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Clinical applications of protein A immunoadsorption in thrombocytopenic disorders
Pergamon
0955-3886(94)00045-x
7iansfus. Sci. Vo1.15, No.4, pp. 423-427, 1994 Copyri t @ 1994 Elsevier Science Ltd Printed in b,$;y~;~r~~~$h;h~g~
Clinical Applications of Protein A Immunoadsorption in Thrombocytopenic Disorders Robert B. Howe, MD* $ Douglas J. Christie, PhDt
Recent reports of a favorable clinical response to immunoadsorption therapy with Staphylococcal protein in AIDSassociated immune thrombocytopenia,‘,’ autoimmune thrombocytopenic purpura,3 alloimmune thrombocytopenia,4 the hemolytic-uremic syndrome’ and thrombotic thrombocytopenic purpura6 have rekindled interest in the application of this therapy and in its mechanism(s) of action. Most reports have only appeared in abstract form and few have addressed the utility of protein A therapy in a systematic manner. Bertram et a1.l reported a favorable response in six of nine patients with HIV-related immune thrombocytopenia. The mean increase in pIateIet count was 95,000/mm3; four individuals achieved normal platelet counts. At the time of their report these responses had been sustained for 5 months. No significant changes in total serum IgG levels or platelet-associated IgG (PAIg) were noted; however, circulating immune complexes (CIC) were reduced. They treated a relatively small volume (250 mL) of plasma using the off-line technique. More recently’ these investigators reported a 55% (16129) response rate to protein A treatment of HIVassociated thrombocytopenia. Twelve patients sustained remissions of over 8 ‘Professor of Medicine and tAssociate Professor of Medical Technology, Box 480, University of Minnesota Health Ckk;, 420 Delaware Street SE, Minneapolis, MN 55455,
*Author for correspondence.
months. Interestingly, they could not account for the diminution in plasma levels of CIC, platelet-directed IgG (PDIg), or PAIg by the amount of plasma adsorbed per treatment. Anti-F(ab’J, levels increased over the 3 months following therapy. Preliminary reports have suggested that a similar benefit might be conferred thrombotic with patients upon thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) in cancer patients5 and in patients with the spontaneous l-TP-HUS6 Twenty-one patients with cancer-associated, mitomycin C-induced HUS were reported by Lesesne et al. in 1989.5 Ten of the 21 patients demonstrated resolution of thrombocytopenia, improvement in anemia and stabilization of renal function, but since criteria defining these responses and specific data were not given, the claims are difficult to evaluate. The authors also failed to provide specific treatment regimens, such as frequency and total number of treatments and volume of blood treated, and whether the blood was treated “on” or “off-line”. No data on immunoglobulin or CIC changes were given. Gaddis et al.” reported in abstract form on ten females and one male aged 17 to 62 with classical TTP. All had failed plasmapheresis and other therapies including glucocorticoids, vincristine, anti-platelet drugs and splenectomy. Eight of the patients had complete resolution of their TTP. Only two
424
Transjus. Sci.
Vol. 15, No. 4
patients required more than six treatments; however, both had a complete response. We must await full publication and confirmatory studies in order to evaluate the utility of protein A immunoadsorption in this syndrome. Snyder et aL3 accumulated 72 cases of patients with refractory autoimmune thrombocytopenic purpura (ITP) who had failed at least two standard forms of therapy. Many had not been splenectomized. Following an average of six immunoadsorption treatments 46% had a complete or partial remission. Resultant platelet counts were normal (complete remission) or at levels not associated with risk of bleeding (partial remission). Spleen intact patients had an equal chance of response as splenectomized subjects, suggesting that this therapy may obviate the necessity for splenectomy. Importantly, these remissions have been sustained for over 2 years, suggesting some more fundamental therapeutic effect than simple antibody removal. Unfortunately, this study was not randomized or controlled, but a prospective study, perhaps comparing protein A therapy with splenectomy, would appear warranted. Christie and Howe4 reported on ten patients with thrombocytopenia secondary to bone marrow failure who were refractory to platelet transfusion, most of whom had been alloimmunized. The study was non-randomized and the underlying diagnoses were polyglot. Platelet-directed antibodies were demonstrated in 8/10. All but one were refractory to platelet transfusion with random-donor, single-donor and HLAmatched platelets. Following treatment with protein A there was improved responsiveness to platelet transfusion, as measured by platelet count, posttransfusion corrected count increments requirements. (CCI), ‘and transfusion Titers of PDIg were reduced following treatment. Studies have now been completed on 12 such patients with bone marrow failure who have become refractory to platelet transfusion. Seven are male and
