Danuta Kowalczyk

Effect of the immunomodulating agent, pentoxifylline, in the treatment of sepsis in prematurely delivered infants: a placebo-controlled, double-blind trial.

OBJECTIVE To evaluate the influence of the methylxanthine derivative, pentoxifylline, on plasma levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6 in prematurely delivered infants with generalized bacterial infections and to assess the effect of this immunomodulating drug on the clinical outcome in newborns with sepsis. DESIGN A prospective, randomized, double-blind trial. SETTING The neonatal intensive therapy units in university teaching hospitals. PATIENTS One hundred patients with sepsis admitted during a 1.5-yr period. INTERVENTIONS Patients were randomly assigned to receive pentoxifylline (pentoxifylline group) in a dose of 5 mg/kg/hr for 6 hrs on 6 successive days or an identically presented placebo (placebo group). MEASUREMENTS AND MAIN RESULTS Only infants with sepsis confirmed by positive blood culture were recruited into the study. There were no significant differences at randomization between the pentoxifylline and placebo groups with regard to the birth weight, gestational age, gender, Apgar score, hypotension, neutropenia, thrombocytopenia, metabolic acidosis, plasma levels of cytokines, and occurrence of shock. Plasma levels of TNF, IL-1, and IL-6 were evaluated before and after the drug or placebo administration on the first, third, and sixth days of therapy. Cytokines were determined by immunoenzymetric test EASIA (TNF) and Endogen Interleukin-Elisa (IL-1, IL-6). The frequency of gram-negative sepsis was similar in both groups (37.5% and 36.8%). Pentoxifylline significantly diminished plasma TNF levels (p = .009) but had no effect on plasma IL-1 levels. Mean plasma IL-6 levels, which were measured in the pentoxifylline group on the 6th day of the study, were significantly lower compared with respective data obtained in the placebo group. Only 1 of 40 infants with sepsis in the pentoxifylline group died, whereas 6 of 38 infants in the placebo group did not survive (p = .046). An increased incidence of disordered peripheral circulation and metabolic acidosis (p = .048), anuria or oliguria (p = .03), disseminated intravascular coagulation (p = .043), and the occurrence of clinical symptoms of necrotizing enterocolitis (p = .025) was observed in the course of sepsis in infants in the placebo group. CONCLUSION Pentoxifylline significantly affects the synthesis of TNF and IL-6 as well as reduces the mortality rate in premature infants with sepsis. The dosage and schedule of drug administration in this study attenuated the severity of the clinical course of sepsis in this group of patients.

Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation

ABSTRACT The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3+ and CD4+) or monocytes (CD33+and CD14+) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.

Haploinsufficiency at the human IFNGR2 locus contributes to mycobacterial disease

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome, the known genetic etiologies of which impair the production of, or the response to interferon-gamma (IFN-γ). We report here a patient (P1) with MSMD whose cells display mildly impaired responses to IFN-γ, at levels, however, similar to those from MSMD patients with autosomal recessive (AR) partial IFN-γR2 or STAT1 deficiency. Whole-exome sequencing (WES) and Sanger sequencing revealed only one candidate variation for both MSMD-causing and IFN-γ-related genes. P1 carried a heterozygous frame-shift IFNGR2 mutation inherited from her father. We show that the mutant allele is intrinsically loss-of-function and not dominant-negative, suggesting haploinsufficiency at the IFNGR2 locus. We also show that Epstein-Barr virus transformed B lymphocyte cells from 10 heterozygous relatives of patients with AR complete IFN-γR2 deficiency respond poorly to IFN-γ, in some cases as poorly as the cells of P1. Naive CD4(+) T cells and memory IL-4-producing T cells from these individuals also responded poorly to IFN-γ, whereas monocytes and monocyte-derived macrophages (MDMs) did not. This is consistent with the lower levels of expression of IFN-γR2 in lymphoid than in myeloid cells. Overall, MSMD in this patient is probably due to autosomal dominant (AD) IFN-γR2 deficiency, resulting from haploinsufficiency, at least in lymphoid cells. The clinical penetrance of AD IFN-γR2 deficiency is incomplete, possibly due, at least partly, to the variability of cellular responses to IFN-γ in these individuals.

