1128 of peripheral blood. Such bodies began to appear the number of parasites reached a peak and became more common as the parasitaemia fell. We also observed smears

Hypothesis IMMUNITY TO INTRA-ERYTHROCYTIC PROTOZA I. A. CLARK E. J. WILLS Division

E. RICHMOND A. C. ALLISON

JOAN

of Cell Pathology, Clinical Research Centre, Harrow, Middlesex

Intra-erythrocytic inclusion

bodies were in Giemsa-stained smears of peripheral blood from mice infected with Babesia microti. These bodies, which were probably the pyknotic remnants of dead protozoa, increased as parasitæmia fell and the animal recovered. It is suggested that a soluble mediator liberated by cells of the host during infection penetrates the red blood-cell and leads to degeneration of the parasite.

Summary

seen

PROTOZOA of the genus Babesia parasitise the red blood-cells of many species of mammals and are a major economic problem in cattle, particularly in wet tropical areas. Babesias provide convenient models for studying immunity against intra-erythrocytic protozoa, having certain technical advantages over plasmodia. Normal Babesia microti (King’s 67 strain) trophozoites are shown in fig. la. While investigating immunity to this parasite in female CBA mice we observed previously unreported intra-erythrocytic inclusion bodies in Giemsa-stained

as

forms which were apparently transitional between normal parasites and the inclusion bodies (fig. lb). The structure of these bodies and the timing of their appearance were reminiscent of the degenerate "crisis forms" described in 1944 by Taliaferro and Taliaferro’ in their studies of the morphology of Plasmodium brazilianum in monkeys. Because of the limited resolution of light microscopy, blood from mice infected with B. microti was also examined by electron microscopy. During the phase of rapidly increasing parasitaemia, when transitional forms and inclusion bodies were very rare, virtually all of the parasites appeared normal (fig. 2a). However, as the parasite numbers declined after the exponential growth phase a number of abnormal forms were seen (fig. 2b); these were both smaller and denser than normal babesia, and sometimes showed myelin-type degeneration of their cytoplasmic membranes. There was good correlation between the frequency of transitional forms seen by light microscopy and degenerate parasites detected by electron microscopy at the different stages of parasitxmia examined. The numbers of transitional forms and inclusion bodies, which are probably the pykotic remnants of dead organisms, are sufficient to account for the fall in parasitsemia as the animal recovers. Thus we believe that an infection of normal CBA mice with B, microti is terminated by the death of the parasites within erythrocytes. There is no general agreement about the mechanism of immunity to intra-erythrocytic protozoa; agglutina-

tion2 or phagocytosis34 of parasites or parasitised cells, and antibody-mediated inhibition of host cell penetrations have all been proposed. None of the mechanisms

D., Kastin, A. J., Escalente-Herrera, A., Gonzalez-Martinez, A., Coy, D. H., Schally, A. V. ibid. (in the press). Comaru, A. M. M., Rodrigues, J., Povoa, L. C., Frances, S., Dimetz, T., Coy, D. H., Schally, A. V. ibid. (in the press). Nishi, N., Arimura, A., Coy, D. H., Vilchez-Martinez, J. A., Schally, A. V. Proc. Soc. exp. Biol. Med. 1975, 148, 1009. De la Cruz, A., De la Cruz. K., Arimura, A., Coy, D. H., VilchezMartinez, J. A., Coy, E. J., Schally, A. V. Fert. Steril. 1975, 26,

5. Gonzalez-Barcena,

6. 7.

8.

894. 9.

Vilchez-Martinez, J. A., Coy, D. H., Arimura, A., Coy, E. J., Hirotsu, Y., Schally, A. V. Biochem. biophys. Res. Comm. 1974, 59,

1232. 10. Schalch, D.