five female; ages range from 12 to 70 years. Eleven of the 12 had failed previous treatment with steroids (11 and intravenous IgG (6 patients), patients). Anti-platelet antibodies were demonstrated in ten of 12. All ten had anti-HLA and three of these had additional antibody specificities. Platelet counts, response to platelet transfusions (post-tranfusion CCI), number of platelet units used, concentration of circulating antibodies, clinical bleeding, and side effects were monitored. Pre-treatment counts averaged 14,000 f 8000 in the responders and 9000 f 8000 in the non-responders. Responders averaged 60,000 + 20,000; non-responders 11,000 + 9000 following treatment. CC1 improved from 1782 f 8985 to 11,825 f 3923 in five responders but did not change (860 f 1106 to 1320 + 2124) in the nonresponders. CC1 could not be calculated in two of the responders because they were receiving platelets by continuous infusion. They did, however, demonstrate decreased platelet utilization. There was less bleeding in all responders. Antibodies, when present, decreased in titer. A prospective randomized trial in patients with a more homogeneous underlying diagnosis will be necessary to substantiate these encouraging results. Despite these encouraging reports and the fact that Staphylococcal protein A has been approved for clinical use in ITP, it has yet to find widespread application. This may be due, in part, to lack of well-controlled clinical trials, but the methodology also suffers from lack of insight into mechanisms of action.’ Some understanding derives from its laboratory use as an immunoadsorbent.” Protein A most efficiently binds IgG classes 1, 2 and 4, but has some affinity for IgG3 and IgM and binds some immune complexes with avidity.” Each protein A molecule can potentially bind three molecules of IgG, but steric hindrance limits that binding to a two to one proportion (Fig. 1). Protein A is too toxic to be administered intravenously, but when chemic-
Clinical Applications of Protein A Immunoadsorption
r
\
Figure 1. Each protein A molecule has three potential binding sites which can be OCCUpied by IgG molecules. However, as shown schematically, steric considerations allow for occupancy of only two at a time.
ally bound to media of silica beads, sepharose, or polyacrylamide, provides a system to which blood or plasma can be exposed safely. When blood or plasma so treated is returned to patients, side effects are usually limited to mild hypotension, dyspnea, pruritus, fever and urticaria. However, occasionally lifethreatening reactions have been reported.‘r9 The mechanisms for this is poorly understood, but may involve kinins and other vasoactive substances, since patients on angiotensin converting enzyme (ACE) inhibitors appear to be
425
more vulnerable.‘O Our patients have experienced none of these serious complications. We speculate that this may be due to the fact that our patients are severely neutropenic. Patients with normal neutrophil counts may be more susceptible to the reaction commonly observed in hemodialysis patients and attributed to complement activation and neutrophil aggregation.” Indeed, other investigators in our laboratory have demonstrated elaboration of activated C5a by exposure of plasma to protein A, employing both neutrophil aggregation and immunoassay methods. Other difficulties in understanding the mechanism of action derive from discrepancies in protein A column capacity and observed amounts of protein removed. Commercial protein A columns contain approx. 200 mg of protein A with a theoretical capacity of 1.5 g of IgG. Approximately 1 g of IgG can be eluted from a column following use. Yet treatment results in an approx. 12% diminution in circulating IgG concentration rather than the predicted 23%. One might speculate that the column modifies the IgG resulting in more efficient removal (Fig. 2). Perhaps the
immune complexes
Patient q
Mononuclear Phagocyte System
Figure 2. Although each protein A molecule bound to silica beads can bind a maximum of two IgG molecules, it is possible that bound IgG molecules might complex with one another. The resultant immune complexes might detach from the protein A, re-enter the circulation, and be cleared by the reticuloendothelial system. This might explain, in part, the observed removal of quantities of IgG greater than the column’s capacity.
426
Transfus.
