Chahrazed El Hamel

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, leading to a variety of symptoms in the unborn child that range from asymptomatic to death in utero. Our objective was to better understand the mechanisms of placental infection by HCMV clinical strains, particularly during the first trimester of pregnancy. We thus characterized and compared the replication kinetics of various HCMV clinical strains and laboratory strains by measuring viral loads in an ex vivo model of first trimester villi and decidua, and used NGS and PCA analysis to analyze the genes involved in cell tropism and virulence factors. We observed that first trimester villi and decidua are similarly permissive to laboratory and symptomatic strains, and that asymptomatic strains poorly replicate in decidua tissue. PCA analysis allowed us to segregate our clinical strains based on their clinical characteristics, suggesting a link between gene mutations and symptoms. All these results bring forth elements that can help better understand the mechanisms that induce the appearance of symptoms or in the congenitally infected newborn.

Background: Congenital CMV infection is the leading cause of neonatal neurological deficit. We herein studied in vitro and ex vivo the potential of the hyperimmune globulin Cytotect CP^® (Biotest, Germany) for congenital infection prevention and treatment. Methods: In vitro neutralization assays were conducted in fibroblasts and retinal epithelial cells on the CMV strains TB40/E and VHL/E to determine the 50% and 90% neutralizing doses (ND50 and ND90). The toxicity was assessed by measuring LDH release. Ex vivo assays were conducted in first-trimester villi explants with the TB40/E strain, namely, neutralization assays, the prevention of villi infection, and the inhibition of viral replication in infected villi. Viability was assessed by β-HCG quantification in supernatants. Results: The in vitro neutralization tests showed that Cytotect CP^®® inhibits the development of infection foci (DN50: 0.011–0.014 U/mL for VHL/E and 0.032–0.033 U/mL for TB40E) without any toxicity. In the ex vivo neutralization assays, the DN50 were 0.011 U/mL on day 7 and 0.093 U/mL on day 14. For the prevention of villi infection, the EC50 was 0.024 U/mL on day 7. Cytotect-CP^® did not inhibit viral growth in infected villi. No impact on villi viability was observed. Conclusions: These results sustained that Cytotect CP^® has the potential to prevent CMV congenital infection.

In normal activated B-cells, Activation Induced-cytidine deaminase (AID) is absolutely required for immunoglobulin (Ig) class switch recombination (CSR) and IGHV somatic hypermutation (SHM). AID is also implicated in the Locus Suicide Recombination (LSR) of the Ig heavy (IgH) locus, resulting in the deletion of the IgH constant part. Chronic lymphocytic leukemia (CLL) is an indolent non-Hodgkin B-cell lymphoma characterized by tumor CLL B-cells that weakly express a B cell receptor (BCR) on their surface. The great majority of CLL tumor B-cells are non class-switched. Searching for abnormalities of IgH locus recombination in CLL, we investigated CSR and LSR in samples from CLL patients (N=47) with high blood tumor cell infiltration (>98%) and in healthy volunteers (HV) as controls (N=9). LSR was detectable at comparable levels in both HV and CLL groups. CSR counts were decreased in CLL samples as expected. As distribution of LSR counts was bimodal, we separated CLL patients in two groups so called LSR ^High and LSR ^Low . LSR ^High CLLs exhibited very weak AID expression and low mutation rate of IgHV region and of the AID off-target PIM1 gene. LSR junction diversity, evaluated using the Shannon index, was increased in LSR ^High CLLs suggesting that LSR was on-going in these cells. Also, shorter telomeres were observed in LSR ^High CLLs suggesting an increased number of past mitosis. Consistently, increased levels of cMYC expression were detected in LSR ^High CLLs and treatment free survival of these cases was markedly decreased. We hypothesized that LSR in LSR ^High CLLs is AID independent and could be due to DNA lesion and inaccurate DSB repair within the IgH locus, which could be accessible to recombination machinery due to increased IgH locus transcription. Altogether, our results indicate the accessibility of IgH locus and the proliferation increase LSR rate in LSR ^High CLLs could be related to cMYC resulting in shorter treatment free survival of patients and point on an AID independent mechanism of IgH recombination.

