Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.

Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus.

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.

Time-serial Assessment of Drug Combination Interventions in a Mouse Model of Colorectal Carcinogenesis Using Optical Coherence Tomography.

Optical coherence tomography (OCT) is a high-resolution, nondestructive imaging modality that enables time-serial assessment of adenoma development in the mouse model of colorectal cancer. In this study, OCT was utilized to evaluate the effectiveness of interventions with the experimental antitumor agent α-difluoromethylornithine (DFMO) and a nonsteroidal anti-inflammatory drug sulindac during early [chemoprevention (CP)] and late stages [chemotherapy (CT)] of colon tumorigenesis. Biological endpoints for drug interventions included OCT-generated tumor number and tumor burden. Immunochistochemistry was used to evaluate biochemical endpoints [Ki-67, cleaved caspase-3, cyclooxygenase (COX)-2, ß-catenin]. K-Ras codon 12 mutations were studied with polymerase chain reaction-based technique. We demonstrated that OCT imaging significantly correlated with histological analysis of both tumor number and tumor burden for all experimental groups (P < 0.0001), but allows more accurate and full characterization of tumor number and burden growth rate because of its time-serial, nondestructive nature. DFMO alone or in combination with sulindac suppressed both the tumor number and tumor burden growth rate in the CP setting because of DFMO-mediated decrease in cell proliferation (Ki-67, P < 0.001) and K-RAS mutations frequency (P = 0.04). In the CT setting, sulindac alone and DFMO/sulindac combination were effective in reducing tumor number, but not tumor burden growth rate. A decrease in COX-2 staining in DFMO/sulindac CT groups (COX-2, P < 0.01) confirmed the treatment effect. Use of nondestructive OCT enabled repeated, quantitative evaluation of tumor number and burden, allowing changes in these parameters to be measured during CP and as a result of CT. In conclusion, OCT is a robust minimally invasive method for monitoring colorectal cancer disease and effectiveness of therapies in mouse models.

Evidence Brief--Trends and projections of obesity among Canadians.

The prevalence of obesity, defined as body mass index (BMI) of 30 kg/m2 or higher for adults and as 2 standard deviations above the World Health Organization growth standard mean for children, has increased in many parts of the world. Obese adults are at an increased risk of certain chronic conditions, including hypertension, type 2 diabetes, cardiovascular diseases and some cancers, and of premature death. Obese children have increased cardiometabolic risk, including dyslipidemia, insulin resistance and elevated blood pressure. Excess childhood body weight that continues into adulthood can affect quality of life, educational attainment and earnings over the lifecourse. The Public Health Agency of Canada has projected an annual direct health care cost (including physician, hospitalization and medication costs) of those categorized as obese in Canada in constant 2001 Canadian dollars. Calculated as $7.0 billion in 2011, this annual direct health care cost is projected to rise to $8.8 billion by 2021, based on simulated average direct health care costs, which are higher among the obese ($2,283) than the overweight ($1,726), the underweight ($1,298) and those at normal weight ($1,284). Canadian estimates from 2006 and 2008 that used different methodologies place the annual economic burden (direct and indirect costs) of obesity between $4.6 billion and $7.1 billion. The purpose of this evidence brief is to show current Canadian obesity prevalence rates and estimates for the future using objectively measured height and weight to calculate BMI. The use of objectively measured height and weight to derive BMI is strongly recommended, especially for children and adolescents, as self- or proxy-reported height and weight tend to underestimate actual weight and consequently BMI and obesity prevalence.

