Francis W. Ballardie

The detection of intracytoplasmic interleukin-1α, interleukin-1β and tumour necrosis factor α expression in human monocytes using two colour immunofluorescence flow cytometry

Abstract Two colour flow cytometry was used to analyse in sity cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86–99% ( n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0°C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1α (IL-1α), IL-1β, tumour necrosis factor α (TNF-α), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1β and TNF-α expression following LPS stimulation, with early peaks in TNF-α (2 h), compared witl IL-1β (4 h), and IL-1α (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.

Quantitative Appraisal of Treatment Options for IgA Nephropathy

IgA nephropathy has an impact on renal health care costs worldwide. The paucity of good clinical trials highlights the uncertainty in determining best treatment and for how long. Ongoing debate still raises questions on why opinions vary but may suggest that current data are not fully understood. The scale of benefit of immunosuppressive drugs in suppressing clinical nephritis or improving outcome is unmatched by use of renin-angiotensin inhibitors alone. By minimizing the use of immunosuppressive drugs, higher risk patients may hazard more ESRD. This review addresses how disparate views have formed, quantifying existing data, to give balance to recommendations.

Volumetric analysis of urinary erythrocytes: a standardized methodology to localize the source of haematuria.

Volumetric analysis of urinary red blood cells (RBCs) was performed in two groups of patients: (a) 91 with glomerulonephritis (GN), 158 with non-glomerular diseases (non-GN) and 53 controls without haematuria; (b) 97 with GN and 41 with non-GN diagnoses and greater than or equal to 2+ haematuria, analysed after modified sample preparation (isotonic dilution and haemolysis to eliminate non-RBC debris). In group A, diagnostic ranges were established for modal, mean and differential (urinary-peripheral blood modal) RBC volumes to differentiate GN from non-GN sources of blood loss according to RBC size. 54% (135/249) of modal volumes and 68% (124/183) with greater than or equal to 2+ haematuria were within a diagnostic range of values, i.e. 40-180 fl (sensitivity), and the source of haematuria was correctly identified in 85% (115/135) and 87% (108/124) of these, respectively (specificity). Of the remainder, with modal volumes less than 40 or greater than 180 fl, 45% (51/114) were from GN and 55% (63/114) from non-GN specimens, along with 94% (48/53) controls without haematuria (non-diagnostic analyses). In contrast, whilst 90% (74/82) of mean volumes greater than or equal to 110 fl and 94% (62/66) of differential volumes greater than or equal to 0 fl were from non-GN specimens, values below these were common to both diagnostic groups so that diagnostic sensitivities were reduced to 38% (82/219) and 34% (66/193), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic indices and therapy in IgA nephropathy: Toward a solution

IgA nephropathy is the primary renal disease with the greatest impact on services. The paucity of trials with high evidence-based standards gives emphasis to issues of the rationalization of therapies. Now that certain treatments are increasingly accepted and others under evaluation, reliable discriminatory tests are essential to define and select patients at high risk of progression before irreversible loss of renal tissue, while avoiding drug exposure in others.

Altered Clearance of N-1 Methylnicotinamide Associated With the Use of Low Doses of Cyclosporine

To improve the monitoring of patients on low doses of cyclosporine there is a need for new tests of tubular function. N-1 methylnicotinamide (NMM) is an endogenous organic cation that is secreted by the proximal tubule and its clearance can be measured. In 27 patients with psoriasis, serial measurements of NMN clearance, plasma aldosterone, plasma chloride, bicarbonate, and magnesium were compared with changes in the radionuclide measurement of glomerular filtration rate and renal blood flow before, during, and after a 3-month course of low-dose cyclosporine (< 5 mg/kg/d). N-1 methylnicotinamide clearance decreased significantly with cyclosporine only (2.5 mg/kg/d, n = 10, P < 0.01). Recovery of NMN clearance lagged behind that of glomerular filtration rate and renal blood flow. Serum magnesium decreased significantly on cyclosporine (2.5 mg/kg, n = 10, P < 0.01; 5 mg/kg, n = 9, P < 0.0001). In the whole group, plasma potassium increased significantly (n = 27, P < 0.02) and plasma aldosterone was inappropriately low. Low doses of cyclosporine in psoriasis cause a reduced clearance of NMN, hypomagnesemia, and a variable hyperchloremic acidosis. Nifedipine may alter these biochemical variables without necessarily improving renal hemodynamics. The delayed recovery of NMN clearance in comparison with renal haemodynamic measurements following cyclosporine therapy suggests that this noninvasive test of tubular function may be a marker of persisting cyclosporine nephrotoxicity and that it should be evaluated further.

Changes in numbers of epidermal cell adhesion molecules caused by oral cyclosporin in psoriasis.

AIM--To determine the effects of a three month course of low dose cyclosporin on the expression of epidermal cell adhesion molecules. METHODS--Eighteen patients with psoriasis were treated for 12 weeks with either 2.5 or 5 mg/kg/day of oral cyclosporin. Biopsy specimens taken from skin before, during, and after cyclosporin treatment were stained immunohistochemically for CD 54 (ICAM-1), CD 29 (beta-1 integrins), and CD18 (beta-2 integrins). RESULTS--There was a highly significant (p < 0.01) clinical response after 12 weeks of cyclosporin as assessed by the Psoriasis Area and Severity Index (PASI) score. The staining of CD 29 on keratinocytes of affected and unaffected psoriatic skin was not affected by cyclosporin. Epidermal CD54 was variably expressed in active psoriatic plaques and changed unpredictably after cyclosporin (p = NS). Staining for CD18 on large epidermal dendritic cells was reduced after cyclosporin (p < 0.02). The expression of CD18 by large epidermal dendritic cells during treatment correlated strongly with the PASI score at that time and one month after stopping cyclosporin (p < 0.02). CONCLUSIONS--Persistence of epidermal staining for CD 54 in psoriasis is compatible with a good clinical response to cyclosporin. Residual staining for CD 18 on large epidermal dendritic cells may be a useful marker for early clinical relapse.

Autoimmunity in nephritis.

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A man who brought the war home with him

Department of General Internal Medicine and Nephrology (F W Ballardie FRCP, R Cowley PhD), Department of Pathology (A Curry PhD, H Denley FRCPath), and Department of Laboratory Medicine (J Denton MSc), University of Manchester and Manchester Royal Infi rmary, Manchester, UK; Department of Neurosciences (J Dick FRCP) and Department of Undergraduate Medical Education (A Redmond FRCP), Hope Hospital, Manchester, UK; Centre for Analytical Sciences, University of Sheffi eld, Sheffi eld, UK (A Cox MPhil); and Curie Research Institute, Paris, France (J-L Guerquin-Kern PhD)

Renal Function Does Not Always Predictably Deteriorate in Blind Insulin-Dependent Diabetics with Nephropathy

Two insulin-dependent diabetic women with severe retinopathy who were referred for the management of nephrotic syndrome are presented. On the basis of clinical risk factors, such as retinopathy and severe hypertension, both patients were expected to develop progressive end-stage renal failure. One woman has shown a gradual decline in creatinine clearance over 7 years while the plasma creatinine of the other has remained normal for 11 years. The unexpectedly good outcome of both these patients may be related to maintaining a normal blood pressure despite neither patient receiving a prolonged course of an angiotensin-converting enzyme inhibitor. Because of the efficacy of current antihypertensive therapy, the outcome of diabetic nephropathy cannot be so easily predicted and needs to be formally reassessed.