E. V. Cowdry

STUDIES ON THE ETIOLOGY OF HEARTWATER I. OBSERVATION OF A RICKETTSIA, RICKETTSIA RUMINANTIUM (N. SP.), IN THE TISSUES OF INFECTED ANIMALS.

A Gram-negative, intracellular, coccus-like microorganism was found in cases of heartwater in the three species which are susceptible to the disease; namely, goats, sheep, and cattle. It was absent in the case of control animals, both normal ones and those dying of some. other diseases. The presence of this microorganism was definitely related to the febrile reaction. It was most easily detected in the renal glomeruli and in the small capillaries of the cerebral cortex but probably occurred throughout the body. The microorganism was a typical endothelial parasite, being restricted in distribution to the endothelial cells of the smaller blood vessels and to portions of such elements which had broken off into the blood stream. It was never observed to cause injury to the cells other than those incident to mechanical distention through accumulation within them of many individuals in large densely packed masses which were characteristically spherical. A typical attribute was the presence of several of these masses within the cytoplasm of a single endothelial cell. In view of the association of this microorganism with heartwater, a disease of ruminants, and thus far the only one in which microorganisms resembling Rickettsiœ have been reported, the designation Rickettsia ruminantium is proposed.

Studies on East Coast Fever. II. Behaviour of the Parasite and the Development of distinctive Lesions in susceptible Animals.

The history of the ticks ( Rhipicephalus appendiculatus ) employed for transmission of East Coast fever is detailed in Part I. During the 1 st day the ticks generally become attached and are partly held in place by a kind of cement substance (Fig. 1). Penetration usually occurs at some time during the 2 nd day , and the cellular elements of the corium quickly respond. There is a local swelling of the capillary endothelium, an infiltration with polymorphonuclear leucocytes and a slight increase in eosinophiles. More cement substance is produced which hardens about the edges. Some of it enters through the wound and spreads out in the corium along lines of least resistance (Fig. 2). Later on the eosinophiles increase greatly in number and in turn give place to macrophages which ingest some of the cement substance. Mast cells also become more numerous. The area of reaction extends 2 or 3 mm. from the tip of the proboscis. Marked cytolysis of fibroblasts, of histiocytes and of the swollen endothelial cells occurs. The latter leads to haemorrhage (Figs. 6, 8, 9). The red blood cells and leucocytes are, however, not destroyed. They are sucked up by the tick, the leucocytes first because they accumulate before the haemorrhages take place. Twenty-five of the sixty-six ticks examined were found to contain parasites in their salivary glands, but no parasites were seen in the sixty-six pieces of skin excised while the ticks were attached. Soon after the parasites enter the blood stream and spread throughout the system the corium of the skin is invaded by them like other parts of the body (Table III). The medium-sized lymphocytes are the principal cells parasitised; but parasites are also found in lymphoblasts, large and small lymphocytes and occasionally in parenchymatous liver cells. We never saw them in vascular endotheial cells. For this reason we are unable to substantiate the view, which has apparently never been seriously questioned, that they undergo schizogony in this situation. The first agamonts were noted in lymphocytes in gland smears the day the fever first exceeded 103° F. The gamonts appeared about a day later and the intra-erythrocytic forms soon after. Taking the average of a number of cases: (1) The temperature first surpassed 103° F. on the 9·8th day after infestation with ticks. (2) The first Koch bodies within lymphocytes were observed on the 14·8th day, that is to say, 5 days after the fever commenced. (3) The first parasites within the red cells were noted 1·6 days later, on the 16·4th day after infestation. The cycle which the parasite undergoes in changing from the agamonts to the intra-erythrocytic forms is illustrated in Diagram II (fully described on p. 36). The alterations in the tissues caused directly or indirectly by the parasitic invasion were studied principally in specimens secured at autopsy from animals killed in extremis . The changes which apparently dominate relate to the lymphatic system. The medium-sized lymphocytes and to a less extent the small and large lymphocytes and lymphoblasts, first in the lymph glands and later in all parts of the body, become infested with Koch bodies which grow in their interior apparently without causing any severe injury. The distribu tion of Koch bodies is recorded in Table III, and their incidence in the lymph glands and in the liver in Tables IV and V. The evidence presented does not support the current conception that the parasites serve as a powerful stimulus to the multiplication of lymphocytes. The enlargement of lymph glands is caused more by oedema than by increase in their number. But there is a marked redistribution of lymphocytes. Their number is reduced in the lymph glands and in the peripheral blood stream and increased in the blood vessels of the liver and some other organs. Perivascular lymphocytic infiltrations are also a notable feature of the reaction in the liver, kidneys, adrenals and several other organs, but they are of rare occurrence in the nervous system. It is uncertain how far they are formed by the emigration of lymphocytes from the blood stream and by the proliferation of lymphocytes previously present perivascularly. Small haemorrhages constitute a second alteration of importance. They are particularly prone to occur in the epicardium and endocardium, but may be found in almost any part of the body. Evidence is lacking that these haemorrhages play a part in the genesis of the perivasoular lymphocytic infiltrations. The latter are far more widespread and are chiefly observed in locations where haemorrhages are seldom seen. Oedema is a third type of reaction. It is constantly found in the lymph glands and occasionally in the lungs, especially in the later stages of the disease. Generally speaking, therefore, the lesions reported in this paper are acute in type. The functional activity of the several organs (except the lymph glands) has not been impaired sufficiently severely or over a time long enough to leave distinct structural changes in the parenchymatous organs; but the specimens examined were not collected from protracted cases of East Coast fever or from animals under suspicion of having undergone repeated attacks of the disease. It is the parasites in the red blood cells which are capable of continuing the cycle if they are ingested by ticks able to act as vectors. We have recog nised three principal groups: (1) small spherical-ovoid parasites which are the first to make their appearance, (2) tailed parasites, and (3) plunip forms, both of which develop later and become more numerous than the spherical-ovoid parasites. Possible stages of transition are represented in Diagram I (p. 37). Though many parasites undergo differentiation and some probably divide, a variable number degenerate within the erythrocytes. We question the validity of the genera into which the family Theileriidae are customarily divided on account of the inadequacy of the data on which they are based.

