Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life.

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1ß, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1ß responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1ß induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Mouse strain and injection site are crucial for detecting linked suppression in transplant recipients by trans-vivo DTH assay.

Chemokine-driven accumulation of lymphocytes, mononuclear and polymorphonuclear proinflammatory cells in antigenic tissue sites is a key feature of several types of T-cell-dependent autoimmunity and transplant rejection pathology. It is now clear that the immune system expends considerable energy to control this process, exemplified by the sequential layers of regulatory cell input, both innate and adaptive, designed to prevent a classical Type IV or 'delayed-type' hypersensitivity (DTH) reaction from occurring in the visual field of the eye. Yet, despite an abundance of in vitro assays currently available to the human T-cell immunologist, none of them adequately models the human DTH response and its various control features. The theme of this article is that it is relatively easy to model the effector side of the human DTH response with xenogeneic adoptive transfer models. However, we show that in order to detect inhibition of a recall DTH in response to colocalized donor antigen (linked suppression)--a characteristic feature of peripheral tolerance to an organ transplant--both the challenge site and the immunocompetence of the mouse adoptive host are critical factors limiting the sensitivity of the trans-vivo DTH test.

Human liver allograft acceptance and the "tolerance assay": in vitro anti-donor T cell assays show hyporeactivity to donor cells, but unlike DTH, fail to detect linked suppression.

Human allograft acceptance is associated with immune regulation, characterized by donor-antigen-linked suppression of delayed-type hypersensitivity (DTH). We wished to determine if "classical" in vitro assays of alloreactivity could also detect linked suppression and thus be useful in the clinical diagnosis of active immune regulation. We analyzed peripheral blood mononuclear cells from a group of eight liver transplant recipients, one of whom had stopped all immunosuppression 4.5 years ago yet continues to have good graft function (graft acceptor). The regulator phenotype was defined as the ability to suppress a DTH response to a recall antigen in the presence of donor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro mixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specifically hyporesponsive to donor compared with third-party B-LCL stimulators. However, in contrast to the linked suppression of DTH seen when a given B-LCL expressed donor-type HLA-B antigens, there was no evidence of linked suppression in vitro, either in CTL, proliferative, or interferon-gamma cytokine release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor beta or interleukin-10, which could restore a strong DTH response to donor B-LCL. We conclude that DTH analysis can readily detect donor antigen-linked suppression in liver transplant recipients. CTL and MLC tests failed to do so. These findings may be relevant to the development of a tolerance assay suitable for use in clinical trials.

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient.

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

Soluble donor HLA class I and beta 2m-free heavy chain in serum of lung transplant recipients: steady-state levels and increases in patients with recurrent CMV infection, acute rejection episodes, and poor outcome.

We determined the concentration of donor sHLA/beta(2)m and total beta(2)m-free heavy chain (HC) in the serum of lung transplant recipients with ELISA assays. While we were unable to detect specific donor beta(2)m-free HCs due to a lack of available antibodies, we could determine if events that led to an increase in the release of beta(2)m-free HC also led to an increase in the release of donor sHLA/beta(2)m, particularly the 36 kDa, proteolytically cleaved form. We found that lung transplants constituitively release donor sHLA/beta(2)m at ng/ml levels. The levels (both of donor sHLA/beta(2)m and total beta(2)m-free HC) were significantly increased in CMV-sero-negative recipients (but not in CMV-sero-positive recipients) at the onset of post-transplant CMV disease. Acute rejection episodes were also associated with an increased release of donor sHLA/beta(2)m, but not of beta(2)m-free HC. However, in patients with particularly poor outcome (i.e., graft loss within 1 year) there was a significant release of beta(2)m-free HC. Analysis of one such patient showed a predominance of 36 kDa forms of donor-sHLA/beta(2)m. Our data are consistent with the hypothesis that the metalloproteinase that cleaves beta(2)m-free HC is active during uncontrolled CMV infection and acute rejection. However, recall responses to CMV and controlled immune responses to donor may result in little or no activation of sHLA class I release.

Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis.

We studied late graft rejection in a patient who had received a kidney transplant 9-10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DR beta 1 locus (donor: DR beta 1*1104; recipient: DR beta 1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient's creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient's peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the development of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DR beta 1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DR beta 1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DR beta 1*1104 and DR beta 1*1101 but not with the recipient DR beta 1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this "indirect" alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DR beta 1*1104 subtype.

HLA (A*0201) mimicry by anti-idiotypic monoclonal antibodies.

