Immunological effect of splenectomy in tumor-bearing rat

Immunologic merit and demerit of splenectomy were studied, using experimental model in rat. Animals: SD rat. Tumor: Metastasizing Rat's Mammary Tumor No. 1 (MRMT-1) originally induced by 3-MC administration to SD rat. Experimental study: Splenectomy was done on day before and after subcutaneous inoculation of 200mg of MRMT-1 at the back of 4 week-old female SD rats. Tumor growth following splenectomy and immunological competency of rat's peripheral lymphocytes, spleen cells and thymus cells was investigated and following results were obtained. When splenectomy was done pre-operatively or on the 2nd or 14th day after tumor inoculation, subsequent tumor growth was inhibited, however, when it was done on the 7th day after the inoculation, tumor enhancement and shortening of survival period were observed. When it was done on the 21st day, tumor growth was almost the same as in the rats without splenectomy. The results of cpm values and SI ratio of PHA-induced blastogenesis, and NK-activity indicated that immunological competency of spleen cells of tumor bearers was reduced during the early tumor-bearing period and the late tumor bearing period, while it was increased in the middle tumor bearing period. Tumor growth following splenectomy was considered to be controlled by immunological competency of spleen cells. Thus, it may not be unreasonable that tumor growth is inhibited on some occasions and it is facilitated on other occasions after splenectomy.     

The importance of the spleen for the immunosuppressive action of cyclosporine in transplantation

The spleen plays an important role in the response of the recipient's immune system to a primarily vascularized graft and cyclosporine treatment is known to alter this response. To investigate the interaction between the splenic immune response and CsA's immunosuppressive actions more thoroughly, Lewis recipients of Brown-Norway heterotopic heart grafts were treated i.p. daily with normal saline or with CsA doses of 0.75, 1.5, or 3.0 mg/kg/day from day 1 through day 50 or until rejection. Rats treated with 3 mg/kg were splenectomized intraoperatively (i.o.) or not splenectomized. Rats in subgroups of the other treatment groups were splenectomized i.o., on day 5, not splenectomized, or the recipient's spleen cells were reinfused after i.o. splenectomy. In non-CsA-treated rats, i.o. splenectomy (median survival time, [MST] = 11 days) and day 5 splenectomy (MST = 11 days) prolonged graft survival minimally in comparison with nonsplenectomized animals (MST = 7 days). Reinfusion of the spleen cells reversed this effect (MST = 7 days). Most interestingly, the immunosuppressive efficacy of 1.5 mg/kg of CsA (MST = 91 days) was reduced by day 5 splenectomy (MST = 24 days) and completely abolished by i.o. splenectomy (MST = 11 days). Spleen cell reinfusion partially restored the effect of CsA treatment (MST = 88 days). Since splenectomy resulted in a complete abrogation of the immunosuppressive efficacy of 1.5 mg/kg CsA, our results support the hypothesis that certain spleen cells augment immunosuppression by CsA. These findings provide additional evidence that the immune system's own regulation of its antigraft response can be an important component of the overall suppression of rejection that is associated with the use of certain immunosuppressive drugs.     

Immunological evaluation of splenectomy in tumor-bearing mice

The effect of splenectomy upon neoplastic outgrowth was examined after inoculation of methylcholanthrene-induced C3H/He murine tumors. Three days or 20 days after tumor inoculation, splenectomy resulted in significant retardation of tumor growth when compared with sham operation, while splenectomy 6, 9, 15 days after tumor inoculation did not alter the tumor outgrowth. These results suggest that spleen might have immunologically negative element in early or late stage of tumor burden. In fact, spleen cells from mice bearing MCA-F tumors for 3 days or 30 days nonspecifically facilitated the tumor outgrowth in Winn assay. The non-specific tumor-enhancing cells were radioresistant (700 rads), capable of phagocytizing carbonyl-iron and adherent to plastic dish suggesting those were tumor enhancing macrophages. On the other hand, spleen cells from tumor-bearing mice for 9 to 15 days specifically reduced the tumor growth in Winn assay, and those cytotoxic cells were radio-sensitive (700 rads) T cell population.     

