Mitigation of therapeutic anti-CD38 antibody interference with fab fragments: How well does it perform?

BACKGROUND: Administration of anti-CD38 antibodies is a state-of-the-art therapy for patients diagnosed with multiple myeloma (MM). However, this treatment frequently leads to pan-agglutination of red blood cells (RBCs) in patients' serological testing making accurate blood typing and timely transfusion of compatible blood a challenging effort. The antigen masking indirect antiglobulin test (AMIAT) is an approach to address this diagnostic challenge. STUDY DESIGN AND METHODS: A new reagent, called DaraEx plus, uses anti-CD38 Fab fragments to mitigate the anti-CD38 antibody interference in serological assays by masking CD38 on the cell surface. Its performance is extensively examined with commercial sera as well as with patient samples, and compared to the current standard method using dithiothreitol (DTT), which denatures the CD38 antigens on test panel erythrocytes. RESULTS: In the Bio-Rad ID System, DaraEx plus effectively mitigated the interference caused by anti-CD38 antibodies in 86% of patient samples tested while DTT was successful in only 68%. Moreover, there was no negative influence on DTT-sensitive blood group systems such as KEL upon DaraEx plus treatment. The agglutination reactions of all tested anti-CD38 antibodies (Daratumumab, Felzartamab, and Isatuximab) were inhibited by DaraEx plus. The treatment was successful only if DaraEx plus was added to the test cells before the sample. Some of the other gel card systems tested showed background reactions with DaraEx plus-treated cells. CONCLUSION: DaraEx plus treatment is straightforward and quick to perform. In the Bio-Rad ID System, it is superior to DTT treatment in the prevention of anti-CD38 antibody interference.

Effect of clinical application of anti-CD38 and anti-CD47 monoclonal antibodies on blood group detection and transfusion therapy and treatment.

BACKGROUND: To investigate the effect of anti-CD38 monoclonal antibodies (mAb) (daratumumab, DARA) and anti-CD47 mAb combined with azacytidine on blood transfusion compatibility tests, transfusion effects in the treatment of multiple myeloma or acute myeloid leukemia and the corresponding management strategy. MATERIALS AND METHODS: Among the 19 patients who were treated with DARA and anti-CD47 mAb, 4 patients with cross matching incompatibility were selected. The ABO blood group, the Rh blood group, irregular antibody screening and direct antiglobulin test (DAT) and cross matching testing were performed before and after the application of mAbs using serological methods. Then, irregular antibody screening and microcolumn gel cross matching tests were performed with donor and recipient erythrocytes and serum treated with DL-dithiothreitol (DTT) and Immucor kit, respectively. The transfusion effect was monitored. RESULTS: 21.05% (4/19) patients had mismatched cross-matching results after mAb treatment. The agglutination intensity of irregular antibody screening tests (3 + ∼ 4 +) after anti-CD47 mAb was higher than that (1 + ∼ 2 +) after DARA. In the DARA group, treating RBCs with 0.2 mol L-1 DTT eliminated the DARA interference with antibody screening. In the anti-CD47 mAb group, the antibody screening, cross-matching test and DAT had been strongly interfered, and using Immucor kit eliminated the interference with antibody screening testing. There was no difference in the transfusion effect. CONCLUSION: The application of mAb drugs led to incompatibility of cross matching tests, and the transfusion effect was not affected.

Efficient neutralization of daratumumab in pretransfusion samples using a novel recombinant monoclonal anti-idiotype antibody.

BACKGROUND: Anti-CD38 antibodies such as daratumumab (DARA) are critical therapies for multiple myeloma and other diseases. Unfortunately, anti-CD38 antibodies cause panreactivity in indirect antiglobulin tests (IATs), complicating blood compatibility testing. The anti-CD38 interference is most often mitigated by treating reagent red blood cells (RBCs) with dithiothreitol (DTT). However, when using the DTT method, not all RBC antibody specificities can be detected (e.g., anti-K), and the DTT method is impractical for some transfusion services. We evaluated the ability of a new anti-idiotype antibody to neutralize DARA in vitro and eliminate the anti-CD38 interference. STUDY DESIGN AND METHODS: A recombinant monoclonal rabbit anti-DARA idiotype antibody ("anti-DARA") was generated. The ratio of anti-DARA required to neutralize DARA in spiked samples was evaluated in IATs performed in gel. IATs performed in tube were used to demonstrate that anti-DARA allows alloantibody detection in the presence of DARA. Plasma samples from 29 patients receiving DARA were treated with a fixed quantity of anti-DARA (120 µg) before performing antibody detection tests (screens) in tube. RESULTS: Anti-DARA used at or above a 1:1 ratio with DARA eliminated the DARA interference with IATs. Anti-DARA allowed detection of both alloanti-E and alloanti-K in the presence of DARA. In 27/29 (93.1%) clinical samples, 120 µg anti-DARA was sufficient to neutralize the DARA in 100 µl patient plasma. DISCUSSION: An anti-DARA:DARA ratio as low as 1:1 is sufficient to neutralize DARA in solution. A fixed amount of anti-DARA eliminates the anti-CD38 interference in most patient samples.

