Early lymphocyte collection for anti-CD19 CART production improves T-cell fitness in patients with relapsed/refractory diffuse large B-cell lymphoma.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeted to the CD19 B-cell antigen form an approved treatment for patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). However, since this therapy is administered after multiple lines of treatment and exposure to lymphotoxic agents, there is an urgent need to optimize this modality of treatment. METHODS: To circumvent the difficulties of harvesting adequate and optimal T cells from DLBCL patients and improve CART therapy, we suggest an earlier lymphopheresis (i.e. at first relapse, before salvage treatment). We conducted a prospective study and evaluated the potential benefit of an earlier lymphopheresis (early group, n = 22) on the clinical outcome of CD19-CART infused DLBCL patients, in comparison with standard lymphopheresis (i.e. at second relapse and beyond; standard group, n = 23). RESULTS: An increased percentage of naïve T cells and increased in vitro T-cell functionality were observed in the early group. Additionally, these cells exhibit a lower exhaustion profile than T cells collected in the standard group. CONCLUSION: While improved T-cell phenotype and function in the lymphopheresis product did not translate into significantly improved clinical outcomes, a trend towards better overall survival (OS) and progression-free survival (PFS) was observed. Early lymphopheresis maximizes the potential of salvage therapies, without compromising CAR T-cell quality.

Modulation of allergic responses by mitochondrial STAT3 inhibitors.

BACKGROUND: Recently, we have shown that mast cell mitochondrial STAT3 could serve as a new target for the regulation of the allergic response as it plays an essential role in immunologically mediated degranulation of mast cells. In the present work, we explored how two recently developed mitochondrial STAT3 inhibitors (Mitocur-1 and Mitocur-3) modulate the allergic response. METHODS: Experiments were performed both in vitro in cultured human/mouse mast cells and with rat basophilic leukemia (RBL) cells and also in vivo in mice. The effect of mitochondrial STAT3 inhibition on mast cell function was determined via checking degranulation and several cytokines secretion levels. RESULTS: Here, we show that treatment of rodent and human cultured mast cells with low concentrations of mitochondrial STAT3 inhibitors had no effect on STAT3 target gene expression. However, these inhibitors caused a significant reduction in mast cell exocytosis and cytokine release, due to a decrease in OXPHOS activity and STAT3 serine 727 phosphorylation. It was also observed in an OVA mouse model of allergic asthma that one of the inhibitors used significantly reduced eosinophilia and neutrophilia compared to the control mice group. Furthermore, it was observed that treatment with this inhibitor resulted in a significant reduction in blood histamine levels in mice after IgE-Ag challenge. CONCLUSION: The present data strongly suggest that the development of mitochondrial STAT3 inhibitors could serve as a potential treatment for allergy-associated diseases.

Pathogenicity and molecular characteristics of infectious bronchitis virus (IBV) strains isolated from broilers showing diarrhoea and respiratory disease.

Abstract 1. The possibility that infectious bronchitis virus (IBV) variants isolated from broilers with enteric and respiratory problems have a different tropism and pathological outcome from those IBV strains causing classical respiratory disease was investigated. 2. IBV variants were isolated from broiler flocks with enteric and respiratory problems in two regions of Brazil. The USP-10 isolate, of enteric origin, was inoculated via the oral oroculonasal routes into IBV-antibody-free broilers and specific pathogen-free (SPF) chickens to determine tissue tropism and pathogenicity and compared with an IBV variant (USP-50) isolated from chickens showing signs of respiratory disease only. 3. Both USP-10 and USP-50 strains caused similar pathological patterns by either route of inoculation. Both variants were detected in respiratory and non-respiratory tissues, including the kidney, intestine and testis. 4. Broilers were more susceptible to infection than SPF chickens, and seroconversion was detected in all of the chicks.

Orchitis in roosters with reduced fertility associated with avian infectious bronchitis virus and avian metapneumovirus infections.

