Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.     

High-Contrast Fluorescence Imaging in Fixed and Living Cells Using Optimized Optical Switches

We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.     

Elimination of Iron Deficiency Anemia and Soil Transmitted Helminth Infection: Evidence from a Fifty-four Month Iron-Folic Acid and De-worming Program

Background

Intermittent iron-folic acid supplementation and regular de-worming are effective initiatives to reduce anemia, iron deficiency, iron deficiency anemia, and soil transmitted helminth infections in women of reproductive age. However, few studies have assessed the long-term effectiveness of population-based interventions delivered in resource-constrained settings.

Methodology/Principal Findings

The objectives were to evaluate the impact of weekly iron-folic acid supplementation and de-worming on mean hemoglobin and the prevalence of anaemia, iron deficiency, and soil transmitted helminth infection in a rural population of women in northern Vietnam and to identify predictive factors for hematological outcomes. A prospective cohort design was used to evaluate a population-based supplementation and deworming program over 54 months. The 389 participants were enrolled just prior to commencement of the intervention. After 54 months 76% (95% CI [68%, 84%]) were taking the iron-folic acid supplement and 95% (95% CI [93%, 98%]) had taken the most recently distributed deworming treatment. Mean hemoglobin rose from 122 g/L (95% CI [120, 124]) to 131 g/L (95% CI [128, 134]) and anemia prevalence fell from 38% (95% CI [31%, 45%]) to 18% (95% CI [12%, 23%]); however, results differed significantly between ethnic groups. Iron deficiency fell from 23% (95% CI [17%, 29%]) to 8% (95% CI [4%, 12%]), while the prevalence of iron deficiency anemia was reduced to 4% (95% CI [1%, 7%]). The prevalence of hookworm infection was reduced from 76% (95% CI [68%, 83%]) to 11% (95% CI [5%, 18%]). The level of moderate or heavy infestation of any soil-transmitted helminth was reduced to less than 1%.

Conclusions/Significance

Population-based interventions can efficiently and effectively reduce anemia and practically eliminate iron deficiency anemia and moderate to heavy soil transmitted helminth infections, maintaining them below the level of public health concern.     

Adherence to Chemoprophylaxis and Plasmodium falciparum Anti-Circumsporozoite Seroconversion in a Prospective Cohort Study of Dutch Short-Term Travelers

Background

We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.

Methods

Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Plasmodium falciparum anti-circumsporozoite antibodies.

Results

Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14–29 days in endemic areas, and concurrent use of DEET (N,N-diethyl-meta-toluamide) were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies.

Conclusion

Travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert.     

Is Consumer Response to Plain/Standardised Tobacco Packaging Consistent with Framework Convention on Tobacco Control Guidelines? A Systematic Review of Quantitative Studies

Background and Objectives

Standardised or ‘plain’ tobacco packaging was introduced in Australia in December 2012 and is currently being considered in other countries. The primary objective of this systematic review was to locate, assess and synthesise published and grey literature relating to the potential impacts of standardised tobacco packaging as proposed by the guidelines for the international Framework Convention on Tobacco Control: reduced appeal, increased salience and effectiveness of health warnings, and more accurate perceptions of product strength and harm.

Methods

Electronic databases were searched and researchers in the field were contacted to identify studies. Eligible studies were published or unpublished primary research of any design, issued since 1980 and concerning tobacco packaging. Twenty-five quantitative studies reported relevant outcomes and met the inclusion criteria. A narrative synthesis was conducted.

Results

Studies that explored the impact of package design on appeal consistently found that standardised packaging reduced the appeal of cigarettes and smoking, and was associated with perceived lower quality, poorer taste and less desirable smoker identities. Although findings were mixed, standardised packs tended to increase the salience and effectiveness of health warnings in terms of recall, attention, believability and seriousness, with effects being mediated by the warning size, type and position on pack. Pack colour was found to influence perceptions of product harm and strength, with darker coloured standardised packs generally perceived as containing stronger tasting and more harmful cigarettes than fully branded packs; lighter coloured standardised packs suggested weaker and less harmful cigarettes. Findings were largely consistent, irrespective of location and sample.

Conclusions

The evidence strongly suggests that standardised packaging will reduce the appeal of packaging and of smoking in general; that it will go some way to reduce consumer misperceptions regarding product harm based upon package design; and will help make the legally required on-pack health warnings more salient.     