1,4-Dithiothreitol treatment ameliorates hematopoietic and intestinal injury in irradiated mice: Potential application of a treatment for acute radiation syndrome.

Radiation exposure poses a significant threat to public health, which can lead to acute hematopoietic system and intestinal system injuries due to their higher radiation sensitivity. Hence, antioxidants and thiol-reducing agents could have a potential protective effect against this complication. The dithiol compound 1,4-dithiothreitol (DTT) has been used in biochemistry, peptide/protein chemistry and clinical medicine. However, the effect of DTT on ionizing radiation (IR)-induced hematopoietic injury and intestinal injury are unknown. The current investigation was designed to evaluate the effect of DTT as a safe and clinically applicable thiol-radioprotector in irradiated mice. DTT treatment improved the survival of irradiated mice and ameliorated whole body irradiation (WBI)-induced hematopoietic injury by attenuating myelosuppression and myeloid skewing, increasing self-renewal and differentiation of hematopoietic progenitor cells/hematopoietic stem cells (HPCs/HSCs). In addition, DTT treatment protected mice from abdominal irradiation (ABI)-induced changes in crypt-villus structures and function. Furthermore, treatment with DTT significantly enhanced the ABI-induced reduction in Olfm4 positive cells and offspring cells of Lgr5+ stem cells, including lysozyme+ Paneth cells and Ki67+ cells. Moreover, IR-induced DNA strand break damage, and the expression of proapoptotic-p53, Bax, Bak protein and antiapoptotic-Bcl-2 protein were reversed in DTT treated mice, and DTT also promoted small intestine repair after radiation exposure via the p53 intrinsic apoptotic pathway. In general, these results demonstrated the potential of DTT for protection against hematopoietic injury and intestinal injury after radiation exposure, suggesting DTT as a novel effective agent for radioprotection.

A blockage monoclonal antibody protocol as an alternative strategy to avoid anti-CD38 interference in immunohematological testing.

BACKGROUND: As CD38 is expressed on red blood cells (RBCs), the plasma of patients on daratumumab (DARA) reacts with the panel cells of pretransfusion tests, masking underlying alloantibodies. The treatment of RBCs with dithiothreitol (DTT) is the most disseminated method to overcome DARA effect on immunohematological tests, but it hampers the identification of potentially harmful antibodies. Our goal was to validate a new strategy, the blockage monoclonal antibody protocol (BMAP), to mitigate the DARA interference on RBCs using anti-CD38 and antihuman globulin. METHODS: Samples of patients receiving DARA were included in the study. Sera were tested using both DTT- and BMAP-treated RBCs, which comprised three steps: 1) titration of monoclonal anti-CD38, 2) treatment of RBCs obtained from donors with anti-CD38, and 3) blockage of anti-CD38-adsorbed RBCs with antihuman globulin. RESULTS: Twenty patients were included in the study. Donor RBCs were treated with anti-CD38 and successfully blocked with antihuman globulin. In 19 patients, DARA-mediated agglutination was eliminated using both DTT- and BMAP-treated RBCs. In one patient, agglutination persisted when tested against the BMAP-treated RBCs, and alloantibodies were identified. Patient samples were mixed with commercial anti-D, -C, -e, -K, -Jka, -Kpb and tested against antigen-positive BMAP-treated RBCs, resulting in detection of these antibodies. CONCLUSION: This study validated a new strategy to minimize the interference of DARA on immunohematological tests. The protocol preserves the integrity of RBC antigens, permitting the detection of antibodies from all blood group systems. The BMAP has potential use in other situations where specific antibodies may interfere with pretransfusion screening.

Validation and cost-effectiveness of an in-house dithiothreitol (DTT) treatment protocol for daratumumab patients in a large tertiary care hospital provides gateway for implementation in smaller community hospitals.