The pathogenesis of infection involving both infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) causes reproductive damage in hens after viral replication in the epithelium of the oviduct, resulting in loss of cilia and degeneration and necrosis of the epithelial and glandular cells. Although IBV has been indicated as a possible cause of the formation of calcium stones in the epididymus of roosters, a definitive association has not been confirmed. This report describes the detection of IBV and aMPV in the testes of roosters from a Brazilian poultry broiler breeder's flock with epididymal stones and low fertility. Samples of testis, trachea, and lungs from breeder males aged 57 wk were positive for IBV by reverse transcriptase-polymerase chain reaction (RT-PCR), and virus isolation and testis samples were also positive for aMPV by RT-PCR. The inoculation of testis samples into embryonated chicken eggs via the allantoic cavity resulted in curled, hemorrhagic, and stunted embryos typical of IBV infection. The allantoic fluid was positive by RT-PCR aimed to amplify the region coding for the S1 subunit of the IBV S gene, but it was not positive for aMPV. Sequence analysis of the amplified fragment revealed a close relationship with European IBV genotype D274, previously unreported in Brazil. These results indicate that IBV and perhaps aMPV are likely to have played a role in the pathogenesis of the testicular disease described and should be regarded as factors that can influence male fertility disease in chickens.

Molecular characterization of infectious bronchitis virus strains isolated from the enteric contents of Brazilian laying hens and broilers.

Infectious bronchitis virus (IBV) is the causative agent of avian infectious bronchitis, which is characterized by respiratory, reproductive, and renal signs. However, the role of IBV as an enteric pathogen in still controversial. In Brazil, antigenic groups of IBV divergent from the Massachusetts serotype used for vaccination schedules in that country have already been demonstrated. The present study aimed to assess the different genotypes of IBV in Brazilian commercial poultry flocks by partial sequencing of the S1 amino-terminus coding region using enteric contents as samples and examine their relationship with the vaccine serotype currently in use. Samples of enteric contents were taken as pools of five birds from each of 18 poultry farms (17 broiler and one laying farm) from five Brazilian states between 2002 and 2006. Birds were presenting watery diarrhea and poor general condition but were without respiratory, renal, or reproductive signs. Conventional antibacterial and anticoccidial therapies were unsuccessful and, furthermore, all samples proved negative for rotavirus, reovirus, and astrovirus. Eleven IBV samples were isolated in embryonated eggs and resulted in S1 sequences. Phylogenetic analysis showed that these segregated into an exclusive cluster, close to serotype D274, but distant from Massachusetts. Mean amino acid identity amongst these Brazilian strains was 94.07%; amongst these and serotypes D274, 4/91, and Massachusetts, mean amino acid identity was 77.17%, 69.94%, and 68.93%, respectively. In conclusion, the presence of genotype variant strains of IBV in Brazilian poultry flocks has been demonstrated and might be the reason for the unsuccessful control of IBV in Brazil. Furthermore, these results also strengthen the implications of IBV in enteric diseases of poultry.

The GnRH receptor gene is preferentially expressed in functioning gonadotroph adenomas and displays a Mae III polymorphism site.

