Epigenetic modulation of immune cells: Mechanisms and implications.

Epigenetic modulation of the immune response entails modifiable and inheritable modifications that do not modify the DNA sequence. While there have been many studies on epigenetic changes in tumor cells, there is now a growing focus on epigenetically mediated changes in immune cells of both the innate and adaptive systems. These changes have significant implications for both the body's response to tumors and the development of potential therapeutic vaccines. This study primarily discusses the key epigenetic alterations, with a specific emphasis on pseudouridination, as well as non-coding RNAs and their transportation, which can lead to the development of cancer and the acquisition of new phenotypic traits by immune cells. Furthermore, the advancement of therapeutic vaccinations targeting the tumor will be outlined.

The role of laboratory medicine in a value-based healthcare system: the example of heart failure patient management in the Italian context.

OBJECTIVE: As of today, healthcare systems worldwide face severe challenges that undermine their sustainability. The value-based healthcare (VBHC) approach has been proposed as a strategic and methodological framework to ensure the delivery of the best patient outcomes with economic efficiency. Through the illustrative example of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) for heart failure (HF) patient management in the context of the Italian National Healthcare system, this article explores the role that in vitro diagnostics (IVDs) can play in enabling value-based care models. SUBJECTS AND METHODS: 14 healthcare professionals representing the relevant professional figures involved in HF patient management met to revise the current HF patient journey and design a new care pathway that, leveraging on BNP/NT-proBNP, reflects the VBHC principles. RESULTS: The literature recognizes the dosage of BNP/NT-proBNP as the gold stan-dard for diagnosing HF. However, as of today, these IVDs are not employed at their full potential regarding HF patient management. A new patient journey is proposed so that patients are diagnosed early and properly monitored in the aftermath of hospitalization, improving outcomes at contained costs. CONCLUSIONS: As testified by the example of HF patient management in Italy, laboratory medicine can represent a lever for adopting value-based care models. Still, large-scale adoption of VBHC will call for structural reforms that revise how healthcare delivery is organized, measured, and reimbursed.

A study of HLA class I and class II 4-digit allele level in Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are represented by rare but life-threatening cutaneous adverse reactions to different drugs. Previous studies have found that in a Han Chinese population from Taiwan and other Asian Countries, a strong genetic association between HLA-class I alleles (B*15:02, B*58:01) and SJS and TEN was induced by carbamazepine and allopurinol, respectively. To identify genetic markers that covered the MHC region, we carried out a case-control association enrolling 20 Caucasian patients with SJS/TEN. Our patient series included 10 cases related to paracetamol, 7 to allopurinol and 3 to different drugs (plaquenil, itraconazol, nabumetone). Healthy controls were represented by 115 Caucasian bone marrow or stem cell donors. The HLA-A*, B*, C*, DRB1*, DQB1*, DQA1* and DPB1* genotyping were determined. The frequencies of HLA-A*33:03 as well as C*03:02 and C*08:01 were significantly higher in SJS/TEN patient subgroup showing allopurinol drug-induced severe cutaneous adverse reactions (SCAR) as compared to controls (28.6% vs 0%, P=0.00002, Pc=0.0011; 28.6% vs 0%, P=0.00002, Pc=0.001; 28.6% vs 0%, P=0.00002, Pc=0.001, respectively). In the same subgroup the frequencies of B*58:01, DRB1*15:02 and DRB1*13:02 alleles, although considerably higher than in control group (42.8% vs 5.2%, P=0.003; 28.6% vs 1.7%, P=0.005; 28.6% vs 3.5%, P=0.037, respectively), appeared no more statistically different after P correction (Pc=0.248; Pc=0.29; Pc=1.00, respectively). In addition, in 10 of the 20 SJS/TEN patient subgroup with paracetamol-induced SCAR no statistically significant association with HLA alleles could be found. However, in the same SJS/TEN patient subgroup showing allopurinol drug-induced SCAR, haplotype analysis indicated that B*58:01, DRB1*13:02 and DRB1*15:02 alleles, that in a single allele analysis lost statistical significance after P correction, may still confer susceptibility, because the B*58:01-DRB1*13:02 and DRB1*15:02-DQB1*05:02 are positively associated with the disease (14.2% vs 0.43%, P= 0.00001, Pc=0.00028; 14.2% vs 0.43%, P=0.00001, Pc=0.00028, respectively). Our results show that in contrast to SCAR-related to paracetamol, where HLA alleles do not appear to be involved, HLA molecules behave as a strong risk factor for SCAR-related to allopurinol even when a limited number of patients are considered.

