{beta}1 and {beta}3 integrin upregulation in rapidly progressive glomerulonephritis

The expression and distribution pattern of ß₁ (₁–₆)and _vß₃ integrins and ICAM-1 and VCAM-1 counter receptorswere evaluated by an immunohistochemical technique on eightrenal samples from patients affected by rapidly progressiveglomerulonephritis (RPGN) of different aetiologies. In all casesintegrins and counterreceptors displayed similar patterns. Ontubular cells of renal cortex, a marked upregulation of ₂ß₁,₃ß₁, ₅ß₁, _vß₃ integrins and VCAM-1was observed with as many as 60–90% of tubular cross-sectionslabelled, while a strong ICAM-1 reactivity was limited to theluminal surface. The same adhesion molecules were also uniformlyexpressed on crescentic cells. In glomeruli, integrin upregulationoccurred only on apparently preserved capillary tufts, i.e.in an early stage of lesion, while collapsed and sclerotic tuftsshowed a reduced integrin expression. In addition a morphometricstudy of extracellular matrix (EM) proteins cellular fibronectinand tenascin showed a 9.56±1.9-fold and 3.35±0.6-foldincrease respectively in these proteins, as compared to normalkidney (P<0.001). The upregulation of _vß₃. on podocytesmight play a role in the adhesion of crescentic cells. An increasedproduction of cytokines, in particular transforming growth factor-ß,might induce augmented deposition of EM proteins and upregulationof ß₁ and ß₃ integrins in RPGN.     

Transient overexpression of TGF-{beta}1 induces epithelial mesenchymal transition in the rodent peritoneum

Epithelial mesenchymal transition (EMT), a process involved in many growth and repair functions, has been identified in the peritoneal tissues of patients who undergo peritoneal dialysis. The sequence of changes in gene regulation and cellular events associated with EMT after TGF-beta1-induced peritoneal fibrosis is reported. Sprague-Dawley rats received an intraperitoneal injection of an adenovirus vector that transfers active TGF-beta1 (AdTGF-beta1) or control adenovirus, AdDL. Animals were killed 0 to 21 days after infection. Peritoneal effluent and tissue were analyzed for markers of EMT. In the animals that were treated with AdTGF-beta1, an increase in expression of genes associated with EMT and fibrosis, such as type I collagen A2, alpha-smooth muscle actin, and the zinc finger regulatory protein Snail, was identified. Transition of mesothelial cells 4 to 7 d after infection, with appearance of epithelial cells in the submesothelial zone 7 to 14 d after exposure to AdTGF-beta1, was demonstrated. This phase was associated with disruption of the basement membrane and increased expression of matrix metalloproteinase 2. By 14 to 21 d after infection, there was evidence of restoration of normal submesothelial architecture. These findings suggest that EMT occurs in vivo after TGF-beta1 overexpression in the peritoneum. Cellular changes and gene regulation associated with EMT are evident throughout the fibrogenic process and are not limited to early time points. This further supports the central role of TGF-beta1 in peritoneal fibrosis and provides an important model to study the sequence of events involved in TGF-beta1-induced EMT.     

Expression of TGF-{beta} receptors type I and II in human glomerulonephritis

Background. Transforming growth factor-{beta} (TGF-{beta}) is a multipotent cytokine involved in the turnover of the extracellular matrix. Signal transduction of TGF-{beta} is regulated via at least five different surface receptors; most of the effects, however, are mediated through the interaction of receptors type I and II (RI and RII). We investigated the glomerular expression of TGF-{beta} and its receptors RI and RII in human glomerulonephritis. Methods. Indirect immunofluorescence using monoclonal and polyclonal antibodies against TGF-{beta} isoforms (1,2,3 and 2,3), TGF{beta}-RI and TGF{beta}-RII has been carried out on 30 consecutive renal biopsies (5 FSGS, 11 IgAN, 4 MCGN, 6 SLE, 2 RPGN) and on normal kidney tissue. Results. Glomerular immunoreactivity for TGF-{beta} isoforms correlated with the severity of proliferative lesions: immunofluorescent signal was absent or tracelike in normal kidneys, FSGS, and IgAN with mild mesangial cellularity, and was highest in IgAN with severe mesangial proliferation., MPGN, RPGN, and SLE. Glomerular positivity for TGF-{beta}-RI and -RII was detectable in SLE and RPGN; a low signal was present also in MPGN. Other types of glomerulonephritis, including focal extracapillary proliferations, and glomeruli of normal kidneys were always negative. Conclusions. Our data show that TGF-{beta} expression is a good indicator of the severity of proliferative glomerular lesions in most, if not all cases and that RI-RII expression occurs at levels detectable with indirect immunofluorescence in limited number of glomerulonephritides, mostly associated with systemic diseases. A complex interaction between TGF-{beta} and its ligand may represent a critical factor conditioning the tissue response to immunological injury.     

