Donald Orlic

The ultrastructure of mouse lymph node venules and the passage of lymphocytes across their walls

Postcapillary venules of mouse lymph nodes are vessels situated between capillaries in the cortex and paracortex and efferent veins in the hilar region. They are characterized by a high endothelium. Their vessel diameters and periendothelial ultrastructure are similar to these same elements in the postcapillary venules of other tissues. The high endothelial cells are variable in shape. Their well-developed organelles are indicative of intense synthetic activity. Distinctive particles, multivesicular bodies with dense matrices, are associated with a prominent Golgi complex. An endothelial cell coat is present at the luminal surface. Many lymphocytes, singly or in groups, are within the venule walls. The majority have traversed the endothelium and lie adjacent to the pericytes, which constitute the major obstacle to lymphocyte emigration from the venules. Most lymphocytes that have not yet passed the endothelial layer lie between two or more endothelial cells. Analysis of serial sections supports the contention that lymphocytes pass between, rather than within endothelial cells.

Protein Absorption by the Intestine of the Fetal Rat in Utero

Horseradish peroxidase (molecular weight, about 40,000) injected into the amniotic sacs in pregnant rats has been identified ultrastructurally, 6 to 18 hours later, within the fetal intestine in the absorptive cells and the underlying vascular endothelium. This indicates that macromolecular protein within amniotic fluid swallowed by the fetus can be absorbed and transported by fetal intestine, and may indicate that physiological compounds can be transported by this enteric route to contribute to fetal development.

Histochemical and radioautographic studies of normal human fetal colon

SummaryTwenty fetal and infant colons ranging from 10 weeks in utero to 20 months postpartum, and 12 adult human colons were examined using histochemical techniuques in conjunction with in vitro radioautography using Na235SO4 as a sulfomucin precursor. Only the sulfated component of mucus in fetal goblet cells was found to differ significantly from adult colonic mucins. In the fetus sulfomucin staining was much weaker than in the adult, and was more intense in the left colon which is the reverse of the adult pattern. Sulformucin was concentrated in the crypts throughout the fetal colon whereas in the adult right colon it predominated in the surface cells. As in the adult, saponification liberated carboxyl groups, possibly belonging to sialic acid, and vicinal hydroxyl groups from fetal mucins suggesting that this procedure hydrolyses an ester linkage between these 2 reactive groups.During the middle trimester of fetal life the colon possesses villi whose constituent cells display alkaline phosphatase in their surface coat. These and other morphological and histochemical similarities to fetal small intestine suggest that the fetal colon may have a limited capacity to absorb materials contained within swallowed amniotic fluid during this period.

ULTRASTRUCTURAL AND AUTORADIOGRAPHIC STUDIES OF ERYTHROPOIETIN-INDUCED RED CELL PRODUCTION*

Many aspects of hematopoiesis remain unclear, such as the chemical and structural nature of events that initiate the process of blood cell maturation. A better understanding of these early events would provide a new basis for explaining how formed elements of the blood emerge to replace aging cells and might clarify one widely held theory, i.e. the derivation of mature elements from “totipotent” stem cells. To this end, several problems of blood cell origin are dealt with in this study. The most important considerations constitute an effort to characterize the initial features of erythropoietin-induced red cell maturation. There is little definitive information on the mode of erythropoietin action. Some investigations point to the late stages of erythroid maturation as those responsive to erythropoietin (Borsook et al., 1962; Gallagher et al., 1963); others to a “primitive” precursor cell as the target element (Alpen & Cranmore, 1959; Erslev, 1964). Similarly, there is considerable disagreement concerning the characteristics of the molecular changes that occur in cells during their response to this factor. Literature can be cited to support the idea that erythropoietin enhances the rate of hemoglobin synthesis (Stohlman et al., 1963), that it leads to a shorter maturation period (Brecher & Stohlman, 1962), or that it initiates the process of erythroid cell differentiation (Kurtides et a!., 1963; Krantz & Goldwasser, 1965). In adult mice splenic red cell production can be altered by changing the environmental or humoral conditions, e.g. lowered oxygen tensions being countered by increased erythropoiesis (Brecher & Stohlman, 1959). Conversely, when the hypoxic stimulus is removed. polycythemic mice show a markedly inhibited erythropoietic activity. In this state virtually no definitive nucleated erythroid elements are seen in the spleens. At this time such spleens are sensitive to exogenous erythropoietin and respond to a single injection by producing a cohort of erythroid elements that undergo a synchronized process of maturation. Histological preparations of splenic tissue reveal the presence of proerythroblasts 24 hr after activation, and erythroblasts at 48 hours. Peak peripheral reticulocytosis occurs between 72-96 hr, after which the erythroid wave subsides (Filmanowicz & Gurney, 196 1 ) . The present study, undertaken to identify the morphological and chemical bases of erythropoietin-induced activity, extends our earlier autoradiographic findings (Orlic et al., 1965a), which for the first time, indicated that the stem cell response to erythropoietin involves an almost immediate or simultaneous incorporation of 3H-thymidine. In addition, this work provides information on the molecular and ultrastructural changes that occur during the early hours of response, as well as later stages when erythropoietin-activated transitional cells are replaced by more mature red cell elements. Identification of erythropoietin-sensitive stem cells is

