Immunosuppression and procedure-related complications in 26 patients with type 1 diabetes mellitus receiving allogeneic islet cell transplantation

BACKGROUND: The success of sirolimus and low-dose tacrolimus in islet cell transplantation has influenced many transplant centers to utilize this novel regimen. The long-term safety and tolerability of this steroid-free immunosuppressive protocol for allogeneic islet transplantation has yet to be determined. METHODS: We transplanted 26 adult patients with long standing type 1 diabetes mellitus between April 2000 and June 2004. Immunosuppression consisted of induction with daclizumab and maintenance therapy with tacrolimus and sirolimus. Adverse events (AEs) in patients were followed and graded using the Common Terminology Criteria for Adverse Events, version 3.0 (National Cancer Institute). RESULTS: To date, the majority of patients were able to remain on the immunosuppression combination for up to 22+/-11 months. Four patients were successfully converted to Mycophenolate Mofetil due to tacrolimus-related toxicity. Withdrawal from immunosuppression was decided in four patients due to hypereosinophilic syndrome, parvovirus infection, aspiration pneumonia, and severe depression, respectively. Six patients required filgrastim therapy for neutropenia. Transient elevation of liver enzymes was observed in most patients early after islet infusion. Increased LDL in 20 patients required medical treatment. CONCLUSION: There was a varying range of AEs, most of them mild and self-limiting; however, some required urgent medical attention. The majority of patients were able to tolerate and remain on this effective regimen. To date, no deaths, cytomegalovirus disease, graft-versus-host disease, or posttransplant lymphoproliferative disease has been observed.     

Attentional load of the primary task influences the frontal but not the temporal generators of mismatch negativity

According to the model hypothesized by N??t?nen and Michie, the generation of the mismatch negativity (MMN) requires a mismatch detection, taking place in temporal areas, followed by the activation of frontal generators, underlying attention switching toward the deviant stimulus. We aimed at verifying whether the activation of temporal and frontal regions is dependent on the amount of attentional resources allocable toward the deviant stimulus. We recorded event-related potentials (ERPs) in nine healthy subjects while reading and during a demanding visual task (Multiple Features Target Cancellation, MFTC). Raw data were further evaluated by Brain Electrical Source Analysis (BESA). During the Reading condition, distraction toward the unattended auditory stimuli was reflected by the enhancement of the N1 response to frequent stimuli and by the elicitation of a P3a response to deviant ones. The MMN distribution was explained by bilateral temporal dipoles. During the MFTC condition, no P3a was detected, while source analysis showed the activation of a right frontal generator. Temporal dipoles showed no change between the two conditions: we thus conclude that the earlier mismatch detection is independent on the attentional load. By contrast, the activation of a right frontal subcomponent occurred only during the high-load task, independently on any actual attention shift reflected by the P3a component. We thus discuss the hypothesis whether the right frontal MMN generator, rather than subserving a simple attention switching toward the deviant stimulus, plays a role in modulating the auditory change detection system ("contrast enhancement" model).     

Autoimmunity in thrombotic thrombocytopenic purpura

In the last few years, an autoimmune hypothesis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) has been proposed often, with variable success because of inconsistent supporting data. We are now aware that at least one subgroup of TTP patients does present with pathogenic autoantibodies (i.e, anti-ADAMTS13); this group consequently is a putative candidate for a curative treatment including plasma exchange (still the cornerstone of TTP treatment), together with corticosteroids or other immunosuppressants. Furthermore, for patients not responding to or relapsing following plasma exchange, the use of stronger immunosuppression (e.g., with the use of the anti-CD20 monoclonal antibody rituximab) should be considered as appropriate. Conversely, few data are actually available regarding the complex relationship between TTP and the antiphospholipid (aPL) syndrome, as well as other autoimmune diseases. Indeed, a correct differential diagnosis should be done on the basis of both different clinical presentations and autoantibody profile. At present, the presence of aPL antibodies should give evidence against a diagnosis of TTP, even though we cannot exclude that aPL antibodies may, in a minority of patients, be associated with a primary endothelial damage ultimately resulting in overt TTP.     