Evaluation of Hepatitis A Vaccine in Post-Exposure Prophylaxis,The Netherlands, 2004-2012

Background

The secondary attack rate of hepatitis A virus (HAV) among contacts of cases is up to 50%. Historically, contacts were offered immunoglobulin (IG, a human derived blood product) as post-exposure prophylaxis (PEP). Amid safety concerns about IG, HAV vaccine is increasingly recommended instead. Public health authorities’ recommendations differ, particularly for healthy contacts ≥40 years old, where vaccine efficacy data is limited. We evaluated routine use of HAV vaccine as an alternative to immunoglobulin in PEP, in those considered at low risk of severe infection in the Netherlands.

Methods

Household contacts of acute HAV cases notified in Amsterdam (2004-2012) were invited ≤14 days post-exposure, for baseline anti-HAV testing and PEP according to national guidelines: immunoglobulin if at risk of severe infection, or hepatitis A vaccine if healthy and at low risk (aged <30, or, 30-50 years and vaccinated <8 days post-exposure). Incidence of laboratory confirmed secondary infection in susceptible contacts was assessed 4-8 weeks post-exposure. In a vaccinated subgroup, relative risk (RR) of secondary infection with estimated using Poisson regression.

Results

Of 547 contacts identified, 191 were susceptible to HAV. Per-protocol, 167 (87%) were vaccinated (mean:6.7 days post-exposure, standard deviation(sd)=3.3) and 24 (13%) were given immunoglobulin (mean:9.7 days post-exposure, sd=2.8). At follow-up testing, 8/112 (7%) had a laboratory confirmed infection of whom 7 were symptomatic. All secondary infections occurred in vaccinated contacts, and half were >40 years of age. In healthy contacts vaccinated per-protocol ≤8 days post-exposure, RR_{ref. ≤15 years} of secondary infection in those >40 years was 12.0 (95%CI:1.3-106.7).

Conclusions

Timely administration of HAV vaccine in PEP was feasible and the secondary attack rate was low in those <40 years. Internationally, upper age-limits for post-exposure vaccination vary. Pending larger studies, immunoglobulin should be considered PEP of choice in people >40 years of age and those vulnerable to severe disease.     

Sero‐taxonomy of skeletal macromolecules in living Terebratulid brachiopods

Antibodies prepared against macromolecules isolated from the shells of three living brachiopod genera have proved to be of considerable taxonomic significance, in that the pattern of cross‐reactivity of all three antisera consistently points to a new interpretation for the evolution of the largest extant brachiopod order, the Terebratulida. This new molecular evidence actually complements rather than contradicts the existing morphology‐based taxonomy, since detailed systematic investigation of the taxa in question has already demonstrated subtle but significant morphological differences in the major taxonomic characters which appear to reflect this new interpretation.

As fragments of skeletal macromolecules, including antigenic determinants, are known to survive for many millions of years within the protected micro‐environments provided by enclosing biominerals, these results suggest that such molecular fossils could well provide important insights on at least the high‐level taxonomic relationships of fossil organisms.     

Phylogenetic implications and diagenetic stability of macromolecules from pleistocene and recent shells of Mercenaria mercenaria (mollusca,bivalvia)

The phylogeny and diagenesis of Pleistocene and Recent bivalves were studied immunologically by use of a conventional antiserum elicited against an EDTA‐soluble macromolecular extract from shells of the modern bivalve mollusc Mercenaria mercenaria. ELISA tests of the antiserum with shell fragments of a wide range of modern bivalves gave taxonomically significant results. The antiserum reacted with palaeoheterodonts and heterodonts but not with representatives of other bivalve subclasses. This phylogenetic reactivity was also apparent in tests with fossil shells, although the specificity and overall strength of the reaction were both reduced. Absorption of the antiserum with etched shell powders of various (palaeo)heterodonts yielded more specific antibody preparations.

Investigations of shell matrix diagenesis, using the anti‐Mercenaria serum, demonstrated that small amounts of original determinants could be detected even in fossils over one million years old. The reactivity of the serum with extracts of fossil Mercenaria decreased with sample age. The relationship between serum reactivity and the degree of amino acid racemization was almost linear. Clearly, the various determinants to which antibodies were elicited were being destroyed at different rates.     

Liposomes and Liposome-like Vesicles for Drug and DNA Delivery to Mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the “power house” of the cell, have also become accepted as the “motor of cell death” reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of “Mitochondrial Medicine” as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.