The immune response to osteochondral allografts in dogs

Dog leukocyte antigen (DLA)-matched and mismatched, fresh and frozen (cryopreserved) osteochondral allografts of the proximal part of the radius were implanted orthotopically in beagles. The systemic and local (intra-articular) immune responses were monitored for eleven months using a 51chromium release assay with donor peripheral-blood lymphocytes as target cells. DLA-mismatched fresh grafts markedly and persistently stimulated the formation of antibody directed against donor cell-surface antigens, while DLA-matched grafts did not elicit systemically detectable antibody. The same general pattern was noted when antibody-dependent cell-mediated cytotoxicity was assayed. There was measurable cell-mediated immunity to donor cells from six weeks after surgery onward, although no distinct pattern or differences between experimental groups were noted. Higher titers of anti-DLA antibody were found in synovial fluid than in serum; in fact, synovial antibody was detectable when systemic antibody was not. Joints that received DLA-mismatched grafts had the highest titer of antibody and the intra-articular response was significantly reduced when the graft had been frozen.     

Mechanisms of immunological eradication of a syngeneic guinea pig tumor. II. Effect of methotrexate treatment and T cell depletion of the recipient on adoptive immunity

The influence of methotrexate on the development of immunity to the line 10 hepatoma was studied in guinea pigs. Chronic methotrexate treatment had no apparent effect on the ability of immune guinea pigs to suppress the growth of inoculated tumor cells. In contrast, the same methotrexate regimen inhibited the development of tumor immunity if started before the 8th day after immunization with a vaccine containing viable line 10 cells admixed with Bacillus Calmette-Guérin (BCG) cell walls. Thus, methotrexate selectively inhibited the afferent limb of the immune response. In adoptive transfer experiments, methotrexate-treated recipient guinea pigs were capable of being passively sensitized with immune spleen cells, indicating that the primary cell-mediated immune response of the recipient was not required for adoptive immunity. The contribution of recipient T cells in adoptive immunity was further investigated in guinea pigs deleted of T cells by thymectomy, irradiation, and bone marrow reconstitution. Despite demonstrable deficiency in T lymphocyte reactions, "B" animals were fully capable of rejecting tumors after transfer of immune cells. These results suggest that the expression of adoptive immunity was independent of recipient T cell participation. In addition, sublethal irradiation of immune spleen cells prior to adoptive transfer abolished their efficacy. Proliferation of transferred immune cells in the recipient may be essential for expression of adoptive immunity.     

Effects of antigenic competition between sperm autoantigens and ovalbumin upon humoral and cell-mediated immunity and the development of autoimmune aspermatogenic orchitis (AIAO) in guinea pigs.

The effects of antigenic competition between ovalbumin and sperm autoantigens have been studied in guinea pigs. There was an inhibition of antiovalbumin antibody production up to 60 days after immunization. The cell-mediated immunity against ovalbumin was also depressed at day 30. The simultaneous immunization with both antigens has no effect upon the humoral and cell-mediated immunity against spermatozoa. During the period of inhibition of the humoral and cell-mediated antiovalbumin response, the number of animals developing autoimmune aspermatogenic orchitis was diminished compared to those immunized with spermatozoa alone. Later on, there was no difference between the two groups. The transient inhibition of the immune response against ovalbumin can be explained by the particulate nature of the autoantigens. The sperm cells may be easily trapped by the dendritic reticular cells of the draining lymph nodes. This in turn could affect T cell recognition at early stages, orienting it predominantly toward the sperm autoantigens. At day 90 the situation returned to that present in animals immunized with ovalbumin alone.     

Bacillus Calmette-Guerin (BCG): Its fight against pathogens and cancer

Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. Since the turn of the 20th century BCG has been used as a vaccine for protection against Mycobacterium tuberculosis (Mtb) and has also been found to have protection against nontuberculosis related pathogens. Recently the role of “trained immunity” has been identified as a possible mechanism for BCG vaccine-mediated immunity to Mtb. Similarly, BCG has been used as an immunotherapy for bladder cancer for more than 40 years, and the underlying mechanisms for BCG-mediated anti-tumor activity is poorly characterized. Several studies have shown that multiple immune pathways contribute to the immune response, and efficacy of intravesicle BCG as a cancer therapy. It is vital that we integrate our understanding of BCG as a vaccine and as a cancer therapeutic to facilitate design of future studies in order to maximize the immunotherapeutic potential of BCG. In this review we will outline the role of BCG as a vaccine, the known immune pathways that are activated by intravesical BCG and outline a potential clinical study integrating BCG vaccination prior to intravesicle instillation of BCG.     

