Low Natural Killer Cell Research Articles

Clinical Effects of Probiotic Bifidobacterium longum BB536 on Immune Function and Intestinal Microbiota in Elderly Patients Receiving Enteral Tube Feeding

Immune system function declines with age. We evaluated the effects of supplementation with the probiotic Bifidobacterium longum BB536 on immune function and intestinal microbiota in the elderly. In a double-blind study, 45 elderly patients fed by enteral tube feeding (mean [SD] age 81.7 [8.7] years) were given BB536 (n = 23) or a placebo powder (n = 22) for 12 weeks and were observed for an additional 4 weeks posttreatment. At week 4, all patients received an influenza vaccination (A/H1N1, A/H3N2, and B). Clinical data were assessed, including body temperature, bowel movements, fecal microbiota, and immunological biomarkers in blood. BB536 intake significantly increased cell numbers of bifidobacteria in fecal microbiota. There was a tendency toward an increase (P = .085 at week 4 and P = .070 at week 16) of serum IgA in the BB536 group compared with the placebo group. BB536 intake did not significantly affect hemagglutination inhibition (HI) titers in response to the influenza vaccine. Natural killer (NK) cell activity decreased significantly in the placebo group during the intervention but not in the BB536 group. Among those subjects with low NK cell activity (<55%, n = 10 for each group), a significant intergroup difference (P < .05) was observed in the changed values from baseline of NK cell activity at weeks 8 and 12. These results shed new light on the potential of long-term ingestion of BB536 in increasing the cell number of bifidobacteria in intestinal microbiota and modulating immune function in the elderly.

A Curious Case of Fever, Diarrhea with Pancytopenia

Introduction: Enteropathy Associated T-Cell Lymphoma (EATL) is a rare form of peripheral T-cell lymphoma with a strong association with celiac disease and has an extremely poor prognosis. Hemophagocyticlymphohistiocytosis (HLH) in association with EATL has been reported very rarely. Case Presentation: A 41 year-old-male with no significant past medical history presented with complaints of fever and diarrhea of three weeks duration. Review of systems was otherwise negative. Family history was significant for celiac disease in siblings. Physical examination was significant for a fever of 100.6°F and hepatomegaly. Labs revealed Hb-7 g/dl, wbc-1.1 K/μl, platelets-28 K/μL, transaminitis and coagulopathy. CT abdomen showed hepatomegaly, mesenteric induration and colonic thickening. An extensive workup done revealed the following: S.Ferritin-28,000 μg/l,S. Fibrinogen-76,NK cell-low, soluble CD25-11250 pg/ml, triglyceride-485 mg/dl, positive tissue transglutaminase and endomysial antibodies with negative blood cultures. Small intestinal biopsy showed severe villous atrophy and increase in number of intra epithelial lymphocytes, suggestive of celiac disease. Bone marrow biopsy with flow cytometry and karyotyping showed extensive lymphohistiocytic infiltrate which was CD30/CD7 positive and amplification of 9q31.3-qter diagnostic of HLH secondary to EATL. The patient was started on targeted chemotherapy but developed intestinal perforation and eventually succumbed to his illness after 10 days. Discussion: EATL is a rare malignancy which accounts for less than 1% of all non-Hodgkin's lymphomas and seen predominantly in middle aged men. It exists in 2 distinct forms (type I & II). Type I (80%)is characterized by a strong association with celiac disease, expression of CD3, CD7, CD103, CD30 by tumor cells and chromosome 9q31.3 gain or 16q12.1 deletion. HLH is a rare condition which comprises of primary (genetic) and secondary (viral infections, autoimmune disease, malignancies). Diagnostic criteria for HLH include either a molecular diagnosis or 5 out of the following 8 criteria. 1) Fever, 2) Splenomegaly, 3) Cytopenias affecting 2 or more cell lines, 4) Hypertriglyceridemia or Hypofibrinogenemia, 5) Hemophagocytosis at biopsy, 6) Low or absent Natural Killer cell activity, 7) S.Ferritin > 500 mcg/l, and 8) Soluble CD25 >2400U/l. Treatment protocols are based on combined chemotherapy regimens to target malignant lymphocytes. Conclusion: EATL is a serious complication of celiac disease and can rarely present as HLH. We emphasize the need for high index of suspicion and systematic work up in patients presenting with fever, organomegaly and pancytopenia because delay in treatment can result in irreversible multi organ failure.

