Lymphopoiesis and lymphocyte recirculation in the sheep fetus.

The production and the circulation of lymphocytes has been examined in the sheep fetus where neither foreign antigen nor immunoglobulins occur. It was found that as the lymphoid organs increased in size during fetal life, the numbers and the output of lymphocytes in the thoracic duct lymph increased. The recirculating pool of lymphocytes was estimated to be 5.5 +/- 1.5 X 10(8) cells in fetal lambs 95-100 days of age, 5.7 +/- 1.2 X 10(9) cells in fetuses 130-135 days of age, and 1.2 +/0 9.3 X 10(10) cells in fetuses near to term. The rate of addition of lymphocytes to the recirculating pool was 3.2 +/- 1.9 X 10(6) cells/h in fetuses of 100 days and 3.4 +/- 0.9 X 10(7) cells/h in fetuses of 130 days of age. Lymphocytes recirculated from blood to lymph in fetuses; labeled cells injected into the blood stream reappeared in the thoracic duct lymph promptly and reached maximum levels around 12-18 h after they were injected. Labeled lymphocytes were detected subsequently in greatest numbers in the lymph nodes, particularly in the mesenteric lymph nodes and in the interfollicular areas of the Peyer's patches. Chronic drainage of thoracic duct lymph from fetuses in utero for periods of up to 36 days had no obvious effects on the growth or development of the fetus and only minimal effects on the content of lymphocytes in the various lymphoid tissues even though the number of cells in the blood and lymph were reduced to between 20-30% of normal levels. Thymectomy done in fetuses about 2 mo befor cannulation of the thoracic duct reduced the output of cells in the thoracic duct to about 25% of normal levels and caused a significant reduction in the content of lymphocytes in the various lymphoid tissues. Thymectomized fetal lambs subjected to thoracic duct drainage for periods up to 2 wk in utero had a similar complement of lymphocytes in their lymphoid tissues to intact thymectomized fetal lambs. Lymphocytes obtained from the thoracic duct lymph of lambs thymectomized 2 mo previously recirculated from blood to lymph when they were injected intravenously, although they did this at a significantly slower rate than did lymphocytes from normal lambs.

Relative rates of transcapillary movement of free thyroxine, protein-bound thyroxine, thyroxine-binding proteins, and albumin.

The rate of appearance of labeled thyroxine (T4) and albumin in lymph from various areas after simultaneous i.v. injection of the labeled substances in conscious ambulatory sheep has been used to estimate the relative rates of transcapillary movement of stable T4 and albumin. Labeled T4 appeared in hepatic lymph at the same rate as albumin. In intestinal and leg lymph, labeled T4 appeared eight and four times as rapidly as albumin indicating that T4 crosses capillaries in these areas independently of and much more rapidly than albumin and other proteins having similar distribution kinetics. The lymph:plasma ratios for all the T4-binding proteins including albumin were very similar in any one area showing that the relative fractional rates of transcapillary movement of these proteins were very similar. Therefore in extrahepatic areas, transcapillary movement of T4 in the protein-bound form was quantitatively much less important than in the free form. The findings support earlier views, recently questioned, that free T4 is of considerable physiological significance.

Activation of omental milky spots and milky spot macrophages by intraperitoneal administration of a streptococcal preparation, OK-432.

Omental milky spots are omentum-associated lymphoid tissues that cede peritoneal macrophages and participate in the immunity of the peritoneal cavity. We studied the changing surface features of milky spots and milky spot macrophages of Wistar rats, following the i.p. administration of OK-432, a killed streptococcal preparation (1 Klinische Einheit (unit) in 5 ml of phosphate-buffered saline) by the use of scanning electron microscopy. OK-432-activated macrophages demonstrated marked surface membrane activity and migrated through the stomata of the milky spot into the peritoneal cavity. The characteristic features of activated milky spots and milky spot macrophages were noted as early as 3 h following the administration of OK-432, and continued to be observed until 7 days after the injection. By 14 days after the injection, the structural integrity of the milky spot was partially lost. The activation of milky spots and milky spot macrophages by OK-432 provides a convenient in vivo system for the monitoring and study of i.p. cellular events.

The thyroxine-binding properties of serum proteins. A competitive binding technique employing sephadex G-25.

