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The banking of arterial allografts in the United kingdom. A technical and clinical review.

The standard graft materials for reconstructive vascular surgery are autologous vessels and synthetic prostheses. However, the clinical results are unsatisfactory when the diameter of the required graft is less than 6 mm and attention has therefore been directed to the possibility of using vascular allografts when autologous conduits are not available. In this review, we consider the evidence that cryopreserved allogeneic vessels might meet this need. Other factors being equal, the literature suggests that arteries will give superior performance to veins when grafted into the arterial circulation. Conventional cryopreservation techniques have been developed and these can provide structurally intact and functional small elastic arteries in animal experiments. We conclude that cryopreservation per se produces only modest injury which may well be recoverable following grafting. However, both experimental and clinical studies suggest that immunological injury is a greater problem. Nevertheless, the general indication is that appropriate risk-benefit analysis in individual cases will justify the clinical use of cryopreserved allograft vessels. Immunosuppression with careful immunological monitoring may be justified in the more desperate clinical situations. Finally we conclude that the existing tissue bank network in the United Kingdom is well placed to provide surgeons with such tissue.

Journal Article↗

IL-2-mediated augmentation of NK-cell activity and activation antigen expression on NK- and T-cell subsets in patients with metastatic melanoma treated with interferon-alpha and DTIC.

Considering that well-defined and comprehensive immunological monitoring is the basis for the evaluation of the obtained immunmodulatory effects, we evaluated NK-cell activity, the number of CD3+ CD4+, CD3+ CD8+ T cells and CD16+ CD56+ NK cells, as well as the expression of activation antigens, CD69, CD38 and HLA-DR on CD56+ NK cells, CD8+ and CD3+ T cells, simultaneously with IL-2 and TNF-alpha production, during chemoimmunotherapy with dacarbazine (DTIC) and interferon-alpha (IFN-alpha) in 39 patients with metastatic melanoma. In the first cycle of therapy, there was a significant rise in NK-cell activity, CD4+ T helper cell number, CD4/CD8 T-cell ratio, and the expression of activation antigens CD69 and CD38, on NK and T cells, respectively. However, in the following cycles there was a significant increase only in activation antigens without an increase in the percent or activity of NK cells. The early, but transient, immunopotentiation, present only in the first cycle of combined DTIC and IFN-alpha therapy, suggests that, in spite of increased IL-2 level, associated with augmented NK-cell activity, this therapy has a limited effect probably owing to the adverse effect of persistently high level of TNF-alpha in metastatic disease.

Adult↗

Generation and testing of a highly specific anti-serum directed against porcine endogenous retrovirus nucleocapsid.

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.

Amino Acid Sequence↗

Orbital inflammatory disease: a diagnostic and therapeutic challenge.

The spectrum of orbital inflammatory disease (OID) ranges broadly from specific disease diagnoses, for example, Wegener's granulomatosis or sarcoidosis, to nonspecific inflammation which may involve one or multiple structures of the orbit. Mimics of idiopathic OID must be considered in a comprehensive differential diagnosis and include malignancies, congenital mass lesions, infectious diseases, and occult or distant trauma. Idiopathic OID may be secondary to an underlying systemic inflammatory disease, which must be diagnosed in order to develop a comprehensive therapeutic plan, or may represent localized pathologic processes without systemic involvement. Evaluation of the patient with suspected OID must include a careful history, physical examination, directed laboratory, and radiologic studies, and may sometimes require tissue for diagnostic studies. Therapeutic options for inflammatory diseases are expanding as biologically targeted agents become available that act on specific segments of the inflammatory cascades. The purpose of this paper is to provide a framework for the evaluation and management of patients with the spectrum of diseases known as OID and to discuss some of the new advances in immunologic monitoring and targeted immune therapies that will likely play an increasingly important role in the care of these patients.

Diagnosis, Differential↗

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 microl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-gamma. Absolute numbers of CD8(+) and CD8(-) (putative CD4(+)) T-cells, NK cells (CD8(+)/CD3(-)) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8(-) and CD8(+) T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-gamma. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8(lo)/CD3(lo-int) lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.