TITRE: Synthèse portant sur les données probantes - Tendances et projections relatives à l'obésité chez les Canadiens. INTRODUCTION: La prévalence de l'obésité ­ soit un indice de masse corporelle (IMC) supérieur ou égal à 30 kg/m2 chez les adultes ou deux écarts-types au-dessus de la médiane de la norme de croissance de l'Organisation mondiale de la santé chez les enfants ­ a augmenté dans de nombreuses régions du monde. Les adultes obèses sont plus susceptibles d'être atteints de certaines affections chroniques, notamment d'hypertension, de diabète de type 2, de cardiopathies et de certains cancers, ainsi que de mourir prématurément. Les enfants obèses présentent aussi un risque cardiométabolique accru (dyslipidémie, résistance à l'insuline et hypertension artérielle). Un excès pondéral pendant l'enfance qui se poursuit à l'âge adulte peut nuire à la qualité de vie, au rendement scolaire et au revenu tout au long de la vie. L'Agence de la santé publique du Canada a estimé les coûts annuels directs, en dollars canadiens constants de 2001, des soins de santé (coûts liés aux médecins, aux hospitalisations et aux médicaments) pour les personnes classées comme obèses au Canada. Évalués à 7,0 milliards de dollars en 2011, ils devraient atteindre 8,8 milliards de dollars d'ici 2021, d'après un calcul à partir des moyennes actuelles qui font qu'ils sont plus élevés chez les obèses (2 283 $) que chez les personnes en surpoids (1 726 $), les personnes en insuffisance pondérale (1 298 $) et les personnes de poids normal (1 284 $). Des estimations canadiennes antérieures, de 2006 et 2008 et obtenues par des méthodologies différentes, ont évalué le fardeau économique annuel (coûts directs et indirects) de l'obésité dans une fourchette allant de 4,6 à 7,1 milliards de dollars. Cette synthèse fondée sur des données probantes vise à présenter les taux de prévalence de l'obésité au Canada à la fois actuels et projetés, à partir d'un calcul de l'IMC reposant sur des mesures objectives de la taille et du poids. L'utilisation de mesures objectives de la taille et du poids pour calculer l'IMC est fortement recommandée, particulièrement chez les enfants et les adolescents, car lorsque la taille et le poids sont autodéclarés ou obtenus par procuration, le poids réel est généralement sous-estimé, ce qui fait que l'IMC et la prévalence de l'obésité le sont également.

Dual optical modality endoscopic imaging of cancer development in the mouse colon.

BACKGROUND AND OBJECTIVE: We utilize a miniature, dual-modality endoscope that combines fluorescence-based surface magnifying chromoendoscopy (SMC) and optical coherence tomography (OCT) to follow the anatomical changes that occur during adenoma development in the mouse colon. MATERIALS AND METHODS: Twenty-five mice were treated with the carcinogen azoxymethane (AOM) to induce tumor development in the distal colon, or were treated with saline as control, and were imaged over six months. OCT detects adenoma number with high sensitivity and specificity and can measure lesion size. In methylene blue-lavaged colons, SMC detects changes in the colonic crypts. SMC images of control mouse colons exhibit reticulated patterns of crypts of equal size, forming either a dot or honeycomb pattern. RESULTS: Images of AOM-treated colons show mild crypt irregularities even in grossly normal tissue. Images of small to medium adenoma exhibit larger crypts, more intense signal, and irregular spacing whereas those of large adenoma have heterogeneous, intense signal and loss of crypt structure. CONCLUSIONS: The combination of OCT and SMC permits the detection of neoplastic events from the earliest stages of crypt irregularities before gross tissue changes are noted, through to measuring the growth of protruding adenoma.

Oblique incidence reflectometry: optical models and measurements using a side-viewing gradient index lens-based endoscopic imaging system.

A side-viewing, 2.3-mm diameter oblique incidence reflectometry endoscope has been designed to obtain optical property measurements of turbid samples. Light from a single-mode fiber is relayed obliquely onto the tissue with a gradient index lens-based distal optics assembly and the resulting diffuse reflectance profile is imaged and collected with a 30,000 element, 0.72 mm clear aperture fiber bundle. Sampling the diffuse reflectance in two-dimensions allows for fitting of the reflected intensity profile to a well-known theoretical model, permitting the extraction of both absorption and reduced scattering coefficients of the tissue sample. Models and measurements of the endoscopic imaging system are presented in tissue phantoms and in vivo mouse colon, verifying the endoscope's capabilities to accurately measure effective attenuation coefficient and differentiate diseased from normal colon.

Novel focused OCT-LIF endoscope.

Combined optical coherence tomography (OCT) and laser-induced fluorescence (LIF) endoscopy has shown higher sensitivity and specificity for distinguishing normal tissue from adenoma when compared to either modality alone. Endoscope optical design is complicated by the large wavelength difference between the two systems. A new high-resolution endoscope 2 mm in diameter is presented that can create focused beams from the ultraviolet to near-infrared. A reflective design ball lens operates achromatically over a large wavelength range, and employs TIR at two faces and reflection at a third internal mirrored face. The 1:1 imaging system obtains theoretically diffraction-limited spots for both the OCT (1300 nm) and LIF (325 nm) channels.

Prevalence, distribution and risk associated with tick infestation of dogs in Great Britain.