Measles and Secondary Hemophagocytic Lymphohistiocytosis

To the Editor: We found interesting the article by Lupo et al. about a case of fatal measles in an immunocompetent 29-year-old woman (Fatal measles without rash in immunocompetent adult, France; http://dx.doi.org/10.3201/eid1803.111300). Perhaps, however, the possible diagnosis of secondary hemophagocytic lymphohistiocytosis (HLH) should also have been considered in that setting. HLH is a potentially fatal hyperinflammatory syndrome characterized by histiocyte proliferation and hemophagocytosis. HLH may be inherited (i.e., primary, familial, generally occurring in infants) or may occur at any age secondary to infection, malignancy, or rheumatologic disease. Secondary HLH is determined according to clinical criteria from the HLH Study Group of the Histiocyte Society, which require >5 of the following for a diagnosis: fever; splenomegaly; cytopenia (affecting >2 cell lineages); hypertriglyceridemia or hypofibrinogenemia; hemophagocytosis in the bone marrow, spleen, or lymph nodes; low or absent natural killer cell cytotoxicity; hyperferritinemia; and elevated levels of soluble CD25. We conducted a PubMed search and found 5 articles that described 6 cases of HLH in patients with measles (1–5). Pneumonia was described in all of them (1–5), and central nervous system involvement was described in 3 (1,4). Four cases occurred in children, 3 of them immunocompetent (1,3–5). The 2 adults were an immunocompetent 18-year-old man who had acute respiratory distress (2) and a 19-year-old man with acute lymphocytic leukemia who had measles pneumonia and acute hemorrhagic leukoencephalitis (1). The only fatal case occurred in an immunocompromised 8-year-old boy with giant-cell pneumonia (3). The identification of hemophagocytosis in bone marrow aspirate represents only 1 of the 5–8 criteria needed for a diagnosis of HLH; conversely, a bone marrow aspirate lacking hemophagocytosis does not rule out the diagnosis of HLH. Still, we believe HLH should be considered for any patient with fever and pancytopenia, especially in the presence of respiratory distress or multiorgan dysfunction. An appropriate therapy could save the patient (Secondary hemophagocytic syndrome in adults: a case series of 18 patients in a single institution and a review of literature; http://dx.doi.org/10.1002/hon.960).

Natural Killer Cell Cytolytic Function in Korean Patients with Adult-onset Still’s Disease

To investigate natural killer (NK) cell proportions, NK cell cytotoxicity, and interleukin 18 (IL-18) expression, in patients with adult-onset Still's disease (AOSD). Forty-five patients with AOSD (active = 22, inactive = 23) and 32 healthy controls were included. The proportions of NK cells among peripheral blood mononuclear cells were assessed by flow cytometry. IL-18 and IL-18-binding protein (IL-18BP) concentrations were measured by ELISA. Twenty-four patients with AOSD and 18 controls were examined for cytotoxic activity of NK cells by co-incubating NK cells with NK-sensitive K562 cells. The association of NK cell function with clinical and laboratory measures was investigated. The proportions of NK cells were significantly lower in patients with active AOSD than in patients with inactive disease and controls. NK cell cytotoxic function was significantly lower in patients with AOSD than in controls. NK cell proportions and cytotoxic functions were reexamined in 11 and 6 patients, respectively, after treatment. Low NK cell proportion and cytotoxic dysfunction were improved with clinical improvements of the patients. IL-18 and IL-18BP levels were much higher in patients with active AOSD than in controls. NK cell cytotoxic functions were consistently low and IL-18 and IL-18BP levels were constantly high in patients with AOSD, regardless of disease activity. Low NK cell proportion, defective cytotoxic function, and elevated IL-18 levels may be significant features of AOSD. After resolution of the acute phase, low NK cell proportion was recovered and NK cell cytolytic function was restored along with clinical improvement. These findings possibly contribute to immunologic abnormalities in AOSD.

Regulation of pregnancy maintenance and fetal survival in mice by CD27low mature NK cells

Uterine natural killer (NK) cells are pivotal for successful mammalian reproduction. However, insights on functionally distinct subpopulations of uterine NK cells are largely elusive. Furthermore, translation of findings from murine into human pregnancy has been overshadowed by the limited number of mutual phenotypic NK cell markers. We here provide evidence that a subset of murine mature NK (mNK) cells present at the feto-maternal interface, identified as CD27(low)DX5(+)CD3(neg), is pivotal in maintaining pregnancy. This mNK subset has low cytotoxic capacity, produces higher amounts of interferon (IFN)-γ, and expresses functional homologs of human NK cell immunoglobulin-like receptors. We further show that bone marrow-derived CD27(low) mNK cells are selectively recruited to the uterus and ameliorate the rate of fetal loss when adoptively transferred into alymphoid RAG2(-/-)/γc(-/-) mice. Additionally, expression of CD27 is down-modulated on mNK cells upon migration to the uterus. Hence, we propose the existence of a regulatory mNK cell subset, which is licensed toward successful pregnancy maintenance at the fetomaternal interface in mice. As CD27(low) NK cells are also present in human decidua, the CD27(low) NK subset may provide a tool to foster translational research in reproduction, aiming to improve pregnancy outcome in humans.