BACKGROUND: Treatment of multiple myeloma with daratumumab (DARA) is increasing fast. Unfortunately, this antibody also attaches to red blood cells (RBCs) and mimics an autoantibody's panreactivity during pre-transfusion testing, necessitating specialized techniques, (e.g. dithiothreitol (DTT)) for alloantibody detection. Many hospitals use a reference lab for such testing, increasing both cost and turn-around time (TAT). Herein, we compare the cost and TAT, pre and post-implementation of an in-house DTT protocol. METHODS: We designed a validation of our in-house DTT protocol from Nov to Dec 2017 with full implementation on January 1, 2018. We retrospectively reviewed all pre-transfusion tests on DARA patients from Feb 2016 to April 2018, pre and post-implementation of in-house DTT testing. Descriptive statistics were used for patient demographics and a Student t-test was used to compare cost and TATs (pre and post-implementation). RESULTS: We identified 49 patients on DARA treatment requiring transfusion. Samples from these patients were sent to the reference lab 104 times and were tested in-house 28 times. The average TAT for the reference lab was 19h25 m compared to our in-house TAT of 5h9m (an average time-savings of 14h16 m). We spent approximately $33,800 ($325 per test) for 104 reference lab samples versus $806.12 (˜$28.79 per test) for in-house testing of 28 samples. CONCLUSION: We provide an easily implementable DTT protocol for pre-transfusion testing community hospitals and beyond. As more monoclonal antibodies are developed and approved for clinical use, the lessons learned with DARA will expand to deal with interference from future targeted therapies.

An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases.

RATIONALE: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. OBJECTIVES: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. METHODS: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. MEASUREMENTS AND MAIN RESULTS: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in ßENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. CONCLUSIONS: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.

α2-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α2M Levels Are Associated with Venous Thromboembolism.

BACKGROUND: Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. METHODS: Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. RESULTS: In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. CONCLUSION: The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk.

Dithiothreitol supplementation mitigates hepatic and renal injury in bile duct ligated mice: Potential application in the treatment of cholestasis-associated complications.

Cholestasis is a disorder characterized by impaired bile flow and accumulation of cytotoxic bile acids in the liver. On the other hand, oxidative stress and its deleterious consequences seem to have a significant role in cholestasis-induced organ injury. Hence, antioxidants and thiol-reducing agents could have potential protective effect against this complication. The current investigation was designed to evaluate the effect of dithiothreitol (DTT) as a safe and clinically applicable thiol-reductant in cholestatic animals. DTT is a dithiol compound which effectively reduces disulfide bonds in glutathione molecule or different proteins and preserves cellular redox environment. Bile duct ligated (BDL) mice were supplemented with DTT-containing drinking water (0.25% and 1% w: v) for 14 days. Blood, liver, kidney, and spleen samples were collected at scheduled time intervals (3, 7, and 14 days after BDL operation). Significant elevation in plasma biomarkers of liver and kidney injury was detected in BDL animals. Liver and kidney injury was also histopathologically evident by necrosis, inflammation, and fibrosis. Furthermore, high levels of reactive oxygen species in addition to lipid peroxidation, depleted glutathione reservoirs, and impaired tissue antioxidant capacity was detected in the liver and kidney of cholestatic animals. It was found that DTT supplementation (0.25% and 1% w:v) alleviated markers of oxidative stress in the liver and kidney. Moreover, liver and kidney histopathological changes and collagen deposition were markedly attenuated by DTT treatment. The beneficial effects of DTT administration in cholestasis and its associated complications might be linked to its ability for preserving cellular redox environment and preventing oxidative stress.

Treatment With Dithiothreitol Improves Bacterial Recovery From Tissue Samples in Osteoarticular and Joint Infections.