Current concerns over the potential impacts of climate change and the increased movement between countries of people and companion animals on the distribution of ectoparasites, highlight the need for accurate understanding of existing prevalence patterns. Without these future changes will not be detected. Here, the distribution and prevalence of tick infestations of domestic dogs in Great Britain were examined. A total of 173 veterinary practices were recruited to monitor tick attachment to dogs in their local areas between March and October 2009. Practices selected five dogs at random each week from those brought to the surgery and undertook a thorough, standardized examination for ticks. Each veterinary practice participated for 3 months before being replaced. Any ticks identified were collected and a sample sent to the investigators for identification, along with a clinical history of the dog. A total of 3534 dogs were examined; 810 dogs were found to be carrying at least one tick. Ixodes ricinus (Linnaeus) (Acari: Ixodidae) was identified in 72.1% of cases, Ixodes hexagonus Leach in 21.7% and Ixodes canisuga Johnston in 5.6% of cases. Five samples of Dermacentor reticulatus (Fabricius) (Acari: Ixodidae) were also found, adding to the growing evidence that an established population of D. reticulatus now exists in south-eastern England. Almost all the ticks found were adults. Overall, 19.2% of the veterinary practices reported no tick detections, 50% reported that ≥14.9% of the dogs seen were infested and 14.6% reported that >50% of the dogs inspected carried ticks. The estimated incidence of tick attachment was 0.013 per day in March (lowest) and 0.096 per day in June (highest). A number of risk factors affected the likelihood of tick attachment on dogs. Gundog, terrier and pastoral breed groups were more likely to carry ticks, as were non-neutered dogs. Dogs with shorter hair were less likely to have ticks, and dogs were most likely to carry a tick in June. This study is of value because, unusually, it presents the results of a randomized sample of dogs and gives a prevalence which is higher than those previously recorded in Great Britain.

Organisational culture: variation across hospitals and connection to patient safety climate.

CONTEXT: Bureaucratic organisational culture is less favourable to quality improvement, whereas organisations with group (teamwork) culture are better aligned for quality improvement. OBJECTIVE: To determine if an organisational group culture shows better alignment with patient safety climate. DESIGN: Cross-sectional administration of questionnaires. Setting 40 Hospital Corporation of America hospitals. PARTICIPANTS: 1406 nurses, ancillary staff, allied staff and physicians. MAIN OUTCOME MEASURES: Competing Values Measure of Organisational Culture, Safety Attitudes Questionnaire (SAQ), Safety Climate Survey (SCSc) and Information and Analysis (IA). RESULTS: The Cronbach alpha was 0.81 for the group culture scale and 0.72 for the hierarchical culture scale. Group culture was positively correlated with SAQ and its subscales (from correlation coefficient r = 0.44 to 0.55, except situational recognition), ScSc (r = 0.47) and IA (r = 0.33). Hierarchical culture was negatively correlated with the SAQ scales, SCSc and IA. Among the 40 hospitals, 37.5% had a hierarchical dominant culture, 37.5% a dominant group culture and 25% a balanced culture. Group culture hospitals had significantly higher safety climate scores than hierarchical culture hospitals. The magnitude of these relationships was not affected after adjusting for provider job type and hospital characteristics. CONCLUSIONS: Hospitals vary in organisational culture, and the type of culture relates to the safety climate within the hospital. In combination with prior studies, these results suggest that a healthcare organisation's culture is a critical factor in the development of its patient safety climate and in the successful implementation of quality improvement initiatives.

Enhanced host immune recognition of mastitis causing Escherchia coli in CD-14 transgenic mice.

Escherchia coli causes mastitis, an economically significant disease in dairy animals. E. coli endotoxin (lipopolysaccharide, LPS) when bound by host membrane proteins such as CD-14, causes release of proinflammatory cytokines recruiting neutrophils as an early, innate immune response. Excessive proinflammatory cytokines causes tissue damage, compromising mammary function. If present, soluble CD-14 (sCD-14) might out compete membrane bound CD-14, lessening the severity of the inflammatory response. To test this hypothesis transgenic mice, expressing sCD-14 in their milk (31 to 316 microg/ml), were evaluated. A cell culture study demonstrated, in the presence of LPS, milk from transgenic mice increased secretion of cytokine IL-8 compared to milk from nontransgenic littermates (18 +/- 3 vs. 35 +/- 2 ng/mL, p < 0.001). To assess protection afforded by the transgene, glands were infused with E. coli. Recovery of bacteria showed no clear relationship between sCD14 concentration and the number of organisms recovered; however, there was a strong relationship between sCD14 concentration and edema (r(2) = 0.999, p < 0.001), as measured by weight of fluid in harvested glands. Highest expressing lines had the least edema, suggesting the presence of sCD14 had an effect on reducing the inflammatory response to E. coli, thus, possibly protecting against gland tissue damage.

