Human secondary immune responses induced by influenza in a serum-free tissue culture system.

Peripheral blood lymphocytes are capable of producing antibody in response to antigenic stimulation by a recall antigen, influenza, during culture for 10 days in medium consisting of 1 part Ham's F-12 and 1 part Iscove's modified Dulbecco's medium supplemented with sodium bicarbonate, bovine crystalline insulin, human transferrin, 2-mercaptoethanol, progesterone, and bovine serum albumin. Anti-influenza antibody levels in the supernatants were determined by enzyme-linked immunosorbent assay. Optimal conditions for production of anti-influenza antibody in this serum-free medium were: influenza concentration, 0.032-0.125 HAU/ml; day of harvest, 10; and cell concentration, 3.0-4.0 X 10(5) cells in 200 microliter of medium per well. Use of serum-free medium will allow examination of the effects of various additives to tissue culture without concern for unknown factors or potential interaction with serum.

Prevalence of bronchial hyperresponsiveness in highly trained athletes.

Previous studies indicated that the prevalence of symptomatic asthma is about 4 to 7 percent. No similar studies exist to suggest the prevalence of asthma in highly trained competitive athletes, since asthma is thought to be an uncommon disease in this population. We became concerned, therefore, when a large number of football players developed symptoms consistent with asthma during preparation in California for the Rose Bowl in December 1981. We studied the team and found 12 percent of the football players admitted to a history of asthma, whereas none of the members of the university basketball team and 7 percent of a group of sophomore medical students and physician assistant students gave a history of asthma. Furthermore, 19 percent of the football players indicated that at some time they had chest tightness, cough, wheezing, or prolonged shortness of breath after exercise; 12 percent of the basketball players and 37 percent of the students indicated such a history. We examined each of these three groups for non-specific bronchial hyperresponsiveness to inhaled methacholine using a modified methacholine bronchoprovocation (MBP) challenge and found that 76 of 151 (50 percent) football players tested had positive tests; 76 percent of those with symptoms had positive results of inhalation tests and 47 percent of those with minimal or no symptoms had positive test results. In addition, four of 16 (25 percent) basketball players and 69 of 167 (41 percent) students had positive MBP tests. These studies indicate that bronchial hyperresponsiveness to inhaled methacholine is much more common in these young adults than has previously been suspected.

Protamine-induced fatal anaphylaxis. Prevalence of antiprotamine immunoglobulin E antibody.

Protamine is used widely to reverse the anticoagulant effects of heparin and to delay the absorption of insulin. Although adverse reactions to protamine are reported infrequently and are usually mild, we recently observed the first fatal case of type I anaphylaxis resulting from protamine. This patient had previously been sensitized to protamine during cardiac catheterization and had high levels of protamine-specific immunoglobulin E in the serum. In a prospective study, we found that 10 of 19 diabetic patients (53%) who had received insulin containing insulin also had high levels of antiprotamine immunoglobulin E. In contrast, none of 27 nondiabetic healthy normal controls or 10 diabetics who had never received protamine or protamine-containing insulin had levels of antiprotamine immunoglobulin E over background. This study underscores the risks of routinely administering protamine to susceptible individuals and the need for alternative therapies.

Human immunoglobulin biosynthesis in a serum-free medium.

We have previously reported that human peripheral blood lymphocytes are able to survive and undergo blastogenesis when cultured under serum-free conditions. Now we report that peripheral blood lymphocytes are capable of producing immunoglobulins in response to polyclonal activation during culture for 10 days in medium consisting of one part Ham's F-12 and one part Iscove's modified Dulbecco's medium supplemented with sodium bicarbonate, bovine crystalline insulin, human transferrin, 2-mercaptoethanol, and bovine serum albumin. Immunoglobulin levels in tissue culture supernatants obtained on day 10 were determined by enzyme-linked immunosorbent assay and were analyzed using a new computer program. Pokeweed mitogen was used as the polyclonal activator, and it stimulated immunoglobulin biosynthesis as well in serum-free medium as in medium that contained fetal calf serum. Immunoglobulin production in serum-free medium began on day 4 and continued through day 9, with peak production on days 5 to 8. Optimal conditions in serum-free medium were: PWM dose, 1:400 to 1:800; day of harvest, 10; and cell concentration, 3.5 X 10(5) cells in 200 microliters of medium per well. Use of this serum-free medium will allow examination of the effects of various additives to tissue culture without concern for unknown factors, components, influences, or potential interaction with serum.

Small heparin fragments regulate the amplification pathway of complement.

Heparin is a highly sulfated, polydisperse and heterogeneous glycosaminoglycan which has been well characterized for its ability to regulate multiple sites in the complement cascade. Although previous studies demonstrated the relationship between degree of sulfation, particularly O-sulfation, and complement inhibiting capacity, they left unclear the relationship between the size of the heparin molecule and its ability to inhibit complement. Therefore, although the structure-activity relationship for heparin is well understood for anticoagulant activity, it is ill defined for the complement system. The present studies were designed to examine depolymerized heparin to determine which fragments were capable of inhibiting amplification pathway activation. We found that as the size of the molecule increases the ability to regulate complement increases; below 1000 Da the fragments were essentially inactive and above 3500 Da they had the same activity as does commercial heparin. Furthermore, we examined the five major tetrasaccharides of heparin and found that the degree of sulfation did correlate with the ability to inhibit complement. These studies have for the first time begun to examine the minimal structural requirements for heparin to regulate complement.

Serious adverse reactions to protamine sulfate: are alternatives needed?

Protamine sulfate is a strongly cationic polypeptide that is used commonly in clinical medicine. It is administered regularly after cardiac catheterization, cardiothoracic and vascular surgical procedures, and less frequently after dialysis and leukapheresis because of its capacity to reverse the anticoagulant activity of heparin. In addition, because it delays the absorption of insulin, protamine is combined with insulin in protamine zinc insulin and neutral protamine Hagedorn insulin. Recently, there have been reports of adverse reactions to protamine (Table I). Although most of these reactions were relatively mild, three were fatal; one was clearly the result of type I anaphylaxis. Reactions occur predominantly in patients who were previously exposed to protamine through protamine-containing insulins or during heparin neutralization. Almost 50% of these patients were diabetic; most of whom received neutral protamine Hagedorn insulin, thereby enhancing their chance for presensitization. In 1983 we encountered three patients who suffered adverse reactions to protamine sulfate. Two of these patients will be presented here; the third patient, who died of IgE-mediated anaphylaxis, has already been reported and therefore is mentioned only briefly. We shall discuss adverse reactions to protamine sulfate and alternatives to the routine use of this drug.