Novel mutation in STXBP2 prevents IL-2–induced natural killer cell cytotoxicity

We have identified dizygotic twins with a novel syntaxin-binding protein 2 (STXBP2) mutation, where cytotoxicity cannot be restored with IL-2. This defines STXBP2 as an absolute requirement for NK cell cytotoxic function.

TNF İnhibitörleri ile Tedavi Edilen Romatoid Artrit Hastalarında Doğal Öldürücü Hücre Toksisitesi

Objective: Patients with low natural killer (NK) cell activity were shown to have a higher risk of cancer development. Increased cancer risk in rheumatoid arthritis (RA) patients treated with anti-tumor necrosis factor (anti-TNF) agents is still being debated. Therefore, we think it is important to show whether anti-TNF therapy has any effects on the NK cell cytotoxicity in these patients. Material and Methods: We included 60 rheumatoid arthritis patients, 31 treated with anti-TNF therapy, 29 treated with other disease modifying antirheumatic drugs (DMARDs), and 51 ankylosing spondylitis (AS) patients, 35 treated with anti-TNF therapy and 16 treated with nonsteroidal anti-inflammatory drugs or conventional DMARDs as a disease control into the study. The results of two healthy control groups each consisting of 32 age- and sex- matched healthy individuals were also included. Results: Median values of NK cytoxicity in patients with RA who were being treated with anti-TNF and patients treated with other DMARDs were 29% and 43%, respectively (p=0.203). When we reanalyzed the results independent of clinical diagnosis, patients using anti-TNF medications and patients treated with other medicine had NK cytotoxicity median values of 32% and 43%, respectively (p=0.277). Conclusion: We suppose that NK cell cytotoxicity may decrease in patients with RA when they use anti-TNF agents, and this may help partly to explain why these patients are more prone to development of cancer and severe infections. We think that this study will encourage new studies with higher number of patients to better clarify effects of anti-TNF treatment on NK cell functions.

Atopic Sensitization is Associated With Severe Lower Respiratory Illness in Children With Pandemic H1N1 Influenza Viral Infection

Although most influenza A virus infections result in self-limited uncomplicated disease, it causes significant morbidity and mortality in children.1 Influenza A accounts for the highest rates of influenza-related hospital admissions, especially in young children and elderly people, and pneumonia is one of the most common complications (10%-26%).2,3 Furthermore, it was previously reported that 35% of influenza A (H1N1)-related deaths in children were due to pneumonia.4 In addition to seasonal influenza A infection, there was an outbreak of pandemic H1N1 influenza A viral infection (pandemic H1N1 influenza) that was first reported in patients from Mexico and the United States in early April 2009. An outbreak of pandemic H1N1 influenza during 2009-2010 was responsible for severe respiratory disease and death, especially in children and elderly people with chronic respiratory diseases, even though its overall impact did not appear to cause more severe disease than seasonal influenza A.5,6 A recently published case series of 122 children with pneumonia due to pandemic H1N1 influenza reported up to a 60% prevalence of underlying medical conditions, including asthma (29%), and described the need for intensive care in 20% of patients and mechanical ventilation in 10%.7 The relationship between viral infection and asthma has been investigated during the last few decades and evidence for such a link has been reported; however, the question of whether all respiratory viruses are equipotent remains unclear. As described in a recent review by Holt et al.8, it was assumed that respiratory syncitial virus (RSV) was overwhelmingly the most important trigger of severe lower respiratory illness (LRI) in the first two years of life, and compelling data on the association between early RSV-induced LRI and subsequent development of persistent asthma have been published from independent prospective cohort studies. More recently, it was revealed that the contribution of human rhinovirus to LRI during infancy and the preschool years was considerably higher than previously believed, and that these infections are strongly linked to the risk of subsequent asthma.8 Although the pathogenic nature of the relationship between asthma and H1N1 viral infection is unclear, several studies have reported that asthma is a risk factor for worsening respiratory conditions in pandemic H1N1 influenza patients.6,7 However, there are several other reports that pandemic H1N1 influenza causes high rates of severe LRI (pneumonia, atelectasis), aggravation of asthma, and admission to intensive care units in patients with exisiting asthma or atopy.6,9,10 In a recent animal study, high concentrations of virus-infected cells were found in the central and lower respiratory tracts of ferrets inoculated with H1N1 influenza virus compared to ferrets inoculated with seasonal influenza virus. This result suggests that pandemic H1N1 influenza replicated more extensively in the lower respiratory tract, causing more severe LRI than that caused by seasonal influenza virus.11 In this issue of Allergy, Asthma & Immunology Research, Kim et al.12 report an increased prevalence of H1N1-induced severe lower respiratory tract disease in children with atopic sensitization. In aforementioned study, a high proportion of atopic children with pandemic H1N1 influenza had atelectasis and required ICU care and oxygen and steroid therapy. Based on these findings and the results of several animal studies, the authors suggest that atopic sensitization combined with pandemic H1N1 influenza infection can induce severe LRI. In addition, eosinophil recruitment into the lungs and enhancement of IL-10 production from CD4+ T cells may play a role in the development of severe LRI observed in pandemic H1N1 influenza patients.13,14 Due to the small sample size, Kim et al.12 could not confirm whether atopic sensitization itself, and not asthma, was associated with pandemic H1N1 influenza-induced severe LRI. Although the authors could not directly confirm that the atopic sensitization itself was the cause of severe LRI in pandemic H1N1 influenza, they demonstrated this relationship using proper statistical analyses. There was a recent study that also supports the notion that pandemic H1N1 influenza greatly increases the risk of severe LRI regardless of the severity of previous asthma or the presence of asthma in patients with atopic sensitization.9 In addition, patients with atopy have been shown to exhibit low natural killer cell activity,15 suggesting that both abnormal innate immune responses in atopic individuals and the affinity of the pandemic H1N1 virus for the respiratory tract may lead to increased susceptibility to severe LRI induced by pandemic H1N1 influenza. Based on several recent studies, including the study by Kim et al.12 reported in this issue, it is suggested that atopic sensitization itself can cause severe LRI. This has highlighted the importance of influenza prevention through vaccination, especially in children with atopic sensitization, even if the patients have no obvious history of clinical asthma or any other allergic disease.