Molecular Mechanism for the Inhibitory Action of Interferon on Hematopoiesis

Spleen cell suspensions obtained from adult mice were separated by Ficoll/Hypaque and Percoll density gradient centrifugation. The enriched erythroblast populations were maintained in liquid culture medium for 8 hours with 10,000 units of murine interferon (IFN) alpha and beta. Exposure of these cell cultures to murine IFN alpha and beta resulted in a 48% to 70% increase in 2-5adenylate synthetase (2-5AS) activity. In parallel studies, adult mice were injected daily for 1 or 2 weeks with recombinant human IFN alpha A/D (rHuIFN alpha A/D) at a dose of 10(6) or 10(7) units/kg body weight. This treatment did not significantly affect body weight but did produce a mean 70% increase in spleen wet weight and a mean 46% increase in number of nucleated cells per spleen. This increase in number of splenic hematopoietic cells did not result in a corresponding increase in number of circulating cells. In fact, during this 1 to 2 week period the hematocrit dropped from 45% to 38% in mice injected with high dose rHuIFN alpha A/D. From these findings we propose that IFN induces an early 2-5AS activity in erythroblasts and megakaryocytes. This 2-5AS activity, which is known to inhibit protein synthesis in other cell systems, is thought to be responsible for the block or prolongation in blood cell maturation observed in the present studies.

An Electron Microscopic Study of Erythropoiesis in Fetal and Neonatal Rabbits

This investigation records the chronology of events relating to erythropoiesis in the late fetal and early neonatal rabbit. The formation of the marrow cavity in the rabbit femur and the changing cell population in the extravascular spaces are described for the period from day 24 of gestation to 8 weeks postpartum. In the fetal liver, erythropoiesis is shown to occur in typical red cell islands. As hepatic red cell production declines, lipid accumulates in the liver, much as fatty infiltration of marrow follows reduction of erythropoiesis in the marrow. The spleen appears to have erythropoietic potential although it does not normally contribute to red cell production in this animal. The spleen also appears to serve as a storage site for large numbers of platelets in the developing rabbit.

Iron-containing cytoplasmic inclusions in mouse bone marrow macrophages

Elongated, tapered inclusions, present in the cytoplasm of macrophages in mouse bone marrow, were studied by electron microscopy. The bone marrow of adult mice that were injected with the hemolytic agent phenylhydrazine, displayed a statistically significant increase in the number of inclusions compared with bone marrow from control animals. Ultrastructural analysis demonstrated that ferritin, a known product of red cell destruction, was resent in these inclusions. It is suggested that the inclusions are derived from the degradation of phagocytosed red cells.

Fetal Absorption of 3H‐Leucine Injected into the Amniotic Sacs of Pregnant Rats

When injected into the amniotic sacs of fetal rats in late pregnancy, 3H‐leucine is rapidly absorbed and incorporated into fetal proteins as determined by liquid scintillation spectrometry. Peak values in the fetal liver and pancreas occur around four hours after injection. Approximately 21% of the radioisotope is retained in the injected fetus at 24 hours. The remainder escapes into the maternal circulation and accounts for a gradual increase in levels in noninjected fetuses from the same litter and for persistently high counts found in maternal serum as late as 24 hours after the injection. Specific activity of the radioisotope in fetal tissues is considerably higher (100‐ to 200‐fold) when 3H‐leucine is injected into the amniotic sac rather than intravenously into the mother. The possible treatment of various fetal disorders by means of intra‐amniotic injections is discussed.