Chronic liposome administration in mice: effects on reticuloendothelial function and tissue distribution

Liposomes have a pronounced tendency to localize in the reticuloendothelial (RE) system, a major host defense system. We have examined, in mice, the effect of chronic i.v. administration of low to moderate liposome doses on drug metabolism, phagocytic index and spleen and liver size. Little effect on the rate of pentobarbital metabolism was noted, except when mice received 80 mg/kg of sphingomyelin-containing liposomes in a series of 10 injections over 3.5 weeks. Impairment of RE phagocytic function was found to be related to liposome size and composition, size and frequency of liposome dose and the presence of lipid peroxides. The effects on tissue distribution of liposome-entrapped [14C]sucrose was also determined in mice receiving chronic liposome injections. In RE blockaded mice there was a consistent trend in favor of decreased liver uptake and increased spleen uptake. No significant uptake of liposome contents was seen in non-RE tissues even in mice with severe RE blockade, indicating that the induction of RE blockade by predosing with empty liposomes may not be a successful strategy for increasing liposome uptake to non-RE tissues. Liver to spleen ratios of [14C]sucrose appeared to be a sensitive method for quantitating RE blockade. Sphingomyelin-containing liposomes produced the greatest RE blockade, distearoylphosphatidylcholine-cholesterol liposomes were intermediate and egg phosphatidylcholine-cholesterol liposomes produced the least impairment in RE function. Liposomes which contained 1% vitamin E as an antioxidant had slightly less effect on RE function than liposomes not containing vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of methylprednisolone on the Fc-receptor function of human reticuloendothelial system in vivo

Abstract. To determine whether the Fc-receptor function of reticuloendothelial system (RES) is modified by corticosteroid administration, we studied the spleen to liver uptake ratios of autologous, ⁹⁹Tc-labelled heatdamaged or IgG-coated erythrocytes, injected intravenously into 10 normal volunteers, 4 h after receiving a single dose of 32 mg of methylprednisolone by mouth.
In standard conditions, quantitative scans indicated that the spleen to liver uptake ratios, calculated per unit area 40 min after the injection of labelled erythrocytes, were 13·4 ± 0·6 and 31·2 ± 1·5 (mean values ± -SEM), for the heat-damaged ( n = 7) and IgG-coated red cells ( n = 5) respectively. Four hours after corticosteroid administration, the spleen to liver uptake ratios were significantly reduced in five of ten volunteers. Abnormal ratios correlated with the Fc-receptor function of monocytes measured in vitro using IgG-coated erythrocytes. Indeed, 2–6 h after methylprednisolone was given, the Fc-receptor binding activity of monocytes isolated from the same five subjects was reduced by at least 50%, spontaneously returning to a rather normal value 4–6 h later. The C3-receptor binding activity of these monocytes remained normal, after otherwise identical experimental conditions.
These results show a transient, specific impairment of the Fc-receptor function of RES after methylprednisolone administration, and may therefore explain, in part, the infectious complications occurring in some patients treated by corticosteroids.     

Kupffer cell function in chronic liver injury and after partial hepatectomy

The reticuloendothelial system (RES) plays an important role in the biological defense system. In the liver, Kupffer cells are the main constituent of the RES, and when their function is impaired postoperative complications may more often occur. By using^99mTc-labeled human serum albumin millimicrospheres (^99mTc-HSA-MM) combined with assessment of single photon emission computed tomography (SPECT), we have attempted to determine the function of Kupffer cells independently of the hepatic blood flow. First, Kupffer cell function in rats with chronic liver injury caused by CCl₄ was studied. The hepatic uptake rate in chronic liver injury was decreased, and a reduced phagocytic activity of the Kupffer cells was noted. The parameter concerning Kupffer cell degradation, the excretion rate (k), was markedly decreased in the early period of chronic liver injury. Changes in Kupffer cell function after 30% and 70% hepatectomy were also studied. After 30% hepatectomy, the excretion rate was decreased on the first postoperative day (POD), and it was increased beyond that found after sham operation on the 3rd POD. In contrast, slower recovery of uptake rate was demonstrated. After 70% hepatectomy, both uptake and excretion rates were markedly reduced, and recovery was prolonged beyond the 5th POD. The hepatic uptake was not parallel with the excretion rate in either experiment. These results suggest that the method that measures the hepatic excretion rate may provide a better assessment of Kupffer cell function than the current uptake measurement with radiolabeled colloid.     