Soluble MHC Ags and anti-Id (anti-anti-MHC) Abs have both been shown to inhibit MHC alloantigen-specific B cell responses in vivo. We hypothesized that some anti-idiotypic Abs function as divalent molecular mimics of soluble HLA alloantigen. To test this idea, we studied two well-defined anti-idiotypic mAbs, T10-505 and T10-938, elicited in syngeneic BALB/c mice by immunization with CRll-351, an HLA-A2,24,28-specific mAb. Each anti-Id induced "Ab-3" Abs in rabbits that cross-reacted with HLA-A2 but not with HLA-B Ags. Furthermore, each anti-Id could bind to and block Ag recognition by Ha5C2.A2, a human homologue of mAb CRll-351. Both anti-Id mAb displayed weak reactivity with the human mAb SN66E3, which recognized an overlapping but distinct determinant of HLA-A2 Ags; neither reacted with human mAb MBW1, which recognized a nonoverlapping HLA-A2 determinant. Amino acid sequence comparison of mAb CRII-351 heavy and light chain variable region complementarity-determining regions (CDRs) with those of mAb Ha5C2.A2 and SN66E3 revealed short regions of homology with both human mAb; a large insert in the light chain CDR1 of mAb SN66E3 distinguished it from both CRll-351 and Ha5C2.A2. The amino acid sequences of mAb T10-505 and T10-938, which differed markedly from each other, revealed no homology to the alpha2 domain sequence of HLA-A*0201 that contains the CRll-351 mAb-defined epitope. We conclude that structurally different anti-Id Abs can mimic a polymorphic conformational epitope of an HLA Ag. In the case of T10-505 and T10-938 mimicry was not based on exact replication of the epitope by the hypervariable loops of the anti-Id mAb.

Soluble HLA class I in epithelial lining fluid of lung transplants: associations with graft outcome.

We hypothesized that the small amounts of donor HLA-A and HLA-B proteins detected in the serum during organ allograft rejection are indicative of higher local releases within the graft itself. We determined the concentrations of total HLA class I (HLA-I) and, in selected cases, specific donor and host HLA-A and HLA-B proteins, in the epithelial lining fluid (ELF) sampled by bronchoalveolar lavage (BAL) of lung transplant recipients (n = 37) and of normal controls (n = 25). We found that 1) HLA-I proteins were enriched in the lung ELF relative to other proteins; 2) the concentration of HLA-I in the ELF of well-functioning transplants was similar to that in normal lungs; 3) HLA-I proteins and total proteins were elevated in the ELF of patients who developed chronic rejection or refractory acute rejection; 4) the concentration of HLA-I was correlated with the percentage of neutrophils but not with the percentage of lymphocytes in the ELF of transplanted lungs; and 5) only the percentage of lymphocytes was elevated in the ELF of transplant patients with active CMV infections. Total HLA-I from the ELF was found to contain a mixture of both donor- and recipient-type HLA-A and HLA-B proteins and the donor-type HLA-A2 was found to be highly enriched in the ELF relative to serum.

Donor-derived human leukocyte antigen class I proteins in the serum of heart transplant recipients.

BACKGROUND: Human leukocyte antigen class I proteins are expressed on most cell types in all organ allografts but are constitutively secreted only by certain organs, for example, the liver. We hypothesized that detectable levels of donor-derived human leukocyte antigen proteins would be released from transplanted cardiac allografts only when the allograft was immunologically stimulated, that is, during rejection and perhaps during viral infection. If so, then the release of donor human leukocyte antigen might be a noninvasive monitor of these events. METHODS: We used an enzyme-linked immunosorbent assay to detect donor-derived human leukocyte antigen-A2 in the serum of 21 human leukocyte antigen-A2 negative recipients of human leukocyte antigen-A2-positive heart transplants. The level of donor human leukocyte antigen-A2 during the first 100 days after transplantation was correlated with the clinical status of the patient. RESULTS: We found little or no donor human leukocyte antigen in the serum of heart transplant recipients whose postoperative clinical course was unremarkable for infection or rejection. We did find donor-derived human leukocyte antigen in the serum of heart transplant recipients transiently in the week immediately after transplantation, continuously from patients in whom chronic rejection was developing, during cytomegalovirus infection, and during some, but not all, acute rejection episodes as determined by endomyocardial biopsy. CONCLUSIONS: These findings are consistent with the hypothesis that the donor human leukocyte antigen serum level reflects vascular diseases, rather than myocardial disease in the transplanted heart. Therefore, the serum level of donor human leukocyte antigen cannot be used as a monitor of cellular infiltration and myocyte damage as currently assessed by endomyocardial biopsy but may be an early indicator of the development of vascular disease such as chronic rejection.

Monitoring of kidney and simultaneous pancreas-kidney transplantation rejection by release of donor-specific, soluble HLA class I.