TGF-{beta}- and angiotensin-II-induced mesangial matrix protein secretion is mediated by protein kinase C

Background: Glomerulonephritis is characterized by the accumulation of extracellular matrix protein within the glomerulus. This process, when allowed to proceed unimpeded, leads to glomerulosclerosis and eventually to cessation of glomerular filtration. There is evidence that protein kinase C (PKC) activation plays an important role in mediating at least some of the effects of TGF-{beta} in vascular smooth-muscle cells. The current study was undertaken to determine whether PKC activity is required for both TGF-{beta} and angiotensin II (Ang II) to induce mesangial cell matrix protein secretion. Methods: PKC was inhibited by two separate methods, and [³H]thymidine incorporation was assessed in both the presence and the absence of PKC inhibition. Conditioned medium from cells stimulated with TGF-{beta} or Ang II was collected and analysed for secreted matrix proteins and sulphated proteins by SDS-polyacrylamide gel electrophoresis and western blotting. Results: Twenty-four-hour incubation of rat mesangial cells with phorbol-12-myristate-13-acetate (PMA) reduced total PKC activity to basal levels. Both TGF-{beta} and Ang II were mitogenic in mesangial cells, and chronic PMA pre-incubation inhibited this DNA synthesis. TGF-{beta} and Ang II-induced sulphated protein secretion into conditioned medium was markedly attenuated in PKC-downregulated cells. Secretion of the specific matrix proteins laminin and fibronectin by mesangial cells stimulated with either TGF-{beta} or Ang II was also diminished in PKC-downregulated cells and in cells pre-incubated with the specific PKC inhibitor, chelerythrine. There was no evidence of generalized cell toxicity or decreased non-specific protein synthesis caused by these PKC inhibitors. Conclusions: PKC is a key intermediary in the process by which TGF-{beta} and Ang II cause DNA synthesis and mesangial cell matrix protein production. Thus, PKC inhibitors deserve further study as potential therapeutic agents for a variety of glomerular diseases.     

Expression of {beta}1 integrins in IgA nephropathy

Aim. To compare the expression of {beta}1 integrins in renal biopsies from patients with IgA nephropathy with that found in normal human kidney. Methods. Thirty renal biopsies from patients with IgA disease plus six control specimens were stained with monoclonal antibodies directed against the &agr;1, &agr;2, &agr;3, &agr;4, &agr;6, &agr;v, and {beta}1 integrin chains using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. The intensity of integrin expression was graded semiquantitatively by a pathologist unaware of the antibody used. Results. Glomerular crescents stained strongly for &agr;3, &agr;v, and {beta}1, but integrin expression was greatly reduced or absent in fibrotic glomeruli. There were no alterations in the intensity of mesangial cell staining for any of the integrins tested. There was accentuated staining for the &agr;2, &agr;5, &agr;v, and {beta}1 chains in areas of interstitial scarring plus &agr;2, &agr;3, &agr;v, and {beta}1 on damaged tubules. Inflammatory cells expressed &agr;4, &agr;5, and {beta}1. Conclusions. In IgA nephropathy the interstitium is the main site of altered {beta}1 integrin expression. Glomerular crescents also express several {beta}1 integrins, but we found no differences in the intensity of integrin expression on mesangial cells. Altered {beta}1 integrin expression may play a role in tubulointerstitial scarring in IgA disease. Thus modulation of integrin expression might attenuate this process. Keywords: {beta}1 integrins; glomerulonephritis; IgA nephropathy; fibrosis      

Laminar Distribution of GABAA Receptor {alpha}1, {beta}2, and {gamma}2 Subunit mRNAs in the Granular and Agranular Frontal Cortex of the Rat during Pre- and Postnatal Development

The expression of mRNAs encoding the three GABA_A receptor subunitsthat are associated with the most abundant benzodiazepine-sensitiveGABA_A receptor in adult cortex, that is, the     

Intrarenal localization of interleukin-1{beta} mRNA in crescentic glomerulonephritis