A competitive binding technique is described for the estimation of the thyroxine (T4)-binding properties of serum proteins in dilute blood serum and lymph. When used in conjunction with an assay for total T4 the following parameters can be estimated: the number of functionally different T4 binding proteins, their individual association constants and binding capacities for T4, the amount of T4 which is bound to each binding species, and the concentration of unbound (free) T4. Both human and sheep serum have three functionally different T4-binding proteins. The association constants for the three human proteins were 9.5x10(9), 1.6x10(8) and 3.1x10(5) 1/mol for T4-binding globulin (TBG), T4-binding prealbumin (TBPA) and serum albumin, respectively. The corresponding sheep proteins, TBG, TBP-2 and albumin, had association constants of 8.9x10(9), 1-4 X 10(8) and 3.5x10(5) 1/mol. Human TBG had a mean binding capacity of 21.3 mug/100 ml and that of ovie TBG was 12.8 mug/100 ml. The other specific binding proteins (TBPA in man and TBP-2 in sheep) had mean binding capacities of 307 and 359 mug/100 ml respectively. Two functionally different T4-binding proteins were identified in rat serum.

Effect of ionic strength and ionic composition of assay buffers on the interaction of thyroxine with plasma proteins.

When plasma proteins are diluted with buffer the ionic strength and ionic composition of that buffer affects the interactions between thyroxine (T4) and its plasma protein-binding sites. Increases in phosphate, chloride or barbiturate ion concentration from 50 to 200 mmol/l caused a significant decrease in the affinity of plasma proteins for T4, and a concurrent increase in the concentration of unbound T4. These results cannot be completely accounted for by changes in ionic strength since at the same ionic strength different anions caused quantitatively different effects on unbound T4 concentration. The degree of depression of T4 binding by the three anions studied was in the order barbiturate greater than chloride greater than phosphate. The results of a systematic study on the composition of diluent buffer systems indicated that when a 50 mM-sodium phosphate-100 mM-NaCl buffer (pH 7-4) was used as a plasma diluent, there were unlikely to be gross changes in the T4-binding properties of plasma proteins with dilution.

The immune response of foetal calves.

The immunological response of foetal calves to tetanus toxoid was investigated. Emphasis was placed on foetal immunocompetence and how this related to responses seen in adult cattle. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts and superficial veins of foetal calves allowed continual monitoring of cellular and humoral changes in efferent lymph and peripheral blood. Foetal calves from 195 to 253 days gestational age had the capacity to mount cell-mediated and humoral immune responses of similar character and magnitude as adult cattle to tetanus toxoid. Intravenous and subcutaneous routes of challenge with tetanus toxoid resulted in specific antibody production which peaked 26 to 31 days after vaccination. Significant tetanus toxoid-stimulated lymphocyte proliferation was present 4 to 6 weeks after vaccination with tetanus toxoid in both a foetus and an adult. After antigenic challenge lymphocytes remained the predominant cell type in efferent prescapular lymph of foetuses and cows while at the same time a marked shift to the left, characterised by band neutrophils and neutrophilic metamyelocytes occurred in peripheral blood. Lymph flow rate increased and cell concentration decreased after antigenic challenge.

The proliferative responses of lymphocytes from foetal calves and adult cattle.

This paper describes the proliferative responses of prescapular lymph lymphocytes and peripheral blood lymphocytes of foetal calves compared with cells of similar origin from adult cattle. Lymph lymphocytes were collected continuously by means of cannulation of efferent lymphatic ducts of the prescapular lymph node of foetal calves and adult cattle. Peripheral blood lymphocytes were collected from the foetus by means of cannulation of superficial veins of the foetus or of the umbilical vessels and from the jugular vein of adults. Foetal lymphocytes in one-way mixed lymphocyte culture stimulated and responded as well as adult lymphocytes. Foetal cells stimulated and responded more to cells from unrelated animals than to cells from their dam. Lymph lymphocytes from foetal calves between 188 and 253 days of gestation proliferated as well as adult lymphocytes and at a high level after stimulation with concanavalin A, phytohaemagglutinin and pokeweed mitogen. Response to stimulation with lipopolysaccharide, soybean agglutinin and wheat-germ agglutinin was variable but generally low and within the same range recorded by adult cells. Proliferation by foetal and adult whole-blood cultures was on occasions as high as that recorded by separated lymphocytes, even though fewer lymphocytes were initially present in the whole-blood cultures. Foetal lymph lymphocytes exhibited lower proliferative responses in autologous lymph plasma than in foetal calf serum or pooled foetal lymph plasma. There was no consistent depression of proliferation by culture medium supplements from pregnant animals. Rabbit serum consistently abrogated responses. Fetuin at final concentrations of greater than 2.5 mg/ml significantly depressed proliferation in foetal and adult lymphocytes from efferent lymph and peripheral blood.

The cellular response of lambs to Brucella ovis before and after birth.

