Autologous tolerogenic dendritic cells for rheumatoid and inflammatory arthritis.

OBJECTIVES: To assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities. METHODS: An unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood. RESULTS: There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high. CONCLUSION: IA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 tolDC but no systemic clinical or immunomodulatory effects were detectable. TRIAL REGISTRATION NUMBER: NCT01352858.

Association of the tyrosine kinase LCK with phospholipase C-gamma 1 after stimulation of the T cell antigen receptor.

Stimulation of the T cell antigen receptor (TCR) activates a protein tyrosine kinase and leads to the tyrosine phosphorylation of phosphoinositide-specific phospholipase C-gamma 1 (PLC gamma 1). The molecular interactions involved in this phosphorylation are not known. After stimulation of the TCR on Jurkat T cells, tyrosine-phosphorylated proteins of 36, 38, 58, and 63 kD coprecipitate with PLC gamma 1. An identical pattern of proteins precipitate with TrpE fusion proteins that contain the Src homology (SH) 2 domains of PLC gamma 1, indicating that these regions of PLC gamma 1 are responsible for binding. TCR stimulation leads to an association between the SH2 domains of PLC gamma 1 and a protein tyrosine kinase, which, by peptide mapping, is identical to p56lck. These studies establish that p56lck associates with PLC gamma 1 as a result of TCR stimulation of Jurkat cells, suggesting that p56lck plays a central role in coupling the TCR to the activation of PLC gamma 1.

The OX-44 molecule couples to signaling pathways and is associated with CD2 on rat T lymphocytes and a natural killer cell line.

The MRC OX-44 molecule, which is expressed on all peripheral leukocytes, identifies the subset of thymocytes capable of proliferating in response to alloantigens and lectins (Paterson, D.J., J.R. Green, W.A. Jefferies, M. Puklavec, and A.F. Williams. 1987. J. Exp. Med. 165:1). When we isolated monoclonal antibodies (mAbs) on the basis of their ability to activate the phosphatidylinositol signaling pathway in RNK-16 cells (a rat leukemia line with natural killer activity), three of the resulting mAbs recognized the OX-44 molecule. Addition of these mAbs to RNK-16 elicits protein tyrosine phosphorylation, generates inositol phosphates, and increases the concentration of cytoplasmic free calcium. These responses require the addition of intact mAb and are not observed with F(ab')2 fragments. One of these mAbs (7D2) is mitogenic for freshly isolated rat splenic T cells and synergizes with a mAb to the T cell antigen receptor in this activation. A 50-60-kD glycoprotein coprecipitates with the OX-44 molecule from RNK-16 cells and rat splenic T cells. Peptide mapping and reprecipitation studies indicate that the coprecipitating molecule is CD2. Thus, the OX-44 molecule can couple to multiple signaling pathways and associates with CD2 on both RNK-16 and rat T cells.

The SH3 domain of p56lck binds to proline-rich sequences in the cytoplasmic domain of CD2.

CD2, a cell surface glycoprotein expressed on T cells and natural killer cells, can couple to signaling pathways that result in T cell proliferation. An Src-like protein tyrosine kinase, p56lck, coprecipitates with CD2, and perturbation of CD2 by monoclonal antibodies results in an increase in the activity of p56lck, suggesting that an interaction with p56lck contributes to CD2-mediated signaling. Herein, we investigate the mechanism by which CD2 associates with p56lck. We demonstrate that CD2 and p56lck associate when coexpressed in nonlymphoid cells, that this association requires the cytoplasmic domain of CD2, and that the SH3 domain of p56lck mediates its interactions with CD2. Using truncation mutants of CD2, we identify two regions in the cytoplasmic domain of CD2 involved in binding p56lck. Each region contains a proline-rich sequence that, in the form of a synthetic peptide, directly binds p56lck. Thus, proline-rich sequences in the cytoplasmic domain of CD2 allow this transmembrane receptor to bind to the SH3 domain of p56lck.

Association of Src-like protein tyrosine kinases with the CD2 cell surface molecule in rat T lymphocytes and natural killer cells.

