCA reactions, has been studied. γ₁ antibody was obtained in all schemes of immunization and its amount in antiserum increased with time. Reagin-like antibody appeared on the 7th day after immunization with sufficient dose of ovalbumin and adjuvant, and on later days with less efficient antigens such as DNP—BSA and DNP—BGG. Maximal amounts of the reagin-like antibody were reached in the early days after immunization and its presence in the circulation had a somewhat transient character; however, it persisted in the circulation for periods of more than 60 days, on occasion.

The time course of antigen-induced histamine release from washed peritoneal mast cells of the immunized mice was also studied. A correlation was observed between the histamine release and the production of reagin-like antibody. It is suggested that the antigen-induced histamine release from washed peritoneal mast cells may be taken as an indication of the production of reagin-like antibody.

Some quantitative aspects of the PCA and histamine release reactions were also studied. PCA reactions were affected similarly by varying either dilution or volume of the antiserum intradermally injected. Histamine release from peritoneal mast cells did not seem to be affected specifically by excess of antigen.

     

The effects of a single injection of progesterone on the oestrous cycle, thyroid gland activity and uterus-plasma concentration ratio for radio-iodide in the rat

1. A single subcutaneous injection of 5 mg, 1·25 mg or 625 μg, but not 312 μg, of progesterone in oil delayed ovulation in the rat by 1 or more days, when injected at the dioestrous stage of a 4-day oestrous cycle.

2. When ovulation was delayed in this way the expected increase in the thyroid-serum concentration ratio for ¹³¹I was also delayed but the ratio did increase when delayed ovulation occurred.

3. A single injection of progesterone resulted in an increase in the uterus-plasma and oviduct-plasma concentration ratios for ¹³¹I; the increase was greatest when steroid was injected at the dioestrous stage of the cycle and was delayed and least when the steroid was given at the pro-oestrous stage.

4. Ovulation was advanced by 1 day when progesterone was injected on the second day of dioestrus in rats showing regular 5-day cycles; this ovulation was not accompanied by an increase in the thyroid-serum concentration ratio. In these experiments a dose of progesterone that failed to advance ovulation produced a rise in uterus-plasma and oviduct-plasma ratio for ¹³¹I but no rise was seen when ovulation was induced, suggesting that oestrogen secretion had been stimulated.

5. 20α-Dihydroprogesterone (pregn-4-en-20α-o1-3-one) was not effective in delaying or advancing ovulation at a dose level of 2·5 mg per rat and had no effect on the uterus-plasma concentration ratio for radio-iodide.

6. These results are discussed in relation to the hypothesis that the increase in thyroid gland activity at the oestrous stage of the cycle is related to the neuro-endocrine changes that lead to ovulation.

     

Immunological studies of phytohaemagglutinin: I. Reaction between phytohaemagglutinin and normal sera

Studies of the precipitation of normal human serum by the kidney bean phytohaemagglutinin, Phaseolus vulgaris (PHA), showed that the main serum components involved were α₂-macroglobulin, β-lipoproteins and γM immunoglobulins. Preparations of α₁-glycoprotein, orosomucoid and some γA immunoglobulins were also precipitated by PHA. Antisera prepared against the PHA—NHS precipitate recognized α₂-macroglobulin, β-lipoproteins and γM immunoglobulins, γG immunoglobulins and at least four other unidentified antigens in the β and α regions. PHA did not react with B or A, O blood group substances.

Concanavalin A, a jack bean agglutinin, precipitated the same proteins which were precipitated by Phaseolus vulgaris PHA but a Dolichos biflorus extract did not react with human serum. Incomplete chemical studies of this interaction suggested that even though the same serum proteins were precipitated by Concanavalin A and PHA, the sugar specificity, if involved, is different for both lectins.

Incomplete studies of the component or components of PHA involved in the PHA—NHS interaction suggested that three cathodally migrating proteins were recognized by both the antisera to PHA and to the precipitate formed by PHA and normal human serum.