Splenectomy and the induction of murine colon cancer

The influence of a functional spleen on induction and growth of cancer is unknown. Both beneficial and detrimental results have been observed in tumor-bearing hosts following splenectomy. We examined the effect of splenectomy, splenic autotransplantation, and splenic preservation on the induction and growth of 1,2-dimethylhydrazine (DMH)-induced murine colon cancer. Following splenectomy there was a significant increase in malignant tumors but no increase in benign tumors. To rule out the possibility that splenectomy increased the carcinogenicity of DMH by decreasing the capacity for DNA repair in colon cells, the units of 06-alkylguanine DNA alkyltransferase were measured in tumor-free and malignant colon tissue from both splenectomized and control rats. This repair protein was chosen because it is known to protect cells from the mutagenic effects of methylating agents. There was no significant difference in the alkyltransferase activity of tumor-free colon vs malignant tumor or between treatment regimens. Thus, the ability of the spleen to protect rats from the induction of malignant colon tumors induced by DMH is most likely due to preservation of immunologic surveillance in the host.     

The tumor--immunological significance of splenectomy for cancer therapy

In animal experiment, 5 X 10(5) MH-134 tumor cells were transplanted s.c. on the back of the C3H/He mice. Three, seven, 14 and 21 days after tumor transplantation, splenectomy or sham operation were performed and the tumor growth and survival days were examined in each group. As the results, the tumor growth was inhibited and the survival days prolonged not only in the group splenectomized three days but also 21 days after tumor transplantation. Clinically, the effect of splenectomy in combination with immunotherapy on cell-mediated immunity and the survival rates were studied in the gastric cancer patients of upper and middle stomach with 90 cases of stage III and 48 cases at stage IV, totalling 138 cases who underwent total gastrectomy during 1965 and 1981. Immunotherapy was conducted with immunomodulator levamisole at a daily dose of 150 mg, three consecutive days every other week. As a result, splenectomy was not effective for advanced gastric cancer at stage III and in the patients spleen should be retained for immunotherapy. Splenectomy for gastric cancer at stage IV, particularly in combination with immunotherapy, produced augmentation of cell-mediated immunity and longer survival as well. Complications caused by splenectomy were small.     

Effect of splenectomy on hepatic regeneration in rats

The effect of splenectomy on hepatic regeneration after partial hepatectomy was investigated in splenectomized and non-splenectomized rats. The results were as follows: The liver weight per body weight after partial hepatectomy increased significantly at 2 and 4 days in splenectomized group compared with non-splenectomized one. The rates of 3H-thymidine uptake in the regenerating liver cells at 24 hours after partial hepatectomy were more enhanced in splenectomized group than in non-splenectomized one. Thymidine kinase activity of the regenerating liver at 24 hours after partial hepatectomy in splenectomized rats was significantly higher than those in non-splenectomized rats. Both mitotic indices and labeling indices at 24 hours after partial hepatectomy in splenectomized rats were significantly higher than those in non-splenectomized group. The cells of mitotic stage were distributed mainly periportal area. From those results, it is suggested that the spleen has some inhibitory factors on hepatic regeneration after partial hepatectomy.     

Autotransplantation of Spleen Mitigates Drug-Induced Liver Damage in Splenectomized Mice

Purpose: The spleen presents numerous functions, including the production of immunoglobulins and blood filtration, removing microorganisms and cellular debris. The spleen also has anatomical and functional relationship with the liver, but there are few studies on this topic. The aim of this study was to assess the effect of splenectomy and autologous spleen transplantation on both filtering functions of spleen and acetaminophen-induced hepatotoxicity. Materials and Methods: Fifty-two BALB/c mice were randomized into four groups: splenectomized; splenectomy and splenic autotransplantation in the greater omentum; sham operated control; and non-operated control. At day 7th, 14th, and 28th after surgery, splenic filtration was assessed by counting Howell-Jolly bodies (HJB) and pitted red cells (PIT). The animals received 400 mg/kg acetaminophen by gavage at day 28^th and after 12 or 24 hours were euthanized for evaluation of splenic and hepatic morphology. Results: The splenectomized group demonstrated reduced filtration of HJB and PIT in all analyzes, while the autotransplanted group developed progressive recovery of function after the 14th day. At day 28 after surgery the implants showed similar histology in comparison to normal spleen. Liver histology showed more intense centrilobular necrosis in splenectomized group in comparison to the others, suggesting a protective role of spleen in acetaminophen-induced liver injury. Conclusions: Splenic implants showed structural and functional recovery, demonstrating the ability of autologous implant to rescue filtering function of intact spleen. Furthermore, the integrity of splenic function appears to influence liver morphology, since the presence of the splenic implants mitigated the effects of chemically-induced liver damage.     