The cell surface molecule CD2 has a signaling role in the activation of T lymphocytes and natural killer cells. Because perturbation of CD2 leads to the appearance of tyrosine-phosphorylated proteins, we investigated the possibility that CD2 associates with cytoplasmic protein tyrosine kinases. As determined by in vitro kinase assays and phosphoamino acid analysis, protein tyrosine kinase activity coprecipitated with CD2 from rat T lymphocytes, T lymphoblasts, thymocytes, interleukin-2-activated natural killer cells, and RNK-16 cells (a rat natural killer cell line). In each case, both p56lck and p59fyn were identified in the CD2 immunoprecipitate. In the thymus, the association between CD2 and these kinases occurred predominately in a small subset of thymocytes that had the cell surface phenotype of mature T cells, indicating that the association is a regulated event and occurs late in T-cell ontogeny. The finding that CD2 is associated with p56lck and p59fyn in detergent lysates suggests that interactions with these Src-like protein kinases play a critical role in CD2-mediated signal transduction.

Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome.

The GRB10 gene encodes a growth suppressor and maps to human chromosome 7p11.2-p13. Maternal duplication (matdup) of this region has recently been associated with Silver-Russell syndrome (SRS), which is characterised by pre- and postnatal growth restriction, craniofacial dysmorphism and lateral asymmetry. Maternal uniparental disomy for chromosome 7 (mUPD7) occurs in approximately 7% of SRS patients. Exposure of a recessive allele due to isodisomy has been ruled out in five mUPD7 cases, suggesting genomic imprinting as the basis for disease. Assuming SRS patients with matdup of 7p11.2-p13 and mUPD7 share a common aetiology, this would implicate a maternally expressed gene from this interval, which is involved in growth inhibition. Murine Grb10 was identified as a maternally expressed gene by subtractive hybridisation using normal and androgenetic mouse embryos. Grb10 maps to the homologous region of proximal mouse chromosome 11, for which mUPD incurs reduced birthweight. A role for GRB10 in SRS was evaluated by determining its imprinting status in multiple human foetal tissues using expressed polymorphisms, and by screening the coding region for mutations in 18 classic non-mUPD7 SRS patients. Maternal repression of GRB10 was observed specifically in the developing central nervous system including brain and spinal cord, with biallelic expression in peripheral tissues. This is in contrast to mouse Grb10, and represents the first example of opposite imprinting in human and mouse homologues. While a role for GRB10 in mUPD7 SRS cases can not be ruled out on the basis of imprinting status, no mutations were identified in the patients screened.

On the renal basis for essential hypertension: nephron heterogeneity with discordant renin secretion and sodium excretion causing a hypertensive vasoconstriction-volume relationship.

We propose herein that there are two functionally abnormal nephron populations in essential hypertension: (1) a group of ischemic nephrons with impaired sodium excretion which chronically hypersecrete renin. Numerically, these ischemic nephrons comprise a minor subgroup since most patients with essential hypertension exhibit no overt evidence of renal insufficiency. (2) In reaction to this, a more numerous group of normal nephrons appears in adaptive hypernatriuresis. They have an increased distal sodium supply and consequently, a chronically suppressed renin secretion. One difference between patients with renovascular hypertension and those with essential hypertension is the intermingling of these two populations of nephrons. In our hypothesis, the adapting hyperfiltering normal nephrons accomplish the hypernatriuresis in response to saline infusion, that is characteristic of all essential hypertension. However, the unsuppressed secretion of renin, that arises from the ischemic nephron population attenuates this compensatory natriuresis in the following ways: (1) by inappropriately acting on the hyperfiltering nephrons to enhance proximal tubular sodium reabsorption; (2) by activating TGF-mediated afferent constriction in these nephrons, and (3) simultaneously, the reactive secretion of renin from ischemic nephrons is diluted by non-participation of the adapting hypernatriuretic nephrons so that plasma renin settles at a level which is insufficient to fully compensate GFR in the ischemic nephrons. These adaptive responses provide a basis for the observation that the inhibition of renin activity with converting enzyme inhibitors in essential hypertension increases renal blood flow and sodium excretion. They also explain why converting enzyme inhibitors can effectively reduce blood pressure, even when renin levels are not absolutely elevated, since any circulating renin imposed upon the adapting hypernatriuretic nephrons inappropriately impairs their sodium excretion. In addition, the theory explains why basal renin secretion is either not suppressed or inadequately suppressed in patients with essential hypertension. As a result, whole kidney homeostatic function is compromised because individual nephrons are responding to their individual stimuli to fulfil their individual need, rather than acting in concert with other nephrons. The net effect of this uncoordinated response is to shift total renal function so that systemic arterial hypertension is sustained by abnormal sodium retention for the inappropriately high plasma renin level, or vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Termination of central sleep apnea episodes by upper airway stimulation using intermittent positive pressure ventilation.

