High-energy, ceramic-disk Yb:LuAG laser amplifier.

We report the first short-pulse amplification results to several hundred millijoule energies in ceramic Yb:LuAG. We have demonstrated ns-pulse output from a diode-pumped Yb:LuAG amplifier at a maximum energy of 580 mJ and a peak optical-to-optical efficiency of 28% at 550 mJ. In cavity dumped operation of a nanosecond oscillator we obtained 1 mJ at up to 100 Hz repetition rate. A gain bandwidth of 5.4 nm was achieved at room temperature by measuring the small-signal single-pass gain. Furthermore, we compared our results with Yb:YAG within the same amplifier system.

Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation.

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured.

Optimising the efficiency of pulsed diode pumped Yb:YAG laser amplifiers for ns pulse generation.

We present a numerical model of a pulsed, diode-pumped Yb:YAG laser amplifier for the generation of high energy ns-pulses. This model is used to explore how optical-to-optical efficiency depends on factors such as pump duration, pump spectrum, pump intensity, doping concentration, and operating temperature. We put special emphasis on finding ways to achieve high efficiency within the practical limitations imposed by real-world laser systems, such as limited pump brightness and limited damage fluence. We show that a particularly advantageous way of improving efficiency within those constraints is operation at cryogenic temperature. Based on the numerical findings we present a concept for a scalable amplifier based on an end-pumped, cryogenic, gas-cooled multi-slab architecture.

Renal failure due to scleroderma with thrombotic microangiopathy developing in a woman treated with carboplatin for ovarian cancer.

Acute renal failure in association with microangiopathic hemolytic anemia and the pathological finding of thrombotic microangiopathy may occur in a number of conditions including hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and systemic sclerosis. Distinguishing between these conditions on clinical grounds may be difficult, and further investigations, including serological tests, are normally helpful. We present a patient who was treated with 5 doses of monthly carboplatin chemotherapy for stage IIb ovarian carcinoma and who subsequently developed acute renal failure and microangiopathic hemolysis together with some cutaneous features of systemic sclerosis. Initial serological tests, including anti-nuclear antibody titers measured using rat hepatocytes, were normal, and renal biopsy showed features of microangiopathic hemolysis, fibrinoid change, patchy tubular atrophy, and concentric intimal proliferation. A clinical diagnosis of diarrhea-negative hemolytic uremic syndrome was made and she was treated with plasma exchange and fresh frozen plasma infusion. However, she remained dialysis-dependent. Several weeks later she died following a cardiac arrest. Post-mortem examination revealed medial hypertrophy, concentric intimal proliferation, and thrombi within the small arteries of the kidneys and lungs. Subsequent results from tests taken at the time of her presentation with acute renal failure revealed a normal von Willebrand factor qualitative distribution, and a positive anti-nuclear antibody titer (using a human cell line) in association with positive autoantibodies to RNA polymerase types I, II, and III. Taken together, the clinical, laboratory, and post-mortem findings were suggestive of a diagnosis of systemic sclerosis. We discuss the differential diagnoses, and the associations between these and malignancy and chemotherapy. Finally, we consider the serological tests used for the diagnosis of systemic sclerosis that were, in this case, initially misleading.

Significant frequencies of T cells with indirect anti-donor specificity in heart graft recipients with chronic rejection.

BACKGROUND: The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. METHOD AND RESULTS: Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. DISCUSSION: iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Assessment of the contribution that direct allorecognition makes to the progression of chronic cardiac transplant rejection in humans.

BACKGROUND: Two populations of T cells contribute to allograft rejection. T cells with direct allospecificity are activated after recognition of intact MHC alloantigens displayed at the surface of donor passenger leukocytes carried within the graft. In contrast, T cells with indirect allospecificity recognize donor alloantigens as processed peptides associated with self (recipient)-MHC class II molecules. In small animal models of transplantation, direct pathway T cells dominate the acute rejection process and are rendered tolerant to the graft after the loss of donor passenger leukocytes. It has been argued that indirect pathway T cells contribute substantially to continual graft damage after passenger cell loss. The purpose of this study was to determine whether donor-specific tolerance could be detected in T cells with direct anti-donor allospecificity in human heart transplant recipients after prolonged graft residence. METHODS AND RESULTS: Alloreactive helper (HTLf) and cytotoxic (CTLf) T cells were enumerated by use of limiting dilution analysis. These assay systems were refined to make them specific for the direct pathway of allorecognition and more sensitive in the case of the HTLf assay. Recipient:anti-donor frequencies were generated in 10 long-term recipients of heart grafts with progressive chronic rejection and compared with those against equivalently HLA mismatched recipient:third-party controls. For HTLf, direct pathway donor-specific hyporesponsiveness was detected in 5 of the 10 recipients (HTLf<1:100,000). Of these 5 recipients, 4 also had low anti-donor CTLf (<1:100,000). In the 5th recipient, although the CTLf was >1:100,000, it was significantly lower than that estimated against the third-party control. CONCLUSIONS: Donor-specific hyporesponsiveness is demonstrated in 50% of recipients in both the HTLf and CTLf compartments of the direct alloresponse. Direct allorecognition therefore appears unlikely to be responsible for the progression of chronic rejection, implicating indirect allorecognition as the predominant immunological driving force. Furthermore, these data have potential implications for graft outcome, adjustment of immunosuppression, and recipient monitoring.