Immunosuppression following surgical and traumatic injury

Severe sepsis and organ failure are still the major causes of postoperative morbidity and mortality after major hepatobiliary pancreatic surgery. Despite recent progress in understanding the immune conditions of abdominal sepsis, the postoperative incidence of septic complications after major visceral surgery remains high. This review focuses on the clinical and immunological parameters that determine the risk of the development and lethal outcome of postoperative septic complication following major surgery and trauma. A review of the literature indicates that surgical and traumatic injury profoundly affects the innate and adaptive immune responses, and that a marked suppression in cell-mediated immunity following an excessive inflammatory response appears to be responsible for the increased susceptibility to subsequent sepsis. The innate and adaptive immune responses are initiated and modulated by pathogen-associated molecular-pattern molecules and by damage-associated molecular-pattern molecules through the pattern-recognition receptors. Suppression of cell-mediated immunity may be caused by multifaceted cytokine/inhibitor profiles in the circulation and other compartments of the host, excessive activation and dysregulated recruitment of polymorphonuclear neutrophils, induction of alternatively activated or regulatory macrophages that have anti-inflammatory properties, a shift in the T-helper (Th)1/Th2 balance toward Th2, appearance of regulatory T cells, which are potent suppressors of the innate and adaptive immune system, and lymphocyte apoptosis in patients with sepsis. Recent basic and clinical studies have elucidated the functional effects of surgical and traumatic injury on the immune system. The research studies of interest may in future aid in the selection of appropriate therapeutic protocols.     

Effect of laparoscopy on immune function

BACKGROUND: Laparoscopic surgery is believed to lessen surgical trauma and so cause less disturbance of immune function. This may contribute to the rapid recovery noted after many laparoscopic operations. Preservation of both systemic and intraperitoneal immunity is particularly important in surgery for sepsis or cancer and so an understanding of the impact of laparoscopy on immune function is relevant. METHODS: Literature on immunological changes following laparoscopy and open surgery was identified from Medline, along with cross-referencing from the reference lists of major articles on the subject. RESULTS AND DISCUSSION: Despite a few contradictory reports, systemic immunity appears to be better preserved after laparoscopic surgery than after open surgery. However, the local intraperitoneal immune system behaves in a particular way when exposed to carbon dioxide pneumoperitoneum; suppression of intraperitoneal cell-mediated immunity has been demonstrated in a number of studies. This feature may be clinically important and should be acknowledged when considering laparoscopic surgery in patients with malignancy or sepsis.     

Induction of an immune response to keyhole-limpet hemocyanin in surgical patients with anergy

Groups of surgical patients, classified as reactive or anergic on the basis of delayed type hypersensitivity skin testing with five recall antigens, were immunized with keyhole-limpet hemocyanin (KLH) alone or KLH together with mediators derived from leukocytes of a KLH immune donor cultured with antigen. Patients with anergy injected with KLH alone did not generate an immune response as judged by a T cell proliferative reaction performed 14 days after immunization. In contrast, leukocytes of patients with anergy immunized with KLH together with the mediators reacted to KLH in vitro in similar numbers and with a magnitude comparable to that given by reactive, hospitalized patients without anergy immunized with KLH alone. These results confirm and extend our previous observations showing that anergy defined as a lack of cell-mediated immunity to recall antigens such as purified protein derivative extends to the generation of a systemic immune response to a neoantigen such as KLH and mediators that could restore a state of delayed hypersensitivity to purified protein derivative could also be instrumental in inducing cell-mediated immunity de novo when injected together with the antigen.     

Immune response and non-specific immunotherapy in melanoma

Tumour-associated antigens and specific immune response to these antigens have been clearly demonstrated in patients with melanoma. Impaired cellular immune reactions are evident in patients with progressive and disseminated disease. Immunotherapy is used to heighten the host resistance and hence prevent recurrence and spread of the tumour. BCG vaccine has been used to produce nonspecific stimulation of the immune system. Preliminary evaluation of the effect of adjuvant systemic BCG therapy suggests that the treatment may have a beneficial effect on patients with early melanoma.     

