Systematic Screening Using Luminex for De Novo C3d Fixing of Class II Donor-Specific Antibodies Is Correlated With Luminex Mean Fluorescence Intensity in Renal Transplant Patients.

OBJECTIVES: Chronic antibody-mediated rejection is the main cause of late kidney graft loss. The presence of donor-specific antibodies in the serum is the main criterion for this diagnosis. Single antigen Luminex assays can identify donor-specific antibodies, and semiquantitative estimates of antibodies can be assessed using mean fluorescence intensity. Recent data have shown that patients whose donor-specific antibodies fix C3d have worse clinical outcomes, implying that C3d-specific Luminex assays may provide useful prognostic data. MATERIALS AND METHODS: We compared C3d donor-specific antibodies with standard immunoglobulin G donor-specific antibody mean fluorescence intensities in a cohort of patients with de novo class II donor-specific antibodies and analyzed subsequent graft survival. The included kidney graft recipients received transplants between 2005 and 2015 and had developed de novo class II donor-specific antibodies. Serum was tested using the standard single antigen Luminex technique and the C3d-fixing antibody-detection system (Immucor, Herentals, Belgium). RESULTS: In our patient cohort, 41/924 patients (4.4%) developed class II donor-specific antibodies, and 65 serum samples were analyzed (at baseline and follow-up). Among these samples, 43 (66%) were negative for C3d donor-specific antibodies. A mean fluorescence intensity threshold of 9000 in the single antigen Luminex assay discerned all negative (from positive) C3d donor-specific antibodies, even when all single-bead results were taken into account. Sixteen patients (39%) had poor outcomes (ie, either creatinine levels had doubled or they had lost their graft) over the median follow-up of 5 years. C3d results were significantly associated with graft survival (P = .04). We found a strong correlation between C3d-fixing antibody positivity and mean fluorescence intensity strength in the setting of de novo class II donor-specific antibodies. CONCLUSIONS: These results further reinforce the paradigm that the higher the mean fluorescence intensity, the more complement activation occurs. Routine C3d testing is thus unnecessary in this setting.

Characterization of foreign materials in paraffin-embedded pathological specimens using in situ multi-elemental imaging with laser spectroscopy.

Pathologists typically encounter many disparate exogenous materials in clinical specimens during their routine histopathological examinations, especially within the skin, lymph nodes, and lungs. These foreign substances may be free extracellular deposits or induce several clinical abnormalities or histopathological patterns. However, pathologists almost never investigate or report the chemical nature of exogenous metals in clinical specimens due to a lack of convenient and available technologies. In this paper, a novel strategy based on laser-induced breakdown spectroscopy (LIBS) technology is evaluated for in situ multi-elemental tissue imaging. The improved procedures allow visualization of the presence of chemical elements contained within paraffin-embedded specimens of medical interest with elemental images that are stackable with conventional histology images. We selected relevant medical situations for which the associated pathology reports were limited to the presence of lymphohistiocytic and inflammatory cells containing granules (a granuloma and a pseudolymphoma) or to lymph nodes or skin tissues containing pigments or foreign substances. Exogenous elements such as aluminum, titanium, copper, and tungsten were identified and localized within the tissues. The all-optical LIBS elemental imaging instrument that we developed is fully compatible with conventional optical microscopy used for pathology analysis. When combined with routine histopathological analysis, LIBS is a versatile technology that might help pathologists establish or confirm diagnoses for a wide range of medical applications, particularly when the nature of external agents present in tissues needs to be investigated.

Cutaneous Collagenous Vasculopathy: Report of Two Cases Presenting as Disseminated Telangiectasias and Review of the Literature.

Cutaneous collagenous vasculopathy is a recently described idiopathic microangiopathy characterized by acquired diffuse cutaneous telangiectasias and specific histological features: dilated capillaries in the superficial dermis, with walls thickened by hyaline material containing collagen IV by immunohistochemistry. The authors describe 2 cases and review all cases reported in the literature to date, 34 cases including our own. Cases were mainly observed in women (sex ratio 0.41), median age 63.5 (16-85). Hypertension and diabetes seem more frequent in these patients than in the general population. Typical clinical presentation is fine hair telangiectasias appearing on the lower limbs and progressing toward the trunk and upper limbs, sparing the face. Facial and neck involvement are however reported. When faced with isolated acquired diffuse cutaneous telangiectasias, clinicians should perform a skin biopsy to rule out cutaneous collagenous vasculopathy.

Conversion to mammalian target of rapamycin inhibitors increases risk of de novo donor-specific antibodies.