OBJECTIVE: Given the central role of the GnRH receptor (GnRHR) in the regulation of the gonadotrophin secretion, it might be implicated directly or indirectly in the pathogenesis of gonadotroph tumours. DESIGN: We determined if GnRHR mRNA was expressed in gonadotroph tumours using RT-PCR and analysed the GnRHR gene for the presence of mutations in its coding region, using direct sequencing of PCR products. Results were analysed according to the pattern of expression of alpha, beta-FSH and beta-LH subunit (SU) genes. SUBJECTS: RNA was extracted from 20 gonadotroph tumours identified by immunohistochemistry (> 10% of stained cells): 9 adenomas were functioning (high serum gonadotrophin levels), 3 were associated with high alpha-SU levels and 8 were nonfunctioning. Genomic DNA was extracted from 64 normal subjects. RESULTS: We found GnRHR mRNA in 12 tumours (60%): 8/9 functioning (88%), 1/3 alpha-secreting (33%) and 3/8 nonfunctioning (37.5%) gonadotroph adenomas. There was a significant association between GnRHR expression and immunostaining for beta-FSH (P = 0.014). The nucleotide sequence of the amplified products was identical to that of human pituitary except for the presence, in 3 functioning adenomas, of a silent C to T transition at nucleotide 453 encoding for the serine residue situated in the second intracellular loop at position 151. Heterozygosity provided evidence that both alleles were transcribed in these tumours. This substitution creates a Mae III restriction site. Genomic DNA from normal subjects were then tested for the presence of this new polymorphism. The frequency of the heterozygosity (18.7%) was not significantly different from that found in gonadotroph tumours (25%) and this new Mae III polymorphism site cannot be used as a tumoural marker. CONCLUSION: The GnRHR gene is preferentially expressed in functioning rather than in nonfunctioning gonadotroph adenomas, but no mutations altering the coding region of the gene were found to further substantiate its role in the pathogenesis of gonadotroph tumours.

Mucocele of the sphenoid sinus: a late complication of transsphenoidal pituitary surgery.

In an 18-month period, 3 cases of sphenoidal mucocele following pituitary surgery were diagnosed at our institution. Only 1 case of this late-onset disorder has yet been reported as a pitfall of the transsphenoidal route. Symptoms include recurrent headache and visual complications. Diagnosis was delayed because of misinterpretation of the magnetic resonance imaging findings, which actually showed the development of a sphenoid mucocele long before clinical symptoms occurred. These 3 cases suggest that attention should be focused not only on the sella turcica, but also on the sphenoid sinus, in analyzing the magnetic resonance imaging data. The treatment consists of endoscopic transnasal marsupialization, since the mucocele is lined by normal epithelium with an inflammatory reaction that will heal with drainage. At the time of surgery, prevention would require either endoscopic control of the mucosal remnants, in case of sinus exclusion, or leaving the sphenoid sinus air-filled under a sealed sella.

Germ-line mutation analysis in patients with multiple endocrine neoplasia type 1 and related disorders.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.

Glycaemic control and development of retinopathy in type 2 diabetes mellitus: a longitudinal study.

Relationships between glycaemic control, hypertension, and development of microangiopathy have been well documented in Type 1 (insulin-dependent) but not in Type 2 (non-insulin-dependent) diabetes mellitus. Therefore, we have investigated these relationships in a cohort of 64 Type 2 patients free of retinopathy (by angiofluorography), who were regularly followed until development of retinopathy or for at least 7 years as outpatients. Glycaemic control was assessed by 1 to 4 HbA1 determinations per year. Retinal status was monitored by annual angiofluorography. Nonproliferative retinopathy developed in 14 patients (cumulative incidence at 13 years: 29.8%) after a mean diabetes duration of 14.3+/-8.9 years (range 2-27). In multivariate analysis (Cox model), mean HbA1 during follow-up (p < 0.001), and hypertension at first examination (p = 0.09) were associated with the development of retinopathy, but age, sex, BMI, diabetes duration, smoking, and fasting blood glucose were not. The relative risk for developing retinopathy (RR) was 7.2 (IC 95%: 1.61-32.4) in patients with a mean HbA1 during follow-up above the median value of the cohort (8.3%) compared with patients with HbA1 during follow-up below this value. RR was 2.5 (IC 0.8-8) in patients with HbA1 at first examination above compared to below the median value (8.4%). RR was 3.0 (IC 0.9-10) in patients treated for hypertension at baseline compared to those without treatment. A sixfold increase in retinopathy prevalence was observed between patients with mean HbA1 in the highest or lowest quartile of mean HbA1 distribution during follow-up. This longitudinal study indicates a strong association between long-term glycaemic control and the development of diabetic retinopathy in Type 2 diabetes.

Diabetes: from phenotypes to genotypes.

