Cyclosporin a treatment in children with minimal change nephrotic syndrome and focal segmental glomerulosclerosis

Summary In a pilot study 23 children with nephrotic syndrome were treated with cyclosporin A (Cs) for 6–45 months. 8 children suffered from steroid dependent minimal change nephrotic syndrome (MCNS) and had experienced at least one course with cytotoxic drugs, but had relapsed thereafter. 2 children had diabetes mellitus type I with nephrotic syndrome and 13 children had steroid resistant focal segmental glomerulosclerosis (FSGS). Cs was started with 100 mg/m2/day in two doses and increased stepwise to obtain a Cs whole blood trough level of 200–400 ng/ml. In steroid dependent MCNS treatment with Cs reduced relapse rate significantly, and prednisone therapy could be stopped completely. After discontinuation of Cs, relapses reoccurred as frequently as before. Renal function remained unimpaired despite repeated Cs treatment courses up to 38 months. In cases of nephrotic syndrome with diabetes type I Cs treatment led to complete remission without changing the insulin requirement. However, after discontinuation of Cs relapses reoccurred. In steroid resistant FSGS 6 children benefited from Cs treatment: 4 went into complete remission, 2 into partial remission. The 2 children with complete remission relapsed but remained Cs responsive. The remaining 7 children with FSGS did not respond to Cs but continued the course of their disease, with two patients rapidly progressing to terminal renal failure. Side-effects of Cs treatment were mild. It is concluded that Cs is an effective agent in steroid dependent MCNS and can be used as an alternative drug in specific cases like steroid toxicity or diabetes mellitus. In steroid resistant FSGS a trial with Cs seems to be warranted since some cases do respond favorably. To avoid nephrotoxicity treatment with Cs should always be monitored closely by determination of blood levels and renal function.Abbreviations MCNS minimal change nephrotic syndrome - FSGS focal segmental glomerulosclerosis - Cs Cyclosporin A     

Single channel recordings of reconstituted ion channel proteins: an improved technique

Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This hydration technique consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K⁺-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K⁺-channel studied displays a conductance _K ⁺ of 145 pS. The single channel conductance in 100 mM Rb⁺ and Na⁺ is _Rb ⁺ = 98 pS and _Na ⁺ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K⁺-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.     

Tissue distribution of the T cell activation antigen Ta1. Serological, immunohistochemical and biochemical investigations.

The recently described Ta1 antigen is expressed by activated T cells in vitro and in vivo, as observed in patients with certain immune-mediated diseases, such as multiple sclerosis. In this paper we report on the tissue distribution of the Ta1 antigen. Serological testing of human tumour cell lines and immunohistochemical analysis of human tissue sections revealed a reactivity of the anti-Ta1 antibody with normal and malignant tissues of the upper gastro-intestinal tract, the biliary tract, exocrine pancreas and kidney. SDS-PAGE analysis of immunoprecipitates from 125I-labelled cells, employing the anti-Ta1 antibody, yielded a 113-115 kD band from three serologically Ta1 positive tumour cell lines, from a serologically Ta1 negative human EBV-transformed B lymphoblastoid cell line, from peripheral blood mononuclear cells (PBMC) and, as previously described, a 105 kD band from PHA activated T cells (Fox et al., 1984). After endoglycosidase F treatment similar bands of 85 kD were precipitated from activated T cells and from tumour cell lines. It is therefore likely that very similar glycoproteins, which differ only modestly in the size of carbohydrate chains, bear the Ta1 epitope on Ta1 positive tissues.     