The effect of the route of administration of Bacillus Calmette-Guerin on regional immunity

In conclusion, under the conditions of the study, oral BCG caused a significant immunopotentiating effect in mesenteric lymph nodes. During the time period studied, the response was regional. However, the most consistent immunopotentiating effect was that shown by the hind-limb injection of BCG. Although there was a more significant regional response from hind-limb injection, there was also a definite tendency toward systemic immunopotentiation. Intraperitoneal BCG did not produce consistent immunopotentiation.     

Impaired immune response by isoniazid treatment during intravesical BCG administration in the guinea pig.

At present, isoniazid (INH) is being used prophylactically to reduce the side effects of intravesical BCG therapy for superficial bladder cancer, although it is not clear whether or not this reduces the antitumor efficacy of BCG. In this study the impact of INH treatment on the immune response after repeated intravesical BCG administration was investigated in guinea pigs. INH was given on the 3 days around each BCG instillation. We found that the administration of INH severely impaired the immunological effects of BCG. The induction of mononuclear cell infiltration in the bladder wall was reduced. Enlargement of the regional lymph nodes (weight and number of cells), and increase of MHC Class II expression on the lymph node cells, normally observed after intravesical BCG administration, were inhibited by INH. Systemic immunity, measured by the DTH reaction in the skin to PPD, was also diminished due to the combined treatment of BCG with INH. When INH was administered during the last 4 of 6 BCG instillations, the immune response to BCG was still impaired. A five-fold increase of the dose of BCG did not overcome the effect of INH. INH probably did not exert a direct suppression of the immune system of the guinea pig as the DNCB skin reactivity was not influenced. Although INH concentrations in the urine were high at the onset of the instillation, in vitro experiments indicated that the effect of INH may not be caused by killing of the BCG organisms shortly after application in the bladder. In conclusion, our data in guinea pigs suggest that the use of INH may impair the immune response to intravesical BCG. As this response may be important for the antitumor effect of BCG, urologists should be cautious with the prophylactic use of INH. The influence on the antitumor efficacy is now investigated in man.     

Inhibition of cell-mediated microcytotoxicity and stimulation of mixed lymphocyte reactivity by mouse pregnancy serum.

Blocking activity of mouse pregnancy serum was investigated using a well defined one-way mixed lymphocyte culture and a cell-mediated microcytotoxicity test. Allogeneic (C57BL (H-2b) X CBA/Ca (H-2k)) and syngeneic (C57BL (H-2b) X C57BL (H-2b)) pregnancy and postpartum sera were found to stimulate rather than inhibit the mixed lymphocyte response. In contrast, the same sera were able to abrogate target cell killing by hyperimmune lymphocytes in a cell-mediated microcytotoxicity test. Although inhibitory activity was present in both allogeneic and syngeneic 18-day pregnancy sera, indicating a non-specific effect, only allogeneic postpartum sera had significant blocking activity when compared with either the equivalent syngeneic group or the normal serum control. The specificity implied by this gained support from preliminary third-party experiments. Blocking activity was found in primiparous as well as multiparous sera. These results provide further evidence for the existence of maternal serum-blocking factors which may operate to protect the conceptus against maternal cell-mediated immunity during pregnancy, and indicate that they may act at the efferent rather than the afferent phase of the immune response.     

Immunostimulation with ibuprofen in chronic osteitis. An experimental study

The present investigation shows that chronic posttraumatic osteitis is associated with markedly elevated prostaglandin E2 (PGE2) serum levels. Synthesis of PGE2 is inhibited by nonsteroidal antiinflammatory drugs, such as ibuprofen, which block cyclooxygenase. Osteitis patients showed significantly decreased PGE2 serum levels during treatment with ibuprofen (1200 mg/day). In addition, a significant increase in soluble interleucin-2 receptor serum levels was observed. PGE2 has significant immunosuppressive effects on cell-mediated immunity. On the other hand, soluble interleucin-2 receptor is a reliable indicator of immune system activation. Ibuprofen seems to be a potent immunostimulating agent for the treatment of chronic posttraumatic osteitis, which is characterized by depression of cell-mediated immunity.     