Sci. Vol. 15, No. 4
anti-anti-plt Ab
suppresses anti-plt Ab
Figure 3. Although protein A is covalently bound to silica beads on the column, in the presence of plasma proteases tiny amounts of protein A with its attached IgG might elute from the column and re-enter the circulation. There the anti-platelet antibodies could be recognized by anti-Id receptors on B cells. Since protein A is mitogenic, this could lead to clonal expansion of a population of B cells programmed to make anti-Id. This could explain the long-term reduction of anti-platelet antibody.
column induces the formation of immune complexes which recirculate and are subsequently cleared by the reticuloendothelial system. Acute removal of antibody cannot explain the prolonged clinical remissions which have been observed3 since extravascular IgG re-equilibrates, new antibody is synthesized, and relatively little antibody is removed by each treatment. Snyder et ~1.‘~ have speculated that antibodies (anti-Id) are induced following protein A therapy. Since protein A is a potent B-cell mitogen13 it is conceivable that tiny amounts of protein A with its attached IgG recirculate, are identified by B cells with anti-Id and that the latter undergo clonal expansion, stimulated by the protein A (Fig. 3). The resultant clone of B cells could then continue to synthesize anti-Id antibody. Unless further laboratory studies elucidate these mechanisms of action protein A therapy will unlikely gain Likewise, wellwide acceptance. designed clinical trials in multiple centers will be necessary to convince clinicians of efficacy.
REFERENCES 1. Bertram JH, Snyder HW Jr, Gill PS, Shulman I, Henry DH, Jenkins D, Kiprov
2.
3.
4.
5.
DD: Protein A immunoadsorption therapy in HIV-related immune thrombocytopenia: A preliminary report. Artif Organs 1988; 12~484-490. Snyder HW Jr, Bertram JH, Channel M, Ernst NR, Balint JP, Jones FR: Reduction in platelet-binding immunoglobulins and improvement in platelet counts in patients with HIV-associated idiopathic thrombocytopenia purpura (ITP) following extracorporeal immunoadsorption of plasma over staphylococcal protein A-silica, Artif Organs 19893 13:71-77. Snyder HW Jr, Cochran SK, Balint JP Jr, Bertram JH, Mittelman A, Guthrie TH Jr, Jones FR: Experience with protein A-immunoadsorption in treatmentresistant adult immune thrombocytopenic purpura. Blood 1992; 79:22372245. Christie DJ, Howe RB, Lennon SS, Sauro SC: Treatment of refractoriness to platelet transfusion by protein A column therapy. Transfusion 1993; 33:234-242. Lesesne JB, Rothschild N, Erickson B, Korec S, Sisk R, Keller J, Arbus M, WOOL ley PV, Chiazze L, Schein PS, Neefe JR: hemolytic-uremic Cancer-associated
Clinical Applications of Protein A Immunoadsorption
syndrome: Analysis of 85 cases from a national registry. / Clin Oncol 1989;
7:781-789. Gaddis T, Guthrie
T, Mittelman A, A immunoadsorption (PAI) in classical thrombotic thrombocytopenia purpura (TTP) refractory to plasma exchange: Report of eleven patients. Blood 1992; 80:63a (abstr.). Jones FR, Balint JP Jr, Snyder HW Jr: Selective extracorporeal removal of immunoglobulin-G and circulating immune complexes: A review. Plasma Ther Transfus Technol1986; 71333349. Goding JW: Use of staphylococcal protein A as an immunological reagent. 1 Immunol Meth 1978; 20:241-253. Smith RE, Gottschall JL, Pisciotta AV: Life-threatening reaction to staphylococcal protein-A immunomodulation. / Howe
R, Sahud M: Protein
427
Clin Apheresis 1992; 7~4-5. 10, Manufacturer’s package insert, Prosorba, Imre Corporation, Seattle, WA, U.S.A. 11. Jacob HS, Craddock PR, Hammerschmidt DE, Moldow CF: Complement-induced granulocyte aggregation: an unsuspected mechanism of disease. New Engl I Med 1980; 302: 789-794. 12. Snyder HW Jr, Balint JP Jr, Jones FR: Modulation of immunity and clinical responses in patients with cancer and autoimmune disease treated by extracorporeal immunoadsorption of plasma with protein A-silica (PROSORBA columns). Semin Hematol 1989; 26:3141. 13. Forsgren A, Svedjelund A, Wigzell H: Lymphocyte stimulation by protein A of Staphylococcus aureus. Eur J Immunol 1976; 6:207-213.
