利用RNA干扰诱导产生的CD8+CD28-抑制性T淋巴细胞的免疫学特性

目的 探讨通过RNA干扰技术诱导产生的CD8+ CD28-抑制性T淋巴细胞(Ts细胞)的免疫学特性.方法 取SD大鼠骨髓,培养分离树突状细胞(DC),设计、合成主要组织相容性复合物(MHC)Ⅰ类小片段干扰RNA(siRNA),以MHC Ⅰ siRNA转染DC.先以Wistar大鼠肠系膜淋巴组织液刺激转染MHCI siRNA的DC,然后将DC与从SD大鼠脾脏分离得到的CD8+T淋巴细胞共同培养,通过磁珠法分离出Ts细胞.分别在由SD大鼠脾脏淋巴细胞(反应细胞)和Wistar大鼠肠系膜淋巴组织细胞(刺激细胞)组成的混合淋巴细胞培养体系中加入数量不等的Ts细胞,检测反应细胞增殖情况;分别以Wistar大鼠肠系膜淋巴组织细胞和卵白蛋白(OVA)刺激SD大鼠脾脏淋巴细胞,然后再按不同比例加入Ts细胞,检测各组脾脏淋巴细胞的增殖情况;在由SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入可溶性重组白细胞介素2(rrIL-2),观察IL-2对Ts细胞功能的影响;采用实时定量聚合酶链反应(PCR)测定Ts细胞中转化生长因子β(TGF-β和γ干扰素(IFN-γ)mRNA的表达,流式细胞仪和实时PCR检测Ts细胞上CD25分子的表达.结果 Ts细胞对SD大鼠脾脏淋巴细胞和Wistar大鼠肠系膜淋巴组织细胞之问的混合淋巴细胞反应(MLR)具有抑制作用,但对于SD大鼠脾脏淋巴细胞和OVA之间的MLR则无抑制作用.在SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入rrIL-2后,SD大鼠脾脏细胞的增殖并无明显增加(P＞0.05).与CD8+CD28+T淋巴细胞和CD8+ T淋巴细胞比较,Ts细胞的TGF-β和IFN-γ mRNA的表达量明显升高(P＜0.01,P＜0.05),而CD25的表达量明显降低(P＜0.05).结论 采用经MHC I siRNA干扰的DC能够诱导CD8+T淋巴细胞产生CD8+ CD28-Ts细胞;Ts细胞在体外具有免疫抑制特性,其免疫抑制作用不被外源性IL-2所逆转,且其免疫调节作用具有抗原特异性.     

Beta2-adrenoreceptor agonist-enhanced recovery of locomotor function after spinal cord injury is glutathione dependent

The beta2-adrenoreceptor agonist, clenbuterol, has been shown to spare spinal cord tissue and enhance locomotor recovery in an experimental model of spinal cord contusion injury. A likely mechanism of neurodegeneration following spinal cord injury involves generation of toxic levels of reactive oxygen species (ROS), e.g., O2-*, H2O2 and OH*, which overwhelm endogenous antioxidants. Agents, such as clenbuterol, that oppose neurodegeneration and improve recovery of locomotor function may possibly act by improving redox status. Consistent with reduced oxidative stress by beta2-agonist treatment following injury, prior blockade of synthesis of the antioxidant tripeptide, glutathione, with buthionine sulfoximine completely inhibited the ability of clenbuterol to enhance locomotor recovery and spare spinal cord tissue. Moreover, at 8 h postinjury, clenbuterol caused an increase in glutathione reductase activity, an indicator of cellular redox status, at the injury site that was also blocked by buthionine sulfoximine. Although clenbuterol improved locomotor recovery only when administered within a therapeutic window of several days postinjury, the accumulation of protein carbonyls in the spinal cord at 1 week postinjury, a consequence of ongoing ROS-mediated neurodegeneration, was also decreased by clenbuterol in a glutathione-dependent manner. Together, these results suggest that activation of beta2-adrenoreceptors during the acute phase of injury stimulates glutathione-dependent antioxidative processes, that lead to reduced oxidative damage and greater locomotor function as the injury evolves during the subacute and chronic phases.     