Acute Disease↗

Effects of subcutaneous interleukin-2 therapy on phenotype and function of peripheral blood mononuclear cells in human immunodeficiency virus infected patients.

In the context of clinical therapy with recombinant human interleukin-2 (IL-2), we monitored immunological alteration in 10 human immunodeficiency virus type-I (HIV-1)-infected individuals, on stable antiretroviral therapy, who had a CD4+ cell count between 200 and 500 cells/mm3. Subcutaneous IL-2 was prescribed thrice weekly (at a dose of 3 x 10(6) IU) for 24 weeks and the patients were followed-up for 32 weeks. IL-2 treatment induced an increase in the CD4+ percentage (P<0.001) and CD4+ cell count (P<0.009). Furthermore. natural killer (NK) cell activity was increased (P<0.001) at week 8 of treatment, whereas lymphokine-activated killer (LAK) cell activity showed a transient, nonsignificant increase at week 8 and was reduced (P<0.001) at 32 weeks. However, the cytotoxic T-lymphocyte (CTL) activity decreased against HIV antigens, and the proliferative response to Candida, IL-2 and phytohaemagglutinin (PHA) declined during the first 8 weeks (P<0.05) and returned to baseline levels after 32 weeks. The HIV RNA level did not change during IL-2 therapy; however, after 8 weeks of follow-up a significant increase (P<0.001) in viral load was observed. In

Adult↗

Vaccine- and immune-based therapy in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) would appear to be an ideal target of T-cell-mediated responses against the cancer cell. The cancer arises in cells that can act as antigen-presenting cells (APCs), CLL cells express tumor antigens, and the cells can be a target of the allogeneic T cells in a graft-versus-leukemia effect. Despite these potential benefits, immune responses against CLL cells have been difficult to elicit. CLL induces immune defects in the host, the tumor cells are inefficient APCs, and therapies given to patients with CLL are themselves immunosuppressive. Successful vaccination approaches in this disease will require steps to overcome these difficulties, including steps to improve the immune defects in this disease, identification of the targets of the immune response to monitor immunologic responses, and improved presentation of antigen.

Animals↗

Allogeneous transplantation of the mitral valve. An open question.

To overcome the disadvantages of mechanical valves and bioprostheses, especially in the mitral position, transplantation of an allogeneous mitral valve was performed in 3 patients. One transplant had to be removed 8 weeks postoperatively, most probably due to maladjustment of the anterior leaflet during surgery. The histology of this specimen showed not clear signs of rejection. In 2 patients, echocardiography showed a normal motion pattern of the mitral valves 6 months postoperatively. Regurgitation was less than 10% of the total stroke volume as calculated by videodensitometry. Both patients were in excellent clinical condition. Mitral valve transplantation can be performed with excellent short-term results. Further studies including immunologic monitoring are necessary to evaluate the long-term behavior of the transplant.

Adult↗

Direct detection of cellular immune responses to cancer vaccines.

The evaluation of cancer immunotherapy is predicated on the hypothesis that markers of tumor antigen-specific T-cell immunity will cone-late with clinical efficacy. Establishing which candidate vaccines should enter large-scale clinical trials will necessitate optimal application of immunologic monitoring assays. Evidence suggests that available techniques are adequate for the direct detection of clinically significant antigen-specific T-cell responses from tissue specimens. To achieve this goal, it is important to have an understanding of individual methods and their limitations, to include appropriate control antigens in the monitoring strategy, and to incorporate statistical considerations into the design and analysis of such studies.

Cancer Vaccines↗

Immune status during albendazole therapy for hydatidosis.

Immunological monitoring of cystic hydatid patients undergoing medico-surgical management has revealed that it is possible to assess individual host responses throughout therapy and subsequently be guided by the results obtained. The analysis has involved the demonstration of variation in haemagglutination antibodies, specific IgG and IgM immunoglobulins and circulating immune complexes before, during, and months after albendazole therapy.

Adolescent↗

Immune reconstitution following stem cell transplantation.