II-1ß is a potent proinflammatory peptide, and inducesexpression of other cytokines which are involved in the immuneresponse. Kidney biopsies from nine patients with crescenticGN were studied by in-situ hybridization to determine the siteof expression of II-1ß mRNA. Biopsies from nine patientswith non-proliferative renal disease were studied as negativecontrols, and tonsillar tissue was studied as a positive control.An II-1ß cDNA probe was ³²P-labelled by random primersand hybridized to paraffin-embedded tissue sections after de-waxing.II-1ß mRNA was expressed in tonsil, but not in negativecontrols. Positive mRNA expression was seen in four of the ninecrescentic biopsies. This was observed in interstitial cellswith morphological characteristics of macrophages adjacent totubular cells, in cells within the glomerular tuft, and in tubularepithelial cells. II-1ß mRNA is expressed in renaltissue in crescentic GN. Tubular and interstitial expressionof II-1ß mRNA appears of equal prominence to glomerularexpression. Intrarenal cytokine synthesis may be involved inthe pathogenesis of crescentic glomerulonephritis.     

Tissue Uptake of 125I-{beta}2-Microglobulin({beta}2-M) in Anephric Animals in the Presence or Absence of Aluminium Intoxication

In long-term haemodialysis patients a new type of amyloidosiscomposed of ß₂-microglobulin (ß₂-M) hasrecently been described. The amyloid deposition has a particularpredilection for articular structures. In the pathogenesis ofthis complication markedly elevated plasma ß₂-M concentrations,such as those observed in anuric patients, have a role. However,other as yet ill-defined factors must also be implicated, possiblecandidates being aluminium intoxication and the widely usedregenerated cellulose (cuprophan) membrane. In the present experimentalstudy, we examined tissue distribution of exogenous ß₂-Mafter i.v. injection of ¹²⁵I-ß₂-M to bilaterally nephrectomisedrats. One hundred and twenty minutes after injection, most radioactivityremained in the vascular compartment. The accumulation in tissueswas weak, and no predilection for a particular tissue becameapparent. Interestingly, chronically aluminium-overloaded, acutelyanephric rats accumulated a significantly greater amount of¹²⁵I-ß₂-M in their spleens than anephric rats withoutprior aluminium intoxication. We then attempted to induce ß₂-M amyloid depositionin rats and mice, some of whom had undergone chronic aluminiumintoxication and subcutaneous implantation of regenerated cellulosefragments for various periods of time. They were subsequentlymade anephric to obtain high plasma ß₂-M concentrations.None of the animals developed ß₂-M amyloidosis inspleen, liver, skin and mechanically altered joint synovium. In conclusion, chronic aluminium intoxication enhances splenicaccumulation of exogenous ¹²⁵I-ß₂-M in anephric rats.The factors required to form ß₂-M-amyloidosis in vivohave still to be defined.     

Identification of post-transplant anti-{alpha}5(IV) collagen alloantibodies in X-linked Alport syndrome

X-linked Alport syndrome (AS) is a heritable disorder whichis associated with mutations in the type IV collagen 5(IV) chaingene (COL4AS) located on chromosome X. Following renal transplantation,an average of 6% of male AS patients develop anti-GBM nephritis.We studied the specificity of the antibodies against type IVcollagen in the serum of a patient with COL4A5 partial deletion.The specificity of these alloantibodies was determined againstcollagenasedigested GBM, as well as against recombinant noncollagenous(NCl) domains of the type IV collagen 1(IV)—6(IV) chainsexpressed in Escherichia coli. Immunoblotting and ELISA demonstratedthat these antibodies bound specifically to the NCl domain of5(IV) collagen. There was no binding to the NCl domain of theother chains, including the Goodpasture antigen. CompetitiveELISA confirmed the results obtained by ELISA and immunoblotting.This patient developed alloantibodies directed against antigenspresent in the grafted kidney, but absent from his Alport kidney.The pathogenesis of post-transplantation glomerulonephritisin the Alport patient studied is thus similar to that of Goodpasturesyndrome, with the exception that the pathogenic antibodiesare targeted to another chain of type IV collagen.     