Optimizing a limiting dilution culture system for quantifying the frequency of interleukin-2-producing alloreactive T helper lymphocytes.

BACKGROUND: The development of sensitive, specific, and reproducible techniques to quantify T cells with direct allospecificity has potential applications in the selection of bone marrow donors and in the monitoring of the antidonor alloresponse in patients after organ transplantation. Such data may provide an objective basis for altering existing immunosuppression, monitoring novel antirejection therapies, and predicting long-term graft outcome. We have previously published a correlation between donor antirecipient T helper frequencies (HTLf) and the severity of acute graft-versus-host disease after bone marrow transplantation. Using the same assay protocol, we have described the development of donor-specific hyporesponsiveness in a proportion of renal transplant recipients. However, several imperfections existed in the protocols used in these studies. Cellular interactions within the stimulator and the responder cell populations, and back stimulation of T cells within the stimulator cell population, could give rise to extraneous interleukin-2 and alter the validity or estimation of derived recipient antidonor HTLf. METHODS: Using peripheral blood mononuclear cells as the responding population and splenic mononuclear cells as the stimulating population, we have examined the possible effects of these cellular interactions on the results of limiting dilution analysis assays for HTLf measurement. RESULTS: These interactions have the ability to alter the validity or estimation of HTLf. We show that by depleting the responder population of HLA class II+ cells and depleting T cells from the stimulating population, these interactions are effectively abrogated. CONCLUSIONS: On the basis of the findings reported here, we describe an optimized HTLf assay which is sensitive, specific, and reproducible. This has obvious applications in the analysis of alloimmune responses in transplantation.

Multiple Cladosporium brain abscesses in a renal transplant patient: aggressive management improves outcome.

Infection remains one of the major complications of nonspecific immunosuppression in renal transplant patients and accounts for significant morbidity and mortality. The incidence of infectious complications has been shown to be related to the degree of immunosuppression and correlated with the total steroid dosage, use of antilymphocyte serum, and number of rejection episodes. We present the case of a patient who received a large cumulative immunosuppressive load as treatment for her original disease and for numerous rejection episodes following renal transplantation, and who later developed multiple brain abscesses. These were shown to be due to the saprophytic black fungus Cladosporium bantianum. This case emphasizes the importance of aggressively pursuing the diagnosis in immunosuppressed individuals--appropriate treatment may be instituted early and may save lives. There have been no previous cases of patients surviving this condition without neurosurgical resection of the lesions.

Detection of donor-specific hyporesponsiveness following late failure of human renal allografts.

Limiting dilution assays to measure the frequency of interleukin-2-secreting peripheral blood T cells were carried out in patients, whose renal allografts had failed due to acute rejection (9 patients) and in patients whose grafts failed more than two years after transplantation without any recent evidence of acute rejection. Using a modified form of the assay we demonstrate that nearly half of 18 patients whose renal transplants had failed after more than two years have low or undetectable HTLp frequencies against donor, but not third-party DR antigens. No such difference was observed in any of the nine patients studied whose transplants were lost from early acute rejection. These results provide the first indication that, as in rodent models of transplantation, T cell unresponsiveness towards donor MHC antigens can occur following prolonged residence of an allograft in humans. Furthermore, the results suggest that chronic rejection may be driven by mechanisms other than direct allorecognition. The assay may be a valuable tool to study the evolution of donor-specific direct T cell alloresponsiveness in patients with well-functioning grafts.

Malignant disease in patients with long-term renal transplants.

The incidence of de novo malignancy was analyzed in 274 renal transplant recipients whose graft had functioned for at least 3 years and who had been followed for 2622 patient-years and individually for up to 29 years. The actuarial incidence and relative risks (RR) of tumor development (compared with National statistics) were calculated. Subgroup analysis was performed according to age, sex, the number of years and type of immunosuppression, and the tumor type. Seventy one tumors occurred in 54 patients. Skin tumors were the most common, followed by lymphoma, renal, bladder, and bronchial carcinoma. The actuarial cumulative risks of tumor development were 18.4% (95% confidence interval [CI] 12.4-24.3%) at 10 years and 49.6% (95% CI 36.3-62.0%) at 20 years. The overall RR of developing a tumor was 6.2 but was higher for men (RR 7.3) than women (RR 4.9). The RR of developing skin cancers, but not other malignancies, increased from 6.6 at 5 years to 20 after > 15 years. There was no evidence that cyclosporine-treated patients had an increased incidence of tumors, indeed the risk may be less in patients treated with cyclosporine and low-dose azathioprine than in those treated with azathioprine and prednisolone alone after more than 5 years.