Bidirectional effects of splenectomy on the growth of syngeneic tumor in mice

The effects of splenectomy on tumor growth following inoculation with a relatively large number of cells (1 X 10(7) ) and a smaller number of cells (5 X 10(5) ) of Meth I tumor were studied. When 1 X 10(7) tumor cells were inoculated, tumor growth in splenectomized mice was depressed, while tumor in sham-operated mice grew progressively. On the contrary, when 5 X 10(5) tumor cells were inoculated, the tumor take was lower in sham-operated than in splenectomized mice. The spleen cells from mice inoculated with either a large or small number of tumor cells, showed an equally potent cytotoxic activity, but no detectable suppressor cell activity. On the other hand, the activity of immunosuppressive factor was detected in sera from mice inoculated with 1 X 10(7) tumor cells, but not in those given 5 X 10(5) cells. The effect of splenectomy on tumor growth is, thus, bidirectional, depending on the dose of tumor cells inoculated.     

Experimental study on preventive effects of lung metastases using LAK cells induced from various lymphocytes--special references to enhancement of lung metastasis after laparotomy stress

In 5 week-old female SD rats, ether anesthesia or laparotomy for 30 minutes was done, followed by iv inoculation of 1 x 10(6) MRMT-1 cells. On day 14, number of lung metastatic nodules was examined. Preventive effect of LAK cells on lung metastases was also observed. LAK cells were induced from regional lymph node lymphocytes (LN), thymus cells (TC) and spleen cells (SC) of the rats on day 7 after tumor inoculation by incubation with 1 micrograms/ml of R-IL2 (TGP-3) for 4 days. 1) Lung metastases were noted in 100% after MRMT-1 intravenous inoculation, while number of lung metastatic nodules was reduced after iv administration of LAK cells. 2) When MRMT-1 cells were inoculated intravenously after laparotomy stress for 30 minutes, number of lung metastatic nodules was significantly increased and LAK cells reduced it significantly. 3) When R-IL2 sc administration for 4 days was accompanied, number of lung metastatic nodules was less than that of without R-IL2 substitution. 4) The antitumor effect of LAK cells was the strongest in LN-LAK, while it was the weakest in SC-LAK. In conclusion, usefulness of LAK cells for preventing lung metastases by surgical stress was indicated. This fact suggested that possibility of adoptive immunotherapy using LAK cells might be useful for prevention of lung metastases after laparotomy.     

Splenectomy Attenuates the Course of Kidney Ischemia-Reperfusion Injury in Rats

Introduction

Renal ischemia-reperfusion injury (IRI) initiates inflammatory response with synthesis of free oxygen radicals, chemokines, and cytokines which attract neutrophils and monocytes, which then differentiate into macrophages and dendritic cells, activating adaptive immune response. The spleen is the main source of both monocytes and lymphocytes. The aim of this study was to assess whether splenectomy performed before or upon IRI affects post-ischemic and long-term renal function.

Methods

Two weeks after right nephrectomy, the left kidney pedicle was clamped for 45 minutes in 24 rats. After the clip insertion, the spleen was removed in 12 animals and the remaining 12 rats underwent sham splenectomy. In the second experiment, splenectomy (n = 9) or sham procedure (n = 9) was performed simultaneously with right nephrectomy, 2 weeks before left kidney ischemia. The excretory function of the kidney was evaluated 48 hours and 7 days after ischemia. In the experimental model of chronic renal failure, 14 days before right nephrectomy, the prolonged 90-minute ischemia was induced in 32 rats with simultaneous splenectomy (n = 16) or sham procedure (n = 16). In long-term observation, the renal function and mortality rate was evaluated.

Results

Kidney function preservation was superior in rats that underwent splenectomy together with renal ischemia when compared to controls. This was further expressed with a 2 times lower mortality rate in splenectomized animals in 6 months observation after prolonged renal ischemia. Renoprotective effect was not observed when splenectomy was performed 2 weeks before IRI.