S., Parlow, A. F., Boon, R. C., Reichlin, S. J. clin. Invest. 1968, 47, 665. 11. Boon, R. C., Schalch, D. S., Lee, L. A., Reichlin, S. Am. J. Obstet. Gynec. 1972, 112, 736. 12. Bermudez, J. A., Kastin, A. J., Schalch, D. S., Lee-Benitez, D. Perez-Pasten, E., Gonzalez-Barcena, D., Arzac, J. P., Schally, A. V. Endocr. Res. Comm. 1974, 1, 477. 13. Kastin, A. J., Arimura, A., Gonzalez-Barcena, D., Coy, D. H., Miller, M. C., Nishi, N., Lee, L., Duron-Huerta, H., Schalch, D. S., Schally, A. V. Int. J. Fert. 1974, 19, 202. 14. Soria, J., Zarate, A., Canales, E. S., Ayala, A., Schally, A. V., Coy, D. H., Coy, E. J., Kastin, A. J. Am.J. Obstet. Gynec. 1975, 123, 195. 15. Kastin, A. J., Schally, A. V., Gual, C., Midgley, A. R., Bowers, C. Y., Diaz-Infante, A. J. clin. Endocr. Metab. 1969, 29, 1046. 16. Kastin, A. J., Schally, A. V., Zarate, A., Arimura, A., GonzalezBarcena, D., Medeiros-Neto, G., Schalch, D. S. Israel J. med. Sci. 1974, 10, 1305. 17. Gonzalez-Barcena, D., Kastin, A. J., Schalch, D. S., Bermudez, J. A., Lee, D., Arimura, A., Ruelas, J., Zepeda, I., Schally, A. V. J. clin. Endocr. Metab. 1973, 37, 481. 18. Arimura, A., Saito, M., Yaoi, Y., Kumasaka, T., Kato, H., Koyama, T., Nishi, N., Kastin, A. J., Schally, A. V. ibid. 1973, 36, 385. 19. Mortimer, C. H., Besser, G. M., Hook, J., McNeilly, A. S. Clin. Endocr. 1974, 3, 19. 20. Arimura, A., Vilchez-Martinez, J. A., Coy, D. H., Coy, E. J., Hirotsu, Y., Schally, A. V. Endocrinology, 1974, 95, 1174.

Fig. 1-(a) Normal B. microti trophozoites in erythrocytes before peak parasitæmia and (b) transitional forms (t) and inclusion bodies (i) after peak parasittemia. Giemsa. Reduced

tofrom

x2800.

Fig. 2 -Electron micrographs of (a) normal B. microti within a red bloodcell before peak parasittemia and (b) condensed degenerate B. microli in red blood-cell after peak parasiteemia. Reduced

to-1 from x 50 000.

1129

postulated would explain the appearance of degenerating parasites in circulating erythrocytes as an animal acquires immunity. Furthermore, there is evidence that antibody cannot enter intact red blood-cells, and it has not been found on the surface of parasites

On the basis of these and later observations, we are in a position to reconsider the pathogenesis of von Willebrand’s disease. Like Macfarlane,2but on considerably more evidence, we wish to propose that the primary defect is vascular, for the following reasons:

within red blood-cells.6 We suggest that certain intracellular parasites are susceptible to a non-antibody soluble mediator liberated by cells of the host animal during the course of infection. This mediator could penetrate the red blood-cell and alter its metabolism in such a way as to inhibit parasite replication, or be directly responsible for the development of the degenerate forms now described. The pronounced cross-immunity between B. microti and certain species of Plasmodiumdespite the absence of cross-reacting fluorescent antibody, implies that comparable mechanisms of immunity operate in the two haemoprotozoa. The presence of "crisis forms" in a plasmodium1 similar to those now described in babesia reinforces this analogy.

(1) There is evidence that normal endothelial cells synthesise and release von Willebrand factor but not factor VIII7 8 and that the factor VIII synthesised contains the same single-polypeptide subunit present in plasma factor-VIII antigen.9 (2) The von Willebrand factor is not synthesised in endothelial cells of patients with the disease. 10 (3) When normal plasma or FRIA (fraction IA after Blomback is injected into patients with variants of von Willebrand’s disease, the bleeding-time is normalised for a short period and so too is factor VIII, somewhat later." However, the factorVIII-related protein, on two-dimensional immunoelectrophoresis, is never in a normal position. (4) When normal plasma or FRIA is injected into patients with quantitative von Willebrand’s disease, a protein which reacts immunologically like normal factor-VIII-related protein is found in plasma, but again in an abnormal, more anodic

so

far

Requests for reprints should be addressed to LA.C., Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA3

Uj. REFERENCES

1 Taliaferro, W. H., Taliaferro, L. G.J. infect. Dis. 1944, 75, 1. 2. Brown, K. N., Brown, I. N. Nature, 1965, 208, 1286. 3. Criswell, B. S., Butler, W. T., Rossen, R. D., Knight, V. J. Immun. 1971,

107, 212. 4. Rogers, R. Int.J. Parasit. 1974, 4, 197. 5. Cohen, S., Butcher, G. A., Crandall, R. B. Nature, 1969, 223, 368. 6. Jerusalem, C., Weiss, M. L., Poels, L.J. Immun. 1971, 107, 260 7. Cox, F. E. Bull. Wld Hlth Org. 1970, 43, 325. 8. Cox, F. E., Turner, S. A. ibid. p. 337.

VON WILLEBRAND DISEASE AS AN

ENDOTHELIAL-CELL ABNORMALITY

J.