Analysis of T cell responses to the autoantigen in Goodpasture's disease.

Goodpasture's disease is a rare form of glomerulonephritis characterized by the production of autoantibodies to the glomerular basement membrane (GBM). In order to understand the development of autoimmunity to the GBM, it is important to examine mechanisms underlying T cell responses to the autoantigen. A MoAb P1, with the same specificity as patients' autoantibodies, was used to affinity-purify the antigen from collagenase-digested human GBM. This material was enriched in the NC1 domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1), known to be the principal target of anti-GBM antibodies, but also contained lower quantities of alpha 4(IV)NC1. In proliferation assays, T cells from 11/14 patients with Goodpasture's disease showed significant responses (SI > or = 2.0) to affinity-purified human GBM. Peak responses were demonstrated at 7 or 10 days at antigen concentrations of 10-30 micrograms/ml. As in other autoimmune disorders, the presence of autoantigen-reactive T cells was also demonstrated in 5/10 healthy volunteers. Tissue typing revealed that all patients possessed HLA-DR2 and/or -DR4 alleles, while normal individuals whose T cells responded possessed DR2 and/or DR7 alleles. The specificity of the T cell response in Goodpasture's disease was further investigated using monomeric components of human GBM purified by gel filtration and reverse phase high performance liquid chromatography (HPLC). Two antigenic monomer pools were obtained, which were shown by amino-terminal sequence analysis to contain alpha 3(IV)NC1 and alpha 4(IV)NC1, respectively. In all patients tested, significant T cell proliferation was observed in response to one or both of these alpha (IV)NC1 domains. These results demonstrate that patients with Goodpasture's disease possess T cells reactive with autoantigens known to be recognized by anti-GBM antibodies.

Heterogeneity amongst natural killer cells revealed by limiting dilution culture; selectivity against virus-infected and tumour cell targets.

Previous studies have suggested that natural killer (NK) cells exhibit heterogeneous cytotoxicity towards different tumour cell targets. No studies have set out to determine whether different NK populations have relative selectivity for virus-infected cells. The aims of this study were to determine if this was the case for short-term clones, and whether there were differences in relative selectivity for particular target cells between clones with NK activity but with different surface phenotypes. Cells from different starting populations [whole peripheral blood lymphocytes (PBL), E-rosette positive or negative, CD16+ or CD3- cells] were grown in limiting dilution culture (LDC) with interleukin-2 (IL-2). The precursor frequency (NK-p) of cells proliferating and exhibiting NK activity towards various virus-infected or uninfected fibroblasts or tumour cell targets was determined by split-well analysis of the LDC. The relative NK-p were similar for different individuals, but were much lower for virus-infected fibroblasts than a tumour cell target. The pattern of cytotoxicity of 757 short-term clones, identified from the LDC, against four to five tumour and virus-infected target cells were analysed. We conclude that there was selective lysis of virus-infected cells by a proportion of NK clones which were predominantly PBL-derived (mainly CD3+). Twenty-six per cent of E(+)-derived clones lysed Molt4 cells only in the absence of phytohaemagglutinin (PHA), and a proportion of PBL- or E(+)-derived clones (up to 44%) lysed uninfected or virus-infected fibroblasts but not Molt4+PHA. Thus, under hese conditions lectin-induced cytotoxicity does not detect total potential cytotoxicity.

NK cells inhibit T-cell responses: LFA3+ but not LFA3- T-cell responses are suppressed.

There is increasing evidence that natural killer (NK) cells have immunoregulatory effects in addition to their ability to lyse tumour and virus-infected target cells. However, much of the evidence to date is based on the reported effect of adding relatively impure NK cell populations to various in vitro cultures, and the effect on T cells has been contradictory. Here we report the inhibitory effect of highly purified CD16+ NK cells on mixed lymphocyte reaction (MLR), using unseparated peripheral blood mononuclear cells (PBMC) and purified T cells as responders. Marked inhibition was observed (up to 75%) which was proportional to the number of CD16+ cells present, and was abrogated by ultraviolet irradiation. In contrast, the addition of CD16+ cells had no effect on the proliferative responses of five CD4+ anti-DR1 alloreactive T-cell clones. To test the relative sensitivity of previously primed versus virgin T cells to NK cell-mediated inhibition, freshly isolated T cells from PBMC were separated into LFA3+ (memory) and LFA3- (virgin) populations. CD16+ cells caused inhibition of proliferation of LFA3+ but not LFA3- cells in an MLR. In addition, the recall response of T cells to influenza was inhibited. These results further illustrate the regulatory potential of CD16+ NK cells, and suggest that previously primed cells are more susceptible to NK-mediated inhibition. However, activated (rather than resting) cells may escape regulation.