The role of tissue macrophages in the induction of proinflammatory cytokine production following intravenous injection of lipoplexes

Recent studies have demonstrated that intravenous administration of a plasmid DNA-cationic liposome complex (lipoplex) induced significant proinflammatory cytokine production in blood and inhibited transgene expression in pulmonary endothelial cells. In this study, we examined the effects of gadolinium chloride (GdCl(3)) pretreatment on the biodistribution and induction of proinflammatory cytokine production and transgene expression after intravenous injection of a lipoplex in mice. GdCl(3) is known to transiently deplete liver Kupffer cells and spleen macrophages after intravenous administration. Intravenous administration of a lipoplex triggers high levels of proinflammatory cytokine production, such as TNF-alpha, IFN-gamma and IL-12 in serum and a large amount of (32)P-labeled lipoplex accumulates in the liver 1 h after intravenous administration. However, pretreatment with GdCl(3) dramatically reduces serum levels of these cytokines and liver accumulation of the lipoplex. RT-PCR analysis showed that mRNA expression of TNF-alpha greatly increases in the liver and spleen after lipoplex injection and that pretreatment with GdCl(3) reduces mRNA expression in these organs. Messenger RNA expression of TNF-alpha in the liver occurs in non-parenchymal cells (sinusoidal endothelial cells and/or Kupffer cells). Inhibition of cytokine production by pretreatment with GdCl(3) leads to recovery of transgene expression in the lung following the second injection of lipoplex, which was reduced following the first injection of lipoplex. Thus, the present study demonstrates that tissue macrophages involving liver Kupffer cells and spleen macrophages are closely involved in TNF-alpha production following i.v. administration of the lipoplex. It is also suggested that avoiding lipoplex uptake and subsequent cytokine production by these cells would be a useful method of maintaining a high level of gene expression in the lung after repeated injections.     

Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes

The relative functional maturity of neonatal mouse spleen T- and B-cell populations was assessed by comparing the ability to respond to the thymic-independent antigen, DNP-Ficoll, or thymic-dependent SRBC by producing antibody in vitro. Although mouse spleen cells responded to DNP-Ficoll at an earlier age than they responded to SRBC or TNP-SRBC, the reason for the lag in the T-dependent response was confounded by the finding of high numbers of suppressor T lymphocytes in the neonatal spleen. Thus, small numbers of neonatal spleen T cells or thymocytes significantly decreased the in vitro antibody response of adult spleen cells. Although B lymphocytes appear to be functionally mature soon after birth, their acitivity may be modulated by an excess of suppressor T cells; e.g., the reconstitution of helper cell function in the neonatal spleen required anti-theta treatment before addition of adult helper cells. Suppressive activity attributable to T cells seems to play a dominant role in determining the ability of the neonatal animal to react positively or negatively to antigenic stimulation.     

Function of reduced-size liver transplant in the rat

We have studied the function of partial orthotopic liver transplantation in the rat by evaluating prothrombin time (PT), liver blood flow, basal and glucose-stimulated insulin secretion and glucose tolerance, and the reticuloendothelial function (RES) in hepatectomized rats subjected to partial liver transplantation. A graft corresponding to 68% of a normal liver was trans-planted to totally hepatectomized rats. Comparison was made between control rats and rats subjected to 32% liver resection. PT was not significantly different in the transplanted group compared with liver-resected and control rats. Laser Doppler flowmetry showed that at 28 days after surgery, blood flow had increased in the transplanted livers. Furthermore, on the third day after transplantation, basal plasma insulin was increased and the plasma insulin response to glucose was exaggerated, suggesting reduced insulin action and impaired insulin degradation. Finally, uptake of radioactive-labeled E. coli bacteria, as a measure of RES function, was not compromised in transplanted animals. Based on these results, we conclude that reduced-size liver transplant in out-bred rats results in fast normalization of liver function after surgery although, immediately after surgery, glucose intolerance is seen.     