γ-射线对Smad 3基因剔除小鼠免疫功能影响的初步分析

目的:观察5．0 Gyγ-射线全身照射对Smad 3基因剔除小鼠免疫组织和外周血淋巴细胞亚群的影响及其机制。方法:应用TUNEL和FCM方法观察照射后不同Smad 3基因型小鼠胸腺、脾脏、淋巴结和外周血淋巴细胞亚群和淋巴细胞凋亡率的变化。结果:(1)照后Smad 3 / 、Smad 3 /-和Smad 3-/-三种基因型小鼠外周血WBC和淋巴细胞绝对数均迅速下降,直至照后10d均未恢复到对照组水平,而Smad 3-/-小鼠恢复速度较快。(2)三种基因型小鼠外周血CD3 细胞照后10d明显降低,分别约为对照组的40．2％、31．7％和49．3％,而CD8 细胞亚群的降低的更为明显,分别约为对照组的10．1％、10．6％和27．4％,Smad 3-/-小鼠降低的幅度小于Smad 3 / 和Smad 3 /-组。(3)小鼠胸腺和脾脏照射后T细胞亚群明显降低,而三种基因型小鼠中以Smad 3-/-小鼠各细胞亚群的降低幅度较小。(4)照后三种基因型小鼠CD19 细胞在免疫组织中降低的幅度不同,在淋巴结中CD19 细胞的降低更为明显。(5)照后小鼠淋巴细胞的凋亡率显著增加,然而无论是在外周血、胸腺,还是脾脏、淋巴结,三种基因型小鼠中均以Smad 3 / 淋巴细胞凋亡率的升高最为明显,而Smad 3-/-小鼠显示出相对的辐射不敏感性。结论:5．0 Gyγ-射线能诱发不同Smad 3基因型小鼠外周血和免疫组织淋巴细胞的明显凋亡,导致外周血WBC、淋巴细胞数量的明显减少和免疫细胞功能亚群的损伤;然而对多种指标观察的结果均首次表明, Smad 3基因敲除后导致了小鼠相对的辐射不敏感性,可能与TGF-β信号转导通路的抑制有关,其调节机制尚需进一步阐明。     

Different lymphocyte compartments respond differently to mitogenic stimulation after thermal injury.

Because of the association between the development of an immunocompromised state and an increased risk of infection, increasing attention has been focused on describing and characterizing the immune consequences of thermal injury. Results of human studies are largely based on the in vitro responsiveness of peripheral blood leukocytes, while splenocytes are generally used in the animal studies. Because the response of lymphocytes from different lymphocyte compartments may vary, we compared the responses of murine peripheral blood, splenic, Peyer's patch, and mesenteric lymph node lymphocytes to a battery of mitogens after thermal injury. Burn-induced immunosuppression was maximal in the splenic lymphocyte compartment, where the responses to all three test mitogens were depressed throughout the 28-day postburn study period. Although the PHA-induced mitogen response of lymphocytes from the other three lymphoid compartments remained suppressed throughout the study period, the response to the mitogens Con-A and PWM generally returned to normal or supranormal levels by the seventh postburn day, Therefore it appears that the effect of a thermal injury on lymphocyte function varies according to the lymphocyte compartment examined and the mitogen tested. These results raise the question of whether animal studies using splenic lymphocytes can be correlated with human studies performed on circulating blood lymphocytes.     