Dialysis Arthropathy, {beta}2-Microglobulin and the Effect of Dialyser Membrane

Patients with dialysis arthropathy had the greatest mean serumß₂-microglobulin (59.5 mg/l) but there was no thresholdconcentration of ß₂-microglobulin above which allpatients developed dialysis arthropathy. Haemodialysis patientswithout dialysis arthropathy and patients on continuous ambulatoryperitoneal dialysis (CAPD) also had grossly elevated valuesof ß₂-microglobulin (47.9 mg/l and 30.7 mg/l respectively).There was a significant positive correlation between durationof treatment and serum ß₂ for the patients treatedby haemodialysis, but this was not the case for patients onCAPD. There was a significant negative correlation between residualurinary volume and serum ß₂-microglobulin for thepatients on haemodialysis without dialysis arthropathy, andalso for patients on CAPD. This was not true for the patientswith dialysis arthropathy. Both duration of treatment and residualurine volume correlated with serum ß₂-microglobulin,and therefore an analysis of covariance was used to take accountof this in comparing the groups. This showed that there wasno difference between serum ß₂-microglobulin in haemodialysispatients with and without dialysis arthropathy. However, CAPDpatients had a significantly lower corrected mean serum ß₂-microglobulinHaemodialysis with cuprophane membranes was associated withan increase in ß₂-microglobulin of 11.5%, whereashaemodialysis with polycarbonate was associated with a decreaseof 6.8% at 6 h. Our results provide circumstantial evidencethat repeated haemodialyses with cupro phane membranes may predisposelong-term haemo dialysis patients to dialysis arthropathy. CAPDpatients have lower ß₂-microglobulin concentrationsand may be less likely to develop dialysis a Long-term prospectivestudies are needed to confirm these assertions.     

Nutritional assessment of children on haemodialysis: value of IGF-I, TNF-&agr; and IL-1{beta}

Background: Protein-energy malnutrition (PEM) is associated with increased morbidity and mortality in haemodialysis (HD) patients. Insulin-like growth factor I (IGF-I) has proved to be a sensitive marker of malnutrition, while interleukin-1 (IL-1{beta}) and tumour necrosis factor (TNF) have been found to be raised in catabolic states. Methods: We have investigated the nutritional status of 17 chronic renal failure (CRF) paediatric patients (8 boys, 9 girls) on maintenance HD. Eight predialysis CRF children (5 boys and 3 girls; mean creatinine 5.1±3.2 mg/dl) and 10 healthy children served as control groups. PEM was defined according to anthropometric measurements (triceps skinfold thickness (TST), mid-arm circumference (MAC), and mid-arm muscle circumference (MAMC). These were correlated with serum IGF-I, IL-1 TNF-&agr; transferrin, and albumin (all sampled before the HD session). Results: In the HD group, TST was reduced in 41.2% of the patients, whereas MAC and MAMC were reduced in 82.4 and 76.5% respectively. TST was depleted in only one of the predialysis CRF children. The degree of reduction in MAC and MAMC were 62.5 and 62.5% respectively. Median serum IGF-I level was decreased in both HD and predialysis CRF patients (205.1 interquartile range (IQR) 194.4 &mgr;g/l and 258.8 IQR 155.0 &mgr;g/l respectively) compared to the healthy children (418.0 IQR 310.5&mgr;g/l) (P=0.0009 and P=0.01 respectively). Within the HD group, IGF-I levels were lower in patients with malnutrition defined according to TST (145.0 IQR 125.5 &mgr;g/l) compared to children with normal TST (201.2 IQR 218.8 &mgr;g.l) (P=0.05). IGF-I levels of the HD patients with malnutrition according to TST was also lower than predialysis CRF patients and healthy children (P=0.04 and P=0.002 respectively). Serum IL-1{beta} was undetectable in all groups. Median serum TNF-&agr; levels were higher in HD and predialysis CRF patients compared to healthy children, albeit statistically insignificant. There was no correlation between TNF-&agr;, transferrin or albumin and anthropometric parameters. Conclusions: Our results support the high prevalence of malnutrition in CRF children, which becomes more pronounced when treatment by HD is initiated. We suggest that determination of IGF-I levels in childhood HD patients in conjunction with anthropometric measurements is useful for identification of malnutrition. We have not been able to demonstrate the catabolic effects of cytokines on this state of protein-energy malnutrition.     