Effects of Anti-CCR4 Antibody (KW-0761) on Regulatory T Cells and Natural Killer Cells in Patients with Cutaneous T-Cell Lymphoma

Abstract 444The CC chemokine receptor 4 (CCR4) is expressed on malignant T-cells in cutaneous T-cell lymphoma (CTCL) and also on the surface of regulatory T cells (T-regs). T-regs also express the transcription factor, foxp3 and suppress effector immune cells, including natural killer (NK) cells. Thus, increased T-regs in the tumor microenvironment is associated with impaired anti-tumor immunity. KW-0761, a defucosylated, humanized monoclonal antibody, binds to CCR4 and induces effective antibody-dependent cellular cytotoxicity (ADCC) against CCR4+ malignant T-cells. The phase I/II clinical trial to determine the safety and efficacy of anti-CCR4 antibody (KW-0761) included a translational component to evaluate its effects on T-regs and NK cells in CTCL patients.Peripheral blood mononuclear cells (PBMCs) were collected from 20 patients [10 with mycosis fungoides (MF) and 10 with Sézary syndrome(SS)] pre- and post-treatment at two centers for flow cytometry analysis of CD3+CD4+CD25+CD127- T-regs, CCR4+ T-regs, and CD3-CD56+CD16+ NK cell subsets. Total RNA was extracted from PBMCs, and foxp3 and CCR4 mRNA were quantified using real-time PCR. The standard 4-hour 51Cr release assay was used to assess the cytotoxicity of NK cells.Fifteen of 20 patients (75.0%) had detectable CD3+CD4+CD25+CD127- T-regs (1.26±1.09 % or 33.79±46.88 /μl) at baseline with 60 –100 % of T-regs positive for CCR4. After 4–6 weeks of treatment with anti-CCR4 antibody (KW-0761), all 15 patients had a decrease in T-reg numbers (0.39±0.49 % or 5.65±8.95 /μl, *p<0.05, Figure 1). CCR4+ T-regs were significantly reduced from an average of 67.2% to 24.6% (p<0.01). In parallel, foxp3 and CCR4 mRNA levels were also significantly decreased from baseline levels (foxp3: from 0.57±0.91 to 0.07±0.08, **p<0.01, Figure 1; CCR4: from 23.40±33.50 to 1.73±2.35, p<0.05). The CD3-CD56+CD16+ NK cells were found at baseline in all 15 patients tested. Of 14 paired samples, 10 (71.4%) had increased NK cells after 4–16 weeks of treatment (pre-treatment: 16.02±15.86 % vs. post-treatment: 22.64±13.93 %, p=0.05, Figure 2) with reduced numbers of T-regs. Five of 6 patients studied also showed a dose dependent increase in NK cell cytotoxicity to target cells by 51Cr release assay (Figure 2). Ten patients with SS had a lower NK cells (13.37±18.48 %) and higher foxp3 (0.81±1.19) and CCR4 mRNA (34.60±39.90) at baseline compared to ten patients with MF (19.04±12.98%, 0.31±0.31; 10.94±20.07) respectively. Seven paired samples from SS patients all had increased NK cells post-treatment with a reduction of T-regs and foxp3 and CCR4 mRNA. Six of 7 SS patients had blood improvement with 3 complete and 3 partial blood responses. [Display omitted] [Display omitted] Our results suggest that in addition to ADCC towards malignant T-cells, the anti-neoplastic activity of the anti-CCR4 antibody (KW-0761) may include a reduction in T-regs in most CTCL patients and a subsequent increase in NK numbers and function in some patients. Follow up studies need to be performed to confirm these findings. Disclosures:Ni:KYOWA HAKKO KIRIN CO., LTD: Research Funding. Kim:kyowa: Consultancy, Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Allos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Consultancy. Duvic:KYOWA HAKKO KIRIN CO., LTD: Research Funding.

