Evaluation of the reticuloendothelial system function by the vascular clearance of chondroitin sulfate iron colloid

Vascular clearance of chondroitin sulfate iron colloid (CSFe) was evaluated as a test for the reticuloendothelial system (RES) in rabbits. Injected CS59Fe was taken up by the RES in the liver (49%) and bone marrow (41%) after 60 min, suggesting its application for the RES function test. The clearance rate (K-value) of CSFe from the blood was calculated by measuring serum Fe concentrations after releasing iron from CSFe at certain intervals after injection. Depending upon different injected doses, K-values were varied and the phagocytic velocity, computed by multiplying K-values by corresponding injected doses, reached a plateau at high doses, indicating the phagocytosis of CSFe by the RES takes a saturation process. Double-reciprocal plotting of the dose and the phagocytic velocity showed a linear relationship, which provided the data on the maximum phagocytic velocity (Vmax), 0.122 mg/kg/min, and the CSFe concentration producing 1/2 Vmax (Kp), 0.426 mg/kg. Thus, this CSFe clearance test can be used for the evaluation of the RES function.     

Relationship between the phagocytic inhibition of the rat reticuloendothelial system and the anticholinesterasic effect of an insecticide, Carbaryl (author's transl)]

Phagocytic activity of the reticuloendothelial system (RES) and blood cholinesterase activity were determined in male rats after veinous administrations of carbaryl and 1-naphthol, a carbaryl metabolite. The various parameters were measured 1, 24, 48 and 72 hours after administration of the following four doses per 100 g body weight : 1.875, 3.75, 7.5 and 15 mumol. 1. Results showed an inhibition of the RES phagocytic activity (clearance of colloidal carbon) after carbaryl administration; although 1.875 mumol/100 g had no effect, the other doses inhibited RES activity, blockade time being a function of the dose given. The phagocytic function had returned to normal 72 hr after carbaryl administration. 2. Reductions in spleen weight and protein content were observed together with the RES blockade. 3. At all four doses, the anticholinesterase effect was already apparent one hour after carbaryl administration. 4. 1-naphthol, one of carbaryl's chief metabolites, had no effect either on the RES or on the different parameters studied. These results show a relationship between the phagocytic inhibition of the reticuloendothelial system and the anticholinesterasic effect by carbaryl. They suggest an inhibition of some esterases of macrophages interfering with the phagocytosis.     

Impaired blood clearance of bacteria and phagocytic activity in vitamin A-deficient rats

The effect of vitamin A deficiency on the functional integrity of the reticuloendothelial system and the phagocytic capacity of circulating polymorphonuclear leukocytes was evaluated in retinoate-cycled vitamin A-deficient rats under conditions such that secondary dietary imbalances were eliminated. Kinetics of blood clearance of 2 X 10(7) Escherichia coli injected intravenously was depressed within 8 days of the withdrawal of retinoic acid; all animals were profoundly affected by Day 12 of deficiency. In vitro, the phagocytic activity of polymorphonuclear leukocytes was similarly affected; by Day 12 of deficiency, phagocytic capacity in all deficient animals was less than 40% of the appropriate control values (P less than 0.01). Animals rendered vitamin A deficient by this procedure also displayed marked susceptibility to endogenous bacterial infection, as judged from the proportion of deficient rats that spontaneously developed bacteremia during the later stages of deficiency. These data together demonstrate unequivocally that reticuloendothelial and polymorphonuclear leukocytic functions are impaired in vitamin A deficiency in the absence of other dietary imbalances.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

Opsonic effect of antiserum and complement on phagocytosis by macrophages from the peritoneal cavity of the Japanese eel, Anguilla japonica

Macrophage exudates in the Japanese eel, Anguillu japonica, were induced by intraperitoneal injection with a mixture of Edwnrdsiella bacterin, proteose peptone and liquid paraffin, and the opsonic effect of antiserum and complement on the phagocytic activity of the macrophages was studied.
The macrophages contained a round nucleus with a nucleolus, and an agranular cytoplasm with projecting processes. These cells showed phagocytosis of sheep red blood cells (SRBC). An opsonic effect of antiserum was recognized in terms of a significant increase of macrophage phagocytic activity against SRBC treated with antiserum relative to that against SRBC treated with inactivated normal serum. Complement, however, did not enhance the phagocytic activity of macrophages.     