The immunological response of lambs to Brucella ovis before and after birth was investigated. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts of foetal lambs allowed continual monitoring of the immune response of a single lymph node. Foetal lambs in the last trimester of pregnancy were shown to mount a strong cell-mediated immune response to B. ovis. Lymphocytes from the challenged lymph node stimulated with B. ovis in vitro usually first reacted significantly and had highest [3H]-thymidine incorporation between 4 and 6 days after primary and secondary challenge, whereas, lymphocytes from the unchallenged node did not exhibit significant [3H]-thymidine incorporation until some 24 h later. Lymphocytes from these lambs challenged as foetuses still exhibited significant [3H]-thymidine incorporation in response to B. ovis for 4 to 5 months after birth. The proportion of surface immunoglobulin-positive cells in efferent prescapular lymph of unchallenged lambs ranged from 0.5 to 2.0% but after B. ovis challenge this proportion ranged from 2.7 to 8.7% between 4 to 6 days after challenge. By 9 to 12 days after challenge, the proportion had declined to pre-challenge values.

Technique for in situ excision of distended samples of greater omentum from small laboratory animals.

A simple and reliable technique is described for in situ excision of distended samples of greater omentum from small laboratory animals. The omental bursa is distended by injecting whipped hen egg white. Filter paper frames then are applied to the selected areas of distended omentum and samples of omental membrane are excised together with the filter paper frames. This sampling technique yields undamaged materials suitable for various research purposes.

Retarded and excessive development of skin appendages in fetal lambs in response to thyroidectomy before wool follicle appearance.

The impact on wool follicles of development in an athyroid environment was studied in a series of twin fetal lambs by surgically thyroidectomizing one of each pair before the appearance of follicle buds and comparing development of epidermal appendages in it with their development in the normal co-twin. Thyroidectomy was undertaken at 51 to 54 days' gestation, i.e. after approximately one-third of the gestation period. Each treated fetus was then replaced in the uterus, allowing pregnancy to continue. Eight pairs of twins were removed at intervals from 67 to 122 days' gestation and skin samples from the thyroidectomized and the intact twins were compared. Micromorphometric examination of the samples was used to assess quantitatively the effects of thyroid deprivation on wool follicle development. In thyroidectomized fetuses there was a failure of keratinization in primary wool follicles, an absence of secondary follicles, a tendency to excessive follicular branching and sweat gland development, and a paucity of sebaceous gland formation. The density of wool follicles was substantially increased, but the mean cross-sectional area of these follicles was reduced. The effects of very early thyroidectomy imply that the thyroid plays a role in the stimulation and regulation of wool follicle differentiation. To test the reversibility of the effects observed in the skin of thyroidectomized fetuses, grafts from these animals were transplanted to normal, young fetal lambs. Subsequent examination of grafted skin revealed that complete keratinization had occurred but that none of the other abnormal features had been reversed.

The cause of the shorter gestation length for twin calves: A computer simulation.

The possibility that the shorter length of gestation for twin calves is because pregnancy is determined by the earlier maturing twin has been investigated by computer simulation. The simulation indicated that this explanation alone could account for only about half the shortening of gestation for natural twin calves, and it is suggested that the combined actions of both twins is superimposed on the stimulus of the earlier maturing twin to initiate parturition. With mixed-species twins produced by embryo transfer, the immaturity of the more slowly maturing Bos indicus is such that it cannot cooperate with the earlier maturing Bos taurus cotwin to shorten gestation to appreciably less than is normal for single Bos taurus pregancies.

Morpho-physiological function and role of omental milky spots as omentum-associated lymphoid tissue (OALT) in the peritoneal cavity.

The morpho-physiological function and role of milky spots in the greater omentum are reviewed. These milky spots are composed of cellular aggregations of mesenchymal cells, mainly macrophages and lymphocytes, surrounding capillary convolutions termed omental glomeruli. Initial lymphatics of the omentum begin at the milky spots and drain into lymph collectors. The lymphatic capillaries in the omental milky spots take part in the absorption of various substances from the peritoneal cavity. Omental milky spots probably act as the first line of defense in the peritoneal cavity and therefore are immunologically important. In human infants, most of the cells in these milky spots are macrophages (49%); less common are B lymphocytes (29%) and T lymphocytes (12%). Whereas macrophages form clusters near the peritoneal surface of the milky spots and are oriented toward the peritoneal cavity for migration, clusters of B and T lymphocytes are typically found in periarteriolar locations within the milky spots. This cell zonation facilitates phagocytosis and processing of circulating antigens and foreign bodies which emanate from the peritoneal cavity. During inflammation, the number and size of omental milky spots dramatically increase, and some develop germinal centers within the lymphatic follicles and produce antibodies. During intraperitoneal immunotherapy, the omental milky spots and their cellular elements may be activated by intraperitoneal administration of biological response modifiers, and thereby represent an important immunoregulatory system for the peritoneal cavity. Omental milky spots are also closely linked to the dissemination of cancer cells. Thus, intraperitoneally inoculated experimental tumor cells selectively invade the milky spots and proliferate there to form tumor nodules. This occurrence is relevant to clinical practice where nodular metastases to the omentum are common. Omental milky spots are analogous to regional lymph nodes and as such are the omentum-associated lymphoid tissues and participate in intraperitoneal immune reactions.