Different Origins of IgA Antibodies with Various Antigen Specificities Appearing in Rat Bile

Eight lactating and seven non-lactating female rats were immunized in Peyer's patches (Pp) with Escherichia coli 06 carrying type 1 pili. Eight days later the thoracic duct was drained and bile was collected at the same time. During the lymph drainage in the lactating rats, the biliary IgA anti-pili antibodies decreased less than the anti-lipopolysaccharide (LPS) antibodies. The non-lactating rats showed only a very small difference in decrease between the anti-pili and the anti-LPS antibodies. Transfer of mesenteric lymph node cells from male donor rats immunized with the E. coli strain to syngeneic male recipients resulted in the appearance of both IgA anti-pili and anti-LPS antibodies in the bile. This is at variance with the results seen in lactating rats, where only biliary IgA anti-LPS is seen after similar cell transfer. In conclusion, the study shows that in lactating rats a larger proportion of biliary IgA anti-pili antibodies than anti-LPS antibodies is derived from extraintestinal sites, such as the mammary glands or the liver. Thus, this study confirms that the nature of the antigen influences the transfer of the secretory antibody response to different secretions.     

Induction of oligodendrocyte proliferation and remyelination after chronic demyelination. Relevance to multiple sclerosis

Optic nerve and spinal cord tissue from untreated guinea pigs with chronic relapsing experimental autoimmune encephalomyelitis, guinea pigs with experimental autoimmune encephalomyelitis in which the disease was treated with injections of myelin basic protein (MBP) combined with galactocerebroside (GC), and normal guinea pigs, has been studied morphologically, immunocytochemically and morphometrically. MBP/GC treatment induced widespread proliferation of oligodendrocytes and extensive central nervous system (CNS) remyelination in tissue from both sites. Whereas some oligodendrocytes within lesions from treated animals appeared to be derived from surviving cells which underwent mitosis, the frequent occurrence of nests of oligodendrocytes at the periphery of nerve fiber fascicles in optic nerve among perivascular astrocytic elements, raises the possibility that remyelinating oligodendrocytes might possess progenitors located in these regions. Observations from multiple sclerosis lesions showed that oligodendrocyte proliferation and CNS remyelination occur in human subcortical white matter, but to a lesser degree than that seen in the CNS of MBP/GC/treated guinea pigs. Immunocytochemical examination of CNS tissue from experimental autoimmune encephalomyelitis animals confirmed the morphologic identification of oligodendroglia. Preliminary morphometric analysis confirmed the impression of an increase in oligodendroglial cells in MBP/GC-treated animals. This increase was somewhat obscured statistically by a concomitant rise in the number of fibrous astrocytes. In view of the ability of oligodendrocytes to proliferate and produce new myelin in multiple sclerosis, the possibility is raised that an experimental immunologic approach similar to that employed here might have a beneficial effect in the human disease.     

Synaptic and intrinsic responses of medical entorhinal cortical cells in normal and magnesium-free medium in vitro

1. Extracellular recordings were made from slices of hippocampus plus parahippocampal regions maintained in vitro. Field potentials, recorded in the entorhinal cortex after stimulation in the subiculum, resembled those observed in vivo. 2. Washout of magnesium from the slices resulted in paroxysmal events which resembled those occurring during sustained seizures in vivo. These events were greatest in amplitude and duration in layers IV/V of the medial entorhinal cortex and could occur both spontaneously and in response to subicular stimulation. Spontaneous seizure-like events were not prevented by severing the connections between the hippocampus and entorhinal cortex, but much smaller and shorter events occurring in the dentate gyrus were stopped by this manipulation. Both spontaneous and evoked paroxysmal events were blocked by perfusion with the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonovalerate (2-AP5). 3. Neurons in layers IV/V were characterized by intracellular recording. Injection of depolarizing current in most cells evoked a train of nondecrementing action potentials with only weak spike frequency accommodation and little or no posttrain after hyperpolarization. 4. A small number of cells displayed burst response when depolarized by positive current. The burst consisted of a slow depolarization with superimposed action potentials which decreased in amplitude and increased in duration during the discharge. The burst was terminated by a strong after hyperpolarization and thereafter, during prolonged current pulses a train of nondecrementing spikes occurred. The burst response remained if the cell was held at hyperpolarized levels but was inactivated by holding the cell at a depolarized level. 5. Depolarizing synaptic potentials could be evoked by stimulation in the subiculum. A delayed and prolonged depolarization clearly decremented with membrane hyperpolarization and, occasionally, increased with depolarization. 6. Washout of magnesium from the slices resulted in an enhancement of the late depolarization and a reversal of its voltage dependence. Eventually a single shock to the subiculum evoked a large all-or-none paroxysmal depolarization associated with a massive increase in membrane conductance. Similar events occurred spontaneously in all cells tested. The paroxysmal depolarizations, both spontaneous and evoked, were rapidly blocked by 2-AP5. 7. It is concluded that medial entorhinal cortical cells possess several intrinsic and synaptic properties which confer an extreme susceptibility to generation of sustained seizure activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites.