The period of immune deficiency following stem cell transplantation (SCT) results in significant morbidity and mortality. Whilst supportive therapies have partially improved the outcome of infective episodes, disease relapse remains a considerable obstacle to improvement in overall outcomes. An increased understanding of the importance of the immune system in preventing relapse has derived from studies in the allogeneic setting. Increasing awareness of autologous anti-tumor responses has also focused interest on enhancing such activity. Successful application of some of these newer therapies, such as tumor vaccination approaches, may depend critically on reconstitution of functional immune reactivity. Whilst early recovery of innate immunity (myeloid series and natural killer (NK) cells) results in reconstitution of protective immunity against many bacterial pathogens, both the absolute levels and function of T and B lymphocytes remain abnormal for many months or years. Incorporation of T-cell depletion, choice of graft type (both donor and source), development of graft-vs.-host disease and level of residual thymic activity can all influence aspects of the reconstitution process. Advances in immunological monitoring are providing new insights, particularly into the recovery of specific T-cell subsets. This review focuses mainly on recent advances in the understanding of immune reconstitution in the allogeneic setting.

Animals↗

Immunological surveillance in a pseudorabies quarantined herd using gilts and their progeny as sentinels.

Specific pathogen free gilts and their progeny were evaluated to use as sentinels in a pseudorabies virus (PRV) infected herd by immunologically monitoring for PRV seroconversions. Time intervals targeted were pre- and post-PRV vaccinations, herd exposure, and farrowing to finishing. Post-PRV vaccinations, gilts showed low PRV lymphocyte stimulation and humoral responses. Following herd exposure, control gilts PRV seroconverted and PRV vaccinated gilts increased (2 to 4 times) in virus neutralization (VN) titers. Sixty-seven percent (4/6) of the progeny from a control gilt were PRV seropositive at finishing. Progeny from PRV vaccinated gilts were depleted of passive immunity by week 7, and were seronegative until week 9. At finishing 47% (14/30) of them were PRV seropositive indicating exposure to PRV. The VN test was not sensitive enough to detect weak positive serums, noted as positives by latex agglutination (LA) test, ELISA, and Western blots. The gilts and progeny detected PRV, respectively, in the herd housing quarters and in the farrow to finish facilities. A strategy for future sentinel experimental surveillances using primarily the LA test is proposed.

Agglutination Tests↗

Long-term follow-up of patients with chronic inflammatory demyelinating polyradiculoneuropathy, without and with monoclonal gammopathy.

We previously demonstrated differences in presentation and initial clinical course between patients with idiopathic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP-I) and those in whom CIDP was associated with a monoclonal gammopathy of undetermined significance (CIDP-MGUS). We now report the long-term follow-up of 69 patients with CIDP-I and 25 patients with CIDP-MGUS. (i) The clinical course was progressive in most of the CIDP-MGUS patients. CIDP-I patients were more likely to have a monophasic or relapsing course. (ii) Impairment developed more slowly in CIDP-MGUS than CIDP-I patients, with a longer time from onset of deterioration to peak impairment for each episode. (iii) Patients with CIDP-MGUS experienced less severe functional impairment and a lesser degree of measured weakness during their worst episode than did patients with CIDP-I. The primary source of functional impairment in many CIDP-MGUS patients was sensory. In contrast, the deficits in most patients with CIDP-I were primarily motor. (iv) CIDP-MGUS patients experienced a smaller degree of improvement to a poorer functional level after each episode than did patients with CIDP-I. (v) Most patients had a good outcome. However, the strength and functional scores at the time of last follow-up were significantly poorer in CIDP-MGUS than in CIDP-I patients. (vi) The disease was reclassified in seven patients; some patients with CIDP-I developed an MGUS, while some with CIDP-I or CIDP-MGUS developed multiple myeloma or a related malignant lymphoproliferative disease. These findings have implications for patient counselling and long-term prognosis, and underscore the need for long-term clinical and immunological monitoring.

Adolescent↗

Opportunistic Infections: Why Worry?

The proliferation and increased use of genetically manipulated rodents are taxing laboratory animal facilities and diagnostic laboratories. Although serological testing remains the standard for monitoring immunologically intact animals, molecular diagnostics are playing increasing roles in detecting infections in animals that have compromised immune systems. The importance of these assays is particularly evident now that we recognize that genetic manipulation can result in unanticipated phenotypes, including ones with decreased resistance to infectious diseases.

