Reduced development of CD4-8-B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules.

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4-8-B220+ T cell receptor-alpha/beta+ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with beta 2-microglobulin (beta 2m)-deficient mice. Interestingly, these lpr beta 2m-/- mice develop 13-fold fewer DNT cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lpr beta 2m-/- mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in lpr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Marqueurs de la filtration glomérulaire en pédiatrie [Glomerular filtration markers in pediatrics]

The assessment of glomerular filtration rate (GFR) is critical for the diagnosis and management of renal diseases in pediatric nephrology. Ideally, it requires the measurement of the renal clearance of a filtration marker. Inulin, an exogenous marker, is the only compound the excretion of which occurs exclusively by glomerular filtration, with no tubular handling. Therefore, inulin clearance provides the most accurate method to measure GFR and is considered as the "gold standard", at all ages including very premature neonates. However, inulin dearance is cumbersome and alternative methods are used in clinical practice. If urine is available, endogenous creatinine clearance is the most reliable method. When urine collection is difficult to obtain, GFR can be estimated by the plasma concentration of endogenous markers mainly eliminated by glomerular filtration, such as creatinine, or the more recently described cystatin C and beta 2-microglobulin. When the endogenous production of these markers is constant, their plasma concentration reflects glomerular filtration; it increases with decreasing renal function. However, in pediatric patients creatinine production depends on muscle mass, which significantly increases with linear growth, as well as age and gender. Mathematical formulas taking these parameters into account have thus been developed. Among these, the so-called "Schwartz formula" is often used and is a reliable estimate of GFR in children. Finally, radionuclide renal scans can be used to evaluate the separate glomerular function of each kidney.

Synovial fluid examination for the diagnosis of synovial amyloidosis in patients with chronic renal failure undergoing haemodialysis

The diagnosis of synovial amyloidosis is based upon synovial biopsy. Synovial fluid (SF) in seven patients with amyloid arthropathy associated with chronic renal failure undergoing haemodialysis were studied. The SF and synovial samples of 10 consecutive patients with seronegative mono- or oligoarthritis served as controls. Six of the seven patients with amyloid positive synovial biopsy specimens showed amyloid in their SF. No amyloid was found in the synovial tissue or fluid of the 10 patients in the control group, the sensitivity being 87.7%. The finding of amyloid in SF was highly reproducible, showing its presence in the same joint on several occasions. The deposits were Congophilia resistant to potassium permanganate pretreatment, and the immunohistochemical analysis proved that they contained beta 2 microglobulin. The high sensitivity and good reproducibility of the method shows that the finding of amyloid in SF is sufficient for the diagnosis of synovial amyloidosis. It is possible to perform immunohis

Clinical picture of the amyloid arthropathy in patients with chronic renal failure maintained on haemodialysis using cellulose membranes.

The clinical picture of 15 patients (10 male, five female) with amyloid arthropathy secondary to chronic renal failure treated with haemodialysis has been studied. The average period of haemodialysis was 10.8 years. Joint symptoms appeared between three and 13 years after starting haemodialysis. No patient had renal amyloidosis. Early symptoms were varied and often overlapped: knee swelling (seven patients), painful and stiff shoulders (seven), and carpal tunnel syndrome (six) were the most prominent. Follow up showed extension to other joints. Joint effusions were generally of the non-inflammatory type. Radiologically, geodes and erosions of variable sizes were seen in the affected joints, which can develop into a destructive arthropathy. Amyloid was found in abdominal fat in three of the 12 patients on whom a needle aspiration was performed. Four of 12 patients showed changes compatible with amyloid infiltration in the echocardiogram. One patient had amyloid in the gastric muscular layer, another in the colon mucus, and two of four in rectal biopsy specimens. Amyloid deposits showed the presence of beta 2 microglobulin in 10 patients. The clinical and radiological picture was similar to the amyloid arthropathy associated with multiple myeloma. These patients can develop systemic amyloidosis.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

Lenalidomide maintenance after stem-cell transplantation for multiple myeloma.

BACKGROUND: High-dose chemotherapy with autologous stem-cell transplantation is a standard treatment for young patients with multiple myeloma. Residual disease is almost always present after transplantation and is responsible for relapse. This phase 3, placebo-controlled trial investigated the efficacy of lenalidomide maintenance therapy after transplantation. METHODS: We randomly assigned 614 patients younger than 65 years of age who had nonprogressive disease after first-line transplantation to maintenance treatment with either lenalidomide (10 mg per day for the first 3 months, increased to 15 mg if tolerated) or placebo until relapse. The primary end point was progression-free survival. RESULTS: Lenalidomide maintenance therapy improved median progression-free survival (41 months, vs. 23 months with placebo; hazard ratio, 0.50; P<0.001). This benefit was observed across all patient subgroups, including those based on the β(2)-microglobulin level, cytogenetic profile, and response after transplantation. With a median follow-up period of 45 months, more than 70% of patients in both groups were alive at 4 years. The rates of grade 3 or 4 peripheral neuropathy were similar in the two groups. The incidence of second primary cancers was 3.1 per 100 patient-years in the lenalidomide group versus 1.2 per 100 patient-years in the placebo group (P=0.002). Median event-free survival (with events that included second primary cancers) was significantly improved with lenalidomide (40 months, vs. 23 months with placebo; P<0.001). CONCLUSIONS: Lenalidomide maintenance after transplantation significantly prolonged progression-free and event-free survival among patients with multiple myeloma. Four years after randomization, overall survival was similar in the two study groups. (Funded by the Programme Hospitalier de Recherche Clinique and others; ClinicalTrials.gov number, NCT00430365.).