Changes in the reticuloendothelial phagocytic function after partial hepatectomy

The changes in the phagocytic function of the reticuloendothelial (RE) system after 67% hepatectomy in rats were studied by use of 51Cr-endotoxin as the phagocytable material. The humoral opsonic index was significantly increased to approximately 1.2 to 1.8 (control, 1.0 +/- 0.2, mean +/- SD) until the fourteenth postoperative day. In contrast, the phagocytic index was decreased to 0.068 +/- 0.016 (control, 0.103 +/- 0.015) on the first day, then returned to the normal level on the second day. In this early postoperative period, uptake rates of 51Cr-endotoxin in the liver were remarkably decreased to about 50% to 70% of control, whereas those in the spleen and lung were increased two- to threefold of control. From the third to the fourteenth day, the phagocytic index was significantly increased compared with the preoperative level. During this period, the uptake rates in the liver and spleen were within the normal range. These results suggest that the increases in the opsonic index of 67% hepatectomized rats represent the homeostatic response for maintaining or stimulating RE system phagocytic function, and that high or normal phagocytic index, concomitant with the increase in the opsonic index, implies an enhanced or compensatory stage, respectively. The decrease in the phagocytic index despite the high opsonic index is assumed to represent a compromised stage of the RE system.     

Vascular clearance by the reticuloendothelial system--measurements using two different-sized albumin colloids

Normal and reticuloendothelial system (RES) stimulated rats were examined with dynamic liver RES scintigraphy using a computerized gamma camera. 99Tcm-labelled albumin colloid, albures (radius 250 nm) or nanocoll (radius 25 nm), or both were used as test substances to study the kinetics of vascular clearance after RES stimulation. Registrations were made of 30 s per frame for 5 min and 300 s per frame for 15 min or 25 min and a region of interest (ROI) was indicated over the liver. Whole body and liver RES clearance rate constants (k) were calculated from the liver uptake vs time curve. Liver parenchyma blood flow was estimated with 133Xe washout technique. The blood clearance rate constant of albures in non-activated rats was twice that for nanocoll (1.08 +/- 0.05 vs 0.49 +/- 0.02 10(-2)s-1). There was no mutual interaction between the two colloids, implying that they may be eliminated from the blood-stream by slightly different processes. In zymosan-stimulated animals, nanocoll given in a single injection showed a significantly increased k-value. Neither the albures clearance rate constant nor the nanocoll/albures k-value ratio revealed RES macrophage activation. By contrast the nanocoll/albures ratio, calculated for the liver, rose significantly. The final colloid uptake in the liver revealed RES macrophage activation. No changes in liver parenchyma blood flow per g tissue could be registered after administration of zymosan. The nanocoll and albures colloid particles did not impair the normal liver parenchyma blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

表阿霉素-白藜芦醇长循环脂质体的制备及其体外释放

目的制备经聚乙二醇(PEG)修饰的表阿霉素-白藜芦醇长循环脂质体,考察长循环脂质体体外释药行为,并对其抗肿瘤作用进行评价。方法采用薄膜分散法-硫酸铵梯度法制备表阿霉素-白藜芦醇长循环脂质体;采用动态透析法考察其体外释药行为;SRB法评价体外抗肿瘤作用。结果采用薄膜分散法-硫酸铵梯度法制备的长循环脂质体中表阿霉素与白藜芦醇包封率可达89.80%、65.01%。体外释放研究表明,表阿霉素-白藜芦醇长循环脂质体较普通脂质体及原料药释药速度更慢,稳定性更好。体外细胞毒试验显示,长循环脂质体对C6胶质瘤细胞具有较强的抑制作用。结论制备长循环脂质体操作简便、重复性好,包封率较高,体外释放具有缓释特征,体外抗肿瘤作用强。     

