Kenneth W. Hunter

Thermal decomposition based synthesis of Ag-In-S/ZnS quantum dots and their chlorotoxin-modified micelles for brain tumor cell targeting

Cadmium-free silver-indium-sulfide (Ag-In-S or AIS) chalcopyrite quantum dots (QDs) as well as their core-shell structures (AIS/ZnS QDs) are being paid significant attention in biomedical applications because of their low toxicity and excellent optical properties. Here we report a simple and safe synthetic system to prepare high quality AIS and AIS/ZnS QDs using thermal decomposition. The synthetic system simply involves heating a mixture of silver acetate, indium acetate, and oleic acid in dodecanethiol at 170 °C to produce AIS QDs with a 13% quantum yield (QY). After ZnS shell growth, the produced AIS/ZnS QDs achieve a 41% QY. To facilitate phase transfer and bioconjugation of AIS/ZnS QDs for cellular imaging, these QDs were loaded into the core of PLGA-PEG (5k:5k) based micelles to form AIS/ZnS QD-micelles. Cellular imaging studies showed that chlorotoxin-conjugated QD-micelles can be specifically internalized into U-87 brain tumor cells. This work discloses that the scalable synthesis of AIS/ZnS QDs and the facile surface/interface chemistry for phase transfer and bioconjugation of these QDs may open an avenue for the produced QD-micelles to be applied to the detection of endogenous targets expressed on brain tumor cells, or more broadly to cell- or tissue-based diagnosis and therapy.

Preparation of microparticulate β-glucan from Saccharomyces cerevisiae for use in immune potentiation

Aims: To develop a method for the preparation of an immunologically active, homogeneous, nonaggregated, microparticulate β‐glucan‐containing material from the budding yeast Saccharomyces cerevisiae. 
Methods and Results: Using a combination of sonication and spray‐drying, a homogeneous preparation of 1–2‐µ diameter β‐glucan‐containing particles was made from alkali‐ and acid‐insoluble yeast cell wall material. This microparticulate β‐glucan remained in suspension longer and, following oral administration at 0·1 mg kg−1 for 14 d, enhanced phagocytosis of mouse peritoneal macrophages significantly better than did aggregated β‐glucan particles. 
Conclusions: A new sonication and spray‐drying method can be employed to overcome the problem of aggregation of β‐glucan microparticles in aqueous media. 
Significance and Impact of the Study: A microparticulate form of β‐glucan that remains in suspension longer for pharmaceutical applications and has superior immune potentiation characteristics has been developed.

The mouse mammary carcinoma 4T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci

The murine mammary carcinoma 4T1 causes a leukemoid reaction with profound granulocytosis coincident with the production of tumour‐derived growth factors. Here, we study the evolving cellular landscape of primary tumours and metastatic tumour foci and correlate haematopoietic cell infiltration with the production of tumour‐derived chemokines. Flow cytometric analysis of enzyme digested primary tumours at different times after transplantation revealed a progressively increasing CD45+ haematopoietic cell infiltrate consisting predominantly of CD11b+ myeloid cells. Most of these cells had an F4/80+/CD11c+ phenotype, many of which also stained Gr‐1+. Smaller numbers of Gr‐1+CD11b+ granulocytes and lymphoid cells were also identified. Progressive increases in Gr‐1+ granulocytes were observed in enzymatic digests of livers and lungs with metastatic tumour foci. Cultured 4T1 tumour cells expressed mRNA transcripts for the myeloid cell chemokines RANTES, MCP‐1 and KC, and enzymatically digested cells from primary 4T1 tumours partially depleted of CD45+ cells expressed transcripts for these chemokines and also MIP‐1α and MIP‐1β. These data demonstrate that 4T1 tumour‐bearing mice have mixed myeloid cell infiltrates of primary tumours and granulocytic infiltrates of metastatic organs. This pathologic presentation correlated with the expression of tumour‐derived chemokines.

Selection for increased mass-independent maximal metabolic rate suppresses innate but not adaptive immune function

Both appropriate metabolic rates and sufficient immune function are essential for survival. Consequently, eco-immunologists have hypothesized that animals may experience trade-offs between metabolic rates and immune function. Previous work has focused on how basal metabolic rate (BMR) may trade-off with immune function, but maximal metabolic rate (MMR), the upper limit to aerobic activity, might also trade-off with immune function. We used mice artificially selected for high mass-independent MMR to test for trade-offs with immune function. We assessed (i) innate immune function by quantifying cytokine production in response to injection with lipopolysaccharide and (ii) adaptive immune function by measuring antibody production in response to injection with keyhole limpet haemocyanin. Selection for high mass-independent MMR suppressed innate immune function, but not adaptive immune function. However, analyses at the individual level also indicate a negative correlation between MMR and adaptive immune function. By contrast BMR did not affect immune function. Evolutionarily, natural selection may favour increasing MMR to enhance aerobic performance and endurance, but the benefits of high MMR may be offset by impaired immune function. This result could be important in understanding the selective factors acting on the evolution of metabolic rates.