Conjugate vaccines against the sporozoite stage of Plasmodium falciparum were synthesized by covalently coupling the recombinant protein R32 [with the one-letter amino acid code of MDP-[(NANP)15NVDP]2LR] to tetanus toxoid, cholera toxin, choleragenoid, and Pseudomonas aeruginosa toxin A. Conjugates were produced by using adipic acid dihydrazide as a spacer molecule and carbodiimide as a coupling agent. The molar ratio of R32 to carrier protein ranged from 2.5:1 to 8.4:1. These conjugates were found to be stable, nontoxic, and nonpyrogenic. When adsorbed onto Al(OH)3, all conjugates were capable of inducing anti-R32 antibody. Conjugates made with either cholera toxin or Pseudomonas aeruginosa toxin A were significantly more immunogenic than those constructed with tetanus toxoid or choleragenoid. However, the magnitude of the immune response to the R32 moiety was not governed by the antibody response to the carrier protein.     

Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis lipopolysaccharide (LPS) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and ELISA-inhibition experiments with LPS and delipidated polysaccharide fragments (PS-1 and PS-2) prepared from B. pertussis LPS. Monoclonal antibody 9-1-H5 reacted with B. pertussis LPS only, whereas monoclonal antibodies 6-4-H6 and 9-2-A8 reacted with PS-1 and PS-2 as well as B. pertussis LPS. The antibodies did not react with LPS prepared from B. parapertussis and B. bronchiseptica in an LPS-specific ELISA. A monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis LPS. This assay had a detection limit of B. pertussis LPS in concentrations ranging from 0.16 to 0.32 microgram/ml. The assay was also shown to be specific for the detection of whole B. pertussis bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Streptococcus miteor, Haemophilus influenzae, or Legionella pneumophila. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis LPS.     

A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3

A monoclonal antibody raised against SDS-denatured C3 was shown to react with both solid-phase C3a and unfragmented C3. However, in the fluid phase the antibody was found to bind only to C3a and not to native C3. These findings indicated that the antibody could be used in an assay to detect C3a in human EDTA-plasma without prior separation of C3a from native C3. A simple and rapid competition ELISA was developed which monitored soluble C3a. 200 microliter of C3a (8 ng) was absorbed to plastic wells over night at 4 degrees C. Thereafter, 50 microliter of sample and 50 microliter of constant amounts of monoclonal antibody conjugated with beta-galactosidase, were incubated for 60 min at 37 degrees C. After washing, the colour reaction was started by adding nitrophenyl-galactopyridine to the wells. The microtitre plate was incubated at 37 degrees C for 30 min and the staining intensity was quantified at 405 nm. The assay detected both C3a and C3ades arg. A strong correlation was obtained between the new technique and an RIA which used an acid precipitation step for the separation of C3a prior to the determination of C3a (r = 0.9). Significantly higher levels of C3a were detected both in plasma from patients with immune complexes (93 +/- 9 ng/ml; P less than 0.1) and in plasma from patients treated in blood oxygenators (140 +/- 19 ng/ml; P less than 0.05) than in plasma from normal subjects (74 +/- 4 ng/ml). The results were not affected by repeated freezing and thawing of the plasma samples.