Cytokine polymorphisms and Alzheimer disease: possible associations.

Alzheimer's disease (AD) is a degenerative dementia characterized by typical, destructive alterations of neurons (neurofibrillary tangles and amyloid plaques), and glial proliferation. Cytokine-driven inflammatory environment can contribute to the pathogenesis and/or progression of the disease. The aim of the study was to evaluate and compare genotypic and allelic polymorphisms of 13 cytokine genes in 19 Caucasoid AD patients with medium-high level of dementia (assessed by an MMSE < 24) and 20 normal controls affected by non inflammatory neuropsychiatric disease. Polymorphisms in the genes of IL-lA, IL-lB, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-G, TGF-beta, TNF-alpha, and of the cytokine receptors IL-lR, IL-IRA, IL-4RA were investigated. APO-E and ACE gene polymorphisms were carried out in the patient's group only to evaluate a possible association with known genetic risk factors for AD. A highly significant presence of some alleles belonging to anti-inflammatory cytokine genes was found; particularly the C allele for the -590 promoter and T allele for the -1098 promoter of IL-4 appeared in a significantly higher percentage as compared with controls (P < 0.0006 and P < 0.0005, respectively), while a lesser significance was observed for the allele C of the -819 promoter of IL-10 (P < 0.03). Finally, in the group of demented patients for the APO-E gene we found a statistically significant presence of the E4 allele, whereas no difference was found for the polymorphisms of the ACE gene. Our observations corroborate the possible presence of a pro-inflammatory environment in AD patients, partly sustained by the low expression of anti-inflammatory cytokine genes when defined alleles are present. Large cohort studies are necessary in order to assess the real association of some cytokine alleles or haplotypes with AD.

Aflatoxin B1 misregulates the activity of serine proteases: possible implications in the toxicity of some mycotoxin.

Aflatoxins are highly hazardous contaminants of common food and feed. Aflatoxin B1 in particular, the most predominant among aflatoxins, was thoroughly demonstrated to be highly toxic, mutagenic, teratogenic and carcinogenic in many animal species. Besides its established targets and effects, this work investigates on the possible direct interaction between aflatoxin B1 and three major serine proteases, namely elastase, thrombin and trypsin. These proteases belongs to a class of structurally and functionally related proteins pivotal in both direct and indirect regulation of a number of cellular events. Additionally, several pathological processes, including cancer, inflammatory processes and thrombosis, rely upon the subtle equilibrium between these enzymes and their potential modulators: in fact, their misregulation, caused by foreign molecules, could facilitate (or be the cause for) the occurrence of these pathologies. Our results provide the evidence for a reversible binding between AFB1 and these enzymes, likely to have profound implications in the manifestation of aflatoxicosis. Precisely, the toxin behaved as a moderate competitive inhibitor toward the enzymatic activity of the serine proteases in the low micromolar range.

Genetic diversity of Streptococcus suis clinical isolates from pigs and humans in Italy (2003-2007).

Streptococcus suis, a major porcine pathogen, is emerging as a zoonotic agent capable of causing severe invasive disease in humans exposed to pigs or pork products. S. suis infection is rare in industrialised countries and usually arises as sporadic cases, with meningitis the most common clinical presentation in humans. Recent reports of two cases of meningitis in Sardinia and northeastern Italy prompted this first characterisation of Italian S. suis isolates. Fifty-nine S. suis strains, the two recent human strains and 57 swine clinical isolates collected between 2003 and 2007 from different Italian herds and regions, were tested for antimicrobial susceptibility, PCR-screened for virulence and antibiotic resistance genes, and subjected to molecular typing. Phenotypic and genotypic analysis demonstrated an overall high genetic diversity among isolates, the majority of which were resistant to macrolides (78%) and tetracyclines (90%). The erm(B), tet(O), mosaic tet(O/W/32/O), tet(W), and tet(M) genes were detected. The tet(O/W/32/O) gene, the most frequent tet gene after tet(O), had never been described in the genus Streptococcus before. In addition, a virulent cps2, erm(B) tet(O) clone, belonging to sequence type 1 (ST1) of the ST1 complex, was found to be prevalent and persistent in Italian swine herds. Finally, the two human isolates (both ST1) carrying cps2, erm(B) and tet(W) were seen to be closely related to each other.

Sirolimus therapy in liver transplant patients: an initial experience at a single center.