A 52-year-old man presented with pure central sleep apnea syndrome that failed to respond to nasal continuous positive airway pressure (NCPAP) therapy. Intermittent positive pressure stimulation via a nasal mask was instituted using a portable positive pressure ventilator. We found marked reduction in the respiratory disturbance index, number of desaturations, and the number of arousals with this mode of therapy.

Tubulopathy with macroalbuminuria due to diabetic nephropathy and primary glomerulonephritis.

Tubular damage is a recognized feature of both overt diabetic nephropathy and glomerulonephritis. However, the pattern and mechanism of tubular damage in the two clinical settings remain unclear. Two groups of patients with macroalbuminuria (albuminuria > 300 mg/day) were studied. Group 1 comprised 41 patients with biopsy proven primary glomerulonephritis and group 2 comprised 28 patients with clinical diabetic nephropathy due to insulin dependent diabetes mellitus. Serum creatinine, creatinine clearance, glomerular proteinuria (albuminuria and transferrinuria), markers of tubular damage such as urinary excretion of lysosomal enzyme (N-acetyl glucosaminidase), brush border enzymes (leucine aminopeptidase and gamma-glutamyl transferase) and retinol binding protein (tubular protein) were measured. Both groups were comparable in serum creatinine, creatinine clearance, glomerular proteinuria and excretion of N-acetyl-glucosaminidase. However, a significantly higher degree of tubular brush border enzymuria and a lower level of tubular proteinuria were seen in group 1 than in group 2. In group 1, albuminuria correlated to tubular enzymuria and tubular proteinuria. However, there was no correlation in diabetic patients between parameters of glomerular and tubular damage or dysfunction. The data presented suggested that the pattern of tubulopathy is different in patients with comparable degree of macroalbuminuria due to diabetic nephropathy and glomerulonephritis. Moreover, in diabetic nephropathy contrary to glomerulonephritis, markers of tubular damage are unrelated to glomerular proteinuria. This may suggest different mechanisms of tubular damage in the two clinical settings. We recommended that in all patients with proteinuria, particularly those with diabetic nephropathy, markers of renal tubular damage may be useful in monitoring the course of their disease.

Genetic variants of microsomal metabolism and susceptibility to hydrocarbon-associated glomerulonephritis.

Hydrocarbon (HC) exposure can play a role in the development of chronic glomerulonephritis (GN). Interindividual variations in various metabolizing enzymes may influence HC biotransformation, and hence susceptibility to HC-associated GN. We evaluated the role of human genotypic polymorphism in HC-associated GN, in 41 patients (30 male, 11 female) with primary GN (17 diffuse mesangial proliferative GN, 12 focal segmental GN, 11 membranous GN, one membranoproliferative GN) and 60 (46 male, 14 female) healthy controls. Genotypic polymorphisms of (CYP) P450 2D6 (CYP2D6), glutathione S-transferases mu (GSTM1) and theta (GSTT1) and N-acetyltransferase (NAT-2) were determined using polymerase chain reaction analysis of white-blood-cell DNA. HC exposure scores were determined using a validated questionnaire, and were significantly elevated in GN patients compared to controls. While no significant differences in the various genotypic frequencies were observed in the GN group overall, compared to controls, there was a significant increase in GSTM1 null (n = 10) to GSTM1 wild type (n = 1), and NAT fast (n = 10) to slow (n = 1) acetylators, in the membranous GN group compared to controls (p < 0.05). These results suggest a possible role for GSTM1 null and NAT fast acetylator in the development of HC-associated membranous GN.

Substance abuse and the kidney.

Substance abuse has been increasing steadily in the UK and some other countries. Recent evidence suggests more than 40% of young people have tried illicit drugs at some time. There are numerous medical consequences to recreational drug use, and a physician should always consider substance abuse in any unexplained illness. The renal complications of drug abuse are also becoming more frequent, and may encompass a spectrum of glomerular, interstitial and vascular diseases. Although some substances are directly nephrotoxic, a number of other mechanisms are also involved. These effects are often chronic and irreversible, but occasionally acute with possible recovery. The rapid growth of illicit drug use is clearly a major public health problem. We review the commonly used substances of abuse and their associations with renal disease.