BACKGROUND: Prosthetic implants, periprosthetic and osteoarticular tissues are specimens of choice for diagnosis of bone and joint infections. Homogenization is considered the best procedure for treatment of tissues samples, but, it is not always performed in all laboratories. Dithiothreitol (DTT) has been proposed as an alternative treatment to sonication for microbiological diagnosis of prosthetic joint infections. In this study, the applicability of DTT treatment for processing of periprosthetic and osteoarticular tissues for diagnosis of bone and joint infections was evaluated and compared with normal saline solution treatment. METHODS: Periprosthetic tissue samples were collected from 70 consecutive patients (25 infected and 45 not infected). For each patient, samples from the same site were randomly allocated to DTT or saline treatment. Treated samples were centrifuged at 3000 rpm for 10 minutes. Aliquots from the concentrated samples were plated on agar plates and inoculated in broths. Sensitivity and specificity were calculated for each treatment. RESULTS: Microbial growth was observed in samples from 14 and 11 infected patients after DTT and saline treatments, respectively. Concordance between the 2 methods was observed in the 85.7% of cases. Sensitivity was 88.0% for DTT and 72.0% for saline. Specificity was 97.8% and 91.1% for DTT and saline, respectively. Treatment with DTT showed higher sensitivity and specificity with respect to the method routinely used in our laboratory. CONCLUSION: DTT treatment may be considered a practicable strategy for microbiological analysis of tissues for diagnosis of bone and joint infections.

Sulfasalazine-induced renal injury in rats and the protective role of thiol-reductants.

Sulfasalazine is widely used for inflammatory-mediated disorders in human. Renal damage is a serious adverse effect accompanied sulfasalazine administration. No specific therapeutic option is available against this complication so far. Oxidative stress seems to play a role in sulfasalazine-induced renal injury. Current investigation was designed to evaluate the effect of N-acetyl cysteine (NAC) and dithiothreitol (DTT) as thiol reductants against sulfasalazine-induced renal injury in rats. Oral administration of sulfasalazine (600 mg/kg for 14 consecutive days) caused renal injury as judged by increase in serum level of creatinine and blood urea nitrogen. Furthermore, the level of reactive oxygen species and lipid peroxidation were raised in kidney tissue after sulfasalazine administration. Additionally, it was also found that renal glutathione reservoirs were significantly depleted in sulfasalazine-treated animals. Histopathological examination of kidney endorsed organ injury in drug-treated rats. Daily intraperitoneal administration of NAC (250 and 500 mg/kg/day) and/or DTT (15 and 30 mg/kg/day) effectively alleviated renal damage induced by sulfasalazine. Data suggested that thiol reductants could serve as potential protective agents with therapeutic capabilities against sulfasalazine adverse effect toward kidney.

Cellular mechanisms of plasmalemmal sealing and axonal repair by polyethylene glycol and methylene blue.

Mammalian neurons and all other eukaryotic cells endogenously repair traumatic injury within minutes by a Ca²âº-induced accumulation of vesicles that interact and fuse with each other and the plasmalemma to seal any openings. We have used uptake or exclusion of extracellular fluorescent dye to measure the ability of rat hippocampal B104 cells or rat sciatic nerves to repair (seal) transected neurites in vitro or transected axons ex vivo. We report that endogenous sealing in both preparations is enhanced by Ca²âº-containing solutions and is decreased by Ca²âº-free solutions containing antioxidants such as dithiothreitol (DTT), melatonin (MEL), methylene blue (MB), and various toxins that decrease vesicular interactions. In contrast, the fusogen polyethylene glycol (PEG) at 10-50 mM artificially seals the cut ends of B104 cells and rat sciatic axons within seconds and is not affected by Ca²âº or any of the substances that affect endogenous sealing. At higher concentrations, PEG decreases sealing of transected axons and disrupts the plasmalemma of intact cells. These PEG-sealing data are consistent with the hypothesis that lower concentrations of PEG directly seal a damaged plasmalemma. We have considered these and other data to devise a protocol using a well-specified series of solutions that vary in tonicity, Ca²âº, MB, and PEG content. These protocols rapidly and consistently repair (PEG-fuse) rat sciatic axons in completely cut sciatic nerves in vivo rapidly and dramatically to restore long-lasting morphological continuity, action potential conduction, and behavioral functions.

An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells.

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 microl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 microl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100 degrees C for 45 min. After cooling, 100 microl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r(2) > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.

Dithiothreitol reversal of allyl alcohol cytotoxicity in isolated rat hepatocytes.

Reversal by dithiothreitol (DTT) of allyl alcohol cytotoxicity was investigated in isolated rat hepatocytes. Allyl alcohol-induced protein sulfhydryl loss, bleb formation, and cell death were prevented by DTT, when it was added to hepatocytes 30 min after the toxicant. The protective effect of DTT also was demonstrated in cells that were washed after 30 min of exposure to allyl alcohol, indicating that protection was not related to inhibition of allyl alcohol metabolism or inactivation of acrolein. DTT reversed the cell surface protrusions that formed during exposure to allyl alcohol, but reversal of blebbing did not insure that the cells would remain viable. Glutathione disulfide was not formed in allyl alcohol-treated cells, and DTT reversal of cytotoxicity occurred without restoring glutathione levels. Moreover, protection against allyl alcohol toxicity required the continuous presence of DTT. The results suggest that initial events in the toxic process are reversible, and that DTT can prevent cytotoxicity if added to hepatocytes before irreversible damage occurs; however, the mechanism by which DTT exerts its protection is not clear.