P. CAEN

Y. SULTAN

Institut de Recherches

sur

les Maladies du

Sang,

Centre

Hayem, Hôpital Saint-Louis, F-75475 Paris Cedex 10, France

Summary

It is ease

suggested that von Willebrand’s disis primarily a vascular defect,

located mainly in the endothelial cells. IN 1926, von Willebrand’ described an autosomal dominant haemorrhagic disorder which he named "angiohaemophilia" because of an association of prolonged bleeding-time with normal platelet-count and with hsemarthroses. At various times afterwards the primary defect was thought to be in the platelets and even in thevasculature.2 In 1953, the prolonged bleeding-time was found to be associated with a decrease in factor VIII,3and in 1957 Nilsson et al.s showed that the abnormalities could be corrected by transfusions of Cohn fraction I prepared from normal plasma. Since then plasma has been regarded as the site of the primary defect. In 1963, Cornu, Larrieu, and we6 showed that the prolonged bleeding-time and diminished factor VIII could be corrected not only by normal plasma but also by hæmophilic plasma: the effect on factor VIII clotting activity was delayed but it outlasted that on bleedingtime. In consequence, both haemophilia A and von Willebrand’s disease came to be regarded as being due to two different defects in factor VIII.

now

position. 12 Therefore, when normal plasma or fraction IA is injected into a von Willebrand recipient, whether the abnormality in the von Willebrand antigen (or ristocetin cofactor) is quantitative or qualitative,13 it seems that the von Willebrand protein acts first on the endothelial surface of the vessel wall, thereby correcting the bleeding-time temporarily; and that, on the endothelium, the factor is altered into a molecule that is abnormal electrophoretically and functionally.’i i2 We suggest, therefore, that von Willebrand’s disease is due to an abnormality in endothelial cells which prevents them synthesising von Willebrand factor or causes them to synthesise an abnormal factor. If such a relationship between von Willebrand factor and endothelium determines the effectiveness of platelets in securing hæmostasis,13 an explanation is provided for the prolonged bleeding-time in von Willebrand’s disease. The exact relation between such an abnormal factor and factor VIII has still to be determined ; and another unresolved question is whether the factor is present on platelets from patients with von Willebrand’s disease.14 15 Further work with endothelial cells should determine the fate of this hypothesis and may also contribute to understanding of the pathogenesis of arteriosclerosis. 16 17 REFERENCES 1. von Willebrand, E. A. Finska LoäkSänllsk. Hand. 1926, 68, 87. 2. Macfarlane, R. G. Q.Jl. Med 1941,10, 1. 3. Larrieu, M. J., Soulier, J. P. Rev. Heāmat. 1953, 8, 361. 4. Alexander, B., Goldstein, R.J. clin. Invest. 1953, 32, 531. 5. Nilsson, I. M., Blombäck, M., von Francken, R. Acta med. scand.

1957, 159,

35. 6.

Cornu, P., Larrieu, M. J., Caen, J. P., Bernard, J. Br, J. Hémat. 1963, 9,

189. 7. Jaffe, E. A., Hoyer, L. W., Nachman, R. L. J. clin. Invest. 1973, 52, 2757. 8. Sedlak, B. J., Booyse, F. M., Bell, S., Rafelson, M. E. Jr. Unpublished. 9. Jaffe, E. A., Nachman, R. L.J. clin. Invest. 1975, 56, 698. 10. Holmberg, L., Mannucci, P. M., Turesson, I., Ruggen, Z. M., Nilsson, I. M. Scand.J. Hæmat. 1974,13, 33. 11. Sultan, Y., Siméon, J., Caen, J. P.J. clin Path. 1975, 28, 309. 12. Sultan, Y., Siméon, J., Maisonneuve, P., Caen, J. P. Thromb. Diath. hæmorrh. (in the press). 13. Weiss, H. J., Hoyer, L., Rickless, F. R., Varma, A., Rogers, J.J. clin. Invest.

1973, 52, 2708. 14. Bouma, B. N.,

Hordijk-Hos, J. M., De Graaf, S., Sixma, J. J. Nature, 1975, 257, 510. 15. Jeanneau, C., Sultan, Y., Simeon, J., Caen, J. Actualités hémat. 1975, 9, 96. 16. Silwer, J., Cronberg, S., Nilsson, I. M. Acta med. scand. 1966, 180, 475. 17. Bowie, E. J. W., Fuster, V., Owen, C. A. Jr., Brown, A. L. m Abstracts of Vth Congress International Society of Thrombosis and Hæmostasis, Paris, 1975, p. 322.

Immunity to intra-erythrocytic protoza.

1128 of peripheral blood. Such bodies began to appear the number of parasites reached a peak and became more common as the parasitaemia fell. We also...
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