Biochemical markers of basement membrane disturbances and occupational exposure to hydrocarbons and mixed solvents.

To investigate possible mechanisms of hydrocarbon or solvent-induced renal damage, we studied three groups of healthy men employed in a UK manufacturing plant. Group 1 (n = 111) were occupationally exposed to hydrocarbon-based paints, Group 2 (n = 100) were occupationally exposed to petroleum-based mineral oils, and Group 3 (n = 92) had low background occupational exposure to hydrocarbons. Occupational atmospheric exposure levels for toluene, xylene, butanol and oil mist around the time of this study were within UK permissible limits. Group 4 (controls) were males with no known occupational hydrocarbon or solvent exposure (n = 108). Circulating laminin antibodies and the auto-antibody implicated in Goodpasture's syndrome (anti-GBM) were measured, as were serum laminin, a basement membrane turnover marker, and soluble E-selectin, an endothelial activation marker. Group 1 had a significantly greater proportion of subjects with high levels of both anti-laminin antibodies and soluble E-selectin; Group 2 had significantly more subjects with raised anti-GBM antibodies, laminin and soluble E-selectin. Mean levels of soluble E-selectin were increased in Groups 1 and 2. In a small but significant proportion of these workers exposed to hydrocarbons/mixed solvents there are alterations both to basement membranes, resulting in auto-antibody production, and to overlying vascular endothelial cells.

Membranous nephropathy, hydrocarbon exposure and genetic variants of hydrocarbon detoxification.

Modulation of biotransformation by genetic traits may be important in determining environmentally-induced nephrotoxicity. We conducted a case-control study to investigate the role of occupational hydrocarbon exposure, along with polymorphisms of the genes coding for N-acetyltransferase 2 (NAT2) and glutathione S-transferase mu (GSTmu), in the development of idiopathic membranous glomerulonephritis (IMGN). Patients (n=36) with biopsy-proven IMGN were matched with healthy controls for age, gender, and geographical area. Lifetime hydrocarbon exposure was assessed by a validated questionnaire. The polymorphisms of the NAT2 and GSTmu genes (GSTM1) were defined by use of a polymerase chain reaction on white-cell DNA from peripheral blood. Exposure to hydrocarbons was significantly greater in patients with IMGN than in controls (mean+/-SEM hydrocarbon exposure score 11 003+/-2955.7 vs. 4352+/-1418, p<0.02). NAT2 acetylator status was identical in patients and controls with 23 (63.9%) fast and 13 (36.1%) slow acetylators in each group. GSTmu was present in 15 (41.7%) patients and 16 (44.4%) controls. While occupational exposure to hydrocarbons remains a likely factor in its pathogenesis, further work is required to identify the genetic polymorphisms that modulate the risk of IMGN.

Evidence of oxidant injury and tubular damage in early diabetic nephropathy.

Two groups of patients with insulin-dependent diabetes mellitus of > 10 years duration and either persistent normoalbuminuria (group 1, n = 49; albumin excretion < 30 mg/day) or microalbuminuria (group 2, n = 33; albumin excretion 30-300 mg/day) were investigated for evidence of free oxygen radical activity (erythrocytic superoxide dismutase and glutathione peroxidase) and oxidant injury (serum malondialdehyde). Glomerular proteinuria (albuminuria, transferrinuria), tubular proteinuria (retinol-binding protein) and tubular enzymuria (N-acetyl-glucosaminidase and leucine aminopeptidase) were also measured. Healthy controls (n = 38) were matched for age and sex. Groups 1 and 2 were similar in terms of age, sex, duration of diabetes and recent glycaemic control. Serum cholesterol and creatinine were similar in all three groups. Free-radical activity and oxidant injury were significantly higher in groups 1 and 2 than in controls (p < 0.001). Glomerular proteinuria, tubular proteinuria and enzymuria were significantly higher in group 2 than in group 1 and controls (p < 0.01). Group 1 had significantly higher transferrinuria, tubular enzymuria and tubular proteinuria than controls. However, groups 1 and 2 were similar in degree of free oxygen radical generation and oxidant injury. In diabetic nephropathy, oxidant injury and renal tubular damage accompany and may even precede microalbuminuria. The presence of these abnormalities in the absence of glomerular proteinuria favours the hypothesis that alterations first occur in the peritubular microcirculation, which by causing oxidant injury and tubular damage, may initiate diabetic nephropathy.

