Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes

We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN- gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.     

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis. In this study, we developed Etf-1–specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti–Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1–GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Human monocytic ehrlichiosis (HME), one of the most prevalent, life-threatening, and emerging tick-borne diseases in the United States (1, 2) is caused by infection with Ehrlichia chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales. E. chaffeensis replicates within human monocytes-macrophages and causes severe flu-like symptoms accompanied by hematologic abnormalities and hepatitis. Currently, the only HME therapy is the broad-spectrum antibiotic doxycycline, which is effective only if initiated early because delayed initiation (e.g., because of misdiagnosis can lead to severe complications or death). In addition, doxycycline is contraindicated for pregnant women and children or those with drug allergies. The presence of underlying illness or injury, immunosuppression, and coinfection with other tick-borne pathogens can similarly lead to severe complications or death (3). No vaccine exists for HME. Tick-borne diseases have risen dramatically in the past 20 y and continue to rise, underscoring the importance of developing new therapeutic approaches and preventive measures (4).The type IV secretion system (T4SS) is conserved among all rickettsial organisms. The recent elucidation of critical roles of T4SS for E. chaffeensis and Anaplasma phagocytophilum infection (5) may provide potential targets for new approaches against rickettsial diseases. For example, the T4SS effectors Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) are critical E. chaffeensis proteins secreted via T4SS into the host cell cytoplasm, as knockdown of Etf-1 or Etf-2 by transfection of E. chaffeensis with specific antisense peptide nucleic acids significantly inhibits E. chaffeensis infection (6, 7). Secreted Etf-1 localizes to mitochondria and blocks mitochondria-mediated host cell apoptosis to keep the infected host cell alive for bacterial intracellular replication (8). A subpopulation of Etf-1 molecules that are not localized to mitochondria interacts with Beclin 1 (ATG6) and active Rab5 (Rab5-GTP), and induces Rab5-regulated autophagy for E. chaffeensis to acquire catabolites as nutrients (9). Etf-2 directly binds Rab5-GTP on Ehrlichia-containing inclusion membranes and blocks Rab5 GTPase activating protein (RabGAP-5) engagement with Rab5-GTP to prevent Ehrlichia-containing inclusions from maturing into late endosomes and fusing with lysosomes (7).Camelidae produce two types of antibodies: conventional antibodies and heavy-chain–only antibodies (10). The variable domain of the heavy chain of heavy-chain–only antibodies (VHHs) of camelids is the smallest (11 to 15 kDa) antigen-binding fragment relative to conventional antibodies. VHHs are soluble and display long surface loops, which are often larger than those of conventional murine and human antibodies (11, 12). The VHHs can be cloned into bacterial or mammalian expression plasmids (13) to produce a nanobody (Nb), a monomeric variable antibody. VHHs cloned into mammalian expression vectors can produce intracellular Nbs within mammalian cells that are superior to conventional antibodies for modulating intracellular functions because they can operate in the reducing intracellular environment, are proteolytically stable, can target subcellular sites, can penetrate cavities in target antigens, and can bind efficiently to antigens, such as enzyme catalytic sites (13–16). Although the therapeutic potential of Nbs has been investigated for several infectious diseases (14, 17–19), the use of Nbs as a therapeutic agent against intracellular bacteria such as E. chaffeensis has not been reported. In the present study, we developed an intracellular Nb approach to block T4SS effectors within mammalian cells, thereby inhibiting intracellular pathogen infection.Progress in developing effective therapy and investigative approach for obligatory intracellular pathogens has been hindered by many factors, not the least of which is the lack of safe and efficient intracellular delivery methods of macromolecules. Although cyclic peptides are generally unable to cross the cell membrane, some naturally occurring cyclic peptides (e.g., cyclosporine A) possess the unusual ability of crossing the cell membrane by passive diffusion and are orally bioavailable (20). Cyclized Arg-rich cell-permeable peptides (CPPs)—such as cyclo(FΦRRRRQ) or cFΦR4, where Φ is l-2-naphthylalanine—or newer and more effective CPPs, such as CPP9 and CPP12, that include d-arginine or d-phenylalanine, provide rapid and efficient cytosolic delivery of their linked cargo proteins into >95% of cells (21–23). They are not cytotoxic at effective concentrations and have oral and intravenous bioavailability based on preliminary pharmacokinetics in mice (22). The cyclic CPPs (and the CPP-cargo conjugates) bind directly to plasma membrane phospholipids and enter cells by endocytosis (22). They then efficiently escape from the early endosome into the cytosol unlike Tat, which escapes only from late endosomes (22–24).In the present study, we have developed anti–Etf-1 Nbs. We obtained a Nb that blocks Etf-1 functions and demonstrated its effectiveness in combination with a cyclic CPP for inhibition of E. chaffeensis infection in cell culture and in a mouse model. These findings represent a significant advance in developing therapeutic and investigative strategy of obligatory intracellular pathogens.     