Cytokine production in transient hypogammaglobulinemia and isolated IgA deficiency

BACKGROUND Transient hypogammaglobulinemia of infancy and isolated IgA deficiency are characterized by normal numbers of circulating B lymphocytes. It is likely that no single abnormality, but rather different factors, may be relevant for the delayed onset of IgG synthesis in transient hypogammaglobulinemia or for the differentiation defect of B cells in IgA deficiency. These factors may include defective production of cytokines or an abnormal response of B cells to various mediators. Alternatively, some cytokines may act as inhibitory factors of B-cell function. METHODS The ability of peripheral blood mononuclear cells from children with proved or probable transient hypogammaglobulinemia (30 patients) and IgA deficiency (15 patients) to secrete several cytokines on stimulation with phytohemagglutinin in vitro was analyzed. RESULTS An enhanced production of tumor necrosis factor (TNF)-alpha, TNF-beta, and IL-10 was observed in transient hypogammaglobulinemia; whereas secretion of IL-1, IL-4, and IL-6 was essentially similar in the control and patient groups. Increased frequency of mononuclear cells secreting TNF-alpha was seen in the patient groups. Apart from elevated production of TNF-alpha, no other abnormalities in cytokine synthesis in selective IgA deficiency were observed. In vitro observations showed that exogenously added TNF-alpha and TNF-beta inhibited IgG and IgA secretion by pokeweed mitogen-stimulated mononuclear cells. During follow-up of 10 children, normalization of serum IgG level was associated with a decrease in previously elevated TNF-alpha and TNF-beta production, but IL-10 production remained unchanged. CONCLUSION These results suggest that TNF may be involved in the regulation of IgG and IgA production and can be associated with an arrest of IgG and IgA switch of B cells in hypogammaglobulinemia. The balance between TNF and IL-10 may be important for the normal development of IgG-secreting B cells.

Intracellular cytokine production by Th1/Th2 lymphocytes and monocytes of children with symptomatic transient hypogammaglobulinaemia of infancy (THI) and selective IgA deficiency (SIgAD)

Intracellular expression of several cytokines was assessed in lymphocytes and monocytes of children with transient hypogammaglobulinaemia of infancy (THI) and selective IgA deficiency (SIgAD). THI was characterized by an increased frequency of CD3+/CD4+ lymphocytes expressing tumour necrosis factor α (TNF‐α), TNF‐β and interleukin 10 (IL‐10), while in SIgAD elevated numbers of these cells containing TNF‐α and interferon γ (IFN‐γ) were observed. No changes in the number of CD4+ T cells expressing IL‐4 in both diseases were noted. The proportion of CD33+ monocytes containing TNF‐α both in THI and SIgAD was unchanged. The secretion of IL‐12 by peripheral blood mononuclear cells (PBMCs) of patients with THI and SIgAD was significantly elevated and associated with an increased frequency of IL‐12 expressing monocytes in THI but not in SIgAD. IL‐18 secretion was slightly, but not significantly, elevated in both diseases. Intracellular Th1 and Th2 type cytokines within CD3+/CD4+ lymphocytes were also determined in the normal blood donors that showed high or low production of IgG and IgA in vitro. In low producers of IgG an increased proportion of CD3+/CD4+ cells expressing TNF‐α and IFN‐γ was found, while in low IgA responders only elevated TNF‐α positive CD3+/CD4+ cells were observed. These results suggest that THI and SIgAD may represent diseases with an excessive Th1 type response that is associated with an up‐regulation of IL‐12 secretion and, at least in THI, elevated numbers of monocytes expressing intracellular IL‐12. Up‐regulation of IL‐12 may be the essential factor in the patomechanism(s) of these diseases as already described in common variable immunodeficiency (CVID).