Diabetes mellitus comprises a heterogeneous group of diseases which have chronic hyperglycaemia in common as well as the resulting microvascular, macrovascular and neurological complications of this condition. Familial studies have provided strong evidence for the existence of genetic determinants in the different types of diabetes. In particular, monozygotic twin studies have indicated a higher rate of concordance in non-insulin-dependent (NIDDM) than in insulin-dependent diabetes mellitus (IDDM). In IDDM, 8 susceptibility loci have been identified, notably the HLA complex and insulin promotor gene. Rigorous family studies have identified monogenic subtypes representing 10-15% of all NIDDM: MODY2 related to glucokinase gene mutations, MODY1 and MODY3 secondary to mutation of hepatic nuclear factors, and diabetes resulting from deletion or mutation of mitochondrial DNA. Most NIDDM result from polygenic heredity, and susceptibility genes conducive to increased receptivity to deleterious environmental influences are now under investigation, such as beta 3 adrenergic receptor, FABP2 and OB. Precise analysis of phenotypes in the remaining families or systematic screening of the genome could allow the genes of each subtype to be identified. Finally, susceptibility genes for the increased severity and frequency of vascular complications have been identified, such as angiotensin converting enzyme, aldose reductase and aldehyde dehydrogenase genes. This progress has been facilitated by developments in molecular biology.

Microalbuminuria in obese patients with or without hypertension.

OBJECTIVE: To evaluate the prevalence in obese patients of an increased urinary albumin excretion rate (UAER) and the factors involved in this parameter. SUBJECTS: Two hundred and seven nondiabetic obese patients with BMI = 34.7 +/- 5.7 (SD) kg/m2. None had proteinuria or a history of nephropathy or uropathy. Fifty-two had moderate hypertension. A control group of 22 lean healthy subjects was also studied. MEASUREMENTS: The UAER was determined from 24-h urine samples by means of immunonephelemetry laser method. Creatinine clearance was calculated. Glycemia and plasma C peptide at fasting and 120 mine after glucose oral administration, HbA1c, serum fructosamine, plasma total cholesterol and triglycerides, HDL and LDL cholesterol were measured. Food intakes were determined by dietary history. RESULTS: Compared with the control group, the UAER was significantly higher in the obese patients (18.0 +/- 20.1 mg/24 h vs 3.2 +/- 2.8 mg/24 h, P < 0.0001). It was elevated (> 30 mg/24 h) in 25 obese patients (12.1%) and in particular, in 19.2% of the obese patients with hypertension. It was significantly higher in the patients with android or mixed (both android and gynoid) obesity than in those with gynoid obesity (p = 0.050). Log UAER correlated negatively with the duration of hypertension (p = 0.038) and was higher in the patients with familial hypertension than in those without (p = 0.002). Log UAER correlated strongly with log creatinine clearance (p < 0.0001) and fractional albumin clearance (p < 0.0001). It correlated significantly with fasting and 120 min after glucose plasma C peptide concentrations (p = 0.018 and p = 0.046, respectively). Creatinine clearance was significantly higher in the patients with android or mixed obesity than in those with gynoid obesity (p = 0.001). Log creatinine clearance correlated negatively with age (p = 0.046), and log LDL cholesterol (p = 0.025) and positively with log lipid caloric intake (p = 0.014). CONCLUSION: These results show the high prevalence of microalbuminuria in nondiabetic obese patients and suggest the involvement of renal hyperfiltration. Microalbuminuria may be an indicator of familial hypertension in obese subjects. Insulin resistance may be involved in the increase in the UAER. Nutritional factors, particularly lipid intake, may contribute to this increase in the UAER via an increase in glomerular hyperfiltration.