Measurement of varicella-zoster virus (VZV)-specific cell-mediated immunity: comparison between VZV skin test and interferon-gamma enzyme-linked immunospot assay

Cell-mediated immunity (CMI) is critical for the prevention and control of varicella-zoster virus (VZV)-related disease. To assess CMI to VZV, a varicella skin test and interferon-gamma enzyme-linked immunospot (ELISPOT) assay were both performed in healthy volunteers, and the results were compared. A total of 151 subjects were examined: 16 aged 20-29 years, 26 aged 30-39 years, 18 aged 40-49 years, 73 aged 50-59 years, and 18 aged 60-69 years. All were seropositive by a glycoprotein antigen-based enzyme-linked immunosorbent assay (gpELISA). Skin test reactivity was significantly correlated with the ELISPOT count, and both decreased with increasing age, indicating an age-dependent decline in CMI to VZV. In contrast, the antibody titer obtained by the gpELISA did not correlate with skin test reactivity. The results suggest that the skin test and ELISPOT assay are both reliable for assessing CMI to VZV and can easily be applied to screen individuals susceptible to the development of herpes zoster.     

Selenium deficiency in a patient with Crohn's disease receiving long-term total parenteral nutrition

We report a case of selenium deficiency in a patient with Crohn's disease on long-term total parenteral nutrition (TPN). She manifested lassitude of the legs, discoloration of the nail beds, and macrocytosis. Since her plasma selenium level was found to be below the measurable level, we diagnosed this case as selenium deficiency. After intravenous administration of sodium selenite, her symptoms were reversed. Careful attention should be paid to selenium deficiency when a patient receives long-term TPN; supplementary administration of selenium via TPN may be required because selenium is often not routinely added to TPN formulations.     

Effect of glycemic state on postprandial hyperlipidemia and hyperinsulinemia in patients with coronary artery disease

Both postprandial hyperlipidemia and hyperinsulinemia have been thought to play an important role in the development of atherosclerosis, and to be a potent risk factor for cardiovascular event. To examine effects of glycemic state on postprandial hyperlipidemia and hyperinsulinemia in patients with coronary artery disease (CAD), a total of 112 consecutive male patients with angiographically confirmed CAD were loaded with a high-fat and high-glucose test meal. CAD patients were divided into three groups as “non-diabetic”, “prediabetic”, and “diabetic” CAD groups. The serum triglyceride (TG) and remnant-like particle cholesterol (RLP-C) levels at the 6th hour in diabetic CAD group showed significantly higher than non-diabetic CAD group, and the incremental area under the curves (iAUCs) of these levels in diabetic CAD group were significantly greater than non-diabetic CAD group (TG, P = 0.0194; RLP-C, P = 0.0219). There were no significant differences in the iAUCs of TG or RLP-C between prediabetic and non-diabetic CAD group. The AUCs of plasma insulin levels or insulin resistance index (IRI): (AUCs of insulin) × (AUCs of glucose) as the insulin resistance marker were greater in diabetic CAD group than non-diabetic CAD group (insulin, P = 0.0373; IRI, P = 0.0228). The AUCs of serum TG or RLP-C levels showed a correlation with the AUCs of plasma insulin (AUC-TG, r = 0.5437, P < 0.0001; AUC-RLP-C, r = 0.6847, P < 0.0001), and they correlated well with the insulin resistance index (AUC-TG, r = 0.7724, P < 0.0001; AUC-RLP-C, r = 0.7645, P < 0.0001). We found that the insulin resistance showed a close relationship with postprandial hyperlipidemia in CAD patients. Diabetic, but not prediabetic state, may be a risk for postprandial impaired lipid metabolism in CAD patients.     

Assessment of Effects of Air Pollution on Daily Outpatient Visits using the Air Quality Index

Background

The air quality index (AQI) is widely used to characterize the quality of ambient air. Chinese cities officially report the AQI on a daily basis. To assess the possible effects of air pollution on daily outpatient visits, we examined the association between AQI and the daily outpatient count.

Methods

Daily data on outpatient visits to each clinical department were collected from the Z county hospital of Datong City, China. The collection period was between 5 April and 30 June, 2012. Daily AQI data and meteorological information were simultaneously recorded. We compared outpatient counts between the index days and comparison days, and calculated Pearson’s product moment correlation coefficient between outpatient counts and AQI levels.

Results

The average AQI level for index days was significantly higher than that for comparison days. No significant difference was observed in temperature or relative humidity between index days and comparison days. The outpatient counts for pediatrics were significantly higher on index days than on comparison days, and no significant difference was noted in other clinical departments. The outpatient counts for pediatrics positively correlated with the AQI level, and no correlation was noted in other clinical departments.

Conclusion

The present study assessed the association between daily outpatient visits and air pollution using AQI. The results obtained suggest that air pollution could increase the outpatient count for pediatrics.