A quantitative study of the kinetics of blood clearance of P32-labelled Escherichia coli and Staphylococci by the reticuloendothelial system

1. The clearance of P³²-labelled heat-killed E. coli and staphylococci from the blood follows an exponential function of the time, and the bacteria are phagocytized principally by the RES of the liver and spleen. 2. The rates of clearance of equivalent number of E. coli from the blood is rapid in rabbits and slow in mice and appears to be related to the level of antibodies in the serum of these animals. 3. Unlike E. coli, staphylococci are cleared rapidly and efficiently by the RES from the blood of mice which have a sufficient level of serum antibody against these bacteria. 4. The numbers of bacteria, phagocytized by the liver or the spleen respectively, depend upon the rate of clearance and the extent of opsonization of the bacteria. Rapidly cleared, well opsonized E. coli are removed almost exclusively by the liver, while less efficiently phagocytized bacteria are also cleared by the spleen in large numbers. 5. The rate of clearance of E. coli and the efficiency with which they are phagocyted by the RES in mice have been shown to be directly related to the level of antibody in the serum. 6. Treatment of mice with S. typhi or Serratia marcescens endotoxins increases the rate of clearance of E. coli from the blood and the level of antibody against E. coli in the serum. The enhanced clearance of E. coli can be transferred to normal mice by the serum of endotoxin-treated mice.     

Endotoxin pretreatment improves bacterial clearance and decreases mortality in mice challenged with Staphylococcus aureus

We studied the effects of tolerance induced by Escherichia coli-derived LPS on the innate immune response to a subsequent Staphylococcus aureus bacterial challenge. LPS tolerance was induced in wild-type mice by either intraperitoneal or intravenous injection of 2 microg of LPS on 2 consecutive days. Mice were challenged with an intravenous injection of live S. aureus (5 x 10(8) colony-forming units) 2 days after the second LPS dose. LPS-tolerant mice had a diminished serum interferon-gamma response to the bacterial challenge. Bacterial counts in liver and spleen tissues were decreased, and survival was improved after the Staphylococcus challenge in LPS-tolerant mice compared with saline-pretreated control mice. LPS pretreatment by the intravenous route was also associated with a decreased number of bacterial colonies in lung tissue in addition to liver and spleen, suggesting that induction of LPS tolerance was somewhat compartmentalized after intraperitoneal LPS pretreatment. Induction of tolerance seemed to be due to LPS-specific signaling because LPS pretreatment of LPS-nonresponsive C3H/HeJ mice did not provide similar effects after bacterial challenge. Flow cytometric analysis of spleens from LPS-tolerant mice revealed an increase in phagocytic cells (neutrophiles and macrophages) compared with control mice. Ex vivo culture of splenocytes from LPS-tolerant mice demonstrated increased uptake of fluorescein isothiocyanate-tagged ovalbumin, but no difference in either phagocytosis of fluorescein isothiocyanate-labeled Staphylococcus or bactericidal activity could be demonstrated on a per-cell basis. These results show that attenuation of inflammation and mortality during LPS tolerance extends to gram-positive bacterial organisms and suggests that LPS-induced enhancement of the innate immune response may be attributed to increased numbers of phagocytic cells.     