Serum lactic dehydrogenase activity as indication of neoplastic growth and regression.

Summary Lactic dehydrogenase activity of the sera of Swiss and Leaden mice increases promptly after tumor transplantation and decreases with tumor regression.

Malignant properties of cancer cells.

This is an elementary introduction to cancer cells, given as a kind of preface to a monograph by experts on the subject. What I have to say is really a brief summary of my book, Cancer Cells (1955). To distinguish between the important and the less important malignant properties of cancer cells and to present them in the space a t my disposal is difficult. Fortunately, this whole monograph, Cancer Cytology and Cytoclaemistry, is a product of investigators who for years have focused their attention on cancer cells from many angles. By cancer cells, we understand the cells that characterize malignant tumors irrespective of the nature of their cells of origin. In other words, cancer cells include both carcinomas and sarcomas. By malignant (L. malignus, wicked), we mean tumors that grow a t the expense of the body and, unless destroyed, prove fatal. There is much difference of opinion about them. By some workers, ability to spread and produce secondary tumors in other parts of the body is regarded as a criterion of malignancy, but tumors of the central nervous system considered malignant rarely metastasize in this fashion. Such tumors cause death chiefly by the occupation of space needed by nerve cells. Other workers identify the essential feature of malignancy as autonomous cellular multiplication uncontrolled by the usual regulatory mechanisms. These cells, nevertheless, are subject to some control, because the rates of tumor growth sometimes vary between dormancy and rapid increase in size. Having started on its antisocial career, the strain of cells continues to display malignant properties that have been ingrained in its constitution. Assertions are made that, if our knowledge of cell division and resulting growth were only sufficient, we should quickly discover how to combat malignant growth. Consequently, the Committee of the National Research Council that makes grants for cancer research and is financed by the American Cancer Society is called the Growth Committee. Steiner (1954) says: “The attractive statement that the problem of cancer is primarily a problem in growth is both partly true and partly untrue, but it is incomplete and oversimplified.” I t is safe to say that, owing to loss of specialized structures, or of failure to develop them, cancer cells resemble each other more closely than the normal cells from which they arc derived. Before we can evaluate declifferentiation, however, it is necessary to have fairly c!ear ideas about what is understood by the term “differentiation.” In his classic monograph on cellular differentiation, Conklin (1924) defined “differentiation” in the following words: “A11 differentiation is transformation from a more general and homogeneous to a more special and heterogeneous condition. Dedifferentiation, on the Tumor cells often invade neighboring tissues.