Meiotic recombination in normal and clone bulls and their offspring.

Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny. The advent of somatic cell nuclear transfer (SCNT) as a reproductive biotechnology for use in livestock industry has made it easy to bypass these vital steps. However, few studies have been carried out on the impact of SCNT on the reproductive characteristics of cloned animals and, none to date, on the meiotic processes in animals, which were created by circumventing meiosis. In an attempt to assess the impact of cloning by SCNT on the meiotic processes, we undertook an immunocytological comparison of recombination in normal and clone bulls using antibodies raised against the synaptonemal complex protein 3 (SCP3) to label the lateral elements and the mismatch repair protein 1 (MLH1) foci on bivalents as indicators of recombination events. Our studies involving five normal bulls of proven fertility, two SCNT-derived bulls, and four mature offspring of SCNT bulls showed that the mean number of crossing over per spermatocyte for normal bulls (42 +/- 4 SD; ranging from 33 to 56), was not significantly different from that of SCNT-derived bulls (43 +/- 5 SD; ranging from 35 to 56), and the offspring of SCNT-derived bulls (43 +/- 5 SD; ranging from 37 to 58). It would appear that circumventing meiosis to produce these animals does not influence the meiotic processes revealed by MLH1 foci detected in spermatocytes.

Speckled microtubules improve tracking in motor-protein gliding assays.

Gliding assays of motor proteins such as kinesin, dynein and myosin are commonly carried out with fluorescently labeled microtubules or filamentous actin. In this paper, we show that speckled microtubules (MTs), prepared by copolymerizing 98% unlabeled tubulin with 2% rhodamine-labeled tubulin, can be localized to +/-7.4 nm (24 measurements) in images acquired every 125 ms. If the speckled MTs move at about 800 nm s(-1), ten images are sufficient to determine their velocity to a precision of +/-6.8 nm s(-1) (6 microtubules, 24 measurements). This velocity precision is four-fold better than manual methods for measuring the gliding velocity of uniformly labeled MTs by end-point localization. The improved velocity precision will permit the determination of velocity-force curves when one, two and three kinesin motors pull a single load in vitro.

Follicular expression of a human beta-cell leukaemia/lymphoma-2 (Bcl-2) transgene does not decrease atresia or increase ovulation rate in swine.

Transgenic (TG) gilts carrying a human Bcl-2 cDNA transgene driven by mouse inhibin-alpha subunit promoter were produced and evaluated to determine if ectopic expression of Bcl-2 in the ovaries would decrease the frequency of atresia in antral follicles and increase ovulation rate. Immunohistochemical analysis showed that the Bcl-2 transgene protein was expressed in granulosa and theca cells, in 86% of healthy and 54% of atretic follicles analysed in TG prepubertal and Day 50 pregnant gilts combined (n = 24). In contrast, Bcl-2 transgene protein was expressed in only 1.4% of healthy and 0% of atretic follicles in non-TG littermates (n = 13). Real-time reverse transcription-polymerase chain reaction analysis confirmed that human Bcl-2 was expressed in follicles of TG gilts. The atresia rate for the TG and non-TG groups did not differ (P > 0.05) for prepubertal (45 v. 59%) and Day 50 pregnant gilts (53 v. 52%) respectively. The mean +/- s.e.m. ovulation rate did not differ (P > 0.5) between TG (15.9 +/- 0.8, n = 12) and non-TG (16.4 +/- 0.6, n = 7) Day 50 pregnant gilts. The molecular basis of the failure of ectopic Bcl-2 expression to increase the ratio of healthy to atretic follicles is unknown, but it is possible that the activity of the mitochondrial-dependent cell death pathway was not neutralized by ectopic expression of human Bcl-2 or that other cell death pathways compensated for the decreased mitochondrial-dependent cell death.

New gene transfer methods.

The intentional introduction of recombinant DNA molecules into a living organism can be achieved in many ways. Viruses have been making a living by practicing gene transfer for millennia. Recently, man has gotten into the act. The paradigm employed is fairly straightforward. First, a way must be found to move genetic information across biological membrane barriers. Then, presumably, DNA repair mechanisms do the rest. The array of methods available to move DNA into the nucleus provides the flexibility necessary to transfer genes into cells as physically diverse as sperm and eggs. Some of the more promising alternative strategies such as sperm-mediated gene transfer, restriction enzyme-mediated integration, metaphase II transgenesis, and a new twist on retrovirus-mediated gene transfer will be discussed, among other methods.