Sirolimus (SRL) is an mTOR inhibitor that has been shown, in contrast to calcineurin inhibitors (CNI), to inhibit cancers in experimental models. Since February 2005, we introduced SRL in liver transplant patients in group a, in whom the primary disease was hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic or autoimmune liver cirrhosis, and group b, HCC-negative patients who developed posttransplantation cancers de novo. Of 18 patients in group a, 11 received SRL ab initio (subgroup a1), starting for 10 patients at 66.1+/-29.2 days after surgical healing and after 10 days in 1 case; the remaining 7 patients (subgroup a2) received SRL at 31.2+/-24.2 months. Three patients in group b, included 1 with Kaposi's sarcoma, 1 with bladder cancer, and 1 with thyroid cancer. In this group, SRL was introduced at 80.8+/-40.4 months. In all patients but one, who received a single 5 mg loading dose, SRL was started at 2 mg/d and adjusted to 6 to 8 ng/mL blood levels. CNI drugs, present as primary therapy, were gradually tapered to low levels and eventually stopped. The following observations were drawn from this initial experience: (1) 4/21 (19.0%) patients had to discontinue SRL because of early and late side effects: thrombocytopenia (n=2) and headache with leukopenia and leg edema associated with knee joint arthralgia (n=2); (2) 14 patients (11 in group a and 3 in group b) are still on SRL monotherapy; (3) 1 HCC recurrence and 1 de novo pancreatic adenocarcinoma were observed at 14 and 16 months, respectively (at the time of transplantation, both patients were beyond the MIlan HCC criteria), and (4) 1 patient, from subgroup a1, died after 99 days due to pneumonitis and possible relation to SRL lung toxicity. In conclusion, SRL appeared to be an effective immunosuppressant that could be used as monotherapy in liver transplant patients. Any conclusion on SRL anticancer effects can only come from randomized large studies after long follow-up.

Cytotoxic molecule mRNA expression in chronically rejected human kidney allografts.

The pathogenesis of immunological and nonimmunological components that cause chronic kidney allograft nephropathy (CAN), is not yet completely understood. To explore the possible contribution of alloreactive cytotoxic T cells, we analyzed the transcription of cytotoxic molecules such as granzyme B and perforin using semiquantitative RT-PCR on surgically removed grafts obtained from two groups: group 1 (n = 10) were cases of CAN; group 2 (n = 3) had no CAN. Among group 1 kidneys, granzyme-B was expressed in 7 of 10, whereas perforin was detectable in 9 of 10 cases; their detection was not related to the presence of superimposed signs of acute graft lesions. Cytotoxic molecules were never found in group 2 kidneys. These results show that explanted chronically rejected grafts display cytotoxic molecule transcripts in addition to Th2 type cytokines, such as IL-10, IL-3, and IL-6, suggesting that both cellular and humoral alloreactive mechanisms may play important roles in CAN pathogenesis.

A simple procedure for freezing and storing lymphocyte panels in trays.

Lymphocytes from different donors may be frozen in Terasaki trays by a simple procedure. This method provides the tissue typing laboratory with a readily available panel that can be routinely used to test for lymphocytotoxic antibodies sera from patients waiting for kidney transplant, transplanted patients and polytransfused individuals. The advantage of this procedure is that expensive programmed freezing apparatus is not required.

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney.

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.

Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones.

The effectiveness of cyclosporine (CsA) as immunosuppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the "high responder" patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones.

Correlation between angiotensin-converting enzyme gene insertion/deletion polymorphism and kidney graft long-term outcome in pediatric recipients: a single-center analysis.