Historical background of research on mitochondria.

All agree that the turning point iii the development of our knowledge of mitochondria was the discovery reported by Bensley and Hoerr in 1934, that mitochondi’ia, by washing and by centrifugation, (‘all I)e separated from other cytoplasmic components and collected in adequate amounts for chemical analysis. It is my privilege briefly to sketch what we learned about mitochondria before the utilization of this technique as a background for the presentation of the most recent discoveries by others in this symposium. When I first came under the influence of Dr. Bensley at the University of Chicago about 43 years ago, he was already a leader in the investigation of mitochondria. I have 1)een his devoted follower ever since. He often referred us to the historical classic on mitochondria, a beautifully illustrated book by Altmann, published first in 1890. Altmann was well trained in physics and chemistry. His long series of experiments were carefully planned and his results were recorded with meticulous accuracy. It has been the lot of several investigators to report findings only to discover that they had repeated, rather less accurately, Altmann’s work. But he became impressed with the morphological resemblance between mitochondi’ia and h)acteria and called them “l)iol)lasts” (flL6s, life; f3Xa rbs, germ) to indicate his belief that they were elementary vital units existilIg in all cells. This view was received with scant tolerance by others in Germany. At that time the late Prof. F. P. Mall was also working in Leipzig. He described Altmann and his mental state in a graphic way. The idea of bioi)lasts dominated Altmann’s whole being. He would not brook criticism, used to slink into the laboratory by the l)ack door and was called “der Geist”, the ghost. Things went from bad to worse and the end was tragic and of the sort expected. But Bensley was not led astray by the hioblast hypothesis. He fully appreciated the value of Altmann’s objective findings for they revealed the existence of granules, rods and filaments that did resemble bacteria superficially in many different kinds of celis-in other words the presence of fundamental and intriguing cytoplasmic components. Another example of Bensley’s vision i-elates to the description by Michaehs (1899) and Laguesse (1900) of similar structures stained with ,Janus green ill fresh living cells. While others paid no attention to these observations, Bensley studied the paper of Michaelis carefully and noted that the kind of .Janus green advised by Michaelis was the diethyl compound. This he obtained and with it introduced the best technique for the examination of mitochondria in still living cells. Knowing exactly what to look for, he had no difficulty in seeing them in

Combined action of cigarette tar and beta radiation on mice

Periodic beta irradiation from Sr/sup 90/ to distributed areas of the skin of mice in total doses of as much as 20,800 rep when applied concurrently with cigarette tar produces an additive effect in the production of epidermal carcinogenesis. There is no svidence of a synergistic effect between the two carcinogens. Radiation alone has little carcinogenic effect on the skin as applied under the conditions described. Groups with greater percentuges of malignant skin tumors have had shorter lives than those with smaller percentuges or without skin tumors. The percentage of tumors of the lung, leukemia, mamary- gland carcinoma, and hepatoma was greater in groups in which the percentage of malignant tumors of the skin was low. (auth)

The Value of the Study of Mitochondria in Cellular Pathology

THE problem is a wide one, because it has been shown conclusively in the past few years that initoCllondria re almost though not quite coextensive in distribution with living protoplasm. They have attracted the attention of botanists, zoologists, anatomists, physiologists, pathologists and clinicians, who have all studied them from varied points of view. A conservative estimate would place the bibliography of the last decade at about 600 papers, scattered widely in the journals of all countries.2 Any account of the results obtained and the outlook for further investigation is bound to be somewhat narrow and one-sided. A botanist might emphasize the mitochondrial origin of plastids, base thereon the thesis that without mitochondria it is questionable whether we could have any green plants, and go on to philosophise about all life being dependent upon mitochondria. A zoologist would, in all likelihood, devote considerable time to a discussion of the behavior of mitochondria in the developing egg and sperm, their role in fertilization and histogenesis, and the possibility that they may be concerned in the hereditary transmission of certain characteristics.