0955-3886(94)00045-x
7iansfus. Sci. Vo1.15, No.4, pp. 423-427, 1994 Copyri t @ 1994 Elsevier Science Ltd Printed in b,$;y~;~r~~~$h;h~g~
Clinical Applications of Protein A Immunoadsorption in Thrombocytopenic Disorders Robert B. Howe, MD* $ Douglas J. Christie, PhDt
Recent reports of a favorable clinical response to immunoadsorption therapy with Staphylococcal protein in AIDSassociated immune thrombocytopenia,‘,’ autoimmune thrombocytopenic purpura,3 alloimmune thrombocytopenia,4 the hemolytic-uremic syndrome’ and thrombotic thrombocytopenic purpura6 have rekindled interest in the application of this therapy and in its mechanism(s) of action. Most reports have only appeared in abstract form and few have addressed the utility of protein A therapy in a systematic manner. Bertram et a1.l reported a favorable response in six of nine patients with HIV-related immune thrombocytopenia. The mean increase in pIateIet count was 95,000/mm3; four individuals achieved normal platelet counts. At the time of their report these responses had been sustained for 5 months. No significant changes in total serum IgG levels or platelet-associated IgG (PAIg) were noted; however, circulating immune complexes (CIC) were reduced. They treated a relatively small volume (250 mL) of plasma using the off-line technique. More recently’ these investigators reported a 55% (16129) response rate to protein A treatment of HIVassociated thrombocytopenia. Twelve patients sustained remissions of over 8 ‘Professor of Medicine and tAssociate Professor of Medical Technology, Box 480, University of Minnesota Health Ckk;, 420 Delaware Street SE, Minneapolis, MN 55455,
*Author for correspondence.
months. Interestingly, they could not account for the diminution in plasma levels of CIC, platelet-directed IgG (PDIg), or PAIg by the amount of plasma adsorbed per treatment. Anti-F(ab’J, levels increased over the 3 months following therapy. Preliminary reports have suggested that a similar benefit might be conferred thrombotic with patients upon thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) in cancer patients5 and in patients with the spontaneous l-TP-HUS6 Twenty-one patients with cancer-associated, mitomycin C-induced HUS were reported by Lesesne et al. in 1989.5 Ten of the 21 patients demonstrated resolution of thrombocytopenia, improvement in anemia and stabilization of renal function, but since criteria defining these responses and specific data were not given, the claims are difficult to evaluate. The authors also failed to provide specific treatment regimens, such as frequency and total number of treatments and volume of blood treated, and whether the blood was treated “on” or “off-line”. No data on immunoglobulin or CIC changes were given. Gaddis et al.” reported in abstract form on ten females and one male aged 17 to 62 with classical TTP. All had failed plasmapheresis and other therapies including glucocorticoids, vincristine, anti-platelet drugs and splenectomy. Eight of the patients had complete resolution of their TTP. Only two
424
Transjus. Sci.
Vol. 15, No. 4
patients required more than six treatments; however, both had a complete response. We must await full publication and confirmatory studies in order to evaluate the utility of protein A immunoadsorption in this syndrome. Snyder et aL3 accumulated 72 cases of patients with refractory autoimmune thrombocytopenic purpura (ITP) who had failed at least two standard forms of therapy. Many had not been splenectomized. Following an average of six immunoadsorption treatments 46% had a complete or partial remission. Resultant platelet counts were normal (complete remission) or at levels not associated with risk of bleeding (partial remission). Spleen intact patients had an equal chance of response as splenectomized subjects, suggesting that this therapy may obviate the necessity for splenectomy. Importantly, these remissions have been sustained for over 2 years, suggesting some more fundamental therapeutic effect than simple antibody removal. Unfortunately, this study was not randomized or controlled, but a prospective study, perhaps comparing protein A therapy with splenectomy, would appear warranted. Christie and Howe4 reported on ten patients with thrombocytopenia secondary to bone marrow failure who were refractory to platelet transfusion, most of whom had been alloimmunized. The study was non-randomized and the underlying diagnoses were polyglot. Platelet-directed antibodies were demonstrated in 8/10. All but one were refractory to platelet transfusion with random-donor, single-donor and HLAmatched platelets. Following treatment with protein A there was improved responsiveness to platelet transfusion, as measured by platelet count, posttransfusion corrected count increments requirements. (CCI), ‘and transfusion Titers of PDIg were reduced following treatment. Studies have now been completed on 12 such patients with bone marrow failure who have become refractory to platelet transfusion. Seven are male and