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Clinical picture of the amyloid arthropathy in patients with chronic renal failure maintained on haemodialysis using cellulose membranes.

The clinical picture of 15 patients (10 male, five female) with amyloid arthropathy secondary to chronic renal failure treated with haemodialysis has been studied. The average period of haemodialysis was 10.8 years. Joint symptoms appeared between three and 13 years after starting haemodialysis. No patient had renal amyloidosis. Early symptoms were varied and often overlapped: knee swelling (seven patients), painful and stiff shoulders (seven), and carpal tunnel syndrome (six) were the most prominent. Follow up showed extension to other joints. Joint effusions were generally of the non-inflammatory type. Radiologically, geodes and erosions of variable sizes were seen in the affected joints, which can develop into a destructive arthropathy. Amyloid was found in abdominal fat in three of the 12 patients on whom a needle aspiration was performed. Four of 12 patients showed changes compatible with amyloid infiltration in the echocardiogram. One patient had amyloid in the gastric muscular layer, another in the colon mucus, and two of four in rectal biopsy specimens. Amyloid deposits showed the presence of beta 2 microglobulin in 10 patients. The clinical and radiological picture was similar to the amyloid arthropathy associated with multiple myeloma. These patients can develop systemic amyloidosis.

Synovial fluid examination for the diagnosis of synovial amyloidosis in patients with chronic renal failure undergoing haemodialysis

The diagnosis of synovial amyloidosis is based upon synovial biopsy. Synovial fluid (SF) in seven patients with amyloid arthropathy associated with chronic renal failure undergoing haemodialysis were studied. The SF and synovial samples of 10 consecutive patients with seronegative mono- or oligoarthritis served as controls. Six of the seven patients with amyloid positive synovial biopsy specimens showed amyloid in their SF. No amyloid was found in the synovial tissue or fluid of the 10 patients in the control group, the sensitivity being 87.7%. The finding of amyloid in SF was highly reproducible, showing its presence in the same joint on several occasions. The deposits were Congophilia resistant to potassium permanganate pretreatment, and the immunohistochemical analysis proved that they contained beta 2 microglobulin. The high sensitivity and good reproducibility of the method shows that the finding of amyloid in SF is sufficient for the diagnosis of synovial amyloidosis. It is possible to perform immunohis

Heterogeneity of t(4;14) in multiple myeloma. Long-term follow-up of 100 cases treated with tandem transplantation in IFM99 trials.

One hundred de novo multiple myeloma patients with t(4;14) treated with double intensive therapy according to IFM99 protocols were retrospectively analyzed. The median overall survival (OS) and event-free survival (EFS) were 41.4 and 21 months, respectively, as compared to 65 and 37 for patients included in the IFM99 trials without t(4;14) (P<10(-7)). We identified a subgroup of patients presenting at diagnosis with both low beta(2)-microglobulin <4 mg/l and high hemoglobin (Hb) >/=10 g/l (46% of the cases) with a median OS of 54.6 months and a median EFS of 26 months, respectively, which benefits from high-dose therapy (HDT); conversely patients with one or both adverse prognostic factor (high beta(2)-microglobulin and/or low Hb) had a poor outcome. The achievement of either complete response or very good partial response after HDT was also a powerful independent prognostic factor for both OS and EFS.

Role of trabectedin in the treatment of soft tissue sarcoma.

Interest in marine natural products has allowed the discovery of new drugs and trabectedin (ET-743, Yondelis), derived from the marine tunicate Ecteinascidia turbinata, was approved for clinical use in 2007. It binds to the DNA minor groove leading to interferences with the intracellular transcription pathways and DNA-repair proteins. In vitro antitumor activity was demonstrated against various cancer cell lines and soft tissue sarcoma cell lines. In phase I studies tumor responses were observed also in osteosarcomas and different soft tissue sarcoma subtypes. The most common toxicities were myelosuppression and transient elevation of liver function tests, which could be reduced by dexamethasone premedication. The efficacy of trabectedin was established in three phase II studies where it was administered at 1.5 mg/m2 as a 24 h intravenous infusion repeated every three weeks, in previously treated patients. The objective response rate was 3.7%-8.3% and the tumor control rate (which included complete response, partial response and stable disease) was obtained in half of patients for a median overall survival reaching 12 months. In nonpretreated patients the overall response rate was 17%. Twenty-four percent of patients were without progression at six months. The median overall survival was almost 16 months with 72% surviving at one year. Predictive factors of response are being explored to identify patients who are most likely to respond to trabectedin. Combination with other agents are currently studied with promising results. In summary trabectedin is an active new chemotherapeutic agents that has demonstrated its role in the armamentarium of treatments for patients with sarcomas.