大鼠脊髓损伤致截瘫后肠道细菌移位的实验研究

目的：探讨大鼠脊髓损伤致截瘫后是否发生肠道细菌移位。方法：建立大鼠脊髓损伤性截瘫模型，以脊髓损伤性截瘫后12h、24h、48h大白鼠为实验组，未损伤脊髓的正常大白鼠为对照组。在无菌条件下，采集动物下腔静脉血进行内毒素定量测定和细菌培养，采集肝、脾、肠系膜淋巴结、肠腔内容物作细菌培养并进行菌种鉴定。取实验组和对照组各动物的肝、脾、肠系膜淋巴结、空肠、回肠进行病理切片HE染色检查，取空、回肠进行电镜检查。结果：大鼠脊髓损伤致截瘫后24h开始出现内毒素血症，截瘫后48h出现细菌移位。结论：大鼠脊髓损伤致截瘫后将发生肠道细菌移位，提示脊髓损伤截瘫的病人应尽早给予抗生素治疗。     

Progressive increase of CD4(+)/CD45RC(-) lymphocytes after allograft rat lung transplantation: a marker of acute rejection

BACKGROUND: Acute rejection remains one the most serious problems in lung transplantation. Although biopsy has been used for assessing the dysfunction of grafts, it is difficult to determine rejection at an early stage. Lymphocyte infiltration and activation play an important role in acute rejection of transplanted organs, and the dynamic change of lymphocyte subpopulations might be a marker to determine graft rejection after lung transplantation. METHODS: A rat lung transplant model was used. Graft-infiltrating lymphocytes in lung tissues were examined by means of histology, and isolated cells were analyzed by means of flow cytometry. Phenotypes of lymphocytes in the regional and remote lymph nodes, spleen, peripheral blood, and bronchoalveolar lavage fluid were also measured by means of flow cytometry. RESULTS: After allograft transplantation, increased lymphocytes were seen in allografts but not in isografts. In allografts the percentage of T cells increased from day 1 to day 5, whereas that of B cells was decreased. The CD4(+)/CD8(+) ratio decreased in allografts. The proportion of CD4(+)/CD45RC(-) cells increased in the allografts, which was mainly due to the increase of CD45RC(-) cells in the total CD4(+) cells. Similar changes were found in regional mediastinal lymph nodes but not in the mesenteric lymph nodes, spleen, or peripheral blood. Thus this is a specific response to lung allografts. Importantly, CD45RC(-) cells were significantly increased in the bronchoalveolar lavage fluid. CONCLUSION: Significant change of lymphocyte subpopulations is a sign of lymphocyte activation. Increased CD4(+)/CD45RC(-) cells in lung allografts could be an early marker of acute rejection, which can be examined by means of lung lavage and flow cytometry.     

Lymphocyte phenotyping to distinguish septic from nonseptic critical illness

BACKGROUND: Clinical signs and symptoms of sepsis are nonspecific and often indistinguishable from those of nonseptic critical illness. This ambiguity frequently delays the diagnosis of sepsis until culture results can confirm the presence or absence of an infectious organism. Lymphocyte phenotyping can be conducted rapidly and may provide information on the presence of infection before culture results are available. In this study, we hypothesized that lymphocyte phenotype can distinguish between septic and nonseptic critical illness. STUDY DESIGN: C57Bl/6 mice were subjected to either P aeruginosa pneumonia or lipopolysaccharide-induced acute lung injury (ALI). Animals were sacrificed 24 hours postinjury and splenic lymphocytes were harvested. Additionally, 13 patients in a surgical ICU were enrolled in the study. Whole blood was obtained and lymphocytes were isolated by density gradient centrifugation. Lymphocyte phenotype was identified through flow cytometry after labeling lymphocytes for CD3, CD4, CD8, CD20, CD40, CD69, and CD86 with fluorochrome-conjugated antibodies. RESULTS: CD69 expression on B cells and CD8+ splenocytes from septic mice was significantly increased compared with acute lung injury mice (p < 0.001 and p < 0.05, respectively). Similarly, CD4+ and CD8+ lymphocytes from septic patients had a two- to threefold increase in the expression of CD69 compared with nonseptic critically ill patients (p < 0.05). CONCLUSIONS: These data indicated that CD69 expression on lymphocytes may be useful in distinguishing between septic and nonseptic critical illness. Continued investigation into the expression of CD69 during sepsis is warranted.     

Changes in lymphocyte number and phenotype in seven lymphoid compartments after thermal injury.

