An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Serum Levels of Soluble CD26/Dipeptidyl Peptidase-IV in Type 2 Diabetes Mellitus and Its Association with Metabolic Syndrome and Therapy with Antidiabetic Agents in Malaysian Subjects

BackgroundA soluble form of CD26/dipeptidyl peptidase-IV (sCD26/DPP-IV) induces DPP-IV enzymatic activity that degrades incretin. We investigated fasting serum levels of sCD26/DPP-IV and active glucagon-like peptide-1 (GLP-1) in Malaysian patients with type 2 diabetes mellitus (T2DM) with and without metabolic syndrome (MetS), as well as the associations between sCD26/DPP-IV levels, MetS, and antidiabetic therapy.MethodsWe assessed sCD26/DPP-IV levels, active GLP-1 levels, body mass index (BMI), glucose, insulin, A1c, glucose homeostasis indices, and lipid profiles in 549 Malaysian subjects (including 257 T2DM patients with MetS, 57 T2DM patients without MetS, 71 non-diabetics with MetS, and 164 control subjects without diabetes or metabolic syndrome).ResultsFasting serum levels of sCD26/DPP-IV were significantly higher in T2DM patients with and without MetS than in normal subjects. Likewise, sCD26/DPP-IV levels were significantly higher in patients with T2DM and MetS than in non-diabetic patients with MetS. However, active GLP-1 levels were significantly lower in T2DM patients both with and without MetS than in normal subjects. In T2DM subjects, sCD26/DPP-IV levels were associated with significantly higher A1c levels, but were significantly lower in patients using monotherapy with metformin. In addition, no significant differences in sCD26/DPP-IV levels were found between diabetic subjects with and without MetS. Furthermore, sCD26/DPP-IV levels were negatively correlated with active GLP-1 levels in T2DM patients both with and without MetS. In normal subjects, sCD26/DPP-IV levels were associated with increased BMI, cholesterol, and LDL-cholesterol (LDL-c) levels.ConclusionSerum sCD26/DPP-IV levels increased in T2DM subjects with and without MetS. Active GLP-1 levels decreased in T2DM patients both with and without MetS. In addition, sCD26/DPP-IV levels were associated with Alc levels and negatively correlated with active GLP-1 levels. Moreover, metformin monotherapy was associated with reduced sCD26/DPP-IV levels. In normal subjects, sCD26/DPP-IV levels were associated with increased BMI, cholesterol, and LDL-c.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Activity of salivary glands in secreting honey‐elaborating enzymes in two subspecies of honeybee (Apis mellifera L)

The activity of invertase, glucose oxidase and amylase in the cephalic (post‐cerebral) and thoracic salivary glands is determined in Egyptian and Carniolan honeybees (Apis mellifera L). For this purpose, three ages of worker bees are selected for enzyme assays. The results show that the three target enzymes are detected in the two glands during the three worker ages, except invertase, which cannot be detected in the cephalic gland of newly emerged bees of both subspecies. In both glands, the secretion of invertase is highest, followed by amylase and then glucose oxidase. In Carniolan bees, invertase secretion of the cephalic and thoracic glands increases gradually with age. In Egyptian bees, invertase increases with age only in the cephalic gland, whereas, in the thoracic gland, the highest secretion activity is detected in 10–15‐day‐old bees. The highest amounts of glucose oxidase and amylase in the cephalic gland are detected in newly emerged individuals of both Egyptian and Carniolan bees. In the thoracic gland, however, the highest activity of both enzymes is recorded only in newly emerged Egyptian bees. The results are discussed in the light of bee management and biological aspects of the two subspecies.     

Maturation of Human Herpesvirus 6A Glycoprotein O Requires Coexpression of Glycoprotein H and Glycoprotein L

Glycoprotein O (gO) is conserved among betaherpesviruses, but little is known about the maturation process of gO in human herpesvirus 6 (HHV-6). We found that HHV-6 gO maturation was accompanied by cleavage of its carboxyl terminus and required coexpression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cells.