Influence of intracellular thiol and polyamine levels on radioprotection by aminothiols.

We examined the effect of manipulating the levels of two endogenous radioprotectors, glutathione (GSH) and polyamines, on the ability of exogenous aminothiols to protect Chinese hamster ovary cells from the lethal effects of gamma-radiation. Treatment with 0.5 mmol dm-3 buthionine sulfoximine (BSO) for 24 h depleted GSH levels to < 1% of control and significantly sensitized the cells to irradiation in air. Undepleted control cells were protected by WR-1065 (4 mmol dm-3; 30-min preirradiation treatment at 37 degrees C) by 2.09-fold (range 1.98-2.21) at the 10% survival level, whereas BSO-treated cells were protected by a factor of 1.98 (range 1.95-2.14) at this survival level. Thus, GSH depletion had no significant effect on the radioprotective capacity of WR-1065. Treating cells with 1 mmol dm-3 alpha-difluoromethyl ornithine (DFMO) for 48 h depleted the polyamines putrescine and spermidine to very low levels, while spermine was not significantly depleted. DFMO also sensitized cells to aerobic irradiation. WR-1065 protected DFMO-treated cells by 2.29-fold (range 2.08-2.53), whereas undepleted control cells were protected by 2.09-fold (range 1.98-2.21) at the 10% survival level. Thus, WR-1065 appeared to offset the radiosensitizing effect of the DFMO treatment. Cysteamine, on the other hand, protected control and DFMO-treated cells to the same extent. We also examined the effect of combinations of exogenous thiols on radiosensitivity. Cells were treated with WR-1065 (4 mmol dm-3) for 30 min and then with increasing concentrations of dithiothreitol for 5 min prior to irradiation. The protective effects of these two thiols were simply additive.

Antioxidative stress therapy with dithiothreitol tetraacetate. I. Protection against carbon tetrachloride induced liver necrosis.

Dithiothreitol (DTT) is known to prevent or even reverse several deleterious effects of radiation or of chemical agents operating via free radical and oxidative stress. However, its use has been hampered by its chemical instability and toxic properties. In this work, we synthesized and characterized dithiothreitol tetraacetate (DTT-Ac) which is less toxic and chemically stable, and we provided GLC/MS evidence that it is able to rapidly generate fully deacetylated DTT in liver after its administration to rats. Treatment with DTT-Ac simultaneously with CCl4 or 3 h after the hepatotoxin was able to significantly prevent the CCl4-induced liver necrosis at 24 h after poisoning. DTT-Ac administration was able to significantly reduce the intensity of the covalent binding of CCl4 reactive metabolites to microsomal lipids (CB), but it did not prevent the CCl4-induced initiation of a lipid peroxidation (LP) process as evidenced by diene hyperconjugation of microsomal lipids. Results suggest that DTT-Ac protective effects might be due to its in vivo conversion to DTT which in turn would decrease the intensity of CB via different potential mechanisms to be explored. Protection cannot be attributed to decreases in levels of CCl4 reaching the liver or to chain breaking effects on LP.

Determinación de anticuerpos y aloanticuerpos linfocitoxicos en pacientes candidatos a trasplante renal