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.

Synthesis of cetyl myristoleate and evaluation of its therapeutic efficacy in a murine model of collagen-induced arthritis

Cetyl myristoleate (CM) was reported by Diehl and May [J Pharm Sci 83 (1994) 296] to block inflammation and prevent adjuvant-induced arthritis in rats. To verify this earlier work, we have synthesized pure CM and tested its anti-arthritic properties in a collagen-induced arthritis model in DBA/1LacJ mice. Multiple intraperitoneal injections of CM in 450 and 900 mg kg(-1) doses resulted in a significantly lower incidence of disease and caused a modest but significant diminution in clinical signs in those mice that developed arthritis. CM administered in daily oral doses of 20 mg kg(-1) also reduced the incidence of arthritis and caused a small reduction in the clinical signs in mice that developed arthritis. Although the protective effect of CM in collagen-induced arthritis observed in the present study was less dramatic than that reported earlier, our results confirm the anti-arthritic properties of pure CM.

A trade-off between natural and acquired antibody production in a reptile: implications for long-term resistance to disease

Summary Vertebrate immune systems are understood to be complex and dynamic, with trade-offs among different physiological components (e.g., innate and adaptive immunity) within individuals and among taxonomic lineages. Desert tortoises (Gopherus agassizii) immunised with ovalbumin (OVA) showed a clear trade-off between levels of natural antibodies (NAbs; innate immune function) and the production of acquired antibodies (adaptive immune function). Once initiated, acquired antibody responses included a long-term elevation in antibodies persisting for more than one year. The occurrence of either (a) high levels of NAbs or (b) long-term elevations of acquired antibodies in individual tortoises suggests that long-term humoral resistance to pathogens may be especially important in this species, as well as in other vertebrates with slow metabolic rates, concomitantly slow primary adaptive immune responses, and long life-spans.

Mycoplasmal Upper Respiratory Tract Disease Across the Range of the Threatened Mojave Desert Tortoise: Associations with Thermal Regime and Natural Antibodies

Most research of upper respiratory tract disease (mycoplasmal URTD) in the threatened Mojave Desert tortoise (Gopherus agassizii) has worked under the hypothesis that the pathogen, Mycoplasma agassizii, has a relatively consistent and predictable effect on tortoise populations across their natural range. In contrast, we hypothesized that multiple factors influence the prevalence of disease and analyzed biological and environmental variables that vary significantly across the Mojave Desert. We used multiple regression models to analyze associations between mycoplasmal URTD and the genetic structure of 24 tortoise populations, levels of natural antibody (NAb) to M. agassizii in tortoises (one component of the innate immune system), precipitation, and colder thermal regimes. We detected a significant, positive association between mean levels of NAb and seroprevalence to M. agassizii. We hypothesized that NAbs may provide tolerance to mycoplasmal infections and that more tolerant populations may act as host reservoirs of disease. We also detected significant associations between colder winters and mycoplasmal URTD, suggesting that colder winters may depress tortoise immune resistance against M. agassizii or enhance conditions for the growth of M. agassizii.

A New Electrochemical System Based on a Flow-Field Shaped Solid Electrode and 3D-Printed Thin-Layer Flow Cell: Detection of Pb2+ Ions by Continuous Flow Accumulation Square-Wave Anodic Stripping Voltammetry

Here we describe a new and sensitive flow electrochemical detection system that employs a novel flow-field shaped solid electrode (FFSSE). The system was constructed with a 3D-printed thin-layer flow cell (TLFC) and a flat screen-printed FFSSE with USB connection. This interface facilitates continuous flow accumulation square-wave anodic stripping voltammetry (ASV). The flow distribution in the working space of TLFC was simulated using the finite element method (FEM) and the shape and configuration of electrodes were optimized accordingly. We demonstrated the electrochemical determination of Pb2+ using this newly designed TLFC-FFSSE detection system without removal of oxygen from samples. This TLFC-FFSSE based system showed an attractive stripping voltammetric performance compared to a traditional ASV based method. A linear range for detection of Pb2+ was found to be 0.5-100 μg/L (0.5 to 100 ppb) and a detection limit of 0.2 μg/L (0.2 ppb) was achieved in the presence of bismuth as codeposition metal. The system was further applied to detect Pb2+ in biological broths of methane fermentation. The electrochemical detection results were consistent with that obtained from atomic fluorescence spectroscopy (AFS) analysis and the average recovery was found to be 95.5-106.5% using a standard addition method. This new flow electrochemical detection system showed better sensitivity and reproducibility compared to a traditional ASV based method. Such a system offers great potential for on-site and real-time detection of heavy metals where compact, inexpensive, robust, and low-volume analysis is required.

Properties of human blood monocytes. II. Monocytes from healthy adults are highly heterogeneous within and among individuals

Human blood monocytes are known to include subsets defined by the expression of CD14 and CD16 but otherwise are often assumed to be relatively homogeneous. However, we had observed additional heterogeneity that led us to a more extensive examination of monocytes.