Using an HLA-A2-specific ELISA we monitored daily pretransplantation and posttransplantation sera from five kidney and eight simultaneous pancreas-kidney HLA-A2-negative recipients of HLA-A2-positive transplants during hospitalization. We found that, unlike liver transplants, neither kidney nor simultaneous pancreas-kidney transplants continuously secreted donor HLA proteins. However, three of four rejection episodes in kidney recipients and seven of seven rejection episodes in simultaneous pancreas-kidney recipients were accompanied by elevated serum levels of donor sHLA-A2 (> 5 ng/ml). In only one kidney patient was there a release of donor antigen without evidence of rejection, but in the simultaneous pancreas-kidney group most patients had at least one time point of detectable sHLA-A2 without strong evidence of kidney rejection. While total sHLA levels were also elevated during rejection, the rise in donor-specific sHLA was more dramatic when compared to pretransplantation background levels. We hypothesized that the release of donor sHLA class I proteins by transplanted organs might be a systemic indication of rejection in both pancreas and kidney allografts. The detection of donor sHLA in recipient sera could be an important noninvasive monitor of rejection, especially in the pancreas, which is currently difficult to monitor as a single-organ transplant.

Epitope fine specificity of human anti-HLA-A2 antibodies. Identification of four epitopes including a haptenlike epitope on HLA-A2 at lysine 127.

Anti-HLA-A2 CREG antibodies were purified from seven individuals by affinity chromatography. The binding of the purified antibodies to single or multiple amino acid variants of HLA-A2.1 was measured with an inhibition RIA. Substitutions at 10 amino acid residues in the polymorphic alpha 1 and alpha 2 domains were important for human antibody binding; eight of these have previously been shown to be important in the binding of murine anti-HLA-A2 CREG antibodies. Unlike any previously reported murine mAbs, the binding of antibodies from two individuals was eliminated by a substitution at the HLA-A2, -24, -28 shared loop amino acid residue lysine 127. Conversely, when the asparagine at residue 127 on the non-cross-reactive HLA-A3 was replaced with lysine, antibody binding was completely restored. The results further suggest that both lambda- and kappa-containing human antibodies that bind to this region may recognize lysine 127 as a haptenlike epitope. Anti-HLA-A2 antibodies that recognized a conformational epitope defined by changes at glycine 62 in the alpha 1 domain were predominated by lambda light chains whereas those that recognize an epitope defined by a loop residue at tryptophan 107 in the alpha 2 domain were predominated by kappa light chains. The data are consistent with a model of restricted epitope recognition of HLA-A2 by human B cells that is similar to, but distinct from, epitope recognition by mouse B-cell hybridomas, and may help to explain the phenomenon of public or cross-reactive idiotypes in the HLA system.

Estimation of some agents of humoral and cell-mediated immunological response in the families of children with food allergy.

The humoral and cellular agents involved in allergic reactions were examined in 85 persons from 25 families of children with food allergy. The raised level of immunoglobulin M, the lowered levels of C3 and C4 fractions of complement and the increase in the contents of immunological complexes were the abnormalities most frequently met both in food allergic children and their parents. The incidence of the lowered level of IgA in serum was also significant. However, the abnormal results of cellular tests were more often found in the children with food allergy than in their parents. Especially the normal Helper/Suppressor ratio prevailed in the parents (81.6%) as compared to their children (47.6%).

[Subcutaneous administration of recombinant human erythropoietin (R-EPO) in the treatment of anemia in predialysis patients with chronic renal failure]. / Stosowanie podskórne rekombinowanej ludzkiej erytropoetyny (r-Epo) w terapii niedokrwistosci u chorych z przewlekla niewydolnoscia nerek leczonych zachowawczo.

10 anemic (HB less than 9.0 g/dl) predialysis patients with chronic renal failure were treated for three months with s.c. administration of r-Epo. Blood morphological parameters were estimated using hematological autoanalyser Technicon H1. An increase of the mean hemoglobin (Hb) level from 8.39 to 10.57 g/dl was observed. In 8 patients Hb concentration after 3 months therapy ranged from 9.4 to 12.7 g/dl, but in the remaining two of them Hb was lower than 9.0 g/dl. Appearance of a high percentage of hypochromic erythrocytes is probably the most characteristic response to r-Epo treatment. This phenomenon was caused by iron deficiency. A significant increase of serum creatinine and BUN levels were observed in treated patients, without the concomitant decrease of endogenous creatinine clearance. No clinical symptoms suggesting deterioration of the renal function were observed. Subcutaneous therapy with r-Epo appeared an effective and convenient method of treatment of anemia in predialysis patients.