Marzena Lenart

Familial occurrence of warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome

Introduction:Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder with an autosomal-dominant pattern of inheritance and low fatality rate but significant lifelong morbidity.Materials and Methods:A 27-year-old mother of two children has been suffering from severe neutropenia and recurrent infections with the diagnosis of sporadic WHIM syndrome established by sequencing the CXCR4 gene and the finding of a heterozygous 1000 C→T nonsense mutation in the second CXCR4 exon. The first child was an apparently healthy boy delivered at full term. Umbilical cord blood cells were obtained for genetic analysis. Peripheral blood cells were also analyzed at 8 months of life. Both analyses revealed the same mutation as that of his mother. The child was in a good condition, manifesting neutropenia without infections until 11 months of life. He subsequently developed pneumonia requiring a more aggressive treatment. After that, the regular substitution of immunoglobulins (IVIGs) and G-CSF has been preventing serious infections. Six months ago the second boy was delivered who also demonstrated neutropenia without severe infections. Genetic studies using cord blood and also peripheral blood cells in the fourth month showed an identical mutation of the CXCR4 gene as in his mother. Moreover, the mother and her first son demonstrated monocytopenia.Results:The results indicate that genetic defects connected with WHIM syndrome may influence not only the granulocyte, but also the monocytic lineage. Moreover, a perinatal diagnosis of WHIM syndrome made by sequencing the CXCR4 gene should be performed in cases where either parent is known to be affected with this disease.Conclusions:This would facilitate an earlier detection of the deficiency in children, thereby allowing a more comprehensive follow-up and administration of appropriate therapy.

Low hepatitis B virus-specific T-cell response in males correlates with high regulatory T-cell numbers in murine models

Hepatitis B virus (HBV) infection shows significant gender‐related differences in pathogenesis, disease progression, and development of hepatocellular carcinoma. The gender‐associated differences in HBV replication and viral protein levels may be associated with distinct HBV‐specific immune responses in the host. In the present study, we examined the impact of gender on HBV‐specific immune responses in two different mouse models representing transient and persistent hepadnaviral infection; hydrodynamic injection with the HBV genome mimicked acute HBV infection, whereas the efficacy of therapeutic vaccination was studied in the woodchuck hepatitis virus transgenic mouse model. Consistent with previous reports, significantly higher HBV DNA and protein levels were detected in male compared to female mice. Although hydrodynamic injection with the HBV genome resulted in similar numbers of intrahepatic HBV‐specific cluster of differentiation 8–positive (CD8+) T cells, their functionality was significantly reduced in males and correlated with higher numbers of intrahepatic regulatory T cells (Tregs). Similar effects were observed in woodchuck hepatitis virus transgenic mice immunized with a DNA prime‐recombinant adenovirus boost vaccination protocol. Male mice showed functionally suppressed woodchuck hepatitis virus–specific CD8+ T‐cell responses in the liver and significantly higher numbers of intrahepatic Tregs compared to females. Blockade of Treg responses in male mice led to augmented effector functions of specific CD8+ T cells and subsequently improved virus control in both models of transient and persistent hepadnaviral infection. Conclusion: The functionality of virus‐specific CD8+ T cells in male mice was suppressed by intrahepatic Tregs and inversely correlated with levels of hepadnaviral DNA and viral protein; the induction of intrahepatic Tregs by viral replication and/or protein levels may explain the gender‐related differences in the outcomes of HBV infection and limit the success of immunotherapeutic strategies in male patients. (Hepatology 2017;66:69–83).

Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14(++)CD16(-)) monocytes via PI3K/Akt/mTOR-dependent signalling pathway.

Tumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100μg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway.

Analysis of selected aspects of inflammasome function in the monocytes from neonates born extremely and very prematurely

BACKGROUND Inflammasomes regulate activation of caspase-1, which cleaves and activates interleukin (IL)-1β and IL-18, the cytokines that trigger pro-inflammatory and antimicrobial responses. There is very little known about inflammasome function in the subsets of monocytes (MO) isolated from preterm neonates born extremely and very prematurely. METHODS A group of 76 very low birth weight patients without early-onset sepsis was divided into extremely preterm (<28 gestational week) or very preterm (28-32 gestational week) neonates. The first blood sample was collected on the 5th day of life (5th DOL) to analyse MO subsets as well as the intracellular IL-1β expression and supernatant concentration of IL-1β and IL-18. Secondary blood samples were collected within 24h of late-onset sepsis (LOS) development and analysed as above. RESULTS On the 5th DOL, the extremely preterm neonates were characterized by a significantly higher absolute count of MO, in particular in the classical and intermediate subsets, as compared to the very preterm group. The counts of the intermediate and non-classical MO subsets increased during LOS in all neonates. We did not observe significant differences in the intracellular IL-1β expression between the analysed groups. Furthermore, the levels of the analysed cytokines in the MO supernatants were comparable between the extremely and very preterm neonates on the 5th DOL. Finally, a higher level of IL-18 was observed in the supernatant of the extremely preterm group during LOS. CONCLUSIONS During LOS, extremely preterm neonates excrete a higher level of IL-18 cytokines compared to very preterm neonates. Further studies are required to determine whether this observation is a result of a higher count of the circulating MO or is a true reflection of increased inflammasome function in this particular group of newborns.