BACKGROUND: Despite numerous advances in the areas of organ preservation, histocompatibility, and immunosuppression, chronic deterioration of organ allograft function, referred to as "chronic rejection," still remains the main obstacle to long-term graft survival. The common feature of chronic rejection is a concentric generalized graft arteriosclerosis associated with interstitial fibrosis that reflects an allogeneic injury to graft arteries, possibly worsened by other alloantigen-independent risk factors. The presence of the angiotensin I-converting enzyme (ACE) gene-deleted (D) allele has been associated, when in homozygosity, with increased risk of cardiovascular diseases and with an accelerated progression of organ damage in a variety of kidney diseases. In this study, we analyzed whether the insertion/deletion polymorphism of the ACE gene, because of its negative prognostic impact on cardiovascular and renal pathology, could have any influence on kidney graft survival in pediatric recipients. METHODS: DNA was isolated from peripheral blood mononuclear cells from 146 pediatric dialysis patients (mean age: 12.9 years) who received a first kidney graft at our center between December 1985 and July 1997. To rule out any bias due to acute graft losses, only 119 patients who reached a minimum of 12 months of graft survival were considered for statistical analysis. The insertion/deletion polymorphism of the ACE gene was detected using a polymerase chain reaction technique with two flanking primers. RESULTS: The results demonstrated that (i) the distribution of DD and non-DD (ID + II) genotypes was 36.1% (43 patients) and 63.8% (76 patients), respectively; (ii) actuarial graft survival at 7, 8, 9, and 10 years in patients with non-DD genotype was significantly higher than that in patients with DD genotype (7 years: 94.6% vs. 72.4%, P<0.05; 8 years: 94.6% vs. 62%, P<0.025; 9 years: 87.3% vs. 51.4%, P<0.025; 10 years: 76.3% vs. 25.7%, P<0.01). CONCLUSIONS: In conclusion, the above data indicate that DD genotype is associated in pediatric kidney graft recipients with a shorter long-term kidney graft survival and suggest a possible role of this genotype as a cofactor in the progression of nonimmunological injuries leading to chronic kidney graft failure.

Expression of common acute lymphoblastic leukemia antigen (CD 10) by myelinated fibers of the peripheral nervous system.

The common acute lymphoblastic leukemia antigen (CALLA), CD10, is a 100-kDa surface glycoprotein endowed with neutral endopeptidase activity, shared by a number of hemopoietic and non-hemopoietic cells. In this report, immunohistochemical and Western blot techniques, using different anti-CD10 monoclonal antibodies, were utilized to demonstrate that CD10 is also expressed by myelin sheaths of the human peripheral nervous system (PNS), but not of the central nervous system. CD10-positive immunoreactivity appeared to be localized in the outer and inner borders of myelinated fibers, in nodes of Ranvier and in the Schmidt-Lantermann clefts, thus showing a distribution pattern very similar to that of myelin-associated glycoprotein (MAG). The above findings suggest that CD10 antigen, through its enzymatic activity, may have a functional role in the assembly and maintenance of PNS myelin. In addition, it is not known whether CD10, similarly to MAG, may be a target antigen in some PNS immune-mediated disorders.

HLA matching in pediatric recipients of a first kidney graft. A single center analysis.

We retrospectively examined the effect of HLA-A, -B, and -DR serological matching on graft survival in 88 pediatric end-stage renal disease patients who underwent primary renal transplantation. Actuarial graft survivals (GS) at 2 and 6 years in patients with zero DR mismatches (MM) (12 patients) or 1 DR MM (58 patients) were significantly higher than those in patients with 2 DR MM (18 patients) (2-year GS: 100% vs. 90% vs. 59%; 6-year GS: 100% vs. 79% vs. 59%, respectively). Because of the low number of patients in the zero DR MM group, only the GS difference between 1 DR MM and 2 DR MM had a significant result at 1 year (92% vs. 68%). No clear HLA matching effect was obtained in the HLA-A and -B loci. When DR were combined with A or B antigens (0-2 MM vs. 3-4 MM), significantly higher GS at 1, 2, and 6 years persisted for patients with 0-2 MM only in the A, DR group (96%, 94%, and 85% vs. 68%, 63%, and 56%, respectively). It is suggested that avoidance of mismatching for DR alleles at the serological level, in the selection of pediatric recipients of first cadaveric renal transplantation, leads to an improvement of both short- and longterm graft outcome.

Analysis of transcripts of genes located within the HLA-D region in B cells from an HLA-severe combined immunodeficiency individual.

The mRNA levels of LMP7, HLA-DMA, and HLA-DMB genes, all located within the HLA-D region, were analyzed in the "bare lymphocyte" line SJO, which had been shown to be characterized by a transcription defect of class II genes (DP, DQ, and DR). This was done using semiquantitative PCR amplification of cDNA generated from total RNA. The results show that the transcription of the LMP7 gene in SJO cells is equivalent to that in the control B-cell line Raji. In contrast, the HLA-DM gene transcripts were not present in the SJO cell line. This observation indicates that the promoter similarity between the classic class II genes and HLA-DM is sufficient for the SJO defect to extend to the DM loci.

Downregulation of HLA class I antigen expression in CD4+ T lymphocytes from HIV type 1-infected individuals.

