Kim Ahrens

In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone- producing cells

Although organ-specific stem cells possess plasticity that permit differentiation along new lineages, production of endocrine pancreas and insulin-secreting β cells from adult nonpancreatic stem cells has not been demonstrated. We present evidence that highly purified adult rat hepatic oval “stem” cells, which are capable of differentiation to hepatocytes and bile duct epithelium, can trans-differentiate into pancreatic endocrine hormone-producing cells when cultured in a high-glucose environment. These differentiated cells can self-assemble to form three-dimensional islet cell-like clusters that express pancreatic islet cell differentiation-related transcripts detectable by reverse transcription–PCR/nested PCR (e.g., PDX-1, PAX-4, PAX-6, Nkx2.2 and Nkx6.1, insulin I, insulin II, glucose transporter 2, and glucagon) and islet-specific hormones detectable by immunocytochemistry (e.g., insulin, glucagon, and pancreatic polypeptide). In addition, these cells concomitantly lose expression of the hepatocyte protein Hep-par. When stimulated with glucose, these cells synthesize and secrete insulin, a response enhanced by nicotinamide. In a pilot study, the oval cell-derived islet cell-like clusters displayed the ability to reverse hyperglycemia in a diabetic NOD-scid mouse. These results indicate that primary adult liver stem cells can differentiate in a nonlineage-restricted manner. Trans-differentiation into endocrine pancreas could have significant implications for future therapies of diabetes.

Isolate-specific parasite antigens of the Babesia bovis-infected erythrocyte surface.

Bovine erythrocytes taken from in vitro cultures of Babesia bovis parasites from Mexico and the United States were assayed for the presence of new epitopes on the erythrocyte surface. New surface-exposed epitopes were detected by means of a whole-cell antigen capture assay. These epitopes were subsequently demonstrated only on infected erythrocytes by immunofluorescence staining of intact, living cells. Parasite-synthesized antigens were identified on each isolate using a surface-specific immunoprecipitation technique to analyze metabolically-labeled infected erythrocytes. In the Mexico isolate these antigens were 120 kDa and 107 kDa, whereas in the United States isolate polypeptides of 135, 120 and 107 kDa were detected. In each of these assays, reaction of immune sera with the infected erythrocyte surface was found to be isolate-specific.

Gene Expression Profiling during All-trans Retinoic Acid-Induced Cell Differentiation of Acute Promyelocytic Leukemia Cells

Using cDNA microarrays we determined the gene expression patterns in the human acute promyelocytic leukemia (APL) cell line NB4 during all-trans retinoic acid (ATRA)-induced differentiation. We analyzed the expression of 12,288 genes in the NB4 cells after 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours of ATRA exposure. During this time course, we found 168 up-regulated and more than 179 down-regulated genes, most of which have not been reported before. Many of the altered genes encode products that participate in signaling pathways, cell differentiation, programmed cell death, transcription regulation, and production of cytokines and chemokines. Of interest, the CD52 and protein kinase A regulatory subunit alpha (PKA-Rlalpha) genes, whose products are being used as therapeutic targets for certain human neoplasias in currently ongoing clinical trials, were among the genes observed to be markedly up-regulated after ATRA treatment. The present study provides valuable data to further understand the mechanism of ATRA-induced APL cell differentiation and suggests potential therapeutic alternatives for this leukemia.

Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20

The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments. Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining. CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH. This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH. The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question. Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined.

Early exposure to probiotics in a canine model of atopic dermatitis has long-term clinical and immunological effects

Probiotics modulate the immune response and may have protective effects against atopic dermatitis (AD). Clinical trials using dogs with spontaneous disease are limited by confounding factors such as different diets, environments and sensitizations while a more controlled evaluation is possible using experimental models. A validated model of canine AD showed that early exposure to Lactobacillus rhamnosus GG (LGG) significantly decreases allergen-specific IgE and partially prevents AD in the first 6 months of life. This study is a follow-up three years after discontinuation of LGG. Clinical signs were evaluated after allergen challenge with ragweed, timothy, Dermatophagoides farinae. Allergen-specific IgE, IL-10 and TGF-β were measured on the 1st day of challenge, before allergen exposure. Normal dogs were included as controls. Analyses included seven dogs in the non-probiotic and nine in the probiotic litter. For clinical scores, a 2-Group × 9-Time Analysis of Variance showed significant effects of group (p=0.0003, probiotic<controls), time (p<0.0001) and group × time interaction (p<0.0001). IL-10 for all allergens was significantly higher in the control group than probiotics-exposed dogs. Allergen-specific IgE and TGF-β did not differ between litters. Early exposure to probiotics has long-term clinical and immunological effects in this model and larger studies using dogs with spontaneous disease are needed.

DRAQ5-based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

Analysis of cell cycle kinetics offers important information regarding the behavior of normal and neoplastic cells. Most often, cell cycle determinations by flow cytometry (FCM) have been performed using whole‐sample analysis with intercalating dyes like propidium iodide (PI). The cell cycle phase assessment in individual cell subsets in heterogeneous samples is best performed using combined antigen/scatter and DNA analysis. DRAQ5, a novel DNA binding dye that excites at 488 nm and emits in the far red spectra, rapidly penetrates intact live cells while preserving their light scatter properties and expression of surface antigens. We evaluated the ability of this dye to measure cell cycle phases in a variety of clinical hematolymphoid samples.

