Preformed Class Research Articles

The Effect of Simulation Learning Strategy and Education Background on the First Learning Outcomes of Private Students Madrasah Tsanawiyah

The purpose of this study is to determine the impact of simulation learning strategies and educational background on the learning outcomes of fiqh students at Gunung Sitoli's private madrasah tsanawiyah. The method used in this study is quasi-experimental. This method was chosen because the treatment class, both for the simulated learning class and the expository learning class, was a pre-formed class and the students being controlled had an educational background. The results of statistical analysis calculations show that there is an interaction between learning strategies and educational backgrounds, with students with a MI educational background benefiting more from simulation learning strategies than expository learning strategies, and students with an elementary education background benefiting more from expository learning strategies than simulation learning strategies.

P171 Donor specific antibody in simultaneous liver-kidney transplantation: preformed class II HLA antibodies and acute renal and hepatic allograft rejection

P171 Donor specific antibody in simultaneous liver-kidney transplantation: preformed class II HLA antibodies and acute renal and hepatic allograft rejection

Subclinical Antibody-Mediated Rejection

Subclinical Antibody-Mediated Rejection

Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.

The review outlines the diagnosis, prevention strategies, and possible treatment options for acute and chronic antibody-mediated rejection (AMR). Although rare, severe acute AMR (aAMR) usually occurs in patients with high mean fluorescence intensity despite serial dilutions or high-titer preformed class I donor-specific alloantibodies (DSA). The diagnosis is suspected when allograft dysfunction occurs with DSA, diffuse C4d staining, and a microvascular injury, and may be aided by the aAMR score. However, the incidence of and treatment approach to combined T-cell-mediated rejection (TCMR) with DSA present and some but not all features of AMR is yet to be determined. Chronic liver allograft AMR is characterized by low-grade chronic inflammation and progressive fibrosis with DSA, the chronic AMR (cAMR) score may facilitate diagnosis. The 'two-hit' hypothesis, whereby a coexistent insult upregulates human leukocyte antigen class II target antigens on the microvascular endothelium, may explain why suboptimal donors with lower sensitization levels might suffer from acute AMR and those with chronic complications (e.g., recurrent original disease) might be more susceptible to chronic AMR. Although treatment algorithms are needed, prevention is preferable and at a minimum includes transfusion minimization, and medication adherence. Severe acute AMR is rare but diagnosable, and there is need to determine the incidence of and optimal therapy for less severe combined AMR and TCMR. Chronic AMR is likely more common and of significant relevance to long-term allograft survival improvement. The two-hit hypothesis may help to explain the rarity of both findings and shed insight onto future prevention and treatment strategies.

Pretransplant Gelişmiş Hla Sınıf I ve II Panel Reaktif Antikorların Böbrek Allogrefti Üzerine Klinik ve Patolojik Düzeyde Etkileri

Pretransplant Gelişmiş Hla Sınıf I ve II Panel Reaktif Antikorların Böbrek Allogrefti Üzerine Klinik ve Patolojik Düzeyde Etkileri

Clinical impact of preformed donor-specific denatured class I HLA antibodies after kidney transplantation.

Class I single-antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA-sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor-specific antibodies (DSA) using two different assays: an acid-treated SAFB assay (anti-dHLA DSA) and the iBeads assays (SAFB+/iBeads- DSA). Eighty-five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520-13 882]). Anti-dHLA and SAFB+/iBeads- DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500-1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti-dHLA DSA or only SAFB+/iBeads- DSA developed acute clinical antibody-mediated rejection in the first-year post-transplantation, and their five-yr death-censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T-cell flow cytometry cross-match. Therefore, both anti-dHLA DSA and SAFB+/iBeads- DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.

Donor-specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus-infected patients