Valproic Acid Upregulates NKG2D Ligand Expression and Potentially Enhances Natural Killer Cell–mediated Cytolysis of Myeloma

Abstract 5061 Objective:Multiple myeloma (MM) is a plasma cell malignancy. Although high-dose chemotherapy supported by autologous hematopoietic stem-cell transplantation can produce higher response rates and longer survival than standard chemotherapy, MM remains largely incurable by current therapeutic strategies. Clinical and preclinical data have been demonstrated that natural killer (NK) cells have an anti-myeloma effect. However, low NK cell activity affected the successful utilization of NK cell adoptive immunotherapy for myeloma. In this study, we investigated the effect of valproic acid (VPA), as a histone deacetylase inhibitor, on the expression of NKG2D ligands on human myeloma cell lines and primary myeloma cells derived from patients. We also evaluated the sensitivity of myeloma cells to NK cell-mediated lysis before and after treatment with VPA, and the possible underlying mechanisms. Method:ARK, OPM2 myeloma cell lines and primary myeloma cells (n = 4) from patients with myeloma were cultured with 0.5–3mM VPA for 24 to 72 hours. Cells cultured without VPA were as control groups. To evaluate the effect of VPA on NKG2D ligand expression of myeloma cells, mRNA and protein expression of NKG2D ligands (including MICA/B, ULBP1, ULBP2 and ULBP3) of myeloma cells were tested by Real time-PCR and flow cytometry. 51Cr release assay and NKG2D antibody blocking experiments were combined to examine the susceptibility of myeloma cells to NK cell-mediated lysis in response to VPA and the possible killing mechanisms. Results:ARK and OPM-2 myeloma cell lines were treated with various concentrations of VPA. The most prominent upregulations of mRNA expressions of MICA/B, ULBP2 and ULBP 3 were observed in the two tested myeloma cell lines. The enhancement of cell surface expression of MICA/B, ULBP2 and ULBP 3 was detected by flow cytometry after cells treatment with VPA. We also found that VPA upregulated MICA/B expression on OPM2 MM cell line in a time- and dose-dependent manner. These data suggest that VPA could upregulate expression of NKG2D ligands in myeloma cell line at both the mRNA and protein levels. A similar increase of surface expression of NKG2D ligands was obtained in 2–3 mM VPA treated primary MM cells (n=4). NK cell function was studied by 51Cr release assay. VPA treatment of OPM2 showed higher sensitivity to NK cell lysis than untreated cells (specific killing: 72.8±6.2% vs 32.1±3.7% at E/T 10:1, p <0.01). The killing of VPA treatment of patient myeloma cells by NK cells also significantly increased compared with control cells (specific killing: 46.7±5.6% vs 21.6±4.5% at E/T 10:1, n=4, p <0.01). The enhancing effect of VPA was blocked by pretreatment of OPM2 cells with a combination of anti-MICA/B, anti-ULBP1,2,3 mAbs or pretreatment of NK cells with anti-NKG2D mAbs, indicating the contribution of NKG2D-NKG2D ligands interations to VPA-modulated lysis of myeloma. We also found that constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not AKT signaling pathway may be involved in VPA-induced higher NKG2D ligand expression. Conclusion:Histone deacetylase inhibitor valproic acid could upregulate cell surface expression of NKG2D ligands, rendering myeloma cells more sensitive to NK cells, and thereby enhance NK cell–mediated lysis of myeloma. Our findings imply that regulation of the expression of NKG2D ligands by treatment with histone deacetylase inhibitors may be an attractive strategy for immunotherapy of myeloma. Acknowledgments:This work was supported by grants from National Natural Science Foundation of China (81071856 and 30973450 to JS) and funds from Shanghai Tenth People's Hospital (10RD103 and 11SC103 to JS). Disclosures:No relevant conflicts of interest to declare.

Interaction between nk cells and HLA-G1 at the placental interface of HIV-1 infected pregnant women: additional risk factors or physiological association?