Retrovirally transferred genes inhibit apoptosis in an insulin-secreting cell line: implications for islet transplantation

The transplantation of pancreatic islets for the treatment of type I diabetes is hindered by the enormous loss of cells due to early apoptotic events. Genetic engineering of islets with cytoprotective genes is an important strategy aimed to enhance the survival of these cells in the transplant setting. The present study was designed to evaluate and compare the effects of five genes on a cell line derived from insulin-producing beta-cells, NIT-1. Cells were transduced using a Maloney murine leukemia virus (MLV) vector coding for yellow fluorescent protein (YFP) and for one of the following antiapoptotic genes: cFLIP, FADD-DN, BcL-2, PI-9, and ICAM-2. These genes were able to protect NIT-1 cells from cytokine-induced apoptosis to varying degrees ranging from no protection to significant protection equivalent to an optimal dose of a chemical caspase inhibitor. The data demonstrate that cFLIP, FADD-DN, and PI-9 are significantly more effective in protecting NIT-1 cells than BcL-2 and ICAM-2. Additionally, the data show that despite its weak in vitro inhibition of caspase-3, PI-9 affords significant protection against TNF-alpha-induced apoptosis in these cells. These genes may be ideal candidates to augment islet survival following transplantation.     

A novel micro-cell-mediated lympholytic assay for the evaluation of regulatory cells in human alloreactive CTL responses

Since cell-mediated lympholysis (CML), the most commonly used in vitro experimental cytotoxic method for the evaluation of regulatory cells, requires large numbers of cells that are often the limiting factor, we have developed a new micro-cell-mediated lympholytic (m-CML) assay. Various numbers of responding cells were stimulated with equivalent numbers of allogeneic irradiated stimulator cells in the presence of (fivefold) serial dilutions of regulatory cells. On the 8th day, 4-h 51Cr-release assays were performed by adding 5000 labeled target cells from the corresponding stimulators to the cultures.Even though results that were comparable to the macro- (bulk) CML and MLR modulation experiments were obtained with all the cell combinations tested in the m-CML, the combinations with 50,000 responder cells and stimulating cells and dilutions of 25,000 to 40 modulator (regulatory) cells were found to be the most reproducible for assaying regulatory cell potency in vitro. Similarly, expression of the results as percentage inhibition using percent specific lysis values was the simplest method of calculation. This assay was standardized for the evaluation of the inhibitory activity of a variety of regulatory cells, including long-term cultures of cadaver-donor vertebral body bone marrow cells (vDBMC-L), in vitro generated CD8 positive and CD28 negative suppressor T cells and donor chimeric cells isolated from renal transplant recipients who had been perioperatively infused with donor bone marrow cells (DBMC). The results indicate that the m-CML assay is a sensitive and reliable micromethod with at least 10-fold fewer responders, stimulator and modulator cell numbers needed than macro-CML assays for the evaluation of regulatory cells obtained from a variety of immune systems in vitro.     

Transforming growth factor-beta released by PPD-presenting malignant mesothelioma cells inhibits interferon-gamma synthesis by an anti-PPD CD4+ T-cell clone

Besides acting complexly on both normal and tumor cells, transforming growth factor-beta (TGF-beta) can determine the nature of the response to the antigen, strongly inhibiting the differentiation of naive CD4+ T-cells toward a T helper-1 (Th-1) phenotype; in a number of experimental models, TGF-beta also appeared to be a potent immunosuppressant factor. TGF-beta was shown to be released by some human malignant mesothelioma (MMe) cells, which affects the immune response to this tumor. Thus, for a better understanding of the role of TGF-beta in the immune response to MMe cells, we evaluated the production of a Th-1 cytokine (IFN-gamma) and of a Th-2 cytokine (IL-4), following Purified Protein Derivative (PPD) recall antigen presentation by human MMe cells to a class-II major histocompatibility complex (MHC-II)-matched PPD clone (PPD clone). Our data confirm that human MMe cells possess the unusual capability of presenting a common recall antigen to CD4+ lymphocytes but also show that these tumor cells can abrogate Th-1 immune response, as evidenced by a shift in favor of the production of IL-4 over that of IFN-gamma, through a TGF-beta-mediated pathway; only the simultaneous block of TGF-beta1 and beta2 effects can significantly restore a typical Th-1 pattern of cytokine production by PPD clone in response to PPD presentation by MMe. Even though the role of TGF-beta in the promotion of MMe growth should be further and better defined, this effect should be considered when designing new therapeutical approaches aimed at improving the immune response to MMe.     

Effect of predominant methanogenic flora on the outcome of lactose breath test in irritable bowel syndrome patients