Phagocytosis and membrane fluidity: application to the evaluation of opsonizing properties of fibronectin

The uptake of particles by phagocytic cells involves an increase in the membrane fluidity determined by steady-state fluorescence polarization. Binding and endocytosis of target particles is in vivo enhanced by humoral factors called opsonins. In this work, fluorescence polarization was used to detect in vitro the opsonic activity of a plasma protein: fibronectin. The assay is based on the analysis of membrane fluidity variations following the uptake of gelatinized latex beads by phagocytic cells in the presence or in the absence of fibronectin. Using TMA-DPH as fluorescent probe, it was observed that the increase in membrane fluidity was enhanced in presence of fibronectin and depended upon the enhanced in presence of fibronectin and depended upon the opsonic activity was related to the integrity of the molecule. Using this method, the opsonic activity of various plasmas could be also determined.     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.     

Effect of a chronic iodine deficit in the ration on the development of the infectious process]

It was revealed that the infectious process in albino rats kept for 4-5 months on an iodine-deficiency diet was characterised by a tendency to dissemination. The seeding efficiency from the parenchymatous organs increased in such animals significantly, whereas the bactericidal properties of the plasma and the phagocytic activity of blood neutrophils decreased; this was apparently associated with depression of the intracellular metabolism reflected in reduction of the cytochromoxidase and peroxidase in the neutrophils.     

Effect of dietary phosphorus on reticuloendothelial clearance of salmonella organisms in endotoxin treated guinea pigs

Guinea pigs fed purified diets that contained either 0.4% or 1.0% of phosphorus were injected intraperitoneally with crude $\text{Salmonella typhimurium}$ endotoxin. Functional activity of the RES was evaluated after endotoxin administration by determining the initial clearance rates of non-viable ³²P-labeled $\text{Salmonella typhimurium}$. Three days after administration of endotoxin the RES clearance rates were approximately $\text{2}\text{1}\text{2}$ times greater in animals fed 1.0% than in those fed 0.4% of dietary phosphorus. After the RES response had risen to a maximum (5–6 days), there was no difference between dietary groups and RES clearance rates remained high throughout an 18-day period. The increased clearance rate induced by endotoxin involved largely the liver RES. Endotoxin administration led to a decrease in blood inorganic phosphorus in both dietary groups but the concentration in those fed the higher phosphorus level remained significantly higher. It was concluded that the protective action of high phosphorus is mediated at least in part by its beneficial effect on RES function.     

Hemorrhage with blood reinfusion and the relationship between gastroduodenal motility and bile reflux in rats

The relationship between gastroduodenal motility and bile reflux was studied in normal rats and in rats subjected to hemorrhage and blood reinfusion. Bile secretion decreased from 5.3 +/- 0.4 to 4.1 +/- 0.5 microL/(min.100 g rat) (p less than 0.05) during the hypovolemic stress and recovered after blood reinfusion. Gastric bile salt content was low (0.1 +/- 0.03 mumol/(h.100 g rat] during control period and hemorrhage but increased to 0.7 +/- 0.12 mumol/(h.100 g rat) (p less than 0.001) during the 3 h following blood replacement. Marked gastric and duodenal retention of polyethylene glycol was observed immediately after hypovolemia with the former being evident even after 3 h following blood reinfusion, while duodenal emptying recovered rapidly after reinfusion. The frequency of gastric contraction remained unchanged during hemorrhage but decreased after 90 min following blood replacement, whereas the frequency of duodenal contraction abruptly decreased during hemorrhage and recovered after reinfusion. Both gastric and duodenal contractile pressure was significantly decreased during hemorrhage. After reinfusion, the former remained suppressed while the latter was fully recovered within 1 h. Thus, a significant duodenogastric bile reflux observed after reinfusion was due to a higher duodenal contractile pressure, and the uncoordinated gastroduodenal motility with the duodenal motility fully recovered soon after reinfusion while that of the stomach remained suppressed.     