Journal Article↗

Assessment of immunologic status of liver transplant recipients by peripheral blood mononuclear cells in response to stimulation by donor alloantigen.

OBJECTIVE: To determine whether there is a role for assessing peripheral blood mononuclear cell (PBMC) cytokine patterns as a means of measuring the immunologic and clinical status of liver transplant recipients. SUMMARY BACKGROUND DATA: The role of assessing cytokine patterns in the prediction of clinical graft rejection or acceptance remains unclear. The purpose of this study was to examine the cytokine profiles of PBMC stimulated in vitro with donor alloantigen and to correlate prospectively the data with clinical assessment of graft status in orthotopic liver transplant (OLT) recipients. METHODS: PBMCs from OLT recipients were examined for proliferation and cytokine mRNA expression after stimulation by donor alloantigen, third-party alloantigen, or phytohemagglutinin (PHA). mRNA extracted from PBMC was amplified by reverse transcriptase-polymerase chain reaction with oligospecific primer pairs for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN) gamma, tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta. Results were prospectively correlated with each patient's allograft status. RESULTS: Increased IL-4 and TGF-beta and decreased IL-2, IFNgamma, and TNF-alpha mRNA expression by PBMCs in response to donor alloantigen stimulation predicted immunologic graft stability over a minimum 60-day interval compared with mRNA expression of PBMCs from patients with established rejection or those who experienced a rejection episode within a 30-day period (p < 0.05). Stimulation of recipient PBMCs with third-party alloantigens or PHA yielded similar but less specific results. PBMC proliferation to varying antigenic stimulation did not correlate with clinical graft status, nor did cytokine production by unstimulated PBMC. CONCLUSIONS: Prospective assessment of cytokine expression by PBMC from OLT recipients in response to stimulation by donor alloantigen is helpful for predicting the clinical status of the allograft and may be useful in the development of more precise immunologic monitoring protocols.

Adult↗

Combination interleukin-2 and doxorubicin in advanced adult solid tumors: circumvention of doxorubicin resistance in soft-tissue sarcoma?

Based on the likelihood of antitumor interactions between cytokines and cytotoxic drugs, we designed a pilot study to evaluate feasibility, clinical, pharmacologic, and immunologic effects of concomitantly administered subcutaneous (SQ) recombinant interleukin-2 (r-IL-2) and doxorubicin (ADR) in patients with advanced solid tumors (AST). Patients received one injection of ADR alone (70 mg/m2) and 3 weeks later a combination of r-IL-2 (18 MIU/m2 days 1-5 s.q.) and ADR at the same dose either 3-4 h after the first r-IL-2 injection (arm 1) or 2 days after the last r-IL-2 injection (arm 2). The same combination was repeated every 4 weeks according to the evolution of the disease. Pharmacokinetics were assessed over 48 h after injection of ADR alone and after the first ADR-IL-2 cycle and immunologic monitoring at days 1 and 8 of the first ADR-IL-2 cycle. Tumors were measured at baseline, after ADR alone, and after each ADR-IL-2 cycle until progression. Twenty-one adult patients with various AST including 14 soft-tissue sarcomas (STS) entered the study, 11 in arm 1 and 10 in arm 2. All patients were heavily pretreated; 16 had received an anthracycline-containing chemotherapy regimen. Eleven patients were ADR refractory and 1 ADR resistant. Grade 4 neutropenia occurred in 28, 82, and 40% of patients after ADR alone, ADR-IL-2 in arm 1 and ADR-IL-2 in arm 2, respectively. Mucitis was higher in arm 1 (7 of 11 patients) compared with arm 2 (0 of 10) and ADR alone (0 of 21). SQ injections of r-IL-2 did not affect ADR pharmacokinetics. ADR injection in arm 1 prevented IL-2-induced lymphocyte rebounds in all patients but did not alter qualitatively non-major histocompatibility complex-restricted cytotoxicity. There was no response after ADR alone. Two patients, one in each arm, experienced a prolonged (8 and 5 months) objective response after ADR-IL-2. Both had ADR-refractory STS with a local relapse and metastatic metastases. Interestingly, both patients had unusually elevated TNF-alpha levels before and after the first ADR cycle. Combination ADR-IL-2, although toxic, is feasible and manageable with routine clinical support. r-IL-2 enhanced ADR hematologic and extrahematologic toxicities. The two objective responses observed in these heavily pretreated patients refractory to ADR supports the hypothesis of a modulation of ADR resistance, possibly mediated by means of a mechanism involving TNF-alpha. Elevated baseline TNF-alpha levels could be predictive of response to ADR-IL-2 and deserves further investigation.

