Advances in the treatment of lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease that leads to the formation and deposition of immune complexes throughout the body, which are pathogenic for the disease. Different forms of glomerulonephritis can occur in patients with SLE and can contribute significantly to the associated morbidity and, ultimately, mortality from the disease. Over the past two decades, there have been significant strides in our understanding of the disease and in treatments that attempt to control the formation and deposition of anti-DNA auto-antibodies and immune complexes, as well as the subsequent inflammatory cascade mediated through various cellular and humoral pathways leading to progressive renal damage and end-stage renal disease. In this chapter, we review the current understanding of the pathogenesis and treatment of lupus nephritis in its various stages and discuss the experimental and human data regarding some of the potential newer forms of therapy. We discuss data regarding the use of steroids, azathioprine, cyclophosphamide, cyclosporine A, mycophenolate mofetil, gammaglobulin, plasmapheresis, LJP 394, flaxseed oil, bindarit, anti-CD40 ligand, and CTLA4Ig.

Isolation, identification and immunosuppressive activity of SDZ-IMM-125 metabolites from human liver microsomes.

SDZ-IMM-125 N-methyl leucine 9 hydroxylated in the gamma position is a metabolite which was extracted from incubated human liver microsomes and subsequently separated by normal and reverse-phase HPLC. This metabolite was identified by fast atom bombardment mass spectrometry, electrospray-ms/ms mass spectrometry and nuclear magnetic resonance spectroscopy. The in vitro 50% inhibitory concentration, tested against bidirectional mixed lymphocyte reaction was 80 microg/l indicating that this metabolite does not retain in vitro immunosuppressive activity most probably due to the structural modification of SDZ-IMM-125 in the recognized binding region to cyclophilin A reducing its binding affinity relative to the parent drug.

Disposicao cinetica da ciclosporina apos administracao de dose unica: estudo experimental em ratos / Kinetic disposition of the ciclosporin after single dose subcutaneous administration: experimental study in rats

A dose adequada de ciclosporina e aquela que apresenta satisfatorio efeito imunossupressor associado a baixa toxicidade. Na literatura, as doses utilizadas variam de 2,5 mg/kg a 25 mg/kg em trabalhos experimentais de alotransplantes de tecidos e orgaos. Preferencialmente sao administradas pelas vias subcutanea ou intra-muscular, com intervalos de 24,48 horas e ate sete dias. Neste estudo utilizou-se para a administracao de dose unica de ciclosporina A (CSA), a via subcutanea em 21 ratos que foram divididos em tres grupos com sete animais cada, recebendo 2,5 mg/kg (grupo 1), 5,0 mg/kg (grupo 2) e 10,0 mg/kg (grupo 3). Colheram-se amostras de sangue nos tempos 1, 4, 8, 12, 24, 48 e 72 apos a administracao da droga. Efetuou-se ciclosporinemia pelo metodo de radioimunoensaio, Kit SandimmunR monoclonal especifico...

Cross-reactivities of cyclosporin G (NVa2 cyclosporin) and metabolites in cyclosporin A immunoassays.

Immunoassays of cyclosporin A (CsA) have been routinely used to measure CsG. We investigated the cross-reactivities of CsG and its metabolites, as well as the proportion CsG constitutes in relation to total drug measured, for six CsG metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) in the following CsA assays: Sandimmune selective RIA (SS), Sandimmune nonselective RIA (NS), Cyclotrac SP-RIA (CT), fluorescence polarization immunoassay (FPIA), and enzyme immunoassay (EMIT). The cross-reactivity of CsG in these assays was as follows: SS, FPIA, CT, approximately 100%; NS, approximately 40%; EMIT, < 2%. The cross-reactivities of CsG metabolites were investigated in all assays except EMIT and varied among metabolites and assays. The most significant variance was found with the NS assay, where most of the metabolites exhibited cross-reactivities of > 40%. In contrast, in the SS, FPIA, and CT assays, cross-reactivities of < 5% were observed for most of the metabolites. The ranking of cross-reactivities of CsG metabolites in the assays is SS = CT < FPIA < NS. The degree of cross-reactivity did not change significantly when the SS, CT, and FPIA assays were calibrated with CsG instead of CsA--whether parent CsG was present or not. The data suggest that the SS, CT, and FPIA methods would be suitable for the routine monitoring of CsG.

Regulación endocrina de la respuesta inmunitaria: la prolactina y su función / Endocrine regulation of the immune response the prolactin and it is funtion

Tradicionalmente se había considerado al sistema inmunitario como autónomo; sin embargo, pruebas recientes han demostrado que existen comunicaciones bidireccionales entre el sistema inmunitario y el neuroendócrino. Hay influencia de las hormonas gonadales, tiroideas, suprarrenales y pituitarias. De entre éstas, la prolactina ha sobresalido como potencial blanco para modificar la respuesta inmunitaria en algunos estados de enfermedad

New cyclosporin A analogue: synthesis and immunosuppressive activity.