The expression of HLA class I antigens is downregulated in CD4+ T cells following in vitro HIV-1 infection. We determined whether the expression of HLA class I antigens is downmodulated in peripheral blood lymphocytes (PBLs) of HIV-1-positive subjects and whether this defect correlates with disease progression. A cohort of 62 HIV-1-seropositive individuals in different stages of disease was studied. Among these, four subjects were evaluated at yearly intervals for 6 years. The expression of HLA class I, HLA class II, and CD38 antigens was analyzed in PBLs and in CD4+ and CD8+ T lymphocyte subpopulations. The percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in PBLs from HIV-1-positive individuals than in PBLs from HIV-negative controls, proportionally decreased with disease progression, and significantly correlated with the decrease in CD4+ T lymphocytes. Furthermore, the percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in CD4+ T lymphocytes from AIDS patients with respect to CD4+ T lymphocytes from HIV-negative controls and to CD8+ T lymphocytes from HIV-negative controls and AIDS patients. By contrast, the expression of HLA class II and CD38 antigens was upregulated in CD4+ and CD8+ T lymphocytes from HIV-1-positive subjects. The defective expression of HLA class I antigens could impair the lysis of HIV-infected CD4+ cells by virus-specific HLA class I-restricted cytotoxic T lymphocytes and contribute to the progression of disease.

Association between HLA class I antigens and response to interferon therapy in children with chronic HBV hepatitis.

In this preliminary study, children with chronic HBV hepatitis, as was also previously shown for adults, respond to interferon therapy in an HLA class I antigen dependent manner. If this can be confirmed on a large scale, HLA typing may serve as a useful indication of interferon-therapy responders.

Morphology, cytochemical features, and membrane phenotype of HLA-DR+ interstitial cells in the human pancreas.

The morphology, histological distribution, surface, and enzymatic phenotype of pancreatic HLA-DR+ cells were studied on seven human pancreata, removed from cadaver donors. Frozen and paraffin-embedded pancreas sections were stained with a battery of monoclonal antibodies by indirect immunofluorescence, immunoperoxidase, and immunophosphatase techniques. Two type of cells were found to express HLA-DR surface molecules: endothelial cells and nonfibroblastic non-B and non-T interstitial elements. The latter cells, which were localized both in the exocrine and endocrine portions of the organ, were distinguished in two main families (macrophagic and dendritic) according to their morphology, surface phenotype, and lysosomal enzymatic activities. The phenotype of cells belonging to macrophagic cell family was the following: Leu M1+, Leu M2+, Leu M3+, OKM1+, and OKT6-. In addition these cells were positive for the expression of lysosomal enzymes such as alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP). The "dendritic" cell family comprised, among others, cells that were characterized by the presence of numerous finger-like projections, the absence of Leu M1, Leu M2, Leu M3, OKM1, OKT6 surface antigens, and by the negativity for ANAE and AP. These "dendritic looking cells" (DLC) constituted the most represented DR+ cell type within pancreatic islets. The demonstration of dendritic cells within human islets may justify, in humans also, in vitro procedures of intra-islet dendritic cell removal prior to transplantation, in the attempt of islet rejection prevention.

Role of major histocompatibility complex class I expression and natural killer-like T cells in the genetic control of endometriosis.

OBJECTIVE: To evaluate whether the expression of human leukocyte antigen (HLA) class I on eutopic and ectopic endometrial cells modify the susceptibility to lysis mediated by lymphocytes. DESIGN: Evaluation of T lymphocyte cytotoxic activity and HLA class I expression on endometrial cells. SETTING: Subjects were recruited at laparoscopy. PATIENTS: Patients with endometriosis (n = 7). Healthy women as controls (n = 10). MAIN OUTCOME MEASURES: Human leukocyte antigen class I molecule analysis of endometrial cells was carried out by immunofluorescence and flow cytometry. Phenotyping of T lymphocytes was performed to analyze T-cell subsets. Cytotoxicity was performed to determine cytolytic activity against endometrial cells. RESULTS: In vitro culture of endometrial cells down-regulates the expression of HLA class I molecules and enhances the susceptibility to lysis mediated by natural killer (NK)-like T lymphocytes. Cytolytic T-cell clones, expressing the CD94 antigen, are inhibited by the HLA-B7 allele on endometrial cells. Ectopic endometrial cells modulate the expression of HLA class I molecules. CONCLUSIONS: The resistance to lysis of endometrial cells is related to expression of surface HLA class I molecules, which send a negative signal for lysis mediated by NK-like T lymphocytes. The HLA-B7 allele inhibits the cytotoxic activity, suggesting that the growth of ectopic endometrial cells might be under a genetic control.

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)