IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

Genetic and Functional Characterization of Congenital HCMV Clinical Strains in Ex Vivo First Trimester Placental Model.

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.

New therapeutic perspective in the prevention of congenital cytomegalovirus infection.

INTRODUCTION: Hyperimmune globulin Cytotect CP® is a candidate for cytomegalovirus congenital infection prevention. We previously demonstrated its efficacy to prevent villi infection in our first-trimester placenta explants up to day 7, but with an inefficiency at day 14 (Coste-Mazeau et al., Microorganisms, 2021). As this could impact clinical efficacy, we now study the effect of weekly administration of Cytotect CP® on the prevention of villi infection. METHODS: Human embryonic lung fibroblast cells were infected at confluence with the endothelial strain TB40/E. Placentae were collected from voluntary pregnancy terminations (8-14 weeks of gestation) from cytomegalovirus-seronegative women. After 5 days of infection of the cells, villi explants were simultaneously added on sponges with Cytotect CP® at various concentrations. After 7 days, Cytotect CP® was renewed in only half of the plates. Villi were collected at days 7 and 14 with or without medium renewal. We compared the viral load by duplex quantitative PCR cytomegalovirus/albumin and the toxicity by measuring ß-hCG concentrations in the supernatants with and without medium renewal. RESULTS: We did not find any efficacy of Cytotect CP® at day 14 when Cytotect CP® is not renewed, but a regular decrease of the viral load when the immunoglobulins were renewed at day 7, with an EC50 = 0.52 U/mL. We did not observed toxicity of Cytotect CP® with or without renewal of the molecule. CONCLUSION: Cytotect CP® is more effective when renewed at day 7. The prevention of congenital cytomegalovirus infection could be enhanced by reducing the spacing of doses.

Potential of Anti-CMV Immunoglobulin Cytotect CP® In Vitro and Ex Vivo in a First-Trimester Placenta Model.

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 ß-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.

Integron detection for prediction of trimethoprim/sulfamethoxazole susceptibility in children with Enterobacterales urinary tract infections.

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.

Distribution of proteinuria- and albuminuria-to-creatinine ratios in preterm newborns.

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.

Left atrial function in patients with light chain amyloidosis: A transthoracic 3D speckle tracking imaging study.

BACKGROUND: Systemic light chain amyloidosis (AL) is characterized by the extracellular deposition of amyloid fibrils. Transthoracic echocardiography is the modality of choice to assess cardiac function in patients with AL. Whereas left ventricular (LV) function has been well studied in this patient population, data regarding the value of left atrial (LA) function in AL patients are lacking. In this study, we aim to examine the impact of LA volumes and function on survival in AL patients as assessed by real-time 3D echocardiography. METHODS: A total of 77 patients (67±10 years, 60% men) with confirmed AL and 39 healthy controls were included. All standard 2D echocardiographic and 3D-LA parameters were obtained. RESULTS: Fourteen patients (18%) were in Mayo Clinic (MC) stage I, 30 (39%) in stage II, and 33 (43%) in stage III at initial evaluation. There was no significant difference among the MC stages groups in terms of age, gender, or cardiovascular risk factors. As compared to patients in MC II and MC I, those in MC III had significantly larger indexed 3D-LA volumes (MCIII: 46±15mL/m2, MC II: 38±12mL/m2, and MC I: 23±9mL/m2, p<0.0001), lower 3D-LA total emptying fraction (3D-tLAEF) (21±13% vs. 31±15% vs. 43±7%, respectively, p<0.0001), and worse 3D peak atrial longitudinal strain (3D-PALS) (11±9% vs. 18±13% vs. 20±7%, respectively, p=0.007). Two-year survival was significantly lower in patients with 3D-tLAEF <+34% (p=0.003) and in those with 3D-PALS <+14% (p=0.034). Both parameters provided incremental prognostic value over maximal LA volume in multivariate analysis. CONCLUSION: Functional LA parameters are progressively altered in AL patients according to the MC stage. A decrease in 3D-PALS is associated with worse outcome, independently of LA volume.

Normal values of urine total protein- and albumin-to-creatinine ratios in term newborns.

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.

IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition.

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

Specific impairment of proximal tubular cell proliferation by a monoclonal κ light chain responsible for Fanconi syndrome.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Crystal-storing histiocytosis with renal Fanconi syndrome: pathological and molecular characteristics compared with classical myeloma-associated Fanconi syndrome.

BACKGROUND: Crystal-storing histiocytosis (CSH) is a poorly described complication of monoclonal gammopathy featuring histiocyte lysosomal storage of kappa light chain (kappa LC) crystals. Although CSH is usually associated with systemic manifestations, renal involvement is uncommon. METHODS: To investigate the molecular mechanisms implicated in kappa LC crystallization, we performed immunopathological and molecular studies in three patients with CSH and renal Fanconi syndrome (CSH/FS). The V kappa sequences were determined, and resulting molecular models were compared with previously reported myeloma-associated FS kappa LC sequences. RESULTS: All patients presented with chronic tubulo-interstitial nephritis and renal FS with accumulation of monoclonal kappa LC crystals within proximal tubular cells. They showed peri-renal and interstitial infiltration by histiocytes containing eosinophilic crystalline inclusions (pseudo-pseudo-Gaucher cells). LC sequences were determined and assigned to their germline counterparts, in strong homology with previously reported myeloma-associated FS sequences. Comparison of CSH/FS V kappa domain 3D structures with the germline-encoded structures and those from patients with myeloma-associated FS underlined distinct hydrophobic residues exposed to the solvent in two patients, likely favouring the formation of a variant form of crystals that may further resist degradation after phagocytosis. CONCLUSION: Although CSH/FS and myeloma-associated FS are closely related disorders, peculiar mutations in the V domains of CSH/FS monoclonal kappa LCs, different from those in myeloma-associated FS, may account for crystal morphology, predominant accumulation within histiocytes and multiple organ involvement in CSH.

Role of the monoclonal kappa chain V domain and reversibility of renal damage in a transgenic model of acquired Fanconi syndrome.

Acquired Fanconi syndrome (FS) is a complication of monoclonal gammopathies featuring a generalized dysfunction of the proximal tubule of the kidney, due to the storage within proximal tubular cells of a monoclonal immunoglobulin light chain. We engineered transgenic mice in which the endogenous mouse Jkappa cluster was replaced by a human VkappaJkappa rearranged gene cloned from a patient with smoldering myeloma-associated FS. The V region belonged to the VkappaI subgroup and was related to the O2-O12 germ-line gene, a V segment previously found associated with FS and light-chain crystallization in several patients with myeloma. Association of the human VkappaI domain with a mouse kappa constant domain in transgenic animals yielded a nephrotoxicity pattern similar to that observed in patients, strongly suggesting that the whole pathogenic effect of FS light chains can be ascribed to a peculiar structure of the V domain. Morphologic alterations of the kidney tubular cells, which contained rhomboid-shape crystals, were observed in mice, together with alterations of the proximal tubule reabsorption function. Moreover, the number of renal crystalline inclusions was dramatically reduced after conditional deletion of the human VkappaI transgene, showing that proximal tubular lesions are reversible upon suppression of the nephrotoxic light chain secretion.

Cell permeable BH3-peptides overcome the cytoprotective effect of Bcl-2 and Bcl-X(L).

Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-X(L), yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-X(L) failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-X(L).