Objectives: In some countries, third-generation cephalosporins (3GCs) serve as first-line therapy in children with urinary tract infections (UTIs). However, their use may contribute to the emergence of antibiotic resistance, notably among Gram-negative bacteria (GNB). Integrons are bacterial genetic elements involved in antibiotic resistance in GNB. Their absence is associated with >97% susceptibility to trimethoprim/sulfamethoxazole in adults infected with GNB. The objective of this study was to examine the value of integron detection directly from urine samples as a predictive marker of resistance to trimethoprim/sulfamethoxazole in children with GNB-related UTIs. Methods: Children admitted to the Limoges University Hospital’s paediatric emergency department between February 2018 and March 2019 with a suspicion of UTI were eligible for the study. Only confirmed cases presenting a positive urine culture with unique GNB were retained for further study analyses. Integrons were detected directly from urines using real-time PCR. Results: The data of 72 patients were analysed and integrons were detected in 15 urine samples. The negative predictive value of integron detection for resistance to trimethoprim/sulfamethoxazole was 100% as all of the GNB (all were Enterobacterales) isolated from patients with no integrons detected in their urine samples were susceptible to trimethoprim/sulfamethoxazole. Conclusions: The detection of integrons in cases of paediatric patients with suspected UTI could help limit 3GC empirical use and empower an empirical first-line strategy better tailored to the needs of each patient.

Background Urine protein assessment is important when glomerular disease or injury is suspected. Normal values of proteinuria already published for preterm newborns suffer from limitation, with small cohorts of patients. This prospective study was conducted to update the urine total protein- and albumin-to-creatinine ratio values. Methods Urine samples were collected from 231 preterm newborns within the first 48 h (D0–1) and/or between 72–120 h of life (D3–4). Total protein, albumin, and creatinine were measured, their distribution and upper-limit values determined. Results At D0–1 and D3–4, respectively, the median for the total protein-to-creatinine ratio were 80 and 107 mg/mmol (upper-limit values 223 and 289 mg/mmol) in the whole studied population, 149 and 214 mg/mmol in children born before 29 weeks of gestational age, 108 and 130 mg/mmol in those born between 29 and 33 weeks, and 61 and 93 mg/mmol in those born after 33 weeks. For the albumin-to-creatinine ratio, the median were 12 and 17 mg/mmol (upper-limit values 65 and 62 mg/mmol) in the whole studied population, 22 and 50 mg/mmol in children born before 29 weeks, 21 mg/mmol in those born between 29 and 33 weeks, and 8 and 12 mg/mmol in those born after 33 weeks. The use of nephrotoxic drugs and mechanical ventilation seems to influence proteinuria and albuminuria values. Conclusions We report distribution of proteinuria- and albuminuria-to-creatinine in preterm newborns, including the upper-limit values. These values should be taken into account in the detection and diagnosis of glomerular disease and/or injury in daily clinical practice. Graphical abstract

Background It is important to have an accurate assessment of urinary protein when glomerulopathy or kidney injury is suspected. Currently available normal values for the neonate population have limited value, in part because they are based on small populations and obsolete creatinine assays. We have performed a prospective study with the aim to update the normal upper values of the urinary total protein-to-creatinine and albumin-to-creatinine ratios in term newborns. Methods Urine samples were collected from 277 healthy, full-term newborns within the first 48 hours (D0–1) and between 72 and 120 h of life (D3–4). Total protein, albumin, creatinine and osmolality were measured and the upper limit of normal (upper-limit) values determined. Results At D0–1 and D3–4, the upper-limit values for the total protein-to-creatinine ratio were 1431 and 1205 mg/g (162 and 136 g/mol) and those for the albumin-to-creatinine ratio were 746 and 301 mg/g (84 and 34 g/mol), respectively. The upper-limit values were significantly higher at D0–1 than at D3–4 only for the albumin-to-creatinine ratio. Conclusion This study determined the upper limit of normal values for urinary total protein-to-creatinine and albumin-to-creatinine ratios in the largest population of newborns studied to date. These values can therefore be considered as the most clinically relevant data currently available for the detection and diagnosis of glomerular injury in daily clinical practice in this population.

Fanconi syndrome (FS) is a rare renal disorder featuring proximal tubule dysfunction that may occur following tubular reabsorption of a monoclonal light chain (LC), in patients with multiple myeloma. FS may precede the recognition of multiple myeloma by several years. In most cases, crystalline inclusions of monoclonal κ LCs are observed within the lysosomes of proximal tubular cells (PTCs) and probably participate in their functional alteration. To investigate the mechanism implicated in proximal tubule dysfunction, we compared the effects of κ LC-CHEB obtained from a patient with myeloma-associated FS to those of control κ LC-BON obtained from a patient without evidence of FS, on the viability and proliferation of two different PTC lines. Our data suggest that the tubular atrophy in myeloma-associated FS does not result from increased apoptosis of PTCs, but from their impaired capacity to proliferate and renew. Indeed, in vitro incubation of cultured PTCs with physiological amounts of the nephrotoxic κ LC-CHEB was sufficient to cause a depression in DNA synthesis and in cell proliferation. This effect was observed neither with control κ LC-BON nor in the absence of κ LC. The reduced turnover of PTCs may affect tubular repair and regeneration. In addition, the reduced proliferation of myeloma cells producing the same monoclonal κ LC might explain the frequent association of FS with smoldering multiple myeloma.