Thermal injury is associated with dysfunction of host defense systems. The present study used flow cytometric immunofluorescence analyses to investigate changes in number and phenotype of lymphocytes in seven different lymphoid compartments at 2, 6, 12, 24, 48, and 60 days after 50% total body-surface area thermal injury in the rat. Relative to sham-injured control rats, at postburn day 2, significant lymphopenia was observed in the peripheral blood along with depletion of lymphocytes from the spleen and thymus. By day 6 after injury, lymphocytes in the bone marrow and cervical lymph nodes decreased significantly while numbers in the spleen and thymus remained depressed. Splenic and cervical node lymphocyte numbers normalized by day 12, the bone marrow and thymus numbers still were significantly lower than control, and a 6.5-fold increase in number of lymphocytes was observed in the nodes draining the burn wound, pooled axillary, brachial, inguinal, and lumbar lymph nodes. At day 24 after injury, the thymus and bone marrow virtually were depleted of lymphocytes, the mesenteric lymph nodes manifested a significant decrease, and lymphocytes in the nodes draining the burn wound continued to increase in number. This same pattern was maintained on day 48, but numbers of lymphocytes in the mesenteric nodes normalized. At day 60 after injury, lymphocyte numbers in all tissues were normalized, but the spleen and nodes draining the burn wound where increased numbers compared to control persisted. Cell-surface phenotyping was performed on all lymphoid tissues at all time intervals to determine the percentages of lymphocytes comprising the following subsets: Ia+ cells (B cells and activated T cells), T cells, T-Helper/Inducer cells (T-H/I), and T-Suppressor/Cytotoxic (T-S/C) cells. Although changes in lymphocyte subset percentages were complex, they could be divided grossly into two phases. First, all compartments showed significant phenotypic changes in the first six days after burn. With the exception of the nodes draining the burn wound and the blood, this was followed by a return towards normal on day 12. The second phase then ensued with significant phenotypic changes again occurring in most tissues from days 24 to 60 after injury. These studies demonstrate that burn injury results in dramatic alterations in lymphocyte numbers and subset percentages in different lymphoid compartments. Immune alterations observed following thermal injury may be due, in part, to a redistribution of the cellular elements responsible for generation of the immune response.     

Lymphocyte function in patients with interstitial cystitis.

To determine whether there is a primary immunological disorder involved in the etiology of interstitial cystitis, we compared the in vitro function of peripheral blood lymphocytes from 10 patients with interstitial cystitis to those from 10 healthy female controls. The lymphocytes were isolated and incubated in either cell culture medium alone, cell culture medium supplemented with the mitogen phytohemagglutinin or cell culture medium with autologous sterile filtered urine, the latter to detect any immunogenic potential of urine in this disease. We studied the relative proliferation of the phenotypes CD2, CD4, CD8, CD19, CD14 and CD56 by immunofluorescence and flow cytometry. To detect activation of T lymphocytes we also studied expression of HLA-Dr and interleukin-2 receptors. There was no difference in the rate of proliferation of the various lymphocyte phenotypes between the 2 groups in any of the culture media. Similarly, there was no difference between the 2 groups in the degree of lymphocytic activation after incubation. Furthermore, incubation in the presence of autologous urine caused no increase in activation above that seen in control cultures. We also assessed the production of the lymphokines interleukin-1, interleukin-2 and interferon-gamma in the supernatant fluid of the cell cultures. There was no difference in the production of these immunoregulatory cytokines between the control and patient groups, and again urine did not act as a mitogen in either group. We conclude from this study that there is no primary immunological disorder evident in patients with interstitial cystitis, and that the urine does not act as a stimulant to the immune system. This finding casts severe doubt on any theory suggesting that interstitial cystitis is an autoimmune disease or that urine contains an autoantigen causing the disease.     

