• The discovery (1) that there are many substances with the sensitizing properties hitherto believed peculiar to cholesterol and its derivatives, and (2) that sensitizer can be added to <em>antigen</em> in very large quantities, many times those currently used, and yet continue to increase its complement-fixing efficiency with no danger of giving falsely positive <em>tests</em> has made possible the preparation of an <em>antigen</em> much more sensitive than any now available for use in the Wassermann reaction. 100 gm. of dry powdered beef heart muscle are extracted with 500 cc. ether for 15 minutes at 37 degrees C. with shaking.
• After filtration with suction, the ether filtrate is discarded. The powder is then dried and extracted for 3 to 5 days with 500 cc. of 95 per cent alcohol, with intermittent shaking. The mixture is filtered, and the moist powder washed on the filter paper with two portions of alcohol, each 100 cc.
• The alcoholic filtrate and washings are combined and evaporated on the steam bath down to <em>25</em>0 to 300 cc. Cholesterol (0.8 per cent), and sitosterol (0.6 per cent) are then added, and dissolved at 65 degrees -75 degrees C. The excess sensitizer which crystallizes out upon cooling is dissolved just before using by immersing the <em>antigen</em> in a 56 degrees C. bath for a few minutes.
• It is diluted by pouring the saline rapidly into the <em>antigen</em>. A 1:40 dilution is recommended for use in water bath fixation ((1/2) hour at 37 degrees C.), as well as for the short ice <em>box</em> fixation (8 degrees , 4 hours) and a 1:120 dilution for use in the overnight ice <em>box</em> method (16 to 24 hours at 8 degrees C.), as being well beyond its anticomplementary range; the anticomplementary quantities being 1:5 and 1:<em>25</em> respectively.
• There is reason to believe that this <em>antigen</em> possesses almost the maximum sensitivity obtainable. The method of preparation insures its being almost saturated with <em>antigen</em>-lipoids; and more sensitizer could not be added without increasing the turbidity of its dilution in saline to a point where it would interfere with the reading of hemolysis.
• Any further improvement must await the discovery of better sensitizers. Preliminary experiments indicate that this new sensitizer, sitosterol, will find an immediate application, not only in the Wassermann reaction, but also in a more sensitive flocculation <em>test</em> to be described in a following paper.

• Serum, in concentrations greater than 1:<em>25</em>, causes a marked inhibition of complement fixation in general and of the Wassermann reaction in particular. The serum protein is probably adsorbed by the colloidally dispersed lipoid-reagin complexes, forming a protective film which prevents the fixation (adsorption) of complement.
• This inhibition explains the zone phenomenon in complement fixation: a weakly positive serum may give a completely positive reaction in e.g., 1:5 dilution, and yet, because of this serum inhibition, may appear completely negative when <em>test</em>ed as whole serum.
• The greater sensitivity of the ice <em>box</em> <em>test</em> is due (1) to the fact that the serum inhibition just described is less marked at lower temperature; (2) to the prolonged incubation time, making for greater specific fixation; (3) to a more marked non-specific destruction of complement by <em>antigen</em>; and (4) a spontaneous deterioration in the longer ice <em>box</em> <em>test</em>.
• Because of the inhibition by serum protein in high concentration, a quantitative Wassermann technique involving the use of graded quantities of serum is worthless when carried out at 37 degrees C. Even the ice <em>box</em> <em>test</em>, which is less susceptible to this inhibiting effect, will yield a positive reaction with whole serum only when the circulating reagin exceeds a surprisingly high threshold (six to ten times the quantity which could be detected in dilute serum). It is well known that a negative Wassermann, even by a very sensitive <em>test</em>, does not exclude syphilis: it now appears that a negative Wassermann does not exclude circulating reagin.

• Forkhead <em>box</em> P3 (FoxP3) T regulatory (Treg) cells modulate the immune system by blocking other types of T-cells. They maintain tolerance to self-<em>antigens</em> and help in inducing tolerance to foreign <em>antigens</em>. A deregulation of FoxP3 Tregs seems to play an important role in allergic disorders.
• The aim of this work was to study the response of FoxP3 Treg cells and their FoxP3 expression in patients, attending the Allergy Unit and the Chest Outpatient Clinic, Faculty of Medicine, Zagazig University, with allergic airway diseases, before and 1 year after receiving subcutaneous allergen specific immunotherapy (SIT).
• This prospective study was carried out on <em>25</em> patients with allergic airway diseases, confirmed by positive skin <em>test</em>, and that showed clinical improvement one year after SIT. All cases were subjected to total immunoglobulin E quantitation by ELISA. FoxP3 Treg cells frequency and FoxP3 relative fluorescence intensity, as an indicator of Tregs function, were assessed by flowcytometry.
• The results were compared before and after SIT. Twenty five age and sex matched apparently healthy volunteers were enrolled as controls. Our findings demonstrated that in comparison to the control group, the count of FoxP3 T regulatory cells was higher; however, the function was lower among the enrolled patients (P= 0.007 and P< 0.001, respectively). When FoxP3 Tregs were compared in the patients before and one year after SIT, it was found that both the count and FoxP3 expression showed statistically significant increase (P< 0.001).

An inverse correlation was found between FoxP3 Tregs count and FoxP3 expression. It is concluded that patients with allergic airway diseases have increased number of FoxP3 Treg cells but with defective function. SIT plays a role in increasing the number of FoxP3 Tregs and improving their suppressive function, which leads to control of airway inflammation and thus clinical improvement.