Hepatitis C virus (HCV) fibrosis progression after liver transplantation (LT) is accelerated in comparison with fibrosis progression before transplantation. The vast majority of the risk factors for fibrosis progression after LT are not modifiable. With the goal of identifying modifiable risk factors for fibrosis progression, we evaluated the impact of preformed and de novo donor-specific human leukocyte antigen alloantibodies (DSAs) on fibrosis progression after LT in HCV-viremic patients. After blinding, we analyzed all 507 HCV-viremic patients who underwent primary LT from January 2000 to May 2009 and had pretransplant and posttransplant samples available for analysis (86% of the total) for preformed and de novo class I and class II DSAs with a mean fluorescence intensity ≥ 5000 with single-antigen bead technology. Fibrosis was assessed on the basis of indication and protocol liver biopsies; compliance with protocol liver biopsies at 1, 2, and 5 years was ≥80%. Preformed class I DSAs [hazard ratio (HR) = 1.44, P = 0.04] and class II DSAs (HR = 1.86, P < 0.001) were independent predictors of progression to stage 2-4 fibrosis, and de novo DSAs (HR = 1.41, P = 0.07) had borderline significance. In addition, preformed class I DSAs (HR = 1.63, P = 0.03) and class II DSAs (HR = 1.72, P = 0.03) were statistically significantly associated with an increased risk of death. In conclusion, after we controlled for donor and recipient characteristics in multivariate modeling, DSAs were independently associated with fibrosis progression and death after LT in HCV-viremic patients.

The Clinical Relevance of Pre-Formed Anti-HLA and Anti-MICA Antibodies after Cord Blood Transplantation in Children

Preformed anti-HLA antibodies (AHA) are known to be associated with delayed engraftment and reduced overall survival after adult hematopoietic stem cell transplantation. However, limited data is available in pediatric patients. In this study, we explored the role of AHA on clinical outcomes in 70 pediatric patients who received a single unit of HLA mismatch cord blood for hematologic malignancies, immunodeficiencies or metabolic diseases. The presence of AHA was detected in 44% (31/70) of the patients. Preformed class I AHA was associated with an increased occurrence of grade 1–4 acute graft-versus host disease (p<0.05). The presence of anti- major-histocompatibility-complex class I–related chain A antigens (MICA) antibodies was significantly associated with a reduced platelet recovery after transplantation (p<0.05). AHA of class II with the strength of antibody titer measured as the mean fluorescence intensity above 2000 was associated with reduced event-free survival (p<0.05). A reduction of high titer of AHA and anti-MICA antibodies might have to be considered before cord blood transplantation in pediatric patients for better outcomes.

Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation

Preformed donor-specific human leukocyte antigen antibodies (DSAs) are considered a contraindication to the transplantation of most solid organs other than the liver. Conflicting data currently exist on the importance of preformed DSAs in rejection and patient survival after liver transplantation (LT). To evaluate preformed DSAs in LT, we retrospectively analyzed prospectively collected samples from all adult recipients of primary LT without another organ from January 1, 2000 to May 31, 2009 with a pre-LT sample available (95.8% of the patients). Fourteen percent of the patients had preformed class I and/or II DSAs with a mean fluorescence intensity (MFI) ≥ 5000. Preformed class I DSAs with an MFI ≥ 5000 remained persistent in only 5% of patients and were not associated with rejection. Preformed class II DSAs with an MFI of 5000 to 10,000 remained persistent in 23% of patients, and this rate increased to 33% for patients whose MFI was ≥10,000 (P < 0.001). Preformed class II DSAs in multivariable Cox proportional hazards modeling were associated with an increased risk of early rejection [hazard ratio (HR) = 1.58; p = 0.004]. In addition, multivariate modeling showed that in comparison with no DSAs (MFI < 1000), preformed class I and/or II DSAs with an MFI ≥ 5000 were independently correlated with the risk of death (HR = 1.51; p = 0.02).

Alloantibodies in simultaneous liver–kidney transplantation

O'Leary et al. report that the presence of anti-HLA antibodies at the time of simultaneous liver–kidney transplantation is associated with allograft dysfunction and shortened organ and patient survival. Preformed class I alloantibodies were eliminated after transplantation, whereas class II donor-specific antibodies persisted and were associated with poor outcomes.

Class II Alloantibody and Mortality in Simultaneous Liver-Kidney Transplantation

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver-kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody-mediated kidney rejection. To further investigate the risk associated with donor-specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre-SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity≥2,000=positive). Post-SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p=0.006), liver allograft rejection (p=0.002), patient death (p=0.02), liver allograft loss (p=0.02) and renal allograft loss (p=0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR=2.2; p=0.043) and liver allograft loss (HR=2.2; p=0.044). These data warrant reconsideration of the approach to DSA in SLKT.