Temporary articulating methylmethacrylate antibiotic spacer (TAMMAS). A new method of intraoperative manufacturing of a custom articulating spacer.

A temporary articulating antibiotic-impregnated cement spacer for use during the first stage of a two-stage revision of a total knee replacement that had failed because of infection was developed by one of us (W.M.G.). It is simply a knee prosthesis made of methylmethacrylate and antibiotics that is manufactured intraoperatively with use of instruments, medications, and supplies that are already available at most hospitals. This construct allows for motion of the knee during treatment of the infection, thereby reducing the risk of loss of motion after subsequent revision. The technique has been successfully utilized in five patients since 1999 and has now become our standard treatment method.

Safety of a clinical surveillance protocol with 3- and 6-week warfarin prophylaxis after total joint arthroplasty.

The charts of 1869 patients were reviewed for the occurrence of deep venous thrombosis (DVT) and pulmonary embolism after total hip or knee arthroplasty. Prophylaxis consisted of 3 (group 1; n=1235) or 6 (group 2; n=634) weeks low-dose warfarin, pneumatic compression boots worn by patients in the hospital, mobilization on the first postoperative day, and a clinical surveillance protocol. Venous ultrasound or ventilation/perfusion lung scintigraphy (V/Q) was performed only if patients became symptomatic. patients. Twenty-three (1.8%) patients were positive for DVT. Ventilation/perfusion lung scintigraphy was performed on 25 patients, and 5 (0.4%) patients were positive for pulmonary embolism. In group 2, 117 patients were evaluated for DVT, and 19 (3%) patients had positive results determined by ultrasound. Twenty-five patients were evaluated with V/Q and only 1 (0.16%) patient was positive for pulmonary embolism. No patient developed a fatal pulmonary embolism or postphlebitic syndrome. This prophylaxis protocol is an efficient and cost-effective method for the prevention of significant events after surgery.

CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing.

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific B29 (Igbeta/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells. B29 expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced B29 promoters: at conventional CpG and at CC(A/T)GG B29 promoter sites. The pattern of methylated CpG ((m)CpG) and C(m)C(A/T)GG B29 promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of C(m)C(A/T)GG sites in the B29 promoter significantly repressed in vivo transcriptional activity. Also, methylation of a central conserved C(m)CTGG B29 promoter site blocked the binding of early B cell factor. This methylated motif formed DNA-protein complexes with nuclear extracts from all cell types examined. Therefore, C(m)C(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA-protein complex formation.

TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Aberrant expression and localization of the cytoskeleton-binding pp52 (LSP1) protein in hairy cell leukemia.

Non-retractable cell surface projections and cytoskeleton-mediated functional defects are distinguishing features of both hairy cell leukemia (HCL) and neutrophil actin dysfunction (NAD). These defects in NAD neutrophils are attributed to moderate over-expression of pp52 (LSP1), the F-actin-binding, leukocyte-specific phosphoprotein. Here we report that pp52 is similarly elevated in HCL patient PBMCs. Established HCL cell lines exhibited characteristic morphological features like those of fresh HCL cells and showed elevated pp52 levels. The excess pp52 in these HCL cell lines was selectively associated with the F-actin-rich cytoskeletal arrays in surface projections. Treatments producing radical changes in HCL cell shape also altered pp52 expression and intracellular distribution. Alpha interferon (IFNalpha, used to treat HCL) reduced pp52 levels, normalized intracellular pp52 distribution and reverted HCL cells to rounded B cell morphology. Phorbol ester stimulation rapidly generated hyper-phosphorylated pp52 isoforms which translocated from the cytoskeleton to the cytosol prior to the further elongation of surface spikes. This indicates a direct role for phosphorylation in controlling pp52 interactions with the cytoskeleton. Overall, these findings strongly suggest that elevated pp52 expression and/or selective cytoskeletal association contributes to the distinctive morphology of HCL cells.

Pronuclear microinjection.

Last year marked the 20th anniversary of the invention of the term "transgenic" and the development of pronuclear microinjection, a straightforward technique designed to transfer genetic information from nearly any living organism to mammals. After two decades of use, pronuclear microinjection protocols have changed little from the reliable, if not efficient, method described by Gordon and Ruddle. Experience has taught us that once microinjection skills are perfected there are only a few parameters one needs to be concerned about to successfully produce transgenic animals. Those parameters will be discussed, as will some new innovations that promise to finally increase efficiency of pronuclear microinjection methodology.