Neurodegenerative changes detected by neuroimaging in a patient with contiguous X-chromosome deletion syndrome encompassing BTK and TIMM8A genes

Introduction In this study we describe a patient with gross deletion containing the BTK and TIMM8A genes. Mutations in these genes are responsible for X-linked agammaglobulinemia and Mohr-Tranebjaerg syndrome, respectively. X linked agammaglobulinemia is a rare primary immunodeficiency characterized by low levels of B lymphocytes and recurrent microbial infections, whereas, Mohr-Tranebjaerg syndrome is a progressive neurodegenerative disorder with early onset of sensorineural deafness. Material and methods For neuroimaging, the magnetic resonance imaging and magnetic resonance spectroscopy of the brain were performed. Microarray analysis was performed to establish the extent of deletion. Results The first clinical symptoms observed in our patient at the age of 6 months were connected with primary humoral immunodeficiency, whereas clinical signs of MTS emerged in the third year of live. Interestingly, the loss of speech ability was not accompanied by hearing failure. Neuroimaging of the brain suggested leukodystrophy. Molecular tests revealed contiguous X-chromosome deletion syndrome encompassing BTK (from exons 6 through 19) and TIMM8A genes. The loss of the patient’s DNA fragment was accurately localized from 100 601 727 to 100 617 576 bp on chromosome’s loci Xq22.1. Conclusions We diagnosed XLA-MTS in the first Polish patient on the basis of particular molecular methods. We detected neurodegenerative changes in MRI and MR spectroscopy in this patient. Our results provide further insight into this rare syndrome.

Analysis of PD-1 expression in the monocyte subsets from non-septic and septic preterm neonates

Programmed death-1 (PD-1) receptor system represents a part of recently reported immunoregulatory pathway. PD-1 is an immune checkpoint molecule, which plays an important role in downregulating the immune system proinflammatory activity. Until recently, PD-1 expression was not established on immune cells of the preterm infants. The study objectives were to confirm expression of the PD-1 receptors on the monocytes isolated from very low birth weight newborns (VLBW), and to analyze their expression during the first week of life and late-onset sepsis. Peripheral blood mononuclear cells were isolated from 76 VLBW patients without early-onset sepsis on their 5th day of life (DOL). PD-1 expression was determined on the monocyte subsets (classical, intermediate, non-classical) by flow cytometry. In case of late-onset sepsis (LOS), the same analysis was performed. Our results demonstrated that on the 5th DOL, PD-1 receptors were present in all the monocyte subsets. Children, whose mothers had received antenatal steroids, presented higher absolute numbers of non-classical monocytes with PD-1 expression. Infants born extremely preterm who later developed LOS, initially showed a lower percentage of PD-1 receptor-positive intermediate monocytes in comparison to neonates born very preterm. During LOS, we observed a rise in the percentage of classical monocytes with PD-1 expression. In case of septic shock or fatal outcome, there was a higher percentage and absolute count of intermediate monocytes with PD-1 expression in comparison to children without these complications. In conclusion, monocytes from VLBW children express PD-1 receptors. Antenatal steroid administration seems to induce PD-1 receptor expression in the non-classical monocytes. PD-1 might play a role in immunosuppressive phase of sepsis in the prematurely born children with septic shock and fatal outcome.

Comparison of 6B11 mAb and α-GalCer-loaded CD1d dextramers for detection of iNKT cells by flow cytometry

Invariant natural killer T (iNKT) cells are a small population of thymus-derived T cells that are restricted by non-classical MHC class I molecule CD1d and express an evolutionary conserved TCR with an invariant α-chain. The frequency of iNKT cells in peripheral blood is very low, thus, accurate methods to identify and enumerate iNKT cells are needed. The aim of the study was to compare 6B11 mAb or α-GalCer-loaded CD1d dextramers usage in iNKT cell detection. The frequency of CD3+CD56+ lymphocytes is much higher, with statistical significance (p<0,001), than real iNKT cells detected by 6B11 mAb or α-GalCer-loaded CD1d dextramers. The frequency of iNKT cells, recognized by 6B11 mAb or α-GalCer-loaded CD1d dextramers, was in a similar range. Nonetheless, when we compared whether 6B11+ and α-GalCer-loaded CD1d dextramers+ are the same populations, it turned out that by this approach we were able to identify three distinct subsets of iNKT cells: i) 6B11+/α-GalCer-loaded dextramer- cells, ii) 6B11+/α-GalCer-loaded dextramer+ cells, and iii) 6B11-/α-GalCer-loaded dextramer+. Thus, although 6B11 mAb and α-GalCer-loaded dextramers may identify not exactly the same cells, application of these methods seems to give similar results of iNKT cell frequency in peripheral blood. It seems that both approaches for iNKT detection can be used for precise identification of these cells. Moreover, our results indicate that CD3+CD56+ lymphocytes are a heterogeneous population of T cells, expressing activation markers of both NK and T lymphocytes, yet with not well characterized properties.