Adult↗

Effect of rabbit anti-human thymocyte globulin on lymphocyte subpopulations and functions following allotransplantation in the rhesus monkey.

The survival time of skin allografts from RhLA-nonidentical, unrelated donors was increased from a mean of 7.69 days in controls (n = 20) to a mean of 32.53 days in rhesus monkeys (n = 21) receiving a total dose of 250 mg of rabbit anti-human thymocyte globulin (RATG) per kg. Immunological monitoring studies were performed on the peripheral blood of mononuclear cells in control and treated monkeys. After administration of RATG, the percentage of E rosette-forming cells (E-RFC) was greater than 90% depressed, and the percentage of EAC rosette-forming cells was increased 5-fold in the circulation. Significant numbers of RATG-coated cells were detected only during the first week after RATG treatment. The percentage of E-RFC recovered to pretreatment levels within 3 to 4 weeks after RATG treatment, although the absolute E-RFC count remained depressed for 2 to 3 months. In addition, the in vitro proliferative responsiveness to polyclonal mitogens and to allogeneic lymphocytes remained greater than 80% depressed for 2 to 3 months after RATG treatment. The incidence of post-transplant-specific antidonor lymphocyte-mediated cytotoxicity (LMC) was similar in controls (85%) and RATG-treated monkeys (81%), and the appearance of LMC was correlated (r = 0.711) with partial recovery of absolute ERFC counts in the treated group. The appearance and peak of LMC were delayed (P less than 0.001) in RATG-treated monkeys, but preceded and correlated with rejection. Prior to rejection, the serum of RATG-treated monkeys inhibited LMC. Antibody-dependent cellular cytotoxicity appeared after rejection in the majority of recipients in both groups. The appearance and peak of antibody-dependent cell-mediated cytotoxicity (ADCC) were delayed (P less than than 0.001) in RATG-treated monkeys, but did not exhibit a significant correlation with the time of rejection.

Animals↗

Enumeration of Leu2a+, Leu2a+-DR+, and Leu2a+-Leu15+ cells in peripheral blood of renal transplant patients.

Immunological monitoring of peripheral blood cells of renal transplant patients may be helpful for diagnostic and prognostic purposes. We therefore enumerated the percentage of Leu2a+ cells as well as the occurrence of HLA-DR activation markers within this population. Since Leu2a+Leu15+ lymphocytes display suppressor functions in-vitro, this population was included in the study. Blood samples of 50 renal transplant patients within 3 months of transplantation and 41 pretransplant samples were investigated. As controls, peripheral blood cells from 10 healthy volunteers and 20 renal transplant patients with stable graft function more than 6 months after transplantation were examined. Just prior to transplantation the percentages of Leu2a+ cells, as well as the percentages of Leu15+ and DR+ cells within this population, varied considerably. Shortly after transplantation, clinical rejection episodes and cytomegalovirus (CMV) infections correlated with decreased percentages of Leu2a+ cells (P = 0.024 and 0.016, respectively). In nine patients with a decreased percentage of Leu2a+ cells shortly after transplantation but no rejection, the percentage of Leu2a+ cells normalized within 14 days. No correlation was found between increased percentages of Leu15+ or DR+ cells and rejection episodes, although the percentages of Leu2a+Leu15+ and Leu2a+DR+ cells increased significantly shortly before rejection (P less than 0.005). A decreased percentage of Leu2a+ cells, together with increased percentages of Leu15+ and DR+ cells, was seen in association with CMV infections (P less than 0.005). From this study we conclude that low percentages of Leu2a+ cells correlate with rejection episodes or CMV infections, whereas low percentages together with increased percentages of Leu2a+DR+ and Leu2a+Leu15+ cells are associated with CMV infections, especially in patients treated with high doses of cyclosporine.

Adult↗