Isoelectric focusing of urinary metallothionein.

Isoelectric focusing of human urinary metallothionein at a pH range of 4.8 to 7.0 yielded a single protein band with a pI of 5.57 which co-migrated with authentic purified metallothionein I from human liver. Minimum pretreatment of the urine samples (160 ml) was needed. The preparatory steps included sample concentration with the original protein, enriched from 69 +/- 23 micrograms/ml to 2.0 +/- 1.4 mg/ml (+/- SD; n = 9), followed by heat treatment at 80 degrees C for 5 min (2.4 +/- 1.7 mg protein/ml). After focusing, the gels were stained with silver and the lanes were scanned with a laser scanner. Peak areas were used for quantitation with commercial beta 2-microglobulin as a standard. The urinary metallothionein ranged from 1.0 to 2.6 nmol/mmol creatinine, which is comparable with values reached by radio-immunoassay.

Renal dysfunction in patients with sickle cell anemia or sickle cell trait

Patients with sickle cell anemia (Hb SS) or sickle cell trait (Hb AS) may present several types of renal dysfunction; however, comparison of the prevalence of these abnormalities between these two groups and correlation with the duration of disease in a large number of patients have not been thoroughly investigated. In a cross-sectional study using immunoenzymometric assays to measure tubular proteinuria, microalbuminuria, measurement of creatinine clearance, urinary osmolality and analysis of urine sediment, we evaluated glomerular and tubular renal function in 106 adults and children with Hb SS (N = 66) or Hb AS (N = 40) with no renal failure (glomerular filtration rate (GFR) >85 ml/min). The percentage of individuals with microalbuminuria was higher among Hb SS than among Hb AS patients (30 vs 8%, P<0.0001). The prevalence of microhematuria was similar in both groups (26 vs 30%, respectively). Increased urinary levels of retinol-binding protein or ß2-microglobulin were detected in only 3 Hb SS and 2 Hb AS patients. Urinary osmolality was reduced in patients with Hb SS or with Hb AS; however, it was particularly evident in Hb SS patients older than 15 years (median = 393 mOsm/kg, range = 366-469) compared with Hb AS patients (median = 541 mOsm/kg, range = 406-722). Thus, in addition to the frequently reported early reduction of urinary osmolality and increased GFR, nondysmorphic hematuria was found in 26 and 30% of patients with Hb SS or Hb AS, respectively. Microalbuminuria is an important marker of glomerular injury in patients with Hb SS and may also be demonstrated in some Hb AS individuals. Significant proximal tubular dysfunction is not a common feature in Hb SS and Hb AS population at this stage of the disease (i.e., GFR >85 ml/min).

Biochemical profile of amniotic fluid for the assessment of fetal and renal development

Creatinine plays a key role in the function and maturation of fetal kidneys throughout pregnancy. It is important to identify other markers that may help in the diagnosis of renal dysfunction. Our aim was to determine the profile of and the correlation between biochemical markers to be used to assess renal function and maturation of the fetus in the amniotic fluid during pregnancy and to determine the distribution of normal values for creatinine, N-acetyl-ß-D-glucosaminidase (NAG), ß2-microglobulin, glucose, urea, sodium, potassium, phosphorus, calcium, uric acid, albumin, and osmolality in three gestational age groups. This was a cross-section study that assessed 115 samples of amniotic fluid during three different periods of pregnancy, i.e., 13 to 20, 27 to 34, and 36 to 42 weeks. Concentrations of creatinine, NAG, urea, potassium and uric acid increased during pregnancy (P<0.05). ß2-Microglobulin, glucose, sodium, phosphorus, calcium, and albumin concentration and osmolality decreased (P<0.05), whereas ß2-microglobulin, glucose and uric acid presented significant correlations with gestational age and creatinine, respectively (r>0.6, P<0.05). Urea, potassium and phosphorus showed mild correlations with both (r>0.5, P<0.05). NAG, sodium, albumin and osmolality did not show significant correlations (r<0.5, P<0.05). These tests confirmed the important role of creatinine in terms of correlation with gestational age. ß2-Microglobulin, glucose and uric acid were significant as markers of function and maturation of fetal kidneys, whereas NAG did not demonstrate a useful role for the assessment of renal maturation.