Yong Chong

Reciprocal Activation of GATA-1 and PU.1 Marks Initial Specification of Hematopoietic Stem Cells into Myeloerythroid and Myelolymphoid Lineages

A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34(+)Sca-1(+)c-Kit(+) MPP fraction. The GATA-1(+) MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1(+) MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1(+) and PU.1(+) MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.

Selective CD27+ (memory) B cell reduction and characteristic B cell alteration in drug-naive and HAART-treated HIV type 1-infected patients

To investigate HIV-1-related B cell disorders, the quantity of CD27 positive (CD27+) B cells and their CD38, CD95, and bcl-2 intensities were examined by flow cytometry analysis in 16 drug-naive patients, 27 highly active antiretroviral therapy (HAART)-treated patients, and 20 uninfected controls. CD27+ B cells have been recognized as memory B cells. The mean percentage of CD27+ B cells was significantly lower in drug-naive patients (11.9%) and in HAART-treated patients (16.1%) than in controls (31.4%) (p < 0.01). The intensities of CD38 and CD95 on CD27+ B cells were higher in drug-naive patients than in controls (p < 0.01 in CD95). The intensity of CD95 on CD27+ B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27+ B cells was equivalent among the three groups. These findings suggest that disturbance of peripheral B cell composition, exemplified by CD27+ B cell reduction, exists in both drug-naive and HAART-treated HIV-1-infected patients. In addition, the augmented apoptotic state of CD27+ B cells found in HAART-treated patients with undetectable viral loads, indicated by CD95 elevation, suggests that some HIV-1-related B cell disorders last for years after effective antiviral therapy.

Genetic evolution and clinical impact in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae

The emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria, particularly Escherichia coli and Klebsiella pneumoniae, is now a critical concern for the development of therapies against bacterial infection. ESBLs consist of three major genetic groups: TEM, SHV, and CTX-M types. Nosocomial infections due to TEM and SHV-producing K. pneumoniae strains were frequently documented until the late 1990s. The number of reports on community-acquired infections caused by CTX-M-producing E. coli strains have dramatically increased over the last decade; however, K. pneumoniae strains, of either the TEM or SHV types, are persistent and important ESBL producers. The spread of ESBL genes is associated with various mobile genetic elements, such as transposons, insertion sequences, and integrons. The rapid dissemination of ESBL genes of the CTX-M type may be related to highly complicated genetic structures. These structures harboring ESBL genes and mobile elements are found in a variety of plasmids, which often carry many other antibiotic resistance genes. Multidrug-resistant CTX-M-15-producing E. coli strains disseminate worldwide. Efficient mobile elements and plasmids may have accelerated the genetic diversity and the rapid spread of ESBL genes, and their genetic evolution has caused an emerging threat to the bacteria for which few effective drugs have been identified.

Clinical impact of fluoroquinolone prophylaxis in neutropenic patients with hematological malignancies

BACKGROUND The routine use of fluoroquinolone prophylaxis in patients with neutropenia and hematological malignancies is controversial. This prophylaxis has been reported to have a positive impact in reducing infection-related mortality, but the consequent development of antibiotic resistance has become a concern. This study assessed the effect of discontinuing quinolone prophylaxis on the etiology and the resistance pattern of blood culture isolates and on the prognosis among febrile neutropenic patients receiving chemotherapy. METHODS The results of blood cultures obtained from febrile neutropenic patients between January 2003 and June 2009 were analyzed; these results were available through a computer database set up in 2003. RESULTS Patients receiving quinolone prophylaxis between 2003 and 2005 showed a lower incidence of Gram-negative bacteria than patients not receiving prophylaxis between 2006 and 2009 (13.5%, n=9 vs. 48.1%, n=75). Interestingly, after discontinuing prophylaxis, approximately 70% of the Gram-negative bacteria isolated were quinolone-resistant, and some were extended-spectrum β-lactamase (ESBL) producers. The frequencies of quinolone-resistant Gram-positive bacteria isolated were similar between the period of quinolone prophylaxis and the period with no prophylaxis (61.1% vs. 64.3%). In both periods, all Gram-positive isolates were sensitive to vancomycin. The infection-related mortality was comparable between patients receiving prophylaxis and those not receiving prophylaxis (1.5%, n=1 vs. 1.3%, n=2). CONCLUSIONS These findings suggest that quinolone prophylaxis for neutropenia does not induce a significant increase in the growth of quinolone- and multidrug-resistant bacteria. Rather, discontinuing quinolone prophylaxis may induce a dramatic increase in the growth of Gram-negative bacteria, including ESBL producers. Our results suggest that the necessity for quinolone prophylaxis in neutropenic patients should be determined based on local antibiotic resistance patterns.