Las pruebas cruzadas para detectar la presencia de anticuerpos linfocitotóxicos en el suero de candidatos a trasplante renal, son decisivas en el estudio pretrasplante. En el presente estudio se reportan los hallazgos en 97 pacientes con insificiencia renal crónica, candidatos a trasplantes y 127 posibles donantes vivos relacionados. Las pruebas incluyerón detección de anticuerpos antilifocitos T y B separadamente a 4,20 y 37 grados contra linfocitos del posible donante y contra las células autólogas. Se incluye además el tratamiento con ditiotreitol (DTT) para diferenciar entre los isotopos IgM e IgG. Los resultados muestran que 40.2 por ciento de los pacientes y 16.5 por ciento (p<=0.04) de los donantes clínicamente sanos tiene anticuerpos que pueden reaccionar con células alogénicas. Los aloanticuerpos se dectectaron en 38 por ciento de los pacientes. La mayoria de los anticuerpos estaban dirigidos contra los linfocitos B (71.7 por ciento) y correspondieron al isotipo IgM (66.7 por ciento), aunque tambien se detectaron tanto auto como aloanticuerpos IgG.No se detectó un efecto significativo de las trasfuciones o embarazos previos en el desarrollo de anticuerpos ni de aloanticuerpos. De otro lado se observó una frecuencia significativamente mayor (p=0.0009) de donantes intrafamiliares con autoanticuerpos positivos, en pacientes tambien positivos para autoanticuerpos que de receptores negativos para autoanticuerpos positivos que fueron trasplantados con riñones provenientes de sus donantes intrafamiliares o de cadáver, tienen sus injertos funcionantes hasta un año después. Los pacientes sin autoanticuerpos presentaron una menor sobrevida de sus injertos especialmente en el caso de los injertos de cadáver, de los cuales soló sobrevivió la tercera parte después de un año. Los resultados hacen énfasis en la necesidad de realizar las pruebas cruzadas previas al trasplante en condiciones que permitan obtener la mayor información posible sobre los anticuerpos linfocitotóxicos presentes en el suero de los pacientes candidatos a trasplante renal.

The adverse impact on liver transplantation of using positive cytotoxic crossmatch donors.

Because of the liver graft's ability to resist cytotoxic antibody-mediated rejection, it has become dogma that the conventional transplant crossmatch used to avoid hyperacute rejection of other organs is irrelevant to the liver. We examined this hypothesis in a consecutive series of adult primary liver recipients treated with FK506 and low-dose steroids. Twenty-five of 231 (10.8%) patients received a liver from a cytotoxic-positive crossmatch donor (more than 50% of donor T lymphocytes were killed by dithiothreitol-pretreated recipient serum). The outcome was compared with that of 50 negative crossmatch patients who had their transplantations just before and after the crossmatch positive cases. The one-year graft and patient survivals were 56% and 68%, for positive and 82% and 86% for negative crossmatch patients (P = 0.004, P = 0.03, respectively). The difference between patient and first graft survival was accounted for by retransplantation, which was 4 times more frequent in the positive-crossmatch cases. Histologically, failed allografts obtained at the time of retransplantation revealed a spectrum of pathologic findings related to vascular injury. This study showed a higher difficulty of intraoperative blood product management, a degraded prognosis, and a poorer average quality of ultimate graft function when liver transplantation was performed against positive cytotoxic crossmatches. In such patients for whom crossmatch-negative donors may never be found because of the broad extent and intensity of sensitization, special therapeutic strategies perioperatively must be evolved if results are to improve.

Autoreactive lymphocytotoxic IgM antibodies in highly sensitized dialysis patients waiting for a kidney transplant: identification and clinical relevance.

The contribution of different families of lymphocytotoxic antibodies in the serologic reactivity of 45 highly sensitized dialysis patients (HSDP) (panel reactivity antibody value-PRA greater than 80%) was assessed by analyzing patients' sera for the presence of auto- and alloreactive IgM and alloreactive IgG antibodies. A total of 220 sera was screened at different incubation temperatures, before and after treatment with the reducing agent dithiothreitol, against a large variety of cell targets by means of complement dependent cytotoxicity (CDC) and antiglobulin augmented (AHG) CDC assays. The results allowed to subdivide the HSDP under study into four groups: Group 1 consisted of 13 untransplanted patients and 14 patients with a prior failed graft whose PRA values did not change following DTT treatment. Alloreactive IgG antibodies alone, with anti-HLA specificity, were present in the sera of this patient group. Group 2 consisted of 3 untransplanted patients whose sera did not contain any autolymphocytotoxic antibody but appeared completely unreactive to panel lymphocytes following DTT treatment, thus confirming the presence of alloreactive IgM only endowed with antiHLA reactivity. Group 3 consisted of 4 untransplanted and 4 patients with a prior failed graft whose sera were found to contain in addition to autoreactive IgM also alloreactive IgG antibodies. Their PRA values declined after DTT treatment on average from 96.2% to 45% and from 95% to 52.5%, respectively. Group 4 consisted of 6 untransplanted patients whose PRA reactivity to both autologous and panel lymphocytes completely disappeared following DTT treatment, thus indicating that their sera contained exclusively autolymphocytotoxic IgM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)