The relationship between the vascular manifestations of shock produced by endotoxin,trauma, and hemorrhage. II. The possible role of the reticulo-endothelial system in resistance to each type of shock

In studies designed to establish the interrelationship between bacterial endotoxins and the vascular sequelae of hemorrhagic and traumatic shock, the effect of factors known to influence the phagocytic behavior of the reticulo-endothelial system (RES) were investigated. Measures which induced a so called "blockade" of the RES were uniformly associated with an exacerbation of the vascular effects of the endotoxin of E. coli. Such pretreatment also counteracted the cross-tolerance induced by endotoxins against the lethal effects of hemorrhage or drum trauma. The vascular reactions characteristic of irreversible hemorrhagic shock could be simulated by a combination of pretreatment with carbon or proferrin and the infusion of small doses of E. coli endotoxin. An increase in the phagocytic activity of the RES, induced by repeated injections of certain colloids, was associated with an enhanced tolerance of shock. Measurement of carbon clearance values indicated that although an augmented phagocytic capacity was present in rats with induced tolerance to bacterial endotoxins, the development of resistance to trauma was not associated with a comparable change in the phagocytic function of the RES.     

Quantitative reticuloendothelial function of liver and spleen in man

Summary. A method for separate determinations of liver and spleen reticuloendothelial function, using a small-size ⁹⁹Tc^m-antimony sulphide colloid and gamma camera technique, is described. Several methods for achieving blood-background correction are examined, and it is shown that, by a three-compartment model, the use of a specific blood pool tracer can be dispensed with. Hepatic and splenic uptake of the colloid can be described by first order kinetics, and can be calculated to an error less than 5%. In a reference material (n=13), hepatic and splenic clearance was 262 ml/min (100412) and 22 ml/min (0–62), respectively. In cirrhosis (n=7), hepatic clearance was decreased and splenic clearance increased. The results indicate that this method, which is well suited for clinical studies and which is based on a reasonable physiologic model, in cirrhosis of the liver demonstrates a decreased hepatic reticuloendothelial function with (compensatory?) increase in that of the spleen.     

Augmented delayed footpad reaction in thymus cell-depleted mice induced by cholera toxoid

One hundred microgram of cholera toxoid was injected intravenously into DDD and AKR mice and its effects on lymphoid tissues and immune responses against sheep erythrocytes (SRBC) were examined at various times after the injection. (1) A remarkable reduction of thymus cells was revealed from day 1 to 7 and from day 1 to 4 in DDD and AKR mice, respectively. (2) Cholera toxoid exhibited only slight effects on the numbers of spleen cells and peripheral blood leukocytes in both strains. (3) Delayed footpad reactions to SRBC were augmented by a pretreatment with cholera toxoid 4 or 7 days before immunization in both strains. The delayed reactions were not suppressed in the presence of a prominent antibody production and were accompanied by positive macrophage migration inhibition. (4) Antibody production against SRBC, especially of IgG class, was facilitated, when cholera toxoid was given 7 days before the immunization through the footpad in DDD mice. On the other hand, antibody production was suppressed irrespective of immunizing routes and mouse strains, when cholera toxoid was given 1 day before immunization.     

Virus-replicating T cells in the immune response of mice. I. Virus plaque assay of the lymphocytes reactive to sheep erythrocytes

Virus plaque-forming cell assay with vesicular stomatitis virus (VSV), which had been originally introduced by Bloom and his colleagues as a tool for the enumeration of activated lymphocytes, was first applied to the immune response of mice to a widely used antigen, i.e. sheep red blood cells (SRBC). When spleen cells taken from mice previously primed with SRBC were cultured in the presence of the antigen, lymphocytes capable of replicating VSV (antigen-induced virus plaque-forming cells, Ag-V-PFC) were generated in the culture. They seemed to appear as early as 1 day of culture, and the peak was attained by the 2nd day. Most of Ag-V-PFC belonged to T-cell population, since 80-90% of Ag-V-PFC was killed by the treatment of cultured cells with anti-thymocyte serum plus complement. In vitro generation of Ag-V-PFC seemed to be highly cross-reactive (about 40%) with a related antigen (horse red blood cells). Ag-V-PFC detected in the present experiment may not represent helper T cells, effector T cells, or their precursors because of the following: (a) The generation of Ag-V-PFC was completely suppressed by the addition of anti-SRBC mouse serum in the culture, though the helper activity was apparently augmented by the same treatment. (b) Development of Ag-V-PFC was almost completely suppressed by the pretreatment of mice with cyclophosphamide 2 days before immunization, by which delayed-type hypersensitivity (DTH) was markedly augmented. (c) After the immunization of mice, Ag-V-PFC began to develop just when the level of DTH declined, at which point helper activity of the spleen cells also diminished. A possible role of Ag-V-PFC in the immune response was discussed.     