Markers for cell-mediated immune response are elevated in cerebrospinal fluid and serum after severe traumatic brain injury in humans

The brain is believed to be an immunologically privileged organ, sheltered from the systemic immunological defense by the blood-brain barrier (BBB). However, there is increasing evidence for a marked inflammatory response in the brain after traumatic brain injury (TBI). Markers for cellular immune activation, neopterin, beta2-microglobulin (beta2M), and soluble interleukin-2 receptor (sIL-2R), were measured for up to 3 weeks in cerebrospinal fluid (CSF) and serum of 41 patients with severe TBI in order to elucidate the time course and the origin of the cellular immune response following TBI. Neopterin gradually increased during the first posttraumatic week in both CSF and serum. Concentrations in CSF were generally higher than in serum, suggesting intrathecal release of this marker. beta2M showed similar kinetics but with higher serum than CSF concentrations. Nonetheless, intrathecal release as assessed by the beta2M index could be postulated for most of the patients. The mean levels of sIL-2R in both CSF and serum were elevated during the whole study period, serum concentrations being up to 2 x 10(4) times higher than in CSF. No significant intrathecal production of sIL-2R could be detected. The present data shows that severe TBI leads to a marked cell-mediated immune response within the brain and in the systemic circulation. In the intrathecal compartment the activated cells appear to be predominantly of the macrophage/microglia lineage, while the immune activation in the systemic circulation seems to involve mainly T-lymphocytes.     

Analysis of antibody production induced by allogeneic tumor cells inoculated into the anterior chamber of the eye

Humoral immune responses to alloantigens on P815 mastocytoma cells placed into the anterior chamber of murine eyes were studied. The results show that: (1) the amount of specific serum antibody produced by recipients of intracamerally injected P815 cells is directly proportional to the degree of immunogenetic disparity between the recipient and the injected cells; (2) destruction of allogeneic intraocular tumors is mediated, at least in part, by specific antibodies; and (3) retention of allogeneic tissue within the anterior chamber for 3 to 4 days after inoculation is essential to the elicitation of specific antibody formation. We conclude that alloantigens introduced into the anterior chamber are presented to the systemic immune apparatus in a very unique manner resulting in the preferential stimulation of specific antibody production without awakening specific cell-mediated immunity.     

Immunologic abnormalities in head and neck cancer

We consider this study only a preliminary exploration into the importance of host immunity in neoplasms of the head and neck. It is evident that a dysfunction of the immune system did occur in these patients, and although regional in origin, these tumors did effect some alterations in the systemic cellular immune function as measured by DNCB reactivity. Sequential variations in the degree of effect on immunity occurred and these alterations were correlated with the patient's subsequent clinical course. Conversion from positive to negative reactions was observed if control of the tumor was not achieved. Therefore, it is very unlikely that a pre-existent defect in immunity was the principal determinant of disease progression.It must be emphasized that these evaluations are concerned with nonspecific immunity and not with immunity to tumor-specific antigens. Certainly both specific and nonspecific immune reactions are very important in other non-neoplastic bacterial and viral diseases. Studies are now under way to evaluate tumor-specific immune reactions and to determine if nonspecific stimulants of immunity such as BCG can alter the immunologic reactivity and, therefore, affect the clinical course of patients with neoplasms of the head and neck.     

重组hIFN-α-2b-BCG对小鼠原位膀胱肿瘤免疫效应的研究

目的 探讨重组hIFN-α-2b-BCG对原位膀胱肿瘤小鼠体内抗肿瘤免疫反应及其作用机制.方法 构建C57BL/6原位膀胱肿瘤小鼠模型,采用流式细胞仪对小鼠膀胱灌注治疗后的淋巴细胞亚群进行分析,并测定小鼠血清中mTNF-α和mIL-12的水平,了解小鼠全身免疫状况.肿瘤组织冰冻切片进行T细胞亚群的免疫组织化学分析,免疫组化检测肿瘤Fas表达,了解膀胱局部免疫反应. 结果 重组BCG灌注治疗后小鼠外周血CD4+ T细胞比例明显增高,CD8+ T细胞比例无明显变化,CD4+ /CD8+比例为2.63,与野生BCG组(2.10)比较差异有统计学意义(P＜0.05).荷瘤小鼠外周血中Th1型细胞因子mTNF-α和mIL-12灌注治疗后比PBS对照组大幅提高,重组BCG组mTNF-α为806 pg/ml,mIL-12为860 pg/ml,与野生BCG组及野生BCG联合IFN组比较差异无统计学意义(P＞0.05).重组BCG组瘤组织内CD3、CD4和CD5检测强阳性,显著高于PBS对照组(P＜0.05),重组BCG组和野生BCG加IFN组瘤组织CD4+、重组BCG组CD8+检测值显著高于野生BCG组(P＜0.05).BCG灌注后荷瘤小鼠膀胱肿瘤表达Fas明显高于PBS对照组(P＜0.05). 结论 重组hIFN-α-2b-BCG具有在小鼠体内调节系统及局部免疫能力的作用,纠正荷瘤小鼠淋巴细胞亚群的比例失调,增强局部淋巴细胞浸润,具有增强Th1型细胞因子产生作用,上调荷瘤小鼠Fas的表达,诱导对膀胱肿瘤细胞的免疫攻击.