Conclusions

The results suggest a detrimental influence of the spleen on the development of renal IRI.     

Bidirectional effects of splenectomy on the growth of syngeneic tumor in mice

The effects of splenectomy on tumor growth following inoculation with a relatively large number of cells (1×10⁷) and a smaller number of cells (5×10⁵) of Meth I tumor were studied. When 1×10⁷ tumor cells were inoculated, tumor growth in splenectomized mice was depressed, while tumor in sham-operated mice grew progressively. On the contrary, when 5×10⁵ tumor cells were inoculated, the tumor take was lower in sham-operated than in splenectomized mice. The spleen cells from mice inoculated with either a large or small number of tumor cells, showed an equally potent cytotoxic activity, but no detectable suppressor cell activity. On the other hand, the activity of immunosuppressive factor was detected in sera from mice inoculated with 1×10⁷ tumor cells, but not in those given 5×10⁵ cells. The effect of splenectomy on tumor growth is, thus, bidirectional, depending on the dose of tumor cells inoculated.     

Splenectomy in the rat. Immediate and late effects on clearance of gram-negative bacteria and on monocyte activation as expressed by thromboplastin synthesis

Young rats (weight 190-200 g) were subjected to splenectomy or sham laparotomy and gram-negative bacteraemia was induced by caecal perforation or by injection of viable Escherichia coli intraperitoneally (10(9) bacteria) or intravenously (2 X 10(8]. Clearance of bacteria was significantly less in the splenectomized than in the sham-laparotomized rats, irrespective of the mode of microbial inoculation. Monocyte thromboplastin activity, shown to be a sensitive indicator of gram-negative bacterial presence, was heightened in the asplenic rats. When the infectious challenge was made 15 weeks postoperatively, however, no significant difference in bacterial clearance or in monocyte thromboplastin values was found between splenectomized rats and controls. Nor was increased susceptibility to gram-negative bacteria found in asplenic rats when both operation (splenectomy vs. sham laparotomy) and bacterial challenge were performed at a late age (weight 530-580 g). The postsplenectomy clearance of gram-negative bacteria thus seemed to be age-dependent.     

Absence of a relationship between the increased survival of skin grafts provoked by additional spleen transplantation and the detection of donor chimeric cells in rats

The goal of this study was to examine the relationship between the effects of spleen transplantation on the acceptance of a skin allograft from the same donor and the detection of donor chimerism in recipient tissues. Male Sprague-Dawley rats and female Wistar rats were used as donors and recipients, respectively. Four experimental groups were established: group C: only skin grafting was performed; group S: recipients were splenectomized before skin grafting; group 1: skin grafts were performed immediately after splenectomy of recipients and spleen transplantation, and group 2: splenectomy of the recipients and spleen grafting were performed 48 h before skin grafting. All animals were sacrificed 14 weeks after surgery. The rate of acceptance of skin grafts was significantly higher in animals that received both skin and spleen transplantations than in group C (p = 0.03) or in group S (p = 0.04). Detection of donor chimeric cells was significantly more frequent in rats after spleen transplantation than in group C (p = 0.03). However, the detection of chimeric cells was not related to the acceptance of skin grafts. To conclude, the beneficial effect of spleen auxiliary transplantation on allograft survival was not related to the detection of long-term chimerism in the recipient's tissues.     

Splenectomy, suppressor cell activity, and survival in tumor bearing rats

Young female rats of the Fischer strain were subjected to splenectomy at 10 weeks of age. Three weeks later, these animals and a comparable group of intact animals were inoculated subcutaneously with 3 × 10⁶ cells of the syngeneic Ward colon carcinoma. Tumor growth was recorded by serial measurement. Lymph node cells from control, intact tumor-bearing, and asplenic tumor-bearing animals were tested for reactivity in mixed lymphocyte culture (MLC) and for ability to suppress the allogeneic mixed lymphocyte reaction. At 3 weeks after tumor inoculation, lymphocyte reactivity in MLC was somewhat depressed in asplenic tumor-bearing hosts but severely depressed in intact hosts (P < 0.001); suppressor activity was demonstrable in intact hosts but not in asplenic hosts (P < 0.001). At 5 weeks, lymphocyte reactivity in MLC was severely depressed in both groups; suppressor cell activity was present in both groups and differences were no longer significant. Median survival of intact tumor-bearing animals was 97 days after inoculation (P < 0.0001 by the log-rank test), and 120 days in asplenic animals (P < 0.001). We conclude that both depression of lymphocyte reactivity and onset of suppressor activity are delayed in asplenic tumor-bearing animals. Host survival was significantly prolonged.     