Mechanical stress (shaking) and reticuloendothelial function in the chick embryo

Stage 34 (8 day) and 41 (15 day) chick embryos were exposed (3 min) to two separate amounts of mechanical (shaking) stress. Eggs were shaken in the horizontal plane through their long axes. A laboratory shaker (Eberbach) was used to produce the stress and one complete excursion defined as carriage movement through 7.92 cm (3.96 cm in opposite directions). Reticuloendothelial system (RES) activity, as measured by colloidal carbon clearance (CCC), was evaluated following administration via an allantoic vein. Shaking stage 34 or 41 embryos at 100 excursions per minute (EPM) did not affect CCC; shaking them at 300 EPM resulted in an immediate and significant depression of CCC. Stage 41 embryos exposed to 300 EPM and reincubated for either 1, 2 or 4 hours prior to CCC determination reverted to control CCC values sooner than stage 34 embryos subjected to the same regimen. Functional RES response to mechanically induced stress has now been shown for the first time in an embryonic system (at two distinct developmental stages). The response appears to parallel that reported for various adult organisms. Furthermore, we suggest a temporal relationship regarding functional development of the embryonic RES.     

Fetal blood volume responses to fetal haemorrhage: autonomic nervous contribution

The purpose of this study was to determine whether the autonomic nervous system contributes to short-term fetal blood volume restitution following fetal haemorrhage. Unanaesthetized, chronically catheterized fetal sheep averaging 130 days gestation (term = 145-150 days) were studied 5 days after catheter implantation. Blockade of the autonomic nervous system was produced with a bolus intravenous injection of hexamethonium. In 7 blocked fetuses, an average of 17.6% of the initial blood volume was removed over 5 min and this was not different from the 16.5% blood volume removed in 7 control fetuses. However, blood volume restoration averaged 44.6% after 30 min in the blocked fetuses and this was significantly less than the 60.2% volume restoration in the control fetuses (P less than 0.05). Thus blood volume restoration averaged 74% of control in the blocked fetuses. Following blood reinfusion over 5 min, blood volume was transiently elevated in the control group, but averaged 3.4, 4.7, and 5.3% below normal (P less than 0.05) at 10, 20 and 30 min post reinfusion in the blocked group. In addition, arterial pressure was lower after haemorrhage in the blocked group and increased twice that in the control group during reinfusion. Thus, the present data suggest that the fetal autonomic nervous system plays a significant role in mediating the short-term changes in fetal blood volume and vascular pressures following fetal haemorrhage and reinfusion.     

Alpha-1-antitrypsin. Plasma survival studies in the rat of the normal and homozygote deficient forms.

Alpha-1-antitrypsin from normal individuals (Pi type MM) from those with an inherited deficiency of circulatory protein (Pi type ZZ) were labelled with 125I and plasma clearance rates measured in rats either prior to, or following treatment with neuraminidase to remove terminal sialic acid residues. In addition, these proteins and the derivatives were tested for their ability to bind to an hepatic binding protein obtained from rabbit liver membranes that has been shown to be responsible for the clearance of serum asialoglycoproteins. Finally, the two native forms of alpha-1-antitrypsin were treated with galactose oxidase followed by reduction with tritiated potassium borohydride and then analyzed for tritium incorporation in the neutral sugar fraction. The results indicate: (a) clearance from plasma for both forms of alpha-1-antitrypsin is dramatically enhanced upon the loss of terminal sialic acid residues to the liver membrane protein; (b) Z protein does not exhibit terminal galactosyl residues; (c) the low level of Z protein in plasma cannot be accounted for by a faster rate of clearance relative to M protein. The relevance of these findings to the alpha-1-antitrypsin deficiency state are discussed.     

Establishment of a multi-dose study of chondroitin sulfate iron colloid for evaluation of the reticuloendothelial system function.

The assay system of chondroitin sulfate iron colloid (CSFe) was established to evaluate the reticuloendothelial system (RES) function in individual rabbits. In the multi-dose study of CSFe, CSFe was repeatedly administered to each individual rabbit with increasing doses (0.6, 1.2, 2.4, 6.0 mg/kg) at set intervals. Blood samples were serially collected after injection of CSFe and the concentration of CSFe in serum was directly measured as an iron concentration by modifying the previously described assay method [1] to minimize the sample volume. The clearance rate of CSFe at each injected dose was computed by the least-squares method and the double-reciprocal plotting of the doses against the phagocytic velocities by the Lineweaver-Burk method was obtained in each rabbit. The maximum phagocytic velocity (Vmax) and the CSFe concentration producing 1/2 Vmax (Kp) obtained in ten rabbits were 0.129 +/- 0.025 mg/kg per min and 0.417 +/- 0.121 mg/kg (mean +/- S.D.), respectively. The results obtained from this multi-dose study were comparable to our previous results obtained from the mean values of five groups given different doses [1]. The clearance rates of CSFe (0.6, 1.2, 6 mg/kg) decreased after the co-injection of 80 mg/kg of carbon colloid. The calculated Vmax and Kp in 29 rabbits were 0.125 mg/kg per min and 1.167 mg/kg. The Kp was apparently greater than that of the control (Vmax = 0.128 mg/kg per min, Kp = 0.421 mg/kg). Carbon colloid (80 mg/kg) was injected to six rabbits after the completion of the first multi-dose study of CSFe and then the second multi-dose study of CSFe was repeated after 24 h. No differences were found in Vmax and Kp between the two studies as were in the control group (10 rabbits) where saline was injected instead of carbon colloid. These results indicated that carbon colloid (80 mg/kg) gives a competitive and reversible inhibition on the RES. This multi-dose study of CSFe may be applicable for a bed-side analysis of the RES function in a patient.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress

White shrimp Litopenaeus vannamei (also known as Penaeus vannamei) held in 34 per thousand seawater at pH 8.2 were injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 8 x 10(5) colony-forming units (cfu) shrimp(-1), and then transferred to tanks at pH 6.5, 8.2 (control) and 10.1, respectively. After 24-168 h, the mortality of V. alginolyticus-injected shrimp that were transferred to pH 6.5 and pH 10.1 tanks was significantly higher than that of V. alginolyticus-injected shrimp held at pH 8.2. In another experiment, L. vannamei held at pH 8.2 following transfer to pH 6.5, 8.2 (control) and 10.1 for 6, 12, 24, 72 and 120 h were examined for immune parameters, phagocytic activity, and the clearance efficiency of shrimp against V. alginolyticus. The results indicated that the shrimp that were transferred to pH 6.5 and 10.1 showed significantly decreased phenoloxidase (PO) activity, respiratory burst, phagocytic activity, and clearance efficiency against V. alginolyticus over 6-72 h; significantly decreased superoxide dismutase (SOD) activity over 6-24h; and decreased total haemocyte count (THC) over 12-72 h. Shrimp transferred to pH 10.1 showed significantly decreased granular cell counts, and THC after 6h, and decreased SOD activity after 72 h. The immune parameters of shrimp transferred to pH 6.5 and 10.1 returned to the original values after 120 h. However, shrimp transferred to pH 6.5 still maintained lower phagocytic activity, and clearance efficiency against V. alginolyticus, and shrimp transferred to pH 10.1 still maintained lower clearance efficiency against V. alginolyticus. It was therefore concluded that low pH and high pH stress decrease the resistance of white shrimp L. vannamei against V. alginolyticus and decrease several parameters of the immune response.     

Calcium as a possible modulator of Kupffer cell phagocytic function by regulating liver-specific opsonic activity

Recently we described some properties of organ-specific serum opsonins which differentiate between liver- and spleen-specific opsonic activities, and reported that, on dialysis of serum, its liver opsonic activity is enhanced by 2- to 3-fold, whereas spleen-specific activity is reduced by 20-30% of that of control serum (Moghimi, S.M. and Patel, H.M. (1989) Biochim. Biophys. Acta 984, 379-383). This observation suggests that serum contains dialysable factors which regulate liver- as well as spleen-specific opsonic activities. Our results from EGTA-treated serum suggest that dialysable factor(s) could be divalent cations such as Ca2+, Mn2+, Mg2+ or Co2+, and among them, calcium may be the key regulatory factor for liver-specific opsonic activity. The regulatory mechanism of spleen-specific opsonic activity seems to be complex, since addition of dialysate or calcium or magnesium to the dialysed serum does not restore its activity; probably the removal of divalent cations has induced an irreversible conformational change in spleen-specific opsonin. In conclusion, we propose that the blood calcium concentration may play an important role in modulating hepatic phagocytic function by modifying liver-specific opsonic activity in serum. An increase in the physiological concentration of calcium will suppress and a decrease will enhance this opsonic activity.     