Monomeric and dimeric {beta}2-microglobulin may be extracted from amyloid deposits in vitro

Background. There is a controversy as to whether {beta}2-microglobulin ({beta}2M amyloid deposits may be degraded resulting in regression and cure of amyloidosis. We have recently reported a long-term clinical study involving transplanted patients suggesting that there is no resorption of amyloid deposits in vivo, even after correction of the primary cause of amyloidosis. To progress in the study of the solubility of amyloid fibrils we performed an in vitro study with the intent to remove protein constituents from amyloid fibrils and amyloid deposits. Methods. Amyloid fibrils were prepurified from four amyloid deposits surgically obtained from carpal tunnel. They were incubated for 2 h with a phosphate-buffered saline (PBS) solution containing trypsin, collagenase, kallikrein, the three of them, or PBS alone. The experiments were repeated in the presence of the antiprotease &agr;2M). Results. Several bands were observed when the supernatants were run through SDS-PAGE. Western blotting identified in these bands the presence of &agr;2M, light chains of immunoglobulins and {beta}2M in mono- and dimeric form. The same proteins were solubilized with PBS alone. Equivalent results were obtained with crude amyloid deposits; however, {beta}2M presented almost exclusively in monomeric form. Conclusions. These results show that the protein constituents may be recovered from the amyloid fibrils in vitro. They also show that even the more insoluble {beta}2M dimers are resuspended by the action of PBS, with no need for proteases to cleave their attachment to the amyloid deposits.     

Alterations of synovial tissue and their potential role in the deposition of {beta}2-microglobulin-associated amyloid

Background: Beta-2-microglobulin-associated amyloidosis (AB2M) is a frequent complication of long-term dialysis treatment. Uraemic retention of beta-2-microglobulin ({beta}2M) apparently constitutes the basis for AB2M. However, it is unclear why clinical manifestations are largely confined to osteoarticular tissues. It has been speculated that synovial inflammatory changes, induced by uraemia and/or dialysis therapy could predispose this tissue to amyloid deposition. Methods: We investigated which local synovial alterations preceded or paralleled amyloid deposition. Using immunohistology we evaluated synovial leukocyte infiltration (B and T lymphocytes, monocytes/macrophages), cell proliferation, fibroblast activation (de novo expression of &agr;-smooth-muscle actin) the expression of extracellular matrix components (heparan sulphate proteoglycan collagen types I, II, IV), and advanced glycation end-products (AGEs). Results: Synovial AB2M was detected in 20 of 36 chronic peritoneal and haemodialysis patients and none of eight non-uraemic controls. Notably non-AB2M synovial amyloid was present in six additional dialysis and three control patients. Cellular infiltration was largely restricted to patients with advanced AB2M deposits. The infiltrates consisted mainly of macrophages and progressed with increasing degrees of AB2M deposition. In advanced cases they exhibited characteristics of foreign-body reaction. Other infiltrating leukocyte types, altered cell proliferation, or fibroblast activation were absent or uncommon in periarticular tissue of dialysis patients with and without AB2M. Neither dialysis treatment nor the presence of AB2M deposits appreciably altered the qualitative matrix composition in periarticular tissue. AGEs were present in AB2M deposits, the extracellular synovial matrix of dialysis patients (of both, patients with and without AB2M) and, to a lesser degree, in synovia of controls. Conclusions: These data suggest that, except for AGE formation, alterations of none of the parameters assessed, and in particular no inflammatory tissue alterations, precede periarticular AB2M. Rather synovial tissue, possibly modified by AGEs, seems to have an intrinsic propensity for amyloid deposition and inflammatory changes appear to only arise secondary to amyloid deposition. Key words: advanced glycation end-products; amyloidosis; {beta}2-microglobulin; dialysis; immunohistology; lymphocytes; macrophages      

Extracellular collagen modulates the regulation of chondrocytes by transforming growth factor-β1

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-β1, Type-I and type-II collagen, heat denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-β1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [³⁵S]sulfate and [³H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-β1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-β1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-β1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-β1.     

Highly sulphated glycosaminoglycans in articular cartilage and other tissues containing {beta}2 microglobulin dialysis amyloid deposits