Community spread of extended-spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis: a long-term study in Japan

Community-acquired infections caused by extended-spectrum β-lactamase (ESBL)-producing bacteria, particularly CTX-M-producing Escherichia coli, are a rising concern worldwide. There are few data from Japan on the acquisition of ESBLs in the community or the influx of these bacteria into hospitals. Therefore, we examined the prevalence of ESBL carriage in outpatients, in order to estimate the spread of ESBLs in community settings. We analysed bacterial isolates from outpatient samples at our institution over a 9-year period from 2003 to 2011, with respect to epidemiological data on ESBL-producing bacteria and their genotypic features. Out of 5137 isolates, 321 (6.3 %) were ESBL producers, including E. coli, Klebsiella pneumoniae and Proteus mirabilis. The detection rates of the ESBL-producing isolates gradually increased and reached 14.3, 8.7 and 19.6 % for E. coli, K. pneumoniae and P. mirabilis strains, respectively, in 2011. Genotyping analysis showed that many of the strains produced multiple β-lactamases, including TEM, SHV and CTX-M, rather than just CTX-M. The CTX-M-9 group was dominant among the CTX-M genotypes; further, the CTX-M-1 and M-2 groups were also detected (~30 %). This is believed to be the first report from Japan showing a definite increase in ESBL detection in outpatients. In addition, our findings suggest the simultaneous community spread of diverse ESBL genotypes, not an expansion of particular ESBL genes.

Increased frequency of CD27- (naive) B cells and their phenotypic alteration in HIV type 1-infected patients

To investigate HIV-1-related B cell disorders, the quantity of peripheral CD27 negative (CD27-) B cells, their CD38, CD95, and bcl-2 intensities, and their apoptosis susceptibility were examined by flow cytometry analysis in 16 drug-naive patients, 27 HAART-treated patients, and 20 uninfected controls. CD27- B cells have been recognized as naive B cells. The mean percentage of CD27- B cells was significantly higher in drugnaive patients (88.1%) and in HAART-treated patients (83.9%) than in controls (68.6%) (p < 0.01). The intensities of CD38 and CD95 on CD27- B cells were significantly higher in drug-naive patients than in controls (p < 0.01). The intensity of CD95 on CD27- B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27- B cells in drug-naive patients was lower than that of controls. In drug-naive patients, CD27-B cells with high CD38 expression represented low bcl-2 expression. The CD27- B cells of drug-naive patients showed an increased susceptibility to apoptosis, characterized by diminished cell size and a high frequency of annexin-V binding, compared with controls and HAART-treated patients. These findings suggested that HIV-1 infection affects peripheral CD27- (naive) B cells as well as CD27+ (memory) B cells and that CD27- B cells might be activated and rendered highly susceptible to apoptosis by HIV-1 infection. Some phenotypic alterations in CD27- B cells may continue after the reduction of HIV-1 loads by effective antiviral therapy.

Cefepime-resistant Gram-negative bacteremia in febrile neutropenic patients with hematological malignancies.

OBJECTIVES This study was performed to determine the local etiologic pattern of blood culture isolates and antibiotic resistance in febrile neutropenic patients with hematological malignancies. METHODS A total of 142 blood culture isolates from febrile neutropenic patients admitted to our hematology unit were examined, particularly for the detection of cefepime resistance, because cefepime, a fourth-generation cephalosporin, has been used in our unit as initial therapy for febrile neutropenia. RESULTS Among all isolates, 67 (47.2%) were Gram-positive bacteria, the majority of which were fully sensitive to vancomycin. Gram-negative bacteria accounted for 68 (47.9%) of the isolates. Cefepime resistance was seen in 24 (35.3%) of the Gram-negative isolates, and had significantly increased in 2007. The cefepime-resistant isolates primarily consisted of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Approximately 60% of the cefepime-resistant isolates were extended-spectrum β-lactamase (ESBL)-producing organisms. Molecular analysis showed the predominant emergence of CTX-M types. Most of the cefepime-resistant isolates were resistant to third- and various fourth-generation cephalosporins, while having a high susceptibility to carbapenems, particularly meropenem. CONCLUSIONS Cefepime resistance was often detected in the blood culture isolates from febrile neutropenic patients. This result suggests that therapeutic strategies for febrile neutropenia should be modified based on the local antibiotic resistance patterns.