The B-cell Compartment in the Peripheral Blood of Children With Different Types of Primary Humoral Immunodeficiency

The aim of this study was to evaluate the B-cell compartment in the peripheral blood of children with different types of hipogammaglobulinemia: common variable immunodeficiency (CVID), transient hypogammaglobulinemia of infancy (THI), and selective IgA deficiency (SIgAD). We analyzed by flow cytometry the changes in the B-cell subsets with age and showed that children with an early-onset CVID develop similar pattern of B-cell subsets as adult patients with CVID with age, as the levels of memory B cells (CD19+/CD27+) and class-switched memory B cells (CD19+/CD27+/IgD−/IgM−), in contrast to age-matched control group, did not increase with age. Children with SIgAD displayed similar changes as patients with CVID only within the class-switched memory B-cell subpopulation. No significant differences in the level of memory B cells and class-switched memory B cells in children with THI in comparison to age-matched control group were observed. There were no differences in the percentage of immature B cells (CD19+/CD21low) among all studied groups. As B-cell subsets in children with THI were normal during entire period of hypogammaglobulinemia, the persistence of low levels of memory B-cell subsets in some children may facilitate the diagnosis of CVID.

Sepsis caused by Veillonella parvula infection in a 17-year-old patient with X-linked agammaglobulinemia (Bruton's disease).

ABSTRACT A case of a male, 17-year-old, X-linked agammaglobulinemia patient with bacteremia caused by Veillonella parvula, without a defined primary site of infection, is presented. The report demonstrates that V. parvula should not be regarded as a nonpathogenic microorganism, at least not in patients with certain forms of immunodeficiency disease.

The in vitro inhibition of migration of mouse spleen t lymphocytes. I. The response of sensitized t cells to specific antigen and of normal t cells to lymphokines.

Abstract Nylon-wool-purified mouse spleen T lymphocytes possess the ability to migrate in vitro out of capillary tubes. The migration of T cells from mice with contact sensitivity was inhibited in the presence of specific, but not unrelated, antigen. In practice, lymph node cells haptenized in vivo were used as a source of antigen. The indirect test showed that normal T cells can be used as indicator cells since their migration was inhibited in the presence of lymphokinecontaining supernatant. It is suggested that this test can be used as an in vitro correlate of effector T-cell activity in contact sensitivity.

The expression of CD40 on monocytes of children with primary humoral immunodeficiencies

The interactions between CD40 and CD40L (CD154) are critical for effective humoral immune response. CD40 signaling facilitates T lymphocyte dependent B cell proliferation and immunoglobulin isotype switch. The objective of our study was to investigate the CD40 and CD40L expression on peripheral blood mononuclear cells (PBMC) of children with symptomatic transient hypogammaglobulinemia (THI), common variable immunodeficiency (CVID) and selective IgA deficiency (SIgAD). Additionally we studied the production of IL-12 and IL-18 by PBMC stimulated with soluble CD40L. CD40 expression was analyzed on B cells and monocytes, CD40L on activated T lymphocytes, using flow cytometry following staining of the cells with appropriate MAb. We found that CD40 expression on B cells and CD40L on activated T cells were essentially similar in the control and patient groups, while the decreased CD40 expression on monocytes was observed in THI and SIgAD patients compared with normal subjects. The most significant decrease of CD40 expression was observed in THI (37% of positive cells) in comparison with control (81% of positive cells). IL-12, but not IL-18, release by PBMC was increased in THI and CVID, but not in SIgAD. In conclusion we suggest that the decreased expression of CD40 on monocytes of children with THI and SIgAD, but not CVID, may be involved in the pathomechanism of these immunodeficiencies.

Rapid full engraftment and successful immune reconstitution after allogeneic hematopoietic stem cell transplantation with reduced intensity conditioning in Omenn syndrome.

Abstract:  OS is a variant of SCID characterized by generalized erythroderma, alopecia, eosinophilia, and elevated IgE levels. It is fatal unless treated with allogeneic HSCT, which is the only curative approach. However, treatment related complications and graft rejection are major obstacles to the success of treatment. In this report, we describe a patient with OS, complicated by prolonged cytomegalovirus infection, successfully treated by reduced intensity conditioning allogeneic HSCT from sibling donor.