In kidney transplantation, conversion to mammalian target of rapamycin (mTOR) inhibitors may avoid calcineurin inhibitor (CNI) nephrotoxicity, but its impact on post-transplant allo-immunization remains largely unexplored. This retrospective cohort study analyzed the emergence of donor-specific antibodies (DSA) in kidney transplant recipients relative to their immunosuppressive therapy. Among 270 recipients without pretransplant immunization who were screened regularly for de novo DSA, 56 were converted to mTOR inhibitors after CNI withdrawal. DSA emergence was increased in patients who were converted to mTOR inhibitors (HR 2.4; 95% CI 1.06-5.41, P = 0.036). DSA were mainly directed against donor HLA-DQB1 antigens. The presence of one or two DQ mismatches was a major risk factor for DQ DSA (HR 5.32; 95% CI 1.58-17.89 and HR 10.43; 95% CI 2.29-47.56, respectively; P < 0.01). Rejection episodes were more likely in patients converted to mTOR inhibitors, but this difference did not reach significance (16% vs. 7.9%, P = 0.185). Concerning graft function, no significant change was observed one year after conversion (P = 0.31). In conclusion, conversion to mTOR inhibitors may increase the risk of developing class II DSA, especially in the presence of DQ mismatches: this strategy may favor chronic antibody-mediated rejection and thus reduce graft survival.

Predictive diagnostic of chronic allograft dysfunction using urinary proteomics analysis.

BACKGROUND: Kidney transplant Chronic Allograft Dysfunction (CAD), a major cause of long-term graft failure, is currently diagnosed at a late and irreversible stage by graft biopsies. Our goal was to identify predictive urinary biomarkers of CAD before renal lesions appeared by analysis of the urine proteomic profile. METHODS/METHODS: Twenty-nine urinary samples withdrawn three months post-transplant were analyzed by SELDI-TOF technology. CAD development was evaluated by serum creatinine level and confirmed by allograft biopsy one year after transplantation. Comparison of protein profile of both groups revealed 18 biomarkers predictive of CAD occurrence. RESULTS: The biomarker demonstrating the highest diagnostic performance was a protein of 8860 Da that predicted CAD with a sensitivity of 93% and a specificity of 65%. Moreover combination of these biomarkers in two multivariate analyses improved the diagnostic potential of CAD. Relevance of these individual biomarkers and a decisional algorithm constituted of 3 proteins was confirmed in an independent cohort of patients with undetermined CAD status one year post-transplant. CONCLUSIONS: These non invasive biomarkers, detected as soon as three months post-grafting, allowed identification of patients who would develop CAD as late as 4 years after graft. Systematic measurement of these biomarkers would greatly improve the management of immunosuppressive therapy of kidney grafted patients.

An "anaplastic" Kaposi's sarcoma mimicking a Stewart-Treves syndrome. A case report and a review of literature.

Cutaneous angiosarcoma (AGS) developing in a lymphedematous arm, after lymphadenectomy in the context of breast cancer, is the definition of the classical Stewart-Treves syndrome. Like AGS, many tumors such as Kaposi's sarcoma (KS) could develop in chronic lymphedema. We describe the case of a 50-year-old woman who presented with several nodules on the left lymphedematous arm evocative of a Stewart-Treves syndrome, 2 years after a left mastectomy and a homolateral lymphadenectomy. The histological examination revealed an atypical vascular proliferation suggesting AGS, but endothelial atypical cells nuclei were strongly stained by herpes human virus 8 antibody. The final diagnosis was an "anaplastic" KS mimicking a Stewart-Treves syndrome. The total regression of the lesion was obtained by elastic contention and intradermic liposomal doxorubicin. "Anaplastic" KS is a rare histological form of nodular KS, which mimics a cutaneous AGS but classically expresses herpes human virus 8. It is essential to know about this entity, particularly in a lymphedematous arm, to avoid aggressive treatment such as amputation.

Sentinel lymph node biopsy in cutaneous melanoma: outcome after 5-years follow-up.