Anti-inflammatory treatments during the chronic phase of spinal cord injury improve locomotor function in adult mice

Our previous data suggested that ongoing inflammation in the spinal cord 6 weeks following spinal cord injury was detrimental to locomotor function. Others have shown in the acute and sub-acute post-injury phase that microglial/macrophage activation and T regulatory cells are detrimental to recovery. Here, C57BL/6 mice with a moderately severe T9 contusion were injected intravenously daily with minocycline, which reduces microglial/macrophage activation, or with CD25 antibodies, which reduce T regulatory cell function, starting at 6 weeks after injury. Both anti-inflammatory drugs caused an improvement in hindlimb locomotor function over the 2-week treatment, as measured by the Basso Mouse Scale (BMS). The improvement was functionally important, with mice having problems with coordinated stepping (BMS ～6) before treatment to walking essentially normally (BMS >7) at the end of the treatment. The effects diminished within 1 week after termination of the treatments, suggesting an ongoing and dynamic inflammatory process. The area of white matter or the inflammatory markers CD68 for activated microglia/macrophages and CD45 for leukocytes were not different between the groups. These data suggest that inflammation during the chronic phase following spinal cord injury reduces conduction through the epicenter, possibly by release of cytokines, and is amenable to treatment for improved neurological function.     

Detection of immune responses against urinary bladder cancer in sentinel lymph nodes

OBJECTIVE: The lymphatic drainage from a tumour is received in the sentinel node where the immune system encounters tumour derived antigens. We investigated anti-tumoural lymphocyte function in sentinel nodes from patients with urinary bladder cancer. METHODS: In 14 patients undergoing cystectomy due to bladder cancer, radioactive tracer and blue dye were used to identify the sentinel node. Cell suspensions from the tumour, sentinel- and non-sentinel nodes and peripheral blood were analyzed by flow cytometry with antibodies against lymphocyte surface antigens and against the tumour cell marker cytokeratin-20. Reactivity against autologous tumour extract and the mitogen Concanavalin A was tested in proliferation assays with 3H-Thymidine incorporation. Lymphocytes were put in long-term culture with IL-2 and autologous tumour extract. RESULTS: Sentinel nodes were detected in 12 of the 14 patients. Antigen dependent proliferation in response to autologous tumour extract was detected in 6 patients, in 5 cases in sentinel nodes, in the remaining case in a non-sentinel node. Proliferation against Concanavalin A was vigorous in lymph nodes from all patients, whereas tumour infiltrating lymphocytes were unresponsive. Lymphocytes from sentinel nodes could be expanded in vitro. CONCLUSION: Tumour reactive lymphocytes are present in sentinel nodes draining human bladder cancers. These cells display immunologic function upon restimulation in vitro, and provide a promising source for expansion and subsequent adoptive T cell immunotherapy.     

Changes in mixed lymphocyte culture-reactive lymphocytes following alloimmunization of single lymph nodes in sheep.

When an "isolated" single lymph node is challenged with irradiated allogeneic lymphocytes, there is a change in the reactivity of the lymphocytes flowing out of the node when they are cultured in vitro in unidirectional mixed lymphocyte cultures (MLC) against the immunizing lymphocytes. These changes in the reactivity of the recipient lymphocytes are shortlived, follow a set time sequence in relation to the cell traffic changes accompanying the immune response, are the property of small lymphocytes and not blast cells, are exhibited by surface Ig-negative cells, and they are specific for the donor lymphocytes. It is suggested that antigen causes the selective retention of antigen-specific lymphocytes within the stimulated node followed by the proliferation and differentiation of large numbers of antigen-specific cells, which then leave the lymph node as small lymphocytes.     

Tempol, a nitroxide antioxidant, improves locomotor and histological outcomes after spinal cord contusion in rats

We have determined whether the nitroxide antioxidant, tempol, can oppose tissue loss and improve recovery of locomotor function following contusion injury of the spinal cord. Contusion injury was produced in rats at the level of T10 with a weight-drop device and locomotor recovery was determined with the 21-point Basso, Beattie and Bresnahan (BBB) scale. Locomotor function recovered progressively during the 6-week postinjury observation period and was significantly greater in tempol-treated (275 mg/kg i.p., 20 min postinjury) compared to vehicle-treated rats (final BBB scores: 9.1 versus 6.4). Similarly enhanced locomotor recovery was observed with doses of tempol in the range of 138-550, but not 69 mg/kg, and with injection at 48 h postinjury indicating a therapeutic time-window of at least several days. The extent of recovery correlated with measurements of sparing of spinal cord white matter in a region several millimeters in length extending rostrally from the contusion epicenter. In contrast, loss of gray matter was unaffected by tempol treatment. Since tempol acts by scavenging reactive oxygen species (ROS) such as superoxide and hydroxyl radicals, the improved locomotor recovery and spared spinal cord tissue following contusion injury provides evidence of a direct role of ROS-mediated neurodegeneration in spinal cord injury.     