five female; ages range from 12 to 70 years. Eleven of the 12 had failed previous treatment with steroids (11 and intravenous IgG (6 patients), patients). Anti-platelet antibodies were demonstrated in ten of 12. All ten had anti-HLA and three of these had additional antibody specificities. Platelet counts, response to platelet transfusions (post-tranfusion CCI), number of platelet units used, concentration of circulating antibodies, clinical bleeding, and side effects were monitored. Pre-treatment counts averaged 14,000 f 8000 in the responders and 9000 f 8000 in the non-responders. Responders averaged 60,000 + 20,000; non-responders 11,000 + 9000 following treatment. CC1 improved from 1782 f 8985 to 11,825 f 3923 in five responders but did not change (860 f 1106 to 1320 + 2124) in the nonresponders. CC1 could not be calculated in two of the responders because they were receiving platelets by continuous infusion. They did, however, demonstrate decreased platelet utilization. There was less bleeding in all responders. Antibodies, when present, decreased in titer. A prospective randomized trial in patients with a more homogeneous underlying diagnosis will be necessary to substantiate these encouraging results. Despite these encouraging reports and the fact that Staphylococcal protein A has been approved for clinical use in ITP, it has yet to find widespread application. This may be due, in part, to lack of well-controlled clinical trials, but the methodology also suffers from lack of insight into mechanisms of action.’ Some understanding derives from its laboratory use as an immunoadsorbent.” Protein A most efficiently binds IgG classes 1, 2 and 4, but has some affinity for IgG3 and IgM and binds some immune complexes with avidity.” Each protein A molecule can potentially bind three molecules of IgG, but steric hindrance limits that binding to a two to one proportion (Fig. 1). Protein A is too toxic to be administered intravenously, but when chemic-
Clinical Applications of Protein A Immunoadsorption
r
\
Figure 1. Each protein A molecule has three potential binding sites which can be OCCUpied by IgG molecules. However, as shown schematically, steric considerations allow for occupancy of only two at a time.
ally bound to media of silica beads, sepharose, or polyacrylamide, provides a system to which blood or plasma can be exposed safely. When blood or plasma so treated is returned to patients, side effects are usually limited to mild hypotension, dyspnea, pruritus, fever and urticaria. However, occasionally lifethreatening reactions have been reported.‘r9 The mechanisms for this is poorly understood, but may involve kinins and other vasoactive substances, since patients on angiotensin converting enzyme (ACE) inhibitors appear to be
425
more vulnerable.‘O Our patients have experienced none of these serious complications. We speculate that this may be due to the fact that our patients are severely neutropenic. Patients with normal neutrophil counts may be more susceptible to the reaction commonly observed in hemodialysis patients and attributed to complement activation and neutrophil aggregation.” Indeed, other investigators in our laboratory have demonstrated elaboration of activated C5a by exposure of plasma to protein A, employing both neutrophil aggregation and immunoassay methods. Other difficulties in understanding the mechanism of action derive from discrepancies in protein A column capacity and observed amounts of protein removed. Commercial protein A columns contain approx. 200 mg of protein A with a theoretical capacity of 1.5 g of IgG. Approximately 1 g of IgG can be eluted from a column following use. Yet treatment results in an approx. 12% diminution in circulating IgG concentration rather than the predicted 23%. One might speculate that the column modifies the IgG resulting in more efficient removal (Fig. 2). Perhaps the
immune complexes
Patient q
Mononuclear Phagocyte System
Figure 2. Although each protein A molecule bound to silica beads can bind a maximum of two IgG molecules, it is possible that bound IgG molecules might complex with one another. The resultant immune complexes might detach from the protein A, re-enter the circulation, and be cleared by the reticuloendothelial system. This might explain, in part, the observed removal of quantities of IgG greater than the column’s capacity.
426
Transfus.