Altered mRNA and protein expression of filaggrin in the skin of a canine animal model for atopic dermatitis

BACKGROUND Filaggrin is a structural protein that has attracted increasing interest over the past decade for its role in the pathogenesis of human atopic dermatitis (AD). Null mutations in its sequence are considered risk factors in the development of AD. HYPOTHESIS/OBJECTIVES To investigate canine filaggrin mRNA and protein expression in the skin of atopic beagles with experimentally induced AD compared with breed-matched healthy control dogs. METHODS All dogs were environmentally challenged for 3 days consecutively with allergens to which the atopic dogs had been sensitized. Skin biopsy specimens were taken from six healthy and seven atopic beagles before and after allergen challenge. Canine filaggrin mRNA was measured using quantitative real-time PCR. Indirect immunofluorescence was used to localize the filaggrin protein in canine skin. Analysis of variance with Tukeys multiple comparison test (over-time effect) and unpaired Students t-test (treatment effect) were used. Values of P ≤ 0.05 were considered significant. RESULTS Analysis of variance showed a significantly higher expression of filaggrin mRNA in atopic dogs compared with healthy control dogs (P = 0.004 on day 3 and P = 0.01 on day 10) and a decreased mRNA expression on day 3 in healthy control dogs (effect of time, P = 0.006). On blinded evaluation, filaggrin immunofluorescence was distributed homogeneously in the stratum granulosum and the stratum corneum in healthy dogs. Atopic dogs showed a patchy immunofluorescence pattern, which was exacerbated after environmental challenge. CONCLUSIONS AND CLINICAL IMPORTANCE Altered epidermal filaggrin mRNA expression and protein distribution was detected in this experimental model.

A nonsubjective assay for antigenic modifications of the Babesia bovis-parasitized erythrocyte surface

Intracellular protozoan parasites induce numerous alterations in the invaded host cell, including antigenic modifications of the host cell plasma membrane. We have developed a quantifiable, non-subjective assay for the detection of novel antigenic reactivities on the host cell surface using as a model system bovine erythrocytes infected with Babesia bovis. Infected erythrocytes, metabolically labeled with L-[35S]methionine, were sensitized by incubation with bovine immune serum, then were captured in microtiter plates coated with rabbit anti-bovine IgG antibody. This technique enabled specific capture of B. bovis-infected cells with immune infection sera raised against B. bovis but not with similar sera raised against Babesia bigemina. This assay should be easily applicable to the study of other parasitic diseases.

Evaluation of antimicrobial peptides and cytokine production in primary keratinocyte cell culture from healthy and atopic beagles

Increased secretion of antimicrobial peptides and cytokines is present in atopic skin. The purpose of this study was to compare the production of β‐defensin (cBD)3‐like, cathelicidin (cCath) and cytokines in atopic and healthy canine keratinocytes. Seven atopic house dust mites (HDMs) sensitive and five healthy age‐matched beagles were used. Keratinocytes were stimulated for 24 h, and the supernatant was collected. A significantly higher production of cBD3‐like was present at baseline in atopic compared with healthy keratinocytes, but cBD3‐like did not increase after stimulation. IL‐17 and lipopolysaccharide increased cBD3‐like in healthy compared with atopic keratinocytes. cCath increased in both groups after stimulation. Atopic keratinocytes exposed to HDM produced more IL‐8 and keratinocyte‐derived chemokine‐like than healthy keratinocytes. Exposure to HDM induced an increased IL‐8 in atopic keratinocytes and a decreased IFN‐γ in healthy keratinocytes. These results may suggest an over sensitization of atopic keratinocytes and a possible impairment of the cutaneous defense against micro‐organisms.

A comparative study of epidermal tight junction proteins in a dog model of atopic dermatitis.

BACKGROUND Tight junctions (TJ) are important for skin barrier function and could be relevant in modulating allergen penetration in atopic dermatitis (AD). Humans with AD have been described to have decreased expressions of some TJ proteins in the skin. HYPOTHESIS/OBJECTIVES This study aimed to investigate TJ protein expression using an experimental AD model in dogs. METHODS Skin biopsies from six atopic (nonlesional skin) and five normal beagle dogs were stained for TJ proteins [zonula occludens 1 (ZO-1), occludin, claudin-1] by immunohistochemistry. Staining intensity was evaluated both objectively using imaging software and subjectively. Six images/sections were randomized and blindly scored by six investigators for intensity, distribution, integrity and staining pattern. RESULTS The intensity of ZO-1 was significantly decreased in the atopic group objectively (P = 0.010) and subjectively (P = 0.002) relative to the normal group. Occludin was decreased significantly subjectively (P = 0.027) but not objectively. Claudin was not significantly different between groups by either quantification. Additionally, only ZO-1 demonstrated a significantly patchier staining pattern in the atopic group. There was no consistent staining pattern in this study. CONCLUSIONS AND CLINICAL IMPORTANCE ZO-1 and occludin, which have not been described to be associated with the development of AD in humans, could play a role in this atopic dog model. Further investigation on the expression and modulation of TJ proteins and their clinical relevance is needed.