A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N-methyl-4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo in mice was practically the same as that of cyclosporine A used as a standard, whereas the analogue studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood cells in mice.

Comparison of the properties of the CsA analogs monoacetyl CyC (o-acetyl-threonine2 cyclosporin) and methyl-alanyl CsA (N-methyl-L-alanyl6 cyclosporin); monoacetyl cyclosporin is immunosuppressive without binding to cyclophilin.

Cyclosporin (CsA) is an immunosuppressant which binds to cyclophilin (Cyp). The relationship between Cyp binding and immunosuppression has been questioned since one of the analogs of CsA, N-methyl-L-alanyl6 cyclosporin (methyl-alanyl CsA) binds to Cyp but is not immunosuppressive. We compared the immunosuppressive properties of CsA, methyl-alanyl CsA and o-acetyl-threonine2 cyclosporin (monoacetyl CyC), since monoacetyl CyC does not bind to Cyp when tested in cell-free assays and its immunosuppressive properties had not been tested. Cyp is a peptidyl-prolyl isomerase which is abundant in all human tissues, yet the activities of CsA are mostly confined to inhibition of T cell and thymocyte activation, and to neuro- and nephro-toxicity and are independent of inhibition of the isomerase. Activation of thymocytes and of T cells is regulated by the binding of a nuclear factor(s) (NFs) to the NF-AT region (-285 to -255) of the IL-2 promoter. We studied inhibition of binding to the NF-AT region of NFs derived from primary cultures of thymocytes treated with CsA or its analogs. In addition, we compared the effect of CsA and its analogs on the expression of the IL-2 gene in a stably transfected Jurkat-cell line (Fgl 5) which contains three copies of NF-AT and the reporter enzyme beta-galactosidase; and on inhibition of proliferation induced by concanavalin A (Con A) or IL-2. We found that monoacetyl CyC which does not bind to Cyp is immunosuppressive by our criteria when tested in cultured cells due to either a different mechanism of action or to metabolic activation.

The isolation, structural characterization, and immunosuppressive activity of cyclosporin G (NVa2-cyclosporine) metabolites.

Seven cyclosporin G metabolites were isolated by high-performance liquid chromatography from the urine of normal subjects receiving the drug. The structure and purity of the metabolites were assessed by fast atom bombardment/mass spectroscopy, by proton nuclear magnetic resonance (NMR), and by 13C-NMR. The structural modifications of the cyclosporin G metabolites consisted primarily of hydroxylation and demethylation, as is the case for cyclosporin A metabolites. The immunosuppressive activities of the metabolites were tested in three separate in vitro systems: a primary and secondary mixed lymphocyte system, as well as a mitogen stimulated system. In general, the metabolites have immunosuppressive activity of less than 10% of cyclosporin G. The significance of these findings in relation to the therapeutic monitoring of cyclosporin G is discussed.

[Non-immunologic effects of cyclosporins]. / Les effets non-immunologiques des ciclosporines.

Cyclosporins are a group of molecules produced by fungi. The main cyclosporin, cyclosporin A, is widely known for its immunologic properties which have made it the leading drug for prophylactic therapy of transplant rejection. However, cyclosporin A (as well as several of its analogs with or without immunosuppressive properties) has a broader and more varied spectrum of biologic effects both in vivo and in vitro; renal, pancreatic, endothelial, muscle and nerve cells, hepatocytes, keratinocytes, and some microorganisms (plasmodium, schistosoma) are target cells whose metabolism or growth is affected by cyclosporins. Some of these activities are responsible for adverse effects but others may warrant the use of selected cyclosporins in clinical situations other than organ transplants.

Isolation of cross-reacting compounds to Incstar Cyclo-Trac SP RIA in blood samples obtained from cardiac allograft patients on cyclosporine therapy.

To examine the specificity of the Incstar Cyclo-Trac SP RIA kit, individual blood samples from 28 cardiac allograft patients on cyclosporine A (CsA) therapy were extracted and chromatographed by HPLC. Initially, eluates from a pool of the above samples were collected at regular intervals and measured by RIA to locate possible cross-reacting compounds. Unknown cross-reacting materials were detected in a fraction (UNK) that was collected before elution of CsA. For each patient's sample, fraction UNK and the fraction containing CsA were then collected and measured by RIA. In 9 of 28 samples, cross-reactivity was detected in fraction UNK; range 11 to 36%, mean 22 +/- 7.5%. Cross-reactivity was not apparent in fraction UNK of CsA-free blood samples from normal volunteers.

Suppression of immunological response against a novel gene product delivered by implants of genetically modified fibroblasts.