Sci. Vol. 15, No. 4
anti-anti-plt Ab
suppresses anti-plt Ab
Figure 3. Although protein A is covalently bound to silica beads on the column, in the presence of plasma proteases tiny amounts of protein A with its attached IgG might elute from the column and re-enter the circulation. There the anti-platelet antibodies could be recognized by anti-Id receptors on B cells. Since protein A is mitogenic, this could lead to clonal expansion of a population of B cells programmed to make anti-Id. This could explain the long-term reduction of anti-platelet antibody.
column induces the formation of immune complexes which recirculate and are subsequently cleared by the reticuloendothelial system. Acute removal of antibody cannot explain the prolonged clinical remissions which have been observed3 since extravascular IgG re-equilibrates, new antibody is synthesized, and relatively little antibody is removed by each treatment. Snyder et ~1.‘~ have speculated that antibodies (anti-Id) are induced following protein A therapy. Since protein A is a potent B-cell mitogen13 it is conceivable that tiny amounts of protein A with its attached IgG recirculate, are identified by B cells with anti-Id and that the latter undergo clonal expansion, stimulated by the protein A (Fig. 3). The resultant clone of B cells could then continue to synthesize anti-Id antibody. Unless further laboratory studies elucidate these mechanisms of action protein A therapy will unlikely gain Likewise, wellwide acceptance. designed clinical trials in multiple centers will be necessary to convince clinicians of efficacy.
REFERENCES 1. Bertram JH, Snyder HW Jr, Gill PS, Shulman I, Henry DH, Jenkins D, Kiprov
2.
3.
4.
5.
DD: Protein A immunoadsorption therapy in HIV-related immune thrombocytopenia: A preliminary report. Artif Organs 1988; 12~484-490. Snyder HW Jr, Bertram JH, Channel M, Ernst NR, Balint JP, Jones FR: Reduction in platelet-binding immunoglobulins and improvement in platelet counts in patients with HIV-associated idiopathic thrombocytopenia purpura (ITP) following extracorporeal immunoadsorption of plasma over staphylococcal protein A-silica, Artif Organs 19893 13:71-77. Snyder HW Jr, Cochran SK, Balint JP Jr, Bertram JH, Mittelman A, Guthrie TH Jr, Jones FR: Experience with protein A-immunoadsorption in treatmentresistant adult immune thrombocytopenic purpura. Blood 1992; 79:22372245. Christie DJ, Howe RB, Lennon SS, Sauro SC: Treatment of refractoriness to platelet transfusion by protein A column therapy. Transfusion 1993; 33:234-242. Lesesne JB, Rothschild N, Erickson B, Korec S, Sisk R, Keller J, Arbus M, WOOL ley PV, Chiazze L, Schein PS, Neefe JR: hemolytic-uremic Cancer-associated
Clinical Applications of Protein A Immunoadsorption
syndrome: Analysis of 85 cases from a national registry. / Clin Oncol 1989;
7:781-789. Gaddis T, Guthrie
T, Mittelman A, A immunoadsorption (PAI) in classical thrombotic thrombocytopenia purpura (TTP) refractory to plasma exchange: Report of eleven patients. Blood 1992; 80:63a (abstr.). Jones FR, Balint JP Jr, Snyder HW Jr: Selective extracorporeal removal of immunoglobulin-G and circulating immune complexes: A review. Plasma Ther Transfus Technol1986; 71333349. Goding JW: Use of staphylococcal protein A as an immunological reagent. 1 Immunol Meth 1978; 20:241-253. Smith RE, Gottschall JL, Pisciotta AV: Life-threatening reaction to staphylococcal protein-A immunomodulation. / Howe
R, Sahud M: Protein
427
Clin Apheresis 1992; 7~4-5. 10, Manufacturer’s package insert, Prosorba, Imre Corporation, Seattle, WA, U.S.A. 11. Jacob HS, Craddock PR, Hammerschmidt DE, Moldow CF: Complement-induced granulocyte aggregation: an unsuspected mechanism of disease. New Engl I Med 1980; 302: 789-794. 12. Snyder HW Jr, Balint JP Jr, Jones FR: Modulation of immunity and clinical responses in patients with cancer and autoimmune disease treated by extracorporeal immunoadsorption of plasma with protein A-silica (PROSORBA columns). Semin Hematol 1989; 26:3141. 13. Forsgren A, Svedjelund A, Wigzell H: Lymphocyte stimulation by protein A of Staphylococcus aureus. Eur J Immunol 1976; 6:207-213.