Technetium 99m sulfur colloid spleen/liver ratio and other liver tests in the diagnosis of cirrhosis.

The results of seven laboratory tests of liver function, including spleen/liver activity ratios obtained by densitometric analysis of scans, are analyzed in 50 patients with proven Laennec's cirrhosis. In this series, the liver scan not only disclosed the liver gross anatomy and structural abnormality and established the best possible site for biopsy examination, but also, the increased splenic activity served as a useful diagnostic indication of Laennec's cirrhosis. Of 50 proven cases of Laennec's cirrhosis, 41 (82%) had abnormal spleen/liver ratios. An abnormal spleen/liver ratio in combination with abnormal results from any one or two other tests was relatively effective in the detection of cirrhosis. The accuracy is improved if the other laboratory tests are chosen from among tests for serum albumin, serum bilirubin, and SGOT. (Liver abnormalities other than cirrhosis can also present an abnormal spleen/liver ratio.) This simple determination extends the value of the liver scan commonly requested in search of metastases, primary lesions, or inflammatory processes, or in preparation for needle biopsy examination.     

The effects of PEG grafting level and injection dose on gold nanorod biodistribution in the tumor-bearing mice

Gold nanorods have strong absorbance in the near infrared region, which penetrates deeply into tissues, where the absorbed light energy is converted into heat. Therefore, gold nanorods are expected to act as an effective contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. We grafted various amounts of polyethylene glycol (PEG) onto the surface of gold nanorods and investigated the effects of grafting level and injection dose on the biodistribution in the tumor-bearing mice after intravenous injection and enhanced permeability and retention (EPR). Higher PEG grafting levels were advantageous for reticuloendothelial system (RES) avoidance and for suppression of aggregation of the gold nanorods in the circulation. Modification with a PEG:gold molar ratio of 1.5 was sufficient to show both prolonged circulation and the EPR effect. When the injection dose was increased above 19.5 µg of gold, the RES uptake in the liver was saturated and surplus gold nanorods were distributed to other tissues, especially the spleen and the tumor. The results of this study will provide an important basis for the development of cancer therapies mediated by the photothermal effect of gold nanorods.     

Effect of two defined formula diets on jejunal and colonic uptake of hexoses in control and ileal resected rabbits