[Hypophyseal adenomas: functional and tumor evaluation and therapeutic approaches]. / Adénomes hypophysaires: bilan tumoral et fonctionnel et abords thérapeutiques. therapeutic approaches]

Patients with pituitary adenomas present with hypersecretion syndrome(s), and/or pituitary failure(s), and/or signs of mass effect, or incidentally. Pituitary function evaluation, visual acuity and field check-up, and MRI or at least CAT are compulsory for diagnosis, and for therapeutic approach; surgery for Cushing's disease, dopamine agonists for prolactinomas, somatostatin analogs or surgery for thyrotroph adenomas, surgery and/or somatostatin analogs and/or radiotherapy in acromegaly, surgery with additional irradiation in most adenomas of other types, or even expectation in some instances.

Plasma levels of aldosterone versus aldosterone precursors: a way to estimate the malignancy of asymptomatic and nonsecretory adrenal tumors: a French Retrospective Multicentric Study.

The aim of this study was to find out whether the dysfunction of aldosterone pathway, previously proposed as a marker of secretory adrenal carcinoma, is also found in nonsecretory adrenal carcinomas, which pose even more difficult diagnostic problems even for patients with hypertension accompanied or not by hypokalemia. The exploration consisted of using the same method (RIA preceded by a chromatographic step) to determine the plasma levels of the following steroids in the mineral corticosteroid pathway: deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone (18-OHDOC), corticosterone (B), 18 hydroxycorticosterone (18 OH B), and aldosterone. The subjects included 16 adults, each presenting with an endocrinologically asymptomatic adrenal mass associated for some patients with hypokalemia and hypertension (8 with adrenal carcinoma, 2 with adrenal metastasis from other forms of cancer, and 6 adenomas). These results show that even in nonsecretory adrenal carcinoma, there is a dysfunction of the aldosterone pathway, which can be evaluated from the ratio between aldosterone and the substrate of 11 beta hydroxylase (DOC) and its derivative (18-OH DOC). This study suggests that exploration of mineralocorticosteroid pathway can be used as a hormonal marker of adrenal carcinoma for both secretory and non-secretory malignant masses.

Enhanced susceptibility of low-density lipoprotein to in vitro oxidation in type 1 and type 2 diabetic patients.

Macrovascular disease represents a major cause of morbidity and mortality in patients with diabetes mellitus. Low-density lipoprotein (LDL) is involved in the pathogenesis of atherosclerotic lesions, through modifying processes such as oxidation. We examined the in vitro susceptibility to oxidation and the oxidizability of LDL isolated from the plasma of Type 1 and Type 2 diabetic patients. Two groups of diabetic patients (20 Type 1, 20 Type 2) were compared with sex- and age-matched non-diabetic control groups. In vitro oxidation of the purified LDL preparations was assessed by determination of the kinetics for the formation of conjugated dienes (lag phase duration, maximal rate and maximal dienes concentration) and by measurement of thiobarbituric acid-reacting substances (TBARS) in the presence of copper ions. LDL from both Type 1 and Type 2 diabetic patients exhibited a shorter lag phase duration for conjugated dienes formation (94 +/- 14 vs. 108 +/- 20 and 97 +/- 26 vs. 112 +/- 18 min for Type 1 and Type 2 diabetic groups vs. respective control groups, P < 0.05). We also observed an increase in maximal rate of conjugated dienes formation (2.21 +/- 0.55 vs. 1.52 +/- 0.31 and 2.02 +/- 0.55 vs. 1.52 +/- 0.31 nmol/mg LDL/min, P < 0.01) and of maximal production of TBARS (77.9 +/- 11.8 vs. 65.5 +/- 10.4 and 76.7 +/- 9.9 vs. 65.3 +/- 9.4 nmol/mg LDL protein, P < 0.05) in diabetic groups. Our results demonstrate both a higher susceptibility to oxidation and a higher oxidizability of LDL from diabetic patients, as much for Type 1 as Type 2 diabetic subjects with or without pre-existent vascular complications. This enhanced propensity of LDL oxidation in patients with diabetes mellitus could at least partly be attributable to quantitative and qualitative alterations in the chemical composition of LDL and to the glycoxidation process occurring on these lipoproteins.