Axonal remyelination by cord blood stem cells after spinal cord injury

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.     

Pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury

Lipid hydrolysis with subsequent production of eicosanoids and lipid peroxidation are two of the earliest potentially pathochemical events induced in spinal cord tissue by mechanical trauma. Although these membrane lipid disturbances are thought to contribute to the paralysis that occur subsequent to spinal cord injury, such a correlation has not been demonstrated directly. Consequently, the purpose of this study was to test the capacity of alpha tocopherol, the major lipid antioxidant in cellular membranes and a compound that limits the injury-induced lipid hydrolysis and peroxidation in spinal cord tissue, to promote functional recovery in a static loading model of spinal cord injury. After laminectomy, the L2 spinal cord of cats was compressed with 180 g for 5 min. For 5 days before injury and for 5 days postinjury, treated cats received orally 1000 IUD-alpha tocopherol acetate daily. Control cats were similarly injured but untreated. All cats were blindly evaluated weekly for 4 weeks for their neurologic recovery based on an 11 point behavioral scale that assessed walking, running, and stair climbing. By the second postinjury week, alpha tocopherol-pretreated cats demonstrated significantly better recovery than untreated controls. By 4 weeks, treated cats had recovered 72% of their preinjury function as compared with 20% for untreated controls, i.e., a 3.5-fold difference. These results strongly suggest that lipid peroxidation and/or hydrolysis is primarily involved in the genesis of posttraumatic paralysis and that alpha tocopherol exerts its protection of injured spinal cord tissue, at least in part, by its antioxidant and/or antilipolytic activity.     

脊髓损伤患者血清及脑脊液中髓鞘碱性蛋白的动态变化及意义

目的：研究急性脊髓损伤后血清、脑脊液髓鞘碱性蛋白（MBP）的动态变化与脊髓损伤程度、预后情况之间的关系。方法：ELISA法检测47例不同程度脊髓损伤患者伤后不同时间血清和脑脊液MBP含量,结合预后程度进行分析。结果：脊髓完全性损伤组病人伤后12h内即出现脑脊液MBP显著增高,伤后3d达高峰,并持续至伤后第14d,脊髓不完全性损伤组伤后,12h内也出现脑脊液MBP增高,高峰亦为伤后第3d,但各时间点增高幅度较完全性损伤组小,且伤后第14d时即恢复正常;脊髓震荡组不出现脑脊液MBP增高;各组血清检测结果与脑脊液结果平行。结论：血清和脑脊液MBP检测对于判断脊髓损伤程度、估计预后特别是骨髓损伤休克期选择正确的治疗方法具有积极的意义。     

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction

BACKGROUND: Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. METHODS: In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. RESULTS: The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. CONCLUSIONS: These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.     

FTY720 reduces T-cell recruitment into murine intestinal allograft and prevents activation of graft-infiltrating cells