Nuclear Inclusions Suggestive of Virus Action in Salivary Glands of the Monkey, Cebus fatuellus L.∗:

Evidence is fast accumulating that we must recognize a special group of salivary gland viruses. All of them have been discovered by chance. They are so benign that attention was not directed to them by distinctive clinical symptoms. What attracted notice was the extraordinary hypertrophy of certain acinous, or duct, cells accompanied by the formation in their nuclei of inclusions resembling those caused by viruses. The first inclusion-laden cells were reported under the heading of “protozoan-like bodies” in the parotids of 2 infants by Ribbert 1 and in the submaxillary glands of guinea pigs by Jackson. 2 Credit is due to Goodpasture and Talbot 3 for recognizing the close resemblance between the bodies in humans and guinea pigs and for pointing out the similarity of both to the intranuclear inclusions described by Tyzzer 4 in varicella. Lipschütz 5 then rediscovered the intranuclear inclusions in herpes admirably described and illustrated by Kopytowski, 6 and emphasized the great importance of these bodies in “inclusion diseases” in general. But Kuttner and Cole 7 and Kuttner 8 led in the demonstration that the inclusions in guinea pigs are actually caused by a virus. Investigators, while examining the salivary glands of other animals, have been on the lookout for nuclear inclusions with the result that they have been reported in rats, 9 moles, 10 , 11 mice 12 and hamsters. 13 Finally Kuttner and Wang 13 have proved that the intranuclear inclusions in hamsters, mice and wild rats are caused by a virus which is very similar to the submaxillary gland virus of guinea pigs.

Physical and Chemical Properties of Rat Leprosy Bacilli.

It is known that percentages of serum Ca and P are not noticeably altered in human leprosy except in unusual cases or during the lepra reaction. In order to discover the changes, if any, in the lesions themselves a histospectrographic investigation was made of skin lesions obtained from Dr. O. E. Denney at the U. S. Marine Hospital, Carville, La. It was found 1 that the P/Ca ratios in the 5 cases studied were probably 3 times those in normal skins from the same age group. A fair correlation was obtained for the P/Ca ratios with known duration of the disease and volume of leprous cells in the tissues analyzed spectroscopically. The change from the normal may be conditioned by increase in P, decrease in Ca, but probably by both. Perhaps an increase in P may have been occasioned by the tremendous number of bacilli in the lesions. No notable deviations from normal were noted in the Na/Ca, Mg/CA and Fe/Ca ratios. In the hope of relating these observations on mineral constituents more definitely to cells and groups of cells, the technic of microincineration 2 was applied to sections of human leprous nodules also secured from Carville. We found that the lepra cells showed a finely divided white ash, tending in some places to be slightly bluish and something like that observed in cancer cells. 3 However, we were not successful in differentiating the ash resulting from the bacilli on the one hand and from the cells containing them on the other. Consequently we shifted the attack to sections of rat leprosy nodules with which it was obviously more easy to experiment in our laboratory. The strain of organisms was received from Dr. E. L. Walker, 12/11/34.

Morphology of Bacillus of Rat Leprosy.

Conclusions Injections of acid extract of anterior pituitary of cattle have a stimulating effect on the growth of cartilage and bone of growing guinea pigs. The various layers of the cartilage cells become hypertrophie and hyperplastic, and are subsequently quickly calcined and replaced by bone. The epiphyseal line undergoes, therefor, premature closure under the influence of the extract.