We have recently demonstrated the feasibility of genetically modifying autologous primary rat fibroblasts to deliver in vivo a foreign gene product, human growth hormone. However, in this model for gene replacement therapy, all recipient animals developed extremely high titres of antibodies against human growth hormone within two weeks of grafting. We now report on two approaches to suppress this immune-response. First, rats implanted with human growth hormone-secreting rat fibroblasts were treated with an immunosuppressant, cyclosporine A, at 20 mg/kg body weight per day. The production of anti-human growth hormone antibodies in the treated animals was completely blocked during the 12-week course of treatment. Secondly, by using immunologically immature neonatal rats as recipients, the rapid antibody response to the human growth hormone was also avoided. However, after a delay of one month, these rats also developed an extremely high titre of antibodies against the human growth hormone. In comparison, rats in the adolescent, mature and aged groups developed and maintained high titres of antibodies soon after implantation. Therefore, antigenic response against novel gene products can be suppressed either totally by cyclosporine A or temporarily in neonatal animals. The combination of early implantation and subsequent immuno-suppression should be considered in somatic gene therapy for those patients who are negative for cross-reacting-material and may be expected to mount an antigenic response to the replacement gene product.

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Experiencia con cyclosporina a dosis bajas en el trasplante renal / Experience with low dosis cyclosporine in renal trasplantation

Se estudiaron 10 pacientes sometidos a trasplante renal en el Centro Médico Naval, siete de ellos recibieron riñones de donantes no relacionados y tres de donantes relacionados. Todos los pacientes recibieron Cyclosporina y Esteroides como tratamiento inmunosupresor. A todos los pacientes se les realizó dosajes plasmáticos de Cyclosporina, por el método de radioinmunoensayo (RIA), los dosajes se realizaron cuatro veces al día durante los primeros 7 días y luego cada 2 semanas, de los 10 pacientes estudiados, 8 han respondido muy bien a la inmunosupresión con Cyclosporina. En estos pacientes actualmente la dosis de mantenimiento con Cyclosporina fluctúa entre 3.3 a 4 mg/Kg de peso al día y la evolución clínica y función renal es satisfactoria

Chemiluminescence immunoassay of cyclosporine in whole blood.

We describe a chemiluminescent immunoassay (CLI) for measuring cyclosporine in whole blood. Its sensitivity and accuracy are comparable with those of an RIA method that makes use of the same specific monoclonal antibody. The comparison with the RIA method was excellent: y(RIA) = x(CLI) + 11.24 (r = 0.99). In our procedure the samples are incubated with cyclosporin C-hemisuccinate-aminobutyl-N-ethylisoluminol, antibody, and paramagnetic particles coated with second antibody. After magnetic separation and washing, the samples are incubated with 200 microL of NaOH (2 mol/L) at 60 degrees C for 30 min. The chemiluminescence generated by automated serial injections of solutions of microperoxidase and dilute (2 mL/L) H2O2 is measured for 5 s. The data are processed by using a spline fit of log B/Bo log conversion. This method is easy to perform and avoids the hazards and costs associated with isotopic waste disposal. The label is stable for at least three years.

A validation study of selected methods routinely used for measurement of cyclosporine.

We compare four methods for measuring cyclosporine (CyA) in plasma and whole blood of transplant patients: HPLC, RIA with a polyclonal antibody, RIA with a monoclonal antibody, and fluorescence polarization immunoassay (FPIA). The monoclonal RIA procedure correlated acceptably with HPLC, with slope = 1.21, r = 0.97, and Sy,x = +/- 40.1. However, the FPIA, done in three separate instruments, correlated relatively poorly with HPLC, giving slopes of 1.67, 1.51, and 2.32; correlation coefficients of 0.72, 0.43, and 0.83; and Sy,x = +/- 205.4, +/- 334.5, and +/- 222.4. The polyclonal RIA correlated reasonably well with HPLC, with a slope = 1.15, r = 0.90, and Sy,x = +/- 72.6. Values for individual patients with increases both in gamma-glutamyltransferase and creatinine showed very poor correlation between FPIA and HPLC, which suggests that metabolite cross-reactivity with FPIA is significant and unpredictable in patients with liver dysfunction coexisting with renal dysfunction. Evidently, the monoclonal RIA can be substituted for HPLC, if the therapeutic range is adjusted for the 21% higher results obtained by RIA.

Monoclonal antibodies to cyclosporin are representative of the major antibody populations present in antisera of immunized mice.

Two series of mouse antisera raised against cyclosporin (Cs)-carrier conjugates exposing opposite sides of the Cs molecule and more than sixty monoclonal antibodies (mAbs) derived from the same animals were compared in terms of isotype and fine specificity for Cs. The predominant isotypes of the mAbs reflected the in situ distribution of the circulating anti Cs antibodies. The fine specificity of the antibodies was studied by determining their cross-reactivity for a series of Cs-derivatives and Cs-metabolites in competitive ELISA. The antisera raised by different immunizations showed very different cross-reactivity patterns for the Cs-derivatives. However, the in situ anti Cs antibody populations and the majority of mAbs derived from the corresponding animals showed a striking similarity in fine specificity for restricted clusters of residues on the Cs molecule. These results indicate that the mAbs produced against Cs are representative of the major antibody population present in the sera of the mice used for the fusion. By determining the characteristics of antibodies found in the serum of immunized mice it may thus be possible to select animals that are likely to give rise to mAbs of a certain isotype and specificity.