Donor-Specific Antibodies to Class II Antigens Are Associated With Accelerated Cardiac Allograft Vasculopathy

Although a link between donor-specific antibodies against human leukocyte antigens type II (DSA II+) and transplant glomerulopathy has been clearly established, its role in cardiac allograft vasculopathy (CAV) is unclear. Donor-specific antibodies were evaluated using solid-phase single-antigen bead assay before transplantation in 51 heart transplant recipients. Coronary angiography and three-dimensional intravascular ultrasound were performed at baseline and approximately 1 year after the baseline examination. There were 4 (7.8 %), 11 (21.5%), and 2 (3.9%) patients who had DSA against donor class I (DSA I+), DSA II+, or both, respectively. All patients had negative complement-dependent cytotoxic crossmatch. There was accelerated progression of CAV in the DSA II+ group demonstrated by accelerated progression in plaque index (plaque volume/vessel volume) compared to patients with no DSA II+ antibodies (13.8% [12%] vs. -7.9% [37%], P=0.01). The development of any angiographic CAV was also more common in DSA II+ patients as compared to the DSA- patients at 4 years (100% [0%] vs. 64.2% [10%], P=0.05). All other traditional risk factors for CAV or immunosuppression were similar between the groups (P>0.2 for all). This is the first preliminary study demonstrating that heart transplant recipients with preformed class II DSA may be at an increased risk for accelerated CAV as detected by consecutive volumetric three-dimensional intravascular ultrasound.

High MFI Preformed Class I and De Novo Class II Donor Specific Antibodies Accelerate Fibrosis Progression after Liver Transplant in HCV-Infected Patients

Aim: To determine the impact of HLA class I & II DSA on fibrosis progression in HCV-infected patients post-OLT. Methods: Since 1985, our biorepository prospectively collects protocol serum samples from all donors & recipients of OLT in conjunction with a clinical & laboratory research database. 345 consecutive patients with a diagnosis of HCV & confirmed viremia at OLT who had a pre-OLT & a 1-year post-OLT serum sample from 1/01-05/09 were blinded and analyzed for DSA using LABScreen® single antigen beads test. We compared patients with DSA MFI >5000 to patients without DSA (MFI < 1000). Patients undergo yearly protocol liver biopsies. Kaplan-Meier analysis of time to METAVIR fibrosis stage 32 or HCV related liver failure or HCV related death was analyzed. Patients treated for HCV were censored at the time of sustained virologic response (SVR). Results: Patients with preformed MFI >5000 class I DSA (N= 32) had more rapid fibrosis progression post-OLT (figure 1; P=0.02). In addition, patients with de novo MFI >5000 class II DSA (N=26) had more rapid fibrosis progression post-OLT (figure 2; P=0.02). Multivarible Cox regression controlling for donor & recipient age, AA race, CMV, & ACR showed that preformed class I DSA (HR=1.7; P=0.02) & de novo class II DSA (HR=1.7; P=0.02) were independent predictors of fibrosis progression. Although underpowered, analysis of preformed class II & de novo class I DSA did not reveal an association with fibrosis progression.[Figure 1]: No Caption available.[Figure 2]: No Caption available.Conclusion: HCV-infected patients with high MFI preformed class I DSA or high MFI de novo class II DSA experience accelerated fibrosis progression post-OLT.

Fulminant hyperacute rejection after unilateral lung transplantation

Hyperacute rejection (HAR) is a well-known complication in renal and cardiac transplantation, but rare in lung recipients. We present a case of HAR of the lung graft with a fatal outcome of a male patient with preformed class II anti-HLA antibodies.

Binding of H-2Kb-specific Peptides to TAP and Major Histocompatibility Complex Class I in Microsomes from Wild-type, TAP1, and β2-Microglobulin Mutant Mice

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.

Mycoplasma fermentans-derived lipid inhibits class II major histocompatibility complex expression without mediation by interleukin-6, interleukin-10, tumor necrosis factor, transforming growth factor-beta, type I interferon, prostaglandins or nitric oxide.