Background: Human Leucocyte Antigen-G (HLA-G) molecules are involved in the inhibition of cell-mediated immune responses and could promote the propagation of HIV-1 infection across the placental interface thus increasing the risk of vertical transmission. Therefore, the objective of this study was to assess whether the Major Histocompatibility Complex (MHC) - coded molecule HLA-Ginhibits Natural Killer (NK) cell activity thereby, assisting viral penetration across the placental barrier in HIV-1 positive pregnant women. Study Design & Methods: Natural Killer (CD56+) cell activity and placental HLA-G1 expression was assessed using immunohistochemistry and real-time polymerase chain reaction (RT-PCR) techniques, respectively. Studies were performed on a total of fifty five placental samples obtained from HIV-1 infected mothers at birth. Results: Low numbers of NK cells increased risk of vertical transmission [OR = 3.424 (95%CI 0.65-17.89)]. The risk of babies becoming infected increased by 1.3 with every 1 unit increase in HLA-G1 expression. A positive correlation was observed between mothers’ log viral load and transmission of infection to the baby (p = 0.047; 95%CI 1.029-11.499). Conclusion: Low NK cell activity at the placental interface increased the risk of vertical transmission. Maternal viral load remained a strong predictor of viral transmission.

Penicilliosis in Children Without HIV Infection—Are They Immunodeficient?

Penicillium marneffei infection is indigenous to Southeast Asia. Majority of penicilliosis occurs in patients with AIDS, and less commonly with secondary immunodeficiencies. Penicilliosis is rare in otherwise healthy persons, but information on their immunological status is often lacking. From 1996 to 2009, we diagnosed penicilliosis in 5 children. Their clinical features, immunological findings, and genetic studies were analyzed. A systematic review of the English and Chinese literature was performed. Case reports/series on patients <18 years with penicilliosis were included, and patients stated to be human immunodeficiency virus (HIV)-positive excluded. All of our 5 patients were HIV negative. Presentations included fungemia (n = 2), multifocal lymphadenopathy (n = 2), and necrotizing pneumonia (n = 1). Four patients had recurrent mucocutaneous candidiasis. Hyperimmunoglobin E syndrome was diagnosed in 1 patient, while another had functional defect in interleukin-12/interferon-γ axis. Three patients were lymphopenic with low natural killer cell counts, but a specific immune defect was not identified. Systematic review of 509 reports on human penicilliosis identified 32 patients aged 3 months to 16 years with no known HIV infection. Twenty-four patients (75%) had disseminated disease, and 55% died of penicilliosis. Eight patients had primary immunodeficiencies or blood disorders, while 4 others had abnormal immune functions. Immune evaluations of the remaining patients were unstated. Penicilliosis is a severe disease causing high mortality in children. As an AIDS-defining illness, penicilliosis should be regarded as an indicator for underlying immunodeficiency in HIV-negative individuals. Immunological investigations should be performed, especially in those with recurrent infections. Multicentered collaborative studies are needed to collect information on long-term prognosis and define immune defects underlying penicilliosis.

Primary immunodeficiency diseases associated with increased susceptibility to viral infections and malignancies

Primary immunodeficiencies (PIDs) are commonly characterized by an increased susceptibility to specific infections and, in certain instances, a higher than usual incidence of malignancies. Although improved diagnosis and early treatment of PIDs have reduced early morbidity and mortality from infection, the development of cancer remains a significant cause of premature death. The emergence of cancer in patients with PIDs often results from impairments in the immune response that lead to weakened surveillance against oncogenic viruses, premalignant or malignant cells, or both. Here we review the clinical and biologic features of several PIDs associated with enhanced susceptibility to viral infections and cancer, including X-linked lymphoproliferative disease; IL-2-inducible T-cell kinase deficiency; epidermodysplasia verruciformis; warts, hypogammaglobulinemia, infections, and myelokathexis syndrome; autosomal recessive hyper-IgE syndrome; X-linked agammaglobulinemia; and common variable immunodeficiency. It is of importance that we gain in-depth insights into the fundamental molecular nature of these unique PIDs to better understand the pathogenesis of virus-associated malignancies and to develop innovative therapeutic strategies.

Імунофенотипова характеристика двох функціонально різних субпопуляцій натуральних кілерних клітин периферичної крові людини

NK cell cytotoxicity and immunophenotypic characteristics of activated natural killer (NK) cells after co-incubation with K562 target cells in women were investigated. An increase in CD69, HLADR, CD95 expression on target-activated NK cells was demonstrated. Conversely, CD62L expression in NK cells after activation was decreased. The results showed that only a part of NK cells after incubation with K562, became CD69 positive. Moreover, even after lowering of "efector - target" ratio and extensions of time of stimulation, the amount of CD69+NK cells did not exceed 60% (50% +/- 1.7%). Expression of CD69, CD95 and HLADR on NK cells before coincubation didn't correlate with NK cytotoxicity. However, size of population of activated CD69+ NK cells (after stimulation) was correlated with NK cytotoxicity. We also found that the activation capacity after cocultivation with K562 cells is related to loss in CD62L on NK cell's surface. Only CD62L(neg) and less CD62L(low)NK cells had the ability to be activated with K562. In conclusion, we demonstrated that incubation with K562 target cells enhanced the expression of CD69 on the major part of NK cell population and the size of CD69+NK cell population is strongly correlated with NK cytotoxicity.