Abstract：

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.     

Kupffer cell blockade increases mortality during intra-abdominal sepsis despite improving systemic immunity

The effect of Kupffer cell (KC) blockade on systemic immunity during intra-abdominal sepsis was evaluated. Gadolinium chloride, a rare earth metal, reduced KC phagocytosis by 75% when it was given to BALB/c mice for 2 days. Thereafter, control mice and mice with KC blockade underwent either a sham operation or a cecal ligation and puncture. As indicators of systemic cell-mediated immunity, delayed-type hypersensitivity responses to soluble antigen and cellular alloantigen were measured 24 hours after the abdominal operations. The activation of KCs was assessed by their in vitro interleukin 1 production. Control septic mice were profoundly immunosuppressed and demonstrated marked KC activation. Septic mice with KC blockade, however, demonstrated less systemic immune hyporesponsiveness and significantly reduced KC activation, but died more rapidly. We concluded that despite apparent improvement in systemic immunity by KC blockade during intra-abdominal sepsis, the resulting impairment in functional phagocytic integrity predisposes to significantly higher mortality.     

Interference of modern antibacterials with bacillus Calmette-Guerin viability

PURPOSE: We determine the effects of modern antibacterial chemotherapeutics on bacillus Calmette-Guerin (BCG) viability, particularly those of cycloserine, which has been recommended for treating BCG induced sepsis. MATERIALS AND METHODS: The minimal inhibitory concentrations of 31 antibacterial drugs against Connaught BCG strain were determined in vitro by the radiometric BACTEC 460TB method. Minimal inhibitory concentrations were compared with the drug concentrations achievable in blood and urine to estimate systemic or intravesical susceptibility. Susceptibility testing of cycloserine was performed with Connaught, Tice and RIVM BCG strains, using the modified proportion method on Lowenstein-Jensen agar. RESULTS: Connaught BCG was susceptible to quinolones in systemic infections but resistant to beta-lactams, macrolides and some aminoglycosides. It was resistant to pyrazinamide but showed good susceptibility toward the other antituberculosis drugs tested. All 3 BCG strains analyzed were resistant to cycloserine. Most antibacterials may interfere with BCG in the bladder because of high urinary recovery. CONCLUSIONS: Antibacterial drug interference with BCG viability should be avoided during intravesical instillation therapy. In cases of severe complications quinolones rather than cycloserine may be given in addition to standard triple antituberculosis drug therapy or if one of these drugs is not tolerated. Our data may contribute toward enhancing the therapeutic safety and efficacy of intravesical BCG immunotherapy by the appropriate use of antibacterial drugs.     

Role of interleukin-8 in onset of the immune response in intravesical BCG therapy for superficial bladder cancer

In intravesical therapy for superficial bladder carcinoma urothelial cells may, through the production of cytokines, contribute to the bacillus Calmette-Guérin (BCG)-induced local immunological reaction and associated antitumor efficacy. The aim of this study was to investigate such a role for the neutrophil-attracting cytokine interleukin-8 (IL-8). The appearance of IL-8 in patients' urine after BCG therapy was compared with BCG-induced IL-6 and IL-2 and the stability of IL-8 in urine was tested. Compared to IL-6 and IL-2, a rapid induction of IL-8 was observed, occurring after the first BCG instillation. Urinary IL-8 was highly stable, even after 24 h incubation at 37°C. The IL-8 concentration after the first instillation seemed to be associated with subsequent development of an immune response. Consequently, IL-8 seems an attractive candidate for investigation of its prognostic value for a clinical response to BCG therapy.