BACKGROUND: The relationship between hydrogen and methane production is a possible confounding factor in the interpretation of H(2) breath tests (HBT), but is usually disregarded for the interpretation of HBT and, in most instances, only H(2) excretion is measured. The present study was designed to evaluate the effect of predominant fasting methane CH(4) or H(2) production on the outcome of lactose HBT, in a large, homogeneous series of adult patients with irritable bowel syndrome (IBS). PATIENTS AND METHODS: A lactose HBT was performed in 237 IBS patients with predominant fasting methane production (CH(4)>H(2)), recording the outcome of the test, amount of gas excreted and occurrence of clinical symptoms. Data were compared to those of 237 age- and sex-matched IBS patients with low fasting CH(4) excretion. RESULTS: The test was positive in 124 predominant CH(4) producers (52.3%) (PMP), as compared to 201 (84.8%) low methane producers (LMP) (P<0.0001). Peak hydrogen concentration and area under the curve of H(2) were significantly (P<0.001) lower, and the occurrence of symptoms during the test less frequent, in PMP vs LMP patients. During the test, CH(4) excretion doubled in 57/113 (50.4%) patients with negative HBT, and in 49/124 (39.5%) with positive HBT. CONCLUSIONS: Patients with predominant fasting methane production excrete less H(2) than LMP, after an oral load of lactose. The lower prevalence of severe lactose intolerance in PMP, as well as lower incidence of symptoms during the test, is, indeed, related to lower and slower H(2) excretion. The assumption that H(2) excretion is an effective means of quantifying the amount of malabsorbed carbohydrates is questionable in PMP. Methane-producing patients likely have a higher 'false negative' rate as compared to LMP after an oral load of lactose. Nonetheless, as symptoms are related to the amount of gas produced in the colon, HBT identifies patients with 'lactose intolerance', irrespective of the presence of lactose malabsorption, and helps in predicting the effect of lactose-restricted diet.     

Coarse nodular US pattern in hepatic cirrhosis: risk for hepatocellular carcinoma

PURPOSE: To determine the prevalence of the coarse nodular ultrasonographic (US) pattern and its prognostic importance in terms of hepatocellular carcinoma (HCC) risk in hepatic cirrhosis caused by hepatitis B virus (HBV); HBV with hepatitis D virus (HDV), formerly known as hepatitis delta virus; hepatitis C virus (HCV); and alcoholic cirrhosis (ALC) or primary biliary disease (primary biliary cirrhosis [PBC]). MATERIALS AND METHODS: Four hundred two cases of hepatic cirrhosis caused by HBV (94 patients), HDV (100 patients), HCV (100 patients), ALC (63 patients), or PBC (45 patients) were retrospectively reviewed to identify the US pattern present at diagnosis and its possible association with the cause of the disease and subsequent development of HCC during a mean follow-up of 43.9 months +/- 29.9 (SD). Data were analyzed with the chi2, Fisher exact, and log-rank tests and with the Kaplan-Meier method (all two-tailed). RESULTS: The coarse nodular pattern was found in a significantly higher percentage of patients with HDV-related cirrhosis (51%) compared with those with HBV (9%), HCV (9%), ALC (11%), or PBC (9%) (P <.001). This pattern was associated with a significantly increased risk for HCC in patients with cirrhosis and HBV-, HCV-, and ALC-related disease but not in those with HDV-related disease and PBC. CONCLUSION: The coarse nodular pattern is more often seen in patients with HDV-related cirrhosis, and, in this setting (in contrast to HBV-, HCV-, and ALC-related cirrhosis, as well as in PBC), it does not represent an added risk factor for HCC.     

Nonlethal conditioning for the induction of allogeneic chimerism and tolerance to islet allografts

BACKGROUND: We reported that tolerance to skin grafts can be achieved by chimerism induction by way of nonlethal conditioning. In the present study, we evaluated the outcome of islet allografts implanted either simultaneously or after donor bone marrow cell (BMC) infusion when nonlethal conditioning was used. METHODS AND RESULTS: B10 (H-2b) mice were conditioned with antilymphocyte serum (ALS), 100 cGy total body irradiation (TBI), and given 30 x 10(6) allogeneic (B10.BR, H-2k) BMC on day 0. On day 2, cyclophosphamide was given intraperitoneally (IP), followed by a second BMC infusion on day 3. After chimeras were typed for allogeneic BMC engraftment on day 28, animals were rendered diabetic chemically and transplanted under the kidney capsule with islet allografts genetically matched or disparate to the BM. Donor-specific islet grafts were accepted (median survival time [MST] > 180 days, n=6), whereas all major histocompatibility complex (MHC)-disparate third-party BALB/c (H-2d) islet grafts were rejected (MST=13.8 days, n=4). When B10.BR BMC and islets were given simultaneously, graft acceptance (MST >140 days, n=4) was observed. Surprisingly, when MHC-disparate third-party islets (BALB/c) were given together with B10.BR BMC, long-term survival was also observed (MST >100 days, n=3). These findings suggested that conditioning alone at the time of islet implant might induce long-term engraftment without further treatment. However, only chimeric animals accepted a second-set donor-specific graft, whereas all other groups rejected it. CONCLUSION: Our data indicates that stable allogeneic chimerism and islet indefinite survival can be achieved by the use of a nonmyeloablative protocol. The results of the conditioning-only experiments are consistent with the possibility of graft accommodation.