An in vitro technique was used to measure the jejunal and colonic uptake of glucose and galactose in sham-operated control (CONT) rabbits and in animals with a surgical resection of the distal half of the small intestine (RES) performed six weeks previously. CONT and RES were fed ad libitum or pair-fed amounts of standard Purina chow, or ad libitum amounts of Ensure or Flexical HN for two weeks. Flexical NH differs from Ensure in its higher content of carbohydrate, essential fatty acids and polyunsaturated fatty acids but lower total dietary content of fat. The ad libitum chow-fed animals gained weight whereas the animals fed the defined formula diets lost weight. Weight gain was greater in RES than CONT fed chow, and weight loss was greater in RES than CONT fed Ensure or Flexical HN. Despite these differences in body weight, the dry and wet weights of the jejunum, and the adherent mucosal fluid volumes remained unchanged in CONT and RES fed the different diets. The intestinal morphology was unaffected by feeding Ensure or Flexical HN to control animals, whereas the villus cell size, villus and mucosal surface areas were lower in resected rabbits fed these diets than in resected animals fed chow. Glucose uptake was greatest in CONT and RES fed Flexical HN. Glucose uptake was higher in RES than CONT fed chow or Ensure but was lower in RES than CONT fed Flexical HN. In CONT fed Flexical HN, there was a lower value of the apparent Michaelis constant, Km*, and a higher value of the apparent passive permeability coefficient (Pd*) than in animals fed chow or Ensure. In RES there was lower Pd* in Ensure and Flexical HN-fed rabbits than in those fed chow, but the Km* was lower and the maximal transport rate, Jdm, was higher in animals given Flexical HN. The colonic uptake of glucose and the jejunal uptake of galactose was also influenced by feeding Ensure and Flexical HN. These differences in uptake in animals fed Ensure or Flexical-HN could not be explained by differences in body weight gain, since a different pattern of changes in glucose and galactose uptake was observed in CONT or RES rabbits exposed to restricted chow intake.(ABSTRACT TRUNCATED AT 400 WORDS)     

The postnatal development of the white pulp in the rat spleen and the onset of immunocompetence against a thymus-independent and a thymus-dependent antigen.

The spleen of the rat contains 5 well delineated compartments: a central and peripheral part of the periarteriolar lymphatic sheath (PALS), the marginal zone and the follicle with centre and corona. A study was made on the development of these compartments in correlation with the onset of immunocompetence of the spleen. The spleens of animals in the age range from 1-40 days were studied with the light and electron microscopes. Immunocompetence was assessed by measuring serum antibody levels 5 days after intravenous antigen administration. Comparable doses of paratyphoid vaccin (PTV) and sheep red blood cells (SRBC) were used: PTV as a thymus-independent, SRBC as a thymus-dependent antigen. At the first day of life few lymphocytes are present around small arterioles. The marginal zone is first present at 9 days of age. At 14 days of age the typical thymus-dependent area develops. Interdigitating cells, which seem to develop from monocytes, and lymphocytes are present in close apposition. Primary follicles were first seen on 20 days of age, follicle centres at 25 or 30 days of age. The appearance of "typical" dendritic cells coincided with the appearance of follicle centres. The first titre against PTV was seen after antigen administration at 9 days after birth, against SRBC after injection in 14 days old animals. As PTV is a thymus-independent antigen it needs only B-cells for a primary IgM response. The appearance of functional B-cells in sufficient numbers to give a measurable response therefore coincides with the appearance of the marginal zone. Although T-cells are present from birth, the response against SRBC is delayed until the thymus-dependent area has developed. Thus, in the first 2 weeks of life, T-cells are either immature, or they are not able to react, because their microenvironment is not yet adequate.     

The role of fetal calf serum in the primary immune response in vitro

The mode of action of 2-mercaptoethanol (2-ME) on the primary immune response in vitro was investigated. Fetal calf serum (FCS) was preincubated with 2-ME and lyophilized to remove free 2-ME. This 2-ME- treated FCS was able to substitute the function of adherent cells in the primary immune response against sheep red blood cells (SRBC) in vitro; Fractionation of 2-tme-treated FCS on a Sephadex G-100 column showed that 2-ME acted on a high molecular serum component which after activation, could substitute for macrophages. In order to obtain a humoral immune response against SRBC in vitro, spleen cells require selected FCS. These "good" sera could be distinguished from "deficient" sera by their higher content of this 2-ME-activated factor. The height of the in vitro immune response to SRBC was dependent on the amount of activated factor added to the culture medium. FCS normally required in the culture medium could be completely replaced by the factor- containing fraction without deleterious effect on the culture medium. The factor should be added to the spleen cells during the first 24 h of culture and remain there for 72 h in order to obtain an optimal immune response. The factor could be partially absorbed by spleen cells but not by SRBC. The relationship between macrophage, 2-ME, and FCS in eliciting an in vitro primary immune response is discussed.