Mycoplasma cause several diseases in man and animals. Some strains can chronically infect humans, leading to fever or inflammatory syndromes such as arthritis, particularly in immunosuppressed patients. A set of pathogenicity factors shared by many mollicutes may be membrane components that activate macrophages to secrete cytokines and other inflammatory mediators. Mycoplasma-derived high molecular weight material (MDHM) is a macrophage-activating amphiphilic lipid which was purified from Mycoplasma fermentans. We studied the influence of MDHM on the expression of major histocompatibility complex (MHC) class II molecules by mouse resident peritoneal macrophages with an ELISA. Highly purified MDHM at 4 ng/ml and 0.8 microgram/ml crude heat-killed M. fermentans (concentrations chosen to give maximal responses) suppressed interferon (IFN)-gamma-dependent class II MHC induction when added simultaneously with IFN-gamma. MDHM was not toxic and did not result in loss of adherent cells. Kinetic data showed that MDHM first up-regulated, then down-regulated the expression of preformed class II MHC molecules, while expression of Mac-1 and F4/80 antigens remained constant. MDHM-dependent suppression of class II MHC molecule expression resulted in impaired antigen presentation to the helper T cell line D10.G4.1. We further attempted to identify hypothetical products of MDHM-stimulated macrophages as secondary mediators of class II MHC suppression such as were described for lipopolysaccharide (LPS)-stimulated macrophages. Type I IFN, prostaglandins and nitric oxide, all reported to cause down-regulation of class II MHC, could be excluded in this context. Of the cytokines tumor necrosis factor, interleukin (IL)-6, IL-10 and transforming growth factor-beta, only IL-10 inhibited class II MHC expression, although less effectively than MDHM. The involvement of IL-10 was ruled out, as no evidence for its MDHM-dependent formation could be found. Our data suggest that MDHM interferes with class II MHC expression by up-regulating its turnover, and at the same time, inhibits the formation of new class II MHC molecules.

Rat Pit-1 Stimulates Transcription in Vitro by Influencing Preinitiation Complex Assembly

The anterior pituitary-specific transcription factor, Pit-1, activates prolactin, growth hormone, TSHβ, growth hormone receptor genes and autoregulates the pit-1 gene. Its mechanism of transcription activation is unknown. Using immobilized DNA templates and order-of-addition transcription assays, it is shown that Pit-1 is required during pre-initiation complex assembly to activate the prolactin gene in vitro. Using prolactin promoters containing point mutations in the distal TATA box, it is also demonstrated that Pit-1 activation in vitro is not mediated simply by repressing the upstream, alternative promoter. Experiments show that a preformed class II pre-initiation complex is refractory to Pit-1 influence. The data indicate that Pit-1, and perhaps other members of the POU-protein family, activate transcription by influencing the type pre-initiation complex assembled on target promoters.

Kinetics and specificity of peptide-MHC class II complex displacement reactions.

The peptide-induced acceleration of the dissociation of pre-formed complexes of the detergent-solubilized mouse class II molecules IEd and IEk with fluorescein-labeled peptides was investigated using high-performance size exclusion chromatography. While it is generally believed that functional complexes of MHC class II alpha beta heterodimers and peptides have a 1:1 stoichiometry, the data provide qualitative as well as quantitative kinetic evidence that the enhancement of the release of one peptide by a second peptide is due to a two-peptide intermediate. Different combinations of peptides were tested for their ability to accelerate each other's release from IEd. The importance of positive charge for the interaction with IEd was confirmed by the finding that not only dynorphin 1-13 but also poly-L-lysine (14-19 mer) and a peptide corresponding to a mitochondrial presequence (net charge +6) efficiently enhance the release of pre-bound peptides. SDS-PAGE analysis revealed that the efficiently displacing peptides do not stabilize the IEd alpha beta heterodimer at acidic pH, in contrast to the IEd-restricted antigenic peptide HEL 107-116. The data support a mechanism in which the second peptide binds specifically to the pre-formed class II-peptide complex, which, depending on the properties of the peptides involved, leads to the destabilization of the complex and the release of the first peptide.

Expression of HLA-DR mRNA in T cells following activation is early and can precede dna synthesis

The stimulation of T cells is accompanied by the appearance of a number of activation antigens not found on resting T cells. We have studied the expression of one group of such antigens, the HLA class II antigens (DR, DQ and DP), following stimulation with PHA and PMA. Immunofluorescence studies using monoclonal antibodies indicated that cell surface class II determinants were detectable as little as 30 minutes following stimulation, and that their levels remained constant for about 24 hours, after which they began to increase. Studies on the molecule level demonstrated an increase in the steady state levels of mRNA for DR beta by 15 minutes. Immunoblot analysis of resting T cell lysates using a monoclonal antibody reactive with DR detected a polypeptide of approximately 28 KDa, which agrees with the known molecular mass of the light chain of class II molecules on SDS-PAGE, suggesting that the rapid surface expression might have been due to the presence of pre-formed class II polypeptides. These results demonstrate 1) that T cells defined as resting by conventional criteria contain class II polypeptides and 2) that increases in steady state levels of class II mRNA is a very early event which precedes the initiation of DNA synthesis.