The effects of heparin and splenectomy on survival and plasma fibronectin levels in rat peritonitis

Splenectomized patients are predisposed toward developing overwhelming bacterial infections. Administration of heparin is known to improve the survival of animals with intraabdominal sepsis and endotoxemia. The present study evaluates the effect of splenectomy and heparin administration on survival and plasma fibronectin (FN) levels in rats during acute bacterial peritonitis. Peritonitis was induced by cecal ligation and puncture (CLP) in 48 male Sprague-Dawley rats divided into four equal groups (12 each). Eight rats (66.7%) survived 10 days following CLP. When splenectomy was performed simultaneously (CLPS), the survival rate declined to 16.7% (P less than 0.05). Twenty units of heparin given subcutaneously daily for 5 days improved the survival rate to 66.7% following CLPS (P less than 0.05). When heparin was administered following CLP, the survival rate improved to 83.3% (not significantly higher than CLP alone). Plasma FN levels were measured by enzyme-linked immunosorbent assay (ELISA) on Days 0, 1, 2, 4, 7, and 10 following surgery. The plasma FN levels in splenectomized rats (CLPS +/- heparin) and nonsplenectomized rats (CLP +/- heparin) peaked on the first and second postoperative days, respectively. In comparing FN levels, no significant differences were found between the groups except on the second day--the CLPS + heparin group had a significantly lower FN level on Day 2 than CLP +/- heparin. This suggests that heparin confers protection from intraabdominal sepsis not only in animals with normal spleens but also in splenectomized animals. Plasma FN levels are not strongly influenced by heparin administration and concomitant splenectomy.     

The effects of splenectomy and splenic implantation on alveolar macrophage function

The effect of splenectomy on the ability of alveolar macrophages of young and adult rats to phagocytize Pneumococci, Types 3 and 14, and Pseudomonas aeruginosa was studied. Young animals showed a significant (15%) decrease in the phagocytosis of pneumococci type 14, 4 weeks after splenectomy. This depression increased to 30% in 6 weeks' time. Such depression was also noted when young splenectomized rat alveolar macrophages were challenged with Pseudomonas aeruginosa but not with type 3 pneumococci 6 weeks postsplenectomy. Three months following splenectomy in young animals, the rats were grown and they seemed to regain their normal phagocytic activity against pneumococci type 14. Adult rats also showed no alteration in their phagocytic activity against type 3 pneumococci. Autoimplantation of the spleen had a protective effect on the phagocytosis of type 14 pneumococci, and a nonsignificant effect on that of type 3. The present study postulates a modulatory role of the spleen on alveolar macrophage function. Splenectomy may cause the impairment of local lower respiratory immune function, making lungs vulnerable to specific bacterial invasion. Such splenic modulatory effect on alveolar macrophage phagocytic function seems to be age and antigen specific.     

Alterations of the immune system following splenectomy in childhood

After splenectomy due to blunt abdominal trauma, splenectomized children showed a restricted pattern of T-cell immunodeficiency compared to age and sex-matched normal children. Peripheral blood total (CD3) T-cell counts of 11 splenectomized children of 43%, double positive helper (CD4) inducer subpopulation (CD29) cell counts of nine splenectomized children of 7%, and phytohemagglutinin (PHA)-induced T-cell proliferation of 11 splenectomized children of 53,206 c.p.m. were significantly (p less than 0.05) lower than values of normal children (61% CD3 cells, n = 12; 13% CD4CD29 cells, n = 11; 107,832 c.p.m. PHA-induced proliferation, n = 12). The deficit of CD4CD29 cell numbers may be due to impaired maturation of these particular CD4 lymphocytes and may explain diminished PHA-induced proliferation in small children. The significantly higher B-lymphocyte counts of splenectomized children (21%, n = 11; 558 cells/mm3, n = 10) compared with 12 normal children (14%; 329 cells/mm3) may be due to loss of the reservoir function of the spleen.     