Major lymph nodes of the head of the fallow deer (Dama dama) and lymphatic drainage of antlers.

Heads from 10 fallow deer bucks were examined to provide a description of the major lymph nodes in this region. The distribution and size of these nodes were similar to those of sheep and goats. To determine whether there was drainage of lymph from the skin of antlers, and to follow the route of this drainage, a solution containing Evans blue was injected intradermally into the antlers of 2 bucks whilst the animals were anaesthetised. Dye appeared in tracheal lymph ducts 14 to 30 min after injection. The spread of blue colouration in lymphatic ducts and nodes, seen at post-mortem examination, indicated that lymph flowed from antlers laterally into the ipsilateral parotid lymph nodes and from these via medial and lateral retropharyngeal lymph nodes to the tracheal ducts.

The metabolism of chylomicron fatty acids by isolated rat heart-lung preparations.

1. An isolated rat heart-lung preparation is described. This preparation is perfused with the rat's own blood, in the absence of heparin or other anticoagulants, by the sustained efficient contractions of the heart. The preparation has only a small circulating blood volume and is stable for up to 3 hr.2. Chylomicrons which were infused continuously into the circulating blood of the heart-lung preparation were removed from the circulation.3. [(14)C]Palmitic acid incorporated into the chylomicrons was oxidized rapidly to CO(2). Rates of oxidation of chylomicron fatty acids in excess of 3 mg/hr were measured and suggest that in the intact rat, the heart and lungs could account for up to 10% of the chylomicron fatty acids oxidized.4. The infusion of glucose did not reduce the oxidation of the chylomicron fatty acids, whilst the infusion of sufficient quantities of chylomicrons in the absence of glucose spared some of the cardiac glycogen.

Epithelial cell renewal and antibody transfer in the intestine of the foetal and neonatal lamb.

The intestine of the foetal lamb was exposed to large quantities of alpha-globulin (IgG) by prolonged intra-duodenal infusion, and absorption of intact IgG, with transfer to the lymph, continued undiminished, i.e., there was no evidence of closure. The rate of proliferation of the intestinal epithelium of the foetal and newborn lamb was measured using mitotic indices and localised labelling with (3H)-thymidine (TdR). In the foetus, cell division in the crypts occurs at a lower rate than the newborn (p less than 0.001) and there is very slow replacement of the intestinal epithelium. In the newborn lamb, a portion of the small intestine was incubated in vivo with TdR and the progress of labelled cells from the crypts upwards along the villi estimated, using autoradiography of serial biopsies from the same animal. A front of mature, digestive epithelium could be seen advancing up the villi, displacing the immature foetal type of cell which was capable of transfer of intact IgG to the lymphatics of the intestine. The evidence presented supports the hypothesis that immediately after birth the intestinal epithelium of the lamb begins to be replaced by a digestive type of cell, and the layer of cells responsible for absorption of colostral antibodies progressively disappears from the villi, resulting in closure.

The transcapillary exchange of thyroid hormones and thyroxine-binding proteins between blood and tissue fluids.

A study has been made of the relative importance of potien bound and unbound hormone in the exchange of thyroid hormones between blood and interstitial fluid. 2. When [I] tyroxine (or thriiodothyronine) and [I] human serum albumin were injected simultaneously into the circulation of sheep with chronic lymphatic fistulae, the thyroid hormones were removed from the circulation and apperaed in all lymph samples at a greater fractional rate than human serum albumin. 3. The steady-state lymph/plasma concentrations ratios of the two specific thyroxine binding proteins were similar to each other and to those of albmin and total thyroxine. 4. Gel filtration studies indicated that the two specific thyroxine binding proteins, ovine serum albumin and human serum albumin, were all of similar molecular size. 5. Concentrations of unbound thyroxine in plasma and various samples of lymph from the one animal were similar. 6. Increasing the proportion of thyroid hormone that was unbound resulted in an increase rate of equilibration of labelled hormone between blood plasma and lymph. 7. Perfusion of the popliteal lymph node demonstrated that thyroid hormones were removed from lymph during its passage through the node. The amount removed was related to the proportion of hormone in the unbound state.