Function of reticuloendothelial system in splenectomised thalassemics

The activity of reticuloendothelial system (RES) was estimated in 19 patients with beta-thalassemia major, 20 +/- 4 years old, who had undergone successful splenectomy 6 +/- 5 years previously. The kinetics of 125I denatured human serum albumin in low and large doses was applied for this purpose and the parameters derived (effective RES blood flow-ERBF- and maximum phagocytic capacity-PCmax) were compared to those of nonsplenectomized thalassemics, detected in previous works, as well as to those of 13 healthy controls. In splenectomised thalassemics both parameters of RES activity were found significantly lower than those of nonsplenectomized patients (p less than 0.001). Compared to those of healthy controls, PCmax of splenectomised thalassemics was found not to be significantly different, while ERBF was significantly lower (p less than 0.001). No correlation was noted between the above parameters of RES function and the age of the patients, the age at which splenectomy was performed, the time lapsed since the operation, the amount of blood transfused to the patients after splenectomy or their serum ferritin levels. A pilot study performed in 6 out of the 19 splenectomised patients did not reveal any effect of blood transfusion on RES function parameters, by contrast to observations in nonsplenectomized thalassemics. The results of this study suggest that in splenectomised thalassemics the remaining RES reacts to the continuing hemolytic stimulus in a manner different than that of splenic RES of nonsplenectomized patients and account for, at least in part, the predisposition of the former group to infections.     

Studies of reticuloendothelial function in the mouse with model immune complexes. II. Serum clearance, tissue uptake, and reticuloendothelial saturation in NZB/W mice.

We have extended the findings in the accompanying paper by characterizing the serum clearance and tissue uptake of model soluble immune complexes and the saturation of the reticuloendothelial system (RES) by these complexes in normal mice and in mice with murine lupus (NZB/W F1 females). Adult NZB/W or young C3H mice were injected with radiolabeled stable site-specifically cross-linked mouse anti-DNP oligomers as model immune complexes to probe RES function. Blood clearance and uptake by liver, spleen, and kidney were unimpaired in NZB/W mice. To determine if the RES exhibits partial saturation in the NZB/W mice, we deliberately induced a state of RES blockade with heat-aggregated human gamma-glubulin (HAG). With increasing doses of HAG (1 to 4 mg/20 g body weight) both strains similarly showed a progressive increase in RES saturation as measured by reduced liver uptake of model oligomers. Recovery from saturation was complete by 120 min in both strains. At maximal liver saturation there was only a small increase in oligomer uptake by kidney or spleen, the majority of complexes remaining within the circulation. Thus, RES capacity for handling soluble model immune complexes appears unimpaired in NZB/W mice.     

Effect of aflatoxin B1 on the delayed type hypersensitivity and phagocytic activity of reticuloendothelial system in chickens

Efforts were made to see the effect of feeding aflatoxin B₁ at 0.3 ppm level on various aspects of the immune system in chickens. The birds were fed aflatoxin B₁ (AFB₁) mixed ration from 0 to 6 weeks of age and thereafter normal feed was given up to 12 weeks of age. The delayed type hypersensitivity reaction of these birds was assessed by contact sensitivity to Dinitrofluorobenzene. The dietary AFB₁ significantly suppressed the cell mediated immune response at all the three periods tested (30, 45 and 60 days of age). The toxin showed residual effect on immunity as the suppression of cell mediated immunity was maximum three days after the withdrawal of toxin from the feed. The effect of AFB₁ on the phagocytic status of reticulo endothelial system (RES) was assessed by colloidal carbon clearance test at various intervals. The residual effect of toxin was observed on RES too as phagocytic index of AFB₁ fed birds was significantly lowered up to 45 days of age.     

Resveratrol Suppresses Microcirculatory Disturbance in a Rat Model of Severe Acute Pancreatitis

The present study sought to understand the mechanisms of attenuation of severe acute pancreatitis (SAP) by resveratrol (RES). SAP was experimentally induced in rats by injection of 4 % sodium taurocholate in the retrograde pancreatic duct. Three study groups were evaluated: Group I (sham-operated animals), Group II (SAP animals), and Group III (SAP animals treated with RES at 20 mg/kg/body weight, 5 min after induction of SAP). The study outcomes were histopathologic changes and alterations in biochemical markers: plasma renin activity and levels of angiotensin II, endothelin, and nitric oxide in plasma. Biochemical markers were evaluated at 3, 6, and 12 h after induction of SAP. SAP was associated with significant (p < 0.05) histopathologic changes (saponification spots in the intraperitoneal cavity, severe pancreatic edema, blood congestion, varying degrees of necrosis, etc.), as well as with elevation of biochemical markers in blood plasma. RES treatment significantly (p < 0.05) attenuated changes of both histopathologic and biochemical markers induced by SAP. In conclusion, this study provides evidence that RES treatment is a promising therapeutic approach to suppress microcirculatory disturbance in SAP.