Causes, clinical symptoms, and outcomes of infectious diseases associated with hemophagocytic lymphohistiocytosis in Taiwanese adults

Hemophagocytic lymphohistiocytosis (HLH) is an uncommon but a potentially life-threatening condition. Few systematic reviews have been published on the clinical manifestations, causes, and indicators for prognosis of HLH caused by infections. We retrospectively reviewed the medical records of patients diagnosed with HLH documented by bone marrow study at a teaching hospital between 2000 and 2007. HLH was defined according to the HLH-2004 diagnostic guidelines, which include fever; splenomegaly; cytopenia; hypertriglyceridemia; hypofibrinogenemia; and hemophagocytosis evident on pathological examination of bone marrow, spleen, or lymph node tissue; low or absent natural killer cell activity; hyperferritinemia; and high serum levels of soluble CD25. The demographic characteristics, clinical presentations, laboratory results, and final outcomes were recorded. The cause of HLH was diagnosed by microbiological, pathological, serological, and molecular biological methods. Among the studied patients, 66 had HLH because of noninfectious causes and 30 because of infections. Compared with patients with HLH related to noninfectious causes, those with HLH related to infections had lower mortality (70% vs. 47%, p=0.03). The most common causative pathogens causing HLH were virus (41%), mycobacteria (23%), bacteria (23%), and fungi (13%), in that order of frequency. Clinical presentations of HLH were variable and included fever (90%), tachypnea (83%), tachycardia (80%), hepatosplenomegaly (40%), lymphadenopathy (27%), and altered consciousness (23%). Laboratory findings revealed thrombocytopenia in 93%, hyperferritinemia in 90%, elevated serum lactate dehydrogenase levels in 80%, anemia in 67%, and leukopenia in 60% of the patients. Fourteen patients (47%) died. In multivariate analysis, age more than 50 years (p=0.05; odds ratio [OR], 3.46; 95% confidence interval [CI], 1.00-15.73), fever not subsiding within 3 days of diagnosing HLH (p=0.003; OR, 2.38; 95% CI, 1.21-11.25), and occurrence of disseminated intravascular coagulation as a complication (p=0.009; OR, 3.22; 95% CI, 1.68-10.01) were found to be statistically significant indicators of mortality in patients with HLH. The infectious diseases associated with HLH were diverse and resulted in a high mortality rate. Cases in which the patients were aged more than 50 years, developed DIC, and had persistent fever even after 3 days of being diagnosed with HLH showed poor prognosis.

Healthy working school teachers with high effort–reward-imbalance and overcommitment show increased pro-inflammatory immune activity and a dampened innate immune defence

To test whether chronic work stress is accompanied by altered immune functioning, changes in lymphocyte subsets and in lymphocyte production of cytokines were examined in reaction to acute psychosocial stress. Work stress was measured according to Siegrist’s effort–reward-imbalance (ERI) model. ERI reflects stress due to a lack of reciprocity between costs and gains at work. Overcommitment (OC) is conceptualized as a dysfunctional coping pattern mainly characterized by the inability to withdraw from work obligations. Fifty-five healthy teachers (34 women, 21 men, mean age 50.0 ± 8.47 years) were exposed to a standardized laboratory stressor (Trier Social Stress Test). Lymphocyte subset counts and lymphocyte production of tumor-necrosis-factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, -4, -6 and -10 were measured before and after challenge. High levels of ERI and OC were associated with lower natural killer (NK) cell (CD16+/56+) numbers whereas high levels of OC were related to a lower increase in T-helper cells (CD4+) after stress. Furthermore, subjects with higher ERI showed an overall increased pro-inflammatory activity, with higher TNF-α production at both time points and elevated pre-stress IL-6 production. IL-10 production decreased with higher ERI after stress. The ratios of TNF-α/IL-10 and IL-6/IL-10 were significantly increased in subjects high on ERI. Finally, OC was associated with higher IL-2 production post-stress. The present findings suggest a dampened innate immune defence, reflected in lower NK cell numbers together with an increased pro-inflammatory activity in teachers high on ERI and OC. Such pathways could partly be responsible for the increased vulnerability for stress-related diseases in individuals suffering from chronic work stress.

Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis - spontaneous lysis) x 100/(maximum lysis - spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (+/-SD) cytotoxicity of PBMCs from healthy individuals was 20.9 +/- 5.3% and the reference interval was 11.8-31.9%. The mean NK-cell activity of HLH patients (8.3 +/- 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 +/- 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.