μ受体在抗神经生长因子抗体减轻大鼠骨癌痛中的作用

目的 评价μ受体在抗神经生长因子抗体(anti-NGF)减轻大鼠骨癌痛中的作用.方法 实验一健康雌性SD大鼠60只,体重200～220 g,随机分为4组(n=15):假手术组(S组)、假手术+anti-NGF组(SN组)、骨癌痛组(P组)和骨癌痛+anti-NGF组(PN组).P组和PN组于左侧胫骨上段骨髓腔内注射10μl Walker256乳腺癌细胞(1×105个)制备骨癌痛模型;S组和SN组于左侧胫骨上段注射PBS 10μl.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,SN组和PN组鞘内注射anti-NGF 10μg(用生理盐水稀释至10μl),S组和P组鞘内注射生理盐水10μl,2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定自发缩足次数(NSF)、热缩足潜伏期(PWL)和机械性痛阈(PWT).肿瘤细胞接种后21 d时,处死大鼠,取L4.5段脊髓背角和背根神经节,测定μ受体及其mRNA的表达.实验二健康雌性SD大鼠30只,体重200～220 g,随机分为2组(n=15):骨癌痛+anti-NGF组(PN组)和骨癌痛+纳洛酮+anti-NGF组(PNN组).于左侧胫骨上段骨髓腔内注射10μlWalker256乳腺癌细胞(1×105个)制备骨癌痛模型.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,PN组鞘内注射鞘内注入anti-NGF 10μg(生理盐水稀释至25μl);PNN组鞘内注射纳洛酮10μg(生理盐水稀释至25μl),0.5 h后,鞘内注射anti-NGF 10μg(生理盐水稀释至25 μl),2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定大鼠NSF、PWL和PWT.结果 实验一与S组比较,SN组NSF、PWL和PWT差异无统计学意义,SN组和PN组μ受体及其mRNA表达差异无统计学意义(P＞0.05),P组和PN组瘤细胞接种后13～21 d时NSF增加,PWL缩短,PWT降低,P组μ受体及其mRNA表达下调(P＜0.05或0.01);与P组比较,PN组肿瘤细胞接种后18～21 d时NSF减少,PWL延长,PWT升高,μ受体及其mRNA表达上调(P＜0.05或0.01).实验二与PN组比较,PNN组肿瘤细胞接种后18～21 d时NSF增加,PWL缩短,PWT降低(P＜0.05或0.01).结论 anti-NGF减轻大鼠骨癌痛与μ受体的激活有关.     

Splenectomy and renal allograft survival in the rat

The effect of splenectomy on renal allograft survival is not clear. In the rat, spleens isolated from recipients with functioning grafts have been shown to be a major source of cells that are capable of suppressing the rejection response (suppressor T lymphocytes). Thus the removal of the spleen in these allograft recipients could be detrimental to renal allograft survival. This study investigates this hypothesis, and looks for the presence of suppressor cells in other lymphoid organs apart from the spleen. In the rat renal allograft model, donor Lewis spleen cells given to DA recipients intravenously 1 week before transplantation of a Lewis kidney leads to indefinite allograft survival (median survival time (MST) greater than 100 days). Splenectomy before or after pretreatment with donor spleen cells failed to abrogate this effect (MST greater than 100 days). Experiments were performed in which cells or serum were prepared from long-term surviving splenectomized animals which had already been pretreated and transplanted, and then were injected into untreated recipients (adoptive transfer experiments). This was done to determine if cells capable of suppressing graft rejection were present in lymphoid organs outside the spleen in these splenectomized recipients. Thus the IV transfer of 10(8) lymph node cells harvested from splenectomized DA recipients with a long-term surviving LEW graft (LTS), into untreated but lightly irradiated (200 rad) DA recipients resulted in indefinite survival of a fresh Lewis kidney (MST greater than 100 days). In contrast, adoptive transfer of normal DA lymph node cells was ineffective (MST 13 days). Thus splenectomy is not necessarily detrimental to graft survival, as cells capable of preventing graft rejection are found in other lymphoid organs, such as lymph nodes, in splenectomized recipients.