BACKGROUND: Highly sulphated glycosaminoglycans (GAGs) are a common constituentof amyloid deposits and an integral component of articular connectivetissues where ß₂-microglobulin (ß₂M) amyloidis most often found. METHODS: Using alcian blue, magnesium chloride, critical electrolyteconcentration, mucin histochemistry, and immunohistochemistry,the GAGs composition of ß₂M amyloid deposits in jointcapsule and cartilage, carpal, and heart tissues of 22 uraemicpatients was determined. RESULTS: Highly sulphated GAGs were found in ß₂M amyloid depositsnot only within cartilage, where such GAGs are normally foundin high concentration, but also in other articular and extra-articularconnective tissues. Keratan sulphate was often specificallylocalized to ß₂M amyloid deposits in articular cartilageand to a lesser extent in periarticular tissues, with one caseshowing colocalization with systemic vascular amyloid deposition.Other sulphated GAGs, chondroitin 4 and 6 sulphate, dermatansulphate, and heparan sulphate were also identified in tissuescontaining ß₂M amyloid deposits, but with the exceptionof heparan sulphate (identified by mucin histochemistry) werenot specifically localized to the deposits themselves. CONCLUSION: These findings suggest that qualitative or quantitative changesin the composition of highly sulphated GAGs may play a rolein localization of ß₂M amyloid deposits in articularand extra-articular tissues.     

T cell receptor V beta gene usage in HLA-DR1-reactive human T cell populations. The predominance of V beta 8.

The functional specificity and T cell receptor (TCR) V beta gene expression of three class II HLA-DR1-reactive human T cell populations were examined. WP2, a renal allograft-derived, long-term CD4+ T cell line, was specifically cytotoxic for DR1, one of the mismatched antigens present on the allograft. Initial studies of WP2 using six TCR V beta-specific mAb revealed a predominance of T cells expressing a member of the V beta 8 gene family. A smaller, yet significant, number of cells expressed TCR using V beta 5.1. Semiquantitative V beta-specific polymerase chain reaction (PCR) analyses of RNA derived from this T cell line confirmed the presence of V beta 8 and V beta 5.1 messages. The PCR signal for V beta 8 was the strongest, followed by those for V beta 4 and V beta 5. An earlier WP2 culture had a very similar PCR profile, with a dominant signal for V beta 8, although signals for V beta 4 and V beta 5 were considerably lower. Previous PCR analyses of eight other renal allograft-derived, long-term T cell lines, grown under identical in vitro conditions, revealed no other example of predominant usage of V beta 8. We established two replicate long-term, anti-DR1 mixed lymphocyte reactions using PBL from two unrelated normal donors as responder and stimulator. The MLR were given alloantigen every 10 days, and RNA was obtained from the cultured cells immediately prior to each stimulation. PCR analyses of RNA taken at 10-day intervals over a total of 60-70 days indicated that, although the MLR were initially quite heterogeneous with regard to V beta message expression, by the end of the fourth or fifth Ag cycle the predominant PCR signals observed in both MLR were for V beta 8. These results suggest that T cells using V beta 8 gene-encoded segments as part of their TCR may have a selective advantage in responses to DR1.     

Influence of Haemodialysis Membranes on {beta}2-Microlobulin Kinetics:In Vivo and In Vitro Studies

Amyloidosis of the ß₂-microglobulin (ß₂M)type is a recently recognised complication of dialysis. We studiedplasma and ultrafiltrate ß₂M in 36 chronic haemodialysispatients. Long-term dialysis with standard cuprophan membranein non-oliguric patients and with the AN69 polyacrylonitrilemembrane in oliguric patients resulted in lower plasma ß₂Mconcentrations (means±SD, 24.4±6.6 and 33.0±8.2µg/ml, respectively) than with cuprophan in oliguric patients(47.0±13.2 µg/ml, P<0.01). In acute studies, plasma ß₂M (corrected for haemoconcentration)increased, although not significantly, during cuprophan dialysis(n=10) from 40.6±12.2 to 44.8±7.6 µg/mland decreased during AN69 dialyis (n=10) from 39.4±18.0to 24.3±7.1 µg/ml (P<0.02). After 15 min ultrafiltration,ß₂M sieving coefficient in vivo was 0.33 for AN69but near zero for cuprophan. Total mass transfer of ß₂Macross the AN69 membrane during a 4-h dialysis was 142 ±45.8 mg(n=7). AN69, but not cuprophan, membrane fragments incubatedin vitro with normal or uraemic plasma exhibited avid [125-I]ß₂M binding (respectively 12.9% vs 0.47% and 6.09%vs 0.06% of total ß₂M bound at 30 mim, n=6, P<0.001for both). No in vitro generation of ß₂M from wholenormal or uraemic blood could be demonstrated during incubationwith either membrane. In conclusion, at least in some patients, standard cuprophandialysis promotes retention and possibly tissue release of ß₂Min vivo, whereas AN69 dialysis leads to ß₂M removaldue to both transmembrane transfer and, to a lesser extent,membrane binding.