Sentinel lymph node procedure for cutaneous melanoma is largely used and sentinel lymph node status is an important prognostic factor. Few French centers have reported their experience and data. We analysed technique feasibility, recurrence-free and overall-survival rates at 36 and 60 months for the first 62 patients submitted to this technique. The positivity of sentinel lymph nodes was 17.7%. Recurrence-free survival at 36 and 60 months was of 85% and 78% respectively for patients with negative sentinel lymph nodes, whereas the rates were of 45% and 36% respectively for patients with positive sentinel lymph nodes (p = 0.0046). The overall survival rate was of 94% at 36 months and 85% at 60 months for negative patients as opposed to 82% at 36 months and 47% at 60 months for positive patients (p = 0.0019). In our experience, sentinel lymph node technique is a safe procedure with few complications and good pronostic value. This technique allows the identification of patients with a high risk of recurrence who could benefit from early adjuvant therapeutic management. However, these results show that the survival rate of patients with positive sentinel lymph nodes remains significantly lower, even when elective lymph node dissection is performed.

Transforming growth factor-beta in human diabetic nephropathy: effects of ACE inhibition.

OBJECTIVE: Studies in rodent models have suggested that reduction in renal transforming growth factor (TGF)-beta1 may underlie the renoprotective effects of the renin-angiotensin system (RAS) blockade. However, the role of the RAS blockade in abrogating TGF-beta in human disease is unknown. Accordingly, we sought to examine TGF-beta gene expression and biological activity in human renal biopsies, before and after ACE inhibition. RESEARCH DESIGN AND METHODS: RNA was extracted from renal biopsies taken from participants in the Diabiopsies study, a randomized controlled 2-year trial of 4 mg/day perindopril versus placebo that reported a reduction in proteinuria and cortical matrix expansion in type 2 diabetic nephropathy. Biopsies taken at study entry and at 2 years were obtained in 12 patients (6 placebo and 6 taking perindopril). TGF-beta1 and its receptor mRNA were quantified by real-time PCR, and its biological activity was assessed by examining the activation of its intracellular signaling pathway (phosphorylated Smad2) and the expression TGF-beta-inducible gene H3 (betaig-H3). RESULTS: At baseline, TGF-beta1 expression was similar in both placebo- and perindopril-treated groups and was unchanged over a 2-year period in biopsies of placebo-treated subjects. In contrast, perindopril treatment led to a substantial diminution in TGF-beta1 mRNA (mean 83% reduction, P < 0.05). Phosphorylated Smad2 immunolabeling and betaig-H3 mRNA were similarly reduced with ACE inhibition (P < 0.05) but unchanged in the placebo group. No differences were noted in the gene expression of TGF-beta receptor II in biopsies of either placebo- or perindopril-treated subjects. CONCLUSIONS: This study demonstrates that over a 2-year period, treatment with perindopril in patients with type 2 diabetes and nephropathy leads to a reduction in both renal TGF-beta1 gene expression and its downstream activation.

Inflammatory destruction of elastic fibers in acquired cutis laxa is associated with missense alleles in the elastin and fibulin-5 genes.

Cutis laxa (CL) is a condition characterized by redundant, pendulous, and inelastic skin. Acquired CL has been reported in patients with inflammatory diseases. The goal of this study was to investigate whether genetic lesions predispose patients to the development of acquired CL. We report a patient who developed CL following a Toxocara canis parasitism. He later had an aortic root aneurysm that required surgical correction. Histological evaluation showed inflammation followed by destruction of elastic fibers in both the skin and the aorta. Mutational analysis showed that the patient was heterozygous for an inherited fibulin-5 (FBLN5) allele G202R and compound heterozygous for elastin (ELN) alleles A55V and G773D. Western blotting indicated abnormal proteolytic processing of tropoelastin (TE) in patient fibroblasts. The FBLN5 202R allele on the other hand led to increased interaction of FBLN5 and TE and increased deposition of insoluble ELN partially rescuing the deficiency conferred by ELN mutation G773D. We demonstrated that the interaction of ELN and FBLN5 alleles results in elastic fibers susceptible to inflammatory destruction. These results suggest that the pathogenesis of acquired CL involves an underlying genetic susceptibility and highlight the importance of molecular genetic analysis in patients with idiopathic connective tissue disorders.

[Diabetic glomerulopathies]. / Atteintes glomérulaires au cours des etats diabétiques.

The archetypal nodular lesion as described in conventional diabetic glomerulopathies is considered, in the view of recent research advances, as a sub-endothelial microangiopathy developed on a peculiar filtration-type basement membrane (type IV collagen, proteoglycans) and early associated with podocytic and mesangial phenotypic alterations. The molecular pathways leading to glucose pathogenic effect are plural, their interactions, biochemical switch off and regulation mechanisms involving a cellular glucose "operon" are better understood and would allow in the next future to superimpose other therapeutic targets to the hemodynamics changes induced by different angiotensin blockades, future interacting key-controls for the renal microvascular as well as coronary and cerebrovascular risks.