Pregnancy does not adversely affect renal transplant function.

Women with functioning transplanted kidneys often become fertile again. Indeed, renal function, endocrine status and libido rapidly improve after renal transplantation, and 1:50 women of childbearing age become pregnant. However, there is concern regarding the haemodynamic changes of pregnancy, which could lead to a decline in graft function (temporary or permanent). We examined obstetric data and renal parameters in 29 patients and 33 pregnancies. Mean serum creatinine and creatinine clearance remained stable throughout pregnancy and 1 year postpartum. However, there was a significant increase in proteinuria from a mean of 0.45 g/24 h around the time of conception to 1.11 g/24 h at delivery (p<0.05). The proteinuria resolved to baseline levels at 3 months postpartum. We highlight certain parameters to be considered before conception to allow a good obstetric outcome and prolong stable renal function: serum creatinine <150 micromol/l, proteinuria <1 g/day, absence of histological evidence of chronic allograft rejection, controlled blood pressure (140/90) and stability of maintenance immunosuppression.

A case of hepatitis B in a haemodialysis unit.

A patient probably acquired hepatitis B virus (HBV) from a transfused infected unit of fresh frozen plasma. She had been on regular haemodialysis for four months before her infection was discovered. One hundred and seventy-six patients and 304 staff contacts were screened and there were no secondary cases. At donation the infected blood donor was hepatitis B surface antigen (HBsAg) negative but subsequently proved to be anti-hepatitis B core (HBc)-positive and positive for HBV DNA. Consideration should be given to screening blood donations for anti-HBc in addition to HBsAg. Vigilance needs to be maintained on dialysis units against sporadic cases of hepatitis B. All staff should be immunized and be able to demonstrate protective levels of anti-HBs.

A case of resistant Goodpasture's syndrome and staghorn calculus--treatment with bilateral nephrectomy.

We report the case of a 49-year-old female with severe Goodpasture's syndrome, staghorn calculus, and subacute thyroiditis. Despite the use of a combined therapy with corticosteroid, cyclophosphamide, azathioprine and plasma exchange, we were unable to suppress the disease activity or normalize the anti-glomerular basement membrane (GBM) antibody levels. Disease remission with a parallel reduction in anti-GBM antibody titer was only achieved after sequential bilateral nephrectomy.

Chronic bronchiectasis and anti-myeloperoxidase antibody related rapidly progressive necrotizing glomerulonephritis.

We reported two cases of chronic bronchiectasis and rapidly progressive necrotizing glomerulonephritis/severe renal failure which were also positive for anti-myeloperoxidase antibody, and followed their treatment and outcome. Immunosuppressive therapy was complicated by superimposed chest infection in both cases. Nonetheless, cautious use of immunosuppressive and antibiotic therapy reversed dialysis-dependent renal failure in one of the two cases.

Cox-2 inhibitors and other nonsteroidal anti-inflammatory drugs in the treatment of pain in the elderly.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed therapies for acute and chronic pain in the elderly. NSAIDs are effective in treating many disorders, but their use often is limited by toxicities, especially gastrointestinal and renal toxicity. COX-2 inhibitors are a major therapeutic advance, providing the analgesic and anti-inflammatory activity of NSAIDs, with a significant improvement in gastrointestinal safety. These new agents may be ideal therapies for older patients at risk for NSAID-related gastrointestinal toxicity.

Red cell population distributions in healthy dogs.

Red blood cells population distributions were studied in puppies and in mature beagle dogs using the Coulter Channelyzer C-1000. Weekly measurements over a period of six months indicated that only minor fluctuations in population distribution indices occurred. Normal range studies showed that although mean cell volume was similar in young and older dogs, young dogs showed an approximately 12.5 per cent greater volume range due to an increased presence of both larger and smaller cells. The red cell population distributions conformed to a single log normal distribution. A mild anaemia was induced in mature dogs. Compensation for this led to transient changes from the usual log normal shape of the red cell population distribution.