Critical role of natural killer cells in the rejection of human hepatocytes after xenotransplantation into immunodeficient mice

The severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID/Alb-uPA) human liver chimeric mouse model has added a new dimension to studies of liver based human diseases and has important potential for study of human hepatic drug metabolism. However, it remains unclear if natural killer (NK) cell in SCID/Alb-uPA mice has an important negative impact on engraftment and expansion of human hepatocytes after transplantation. Here, we explore the role of mouse NK cells in the rejection of transplanted human hepatocytes in SCID/Alb-uPA mice. We assessed NK cell activity in vivo, using (125)I-iodo-2'-deoxyuridine incorporation assay. Low serum human alpha-1 antitrypsin (hAAT, <10 microg/ml) recipients, representing graft failure, showed resistance to engraftment of MHC class I knockout marrow (indicating high NK cell activity), while NK cell-depleted low hAAT recipients and high hAAT (>100 microg/ml) recipients accepted MHC class I knockout marrow, indicating a correlation between low NK cell activity, in vivo, and high level human hepatocyte engraftment. We also showed that higher level engraftment of human hepatocytes was achieved in both NK cell-depleted SCID/Alb-uPA mice and Rag2(-/-)gammac(-/-)/Alb-uPA (T,B and NK cell deficient) mice compared with untreated SCID/Alb-uPA mice. These results support a critical role for mouse NK cells in the rejection of human hepatocytes xenotransplanted to immunodeficient mice.

Prognostic significance of natural killer cell infiltration in hepatocellular carcinoma

Several studies have Shown the correlation of high numbers of tumor-infiltrating natural killer (NK) cells with a good prognosis for cancer patients. This study was to investigate the impact of NK cell infiltration on the survival and prognosis of patients with hepatocellular carcinoma (HCC) after resection. The proportion of infiltrating NK cells of HCC patients was measured using flow cytometry, and the expression of CD56+ (NK) cells was investigated using immunohistochemistry. Prognostic values of intratumoral and peritumoral NK cell densities were evaluated by Kaplan-Meier method and Cox regression. The level of NK cells was significantly lower in tumor-infiltrating lymphocytes (TIL) of HCC patients than in nontumor-infiltrating lymphocytes (NIL) [(11.8 +/- 8.1)% vs. (18.0+/-7.9)%, P=0.002]. The density of NK cells was also significantly lower in cancer nests than in peritumoral lesions (2.3 +/- 2.6 vs. 8.5 +/- 4.5 cells per field, P<0.001). Patients with low intratumoral NK cells had shorter disease-free survival (P=0.027) and overall survival (P=0.005) than patients with high intratumoral NK cells. In contrast, NK cells in the peritumoral area showed no prognostic significance for either disease-free survival or overall survival. Multivariate Cox proportional hazards analysis showed that intratumoral NK cell density was an independent prognostic factor of prolonged overall survival (hazard ratio = 2.658, P=0.019). Low NK cells infiltration could predict poor prognosis in patients with HCC.

Defective Infiltration of Natural Killer Cells in MICA/B-Positive Renal Cell Carcinoma Involves β2-Integrin-Mediated Interaction

We have explored MICA/B expression and its relationship with innate inflammatory infiltrate in renal cell carcinoma (RCC). The expression of MICA/B, CD16, CD56, and CD68 in 140 RCC lesions contained in a tissue microarray (TMA) was investigated by immunohistochemistry. MICA/B gene and protein expressions in Caki-1 cells were analyzed by reverse transcription-polymerase chain reaction and flow cytometry, respectively. Natural killer (NK) cells were studied by flow cytometry. All the RCC lesions (n = 140) were MICA/B-positive. MICA/B was mainly expressed in the cytoplasm of tumor cells, whereas stromal cells were negative. Renal cell carcinoma lesions showed low NK cell infiltration, although they were rich in CD16+CD56- cells, strongly resembling macrophages. CD16+ macrophage infiltration was more frequently detectable in metastatic lesions compared with primary tumors (P = .0223) and was associated with poor RCC differentiation (P = .007). To investigate mechanisms potentially underlying the lack of NK cells infiltration into MICA/B-positive RCC lesions, we used Caki-1 RCC cells. Caki-1 expressed MICA and MICB genes. However, MICA protein was not detectable in Caki-1 cells, whereas MICB protein was detectable in their cytoplasm and on the cell membrane. Coculture of peripheral blood mononuclear cells with Caki-1, K562, HCT116, respectively, resulted in CD56+CD16+ NK cells deletion without affecting CD56+/CD16- NK subset and immature NK cells generated in vitro from CD34+ cells. Natural killer cell apoptosis seemed to be preferentially triggered by cancer cells because HLA-A0201+ NK cells were only marginally affected by allogeneic HLA-A0201- peripheral blood mononuclear cells. Caki-1 cell-mediated NK cell apoptosis was reduced by an anti-β2-integrin (CD18) monoclonal antibody but was NKG2D-, granule exocytosis-, and caspase-independent.