BACKGROUND: Effective immunosuppression is a critical determinant of graft survival in small-bowel transplantation (SBTx). The present study was designed to determine the potency of FTY720, a newly synthesized immunosuppressant, in rat SBTx and examine the phenotype of graft-infiltrating cells to evaluate its effect on intestinal allografts. MATERIALS AND METHODS: A segment of intestine of Dark Agouti rats was transplanted heterotopically into Lewis rats. The recipients were treated with or without oral FTY720 at a dose of 1 mg/kg per day. Six days after surgery, peripheral blood lymphocytes and lymphocytes from the mesenteric lymph nodes, Peyer's patches, intraepithelial site, and lamina propria of the intestinal allograft were isolated. After the number of lymphocytes in each site was counted, the lymphocyte subpopulations in the intestinal allograft were evaluated by means of a FACScan flow cytometer using several monoclonal antibodies. RESULTS: FTY720 treatment significantly prolonged recipient survival and strongly inhibited rejection histologically in comparison with control rats. FTY720 immunosuppression resulted in a marked reduction of lymphocyte number in the graft epithelium and lamina propria and the proportion of CD8+ and CD25+ cells. FTY720 also significantly decreased T-cell receptors and increased B cells in the graft Peyer's patches. CONCLUSION: FTY720 promoted long-term SBTx recipient survival and maintained the architecture of intestinal allografts. FTY720 immunosuppression may be associated with a reduction of T-cell recruitment subsequent to the redistribution of lymphocyte subpopulations to control the proliferation and activation of graft-infiltrating cells in intestinal allografts.     

Induction of transplantation tolerance in rats by spleen allografts. III. The role of T suppressor cells in the induction of specific unresponsiveness

Heterotopic (WAG x AGUS)F1 spleen allografts survive indefinitely when transplanted to normal AGUS recipients and induce long-term donor-specific unresponsiveness. In this report, we have examined the immune reactivity of spleen graft recipients soon after transplantation, in an attempt to define the immunological mechanisms responsible for the induction of donor-specific unresponsiveness. Unresponsiveness develops as early as one week after splenic transplantation. T cells obtained from the recipient lymph node and spleen exhibit reduced mixed lymphocyte reaction responses to donor (WAG) but respond normally to third-party (PVG) stimulators. In contrast, T cells obtained from the spleen graft are unresponsive to both donor and third-party stimulators. Donor specific T suppressor cells (Ts) appear in the recipient's lymph node and spleen by one week posttransplantation--however, at this time antigen nonspecific suppressor cells predominate in the spleen graft. Only minimal cytotoxic T cell activity could be detected in the spleen graft, with the host spleen and lymph nodes being devoid of cytotoxic T lymphocytes. Sera obtained one or two weeks following splenic transplantation did not contain cytotoxic alloantibodies, and only a very weak response could be detected at one month. These data demonstrate that the unresponsiveness associated with the spontaneous acceptance of spleen allografts is correlated with the early induction of antigen specific Ts in recipient lymphoid tissue and the presence of nonspecific suppressor cells at the graft site.     

Equianalgesic Doses of Subcutaneous but Not Intrathecal Morphine Alter Phenotypic Expression of Cell Surface Markers and Mitogen-induced Proliferation in Rat Lymphocytes

Background: Surgical trauma and opioids are linked with suppression of immune function. Evidence suggests a probable supraspinal action of morphine in altering immune function, although the role of spinal systems have not been evaluated. Therefore, this study compared the effect of equianalgesic doses of subcutaneous and intrathecal morphine on lymphocyte proliferative responses and phenotypic expression of lymphocyte cell surface markers in rats.

Methods: Equianalgesic doses of subcutaneous (10 mg/kg) or intrathecal (30 micro gram, by a chronic intrathecal catheter) morphine were given twice for 5 h (time 0 and 2.5 h). Immediately after the 5-h period or 24 h after the initial injection, spleens were harvested and lymphocytes isolated. Mitogen-induced (phytohemagglutinin, concanavalin A, pokeweed, lipopolysaccharide) lymphocyte proliferation and monoclonal antibody labeling of cell surface markers (T cell, B cell, CD4+, CD8+) were then performed.

Results: Subcutaneous morphine acutely suppressed lymphocyte proliferation to the mitogens phytohemagglutinin, pokeweed, and concanavalin A by 37%, 21%, and 20%, respectively; however, proliferative responses returned to baseline within 24 h. Morphine treatment did not alter the response to lipopolysaccharide. The number of splenic lymphocytes also decreased, whereas the percentage of lymphocytes expressing the CD4+ marker (T helper/inducer cells) modestly increased. Intrathecal morphine did not alter lymphocyte proliferative responses, nor did it change phenotypic expression of cell surface markers.