Hydrophobic bile acids suppress expression of AE2 in biliary epithelial cells and induce bile duct inflammation in primary biliary cholangitis.

Understanding the mechanisms of chronic inflammation in primary biliary cholangitis (PBC) is essential for successful treatment. Earlier work has demonstrated that patients with PBC have reduced expression of the anion exchanger 2 (AE2) on biliary epithelial cells (BEC) and deletion of AE2 gene has led to a PBC-like disorder in mice. To directly address the role of AE2 in preventing PBC pathogenesis, we took advantage of our ability to isolate human BEC and autologous splenic mononuclear cells (SMC). We studied the influence of hydrophobic bile acids, in particular, glycochenodeoxycholic acid (GCDC), on AE2 expression in BEC and the subsequent impact on the phenotypes of BEC and local inflammatory responses. We demonstrate herein that GCDC reduces AE2 expression in BEC through induction of reactive oxygen species (ROS), which enhances senescence of BEC. In addition, a reduction of AE2 levels by either GCDC or another AE2 inhibitor upregulates expression of CD40 and HLA-DR as well as production of IL-6, IL-8 and CXCL10 from BEC in response to toll like receptor ligands, an effect suppressed by inhibition of ROS. Importantly, reduced AE2 expression enhances the migration of autologous splenic mononuclear cells (SMC) towards BEC. In conclusion, our data highlight a key functional role of AE2 in the maintenance of the normal physiology of BEC and the pathogenic consequences of reduced AE2 expression, including abnormal intrinsic characteristics of BEC and their production of signal molecules that lead to the chronic inflammatory responses in small bile ducts.

Natural killer cells regulate T cell immune responses in primary biliary cirrhosis

The hallmark of primary biliary cirrhosis (PBC) is the presence of autoreactive T‐ and B‐cell responses that target biliary epithelial cells (BECs). Biliary cell cytotoxicity is dependent upon initiation of innate immune responses followed by chronic adaptive, as well as bystander, mechanisms. Critical to these mechanisms are interactions between natural killer (NK) cells and BECs. We have taken advantage of the ability to isolate relatively pure viable preparations of liver‐derived NK cells, BECs, and endothelial cells, and studied interactions between NK cells and BECs and focused on the mechanisms that activate autoreactive T cells, their dependence on interferon (IFN)‐γ, and expression of BEC major histocompatibility complex (MHC) class I and II molecules. Here we show that at a high NK/BEC ratio, NK cells are cytotoxic for autologous BECs, but are not dependent on autoantigen, yet still activate autoreactive CD4+ T cells in the presence of antigen presenting cells. In contrast, at a low NK/BEC ratio, BECs are not lysed, but IFN‐γ production is induced, which facilitates expression of MHC class I and II molecules on BEC and protects them from lysis upon subsequent exposure to autoreactive NK cells. Furthermore, IFN‐γ secreted from NK cells after exposure to autologous BECs is essential for this protective function and enables autoreactive CD4+ T cells to become cytopathic. Conclusions: NK cell‐mediated innate immune responses are likely critical at the initial stage of PBC, but also facilitate and maintain the chronic cytopathic effect of autoantigen‐specific T cells, essential for progression of disease. (Hepatology 2015;62:1817‐1827)

Higher incidence of injection site reactions after subcutaneous bortezomib administration on the thigh compared with the abdomen

Subcutaneous (sc) rather than intravenous administration of bortezomib (Bor) is becoming more common for treating multiple myeloma (MM) because scBor results in lower incidence and severity of peripheral neuropathy and has equivalent efficacy. Bor is an irritant cytotoxic agent when it leaks out; therefore, it is recommended that injections of scBor should be rotated among eight different sites on the abdomen and thigh. However, detailed information about injection site reaction (ISR) has not been sufficiently documented. We retrospectively analyzed the incidence and severity of ISR following scBor administration in 15 Japanese patients with MM. Grade 1 ISR occurred following 40 of 158 (25.3%) scBor injections in ten patients, whereas grade 2 ISRs occurred following seven injections (4.4%) in five patients. Five patients did not develop ISR. Of note, grade 2 ISR was documented in 6 of 65 (9.2%) thigh injections but only in 1 of 93 (1.1%) abdominal injections. These data show that grade 2 ISRs were more common in the thigh compared with the abdomen possibly because the thigh contains lesser adipose tissue than the abdomen. Grade 2 ISRs resolved without any sequela within a median of 7 d. scBor administration on the abdomen instead of the thigh should be considered, especially for emaciated patients, because ISR rapidly resolves within the interval before the next injection even if it occurs.