Reasons for Electronic Nicotine Delivery System use and smoking abstinence at 6†months: a descriptive study of callers to employer and health plan-sponsored quitlines.

OBJECTIVE: Describe cigarette smoking abstinence among employer and health plan-sponsored quitline registrants who were not using Electronic Nicotine Delivery Systems (ENDS), were using ENDS to quit smoking or were using ENDS for other reasons at the time of quitline registration. METHODS: We examined 6029 quitline callers aged ≥18 years who smoked cigarettes at registration, and completed ≥1 counselling calls, baseline ENDS use questions and a 6-month follow-up survey (response rate: 52.4%). 30-day point prevalence smoking quit rates (PPQRs) were assessed at 6-month follow-up (ENDS-only users were considered quit). Data were weighted for non-response bias. Logistic regression analyses controlled for participant characteristics and programme engagement. RESULTS: At registration, 13.8% of respondents used ENDS (7.9% to quit smoking, 5.9% for other reasons). 30-day PPQRs were: 55.1% for callers using ENDS to quit, 43.1% for callers using ENDS for other reasons, and 50.8% for callers not using ENDS at registration. Callers using ENDS for other reasons were less likely to quit than other groups (adjusted ORs=0.65-0.77); quit rates did not significantly differ between non-ENDS users and those using ENDS to quit. Among callers using ENDS to quit at baseline, 40% used ENDS regularly at follow-up. CONCLUSIONS: ENDS users not using ENDS to quit smoking were less successful at quitting at 6-month follow-up compared with callers using ENDS to quit smoking and callers who did not use ENDS at programme registration. Incorporating reasons for ENDS use may be important for future studies examining the role of ENDS in tobacco cessation.

Estimating the growth potential of the soil protozoan community.

We have developed a method for determining the potential abundance of free-living protozoa in soil. The method permits enumeration of four major functional groups (flagellates, naked amoebae, testate amoebae, and ciliates) and it overcomes some limitations and problems of the usual 'direct' and 'most probable number' methods. Potential abundance is determined using light microscopy, at specific time intervals, after quantitative re-wetting of air-dried soil with rain water. No exogenous carbon substrates or mineral nutrients are employed, so the protozoan community that develops is a function of the resources and inhibitors present in the original field sample. The method was applied to 100 soil samples (25 plots x 4 seasons) from an upland grassland (Sourhope, Southern Scotland) in the UK. Median abundances for all four functional groups lie close to those derived from the literature on protozoa living in diverse soil types. Flagellates are the most abundant group in soil, followed by the naked amoebae, then the testate amoebae and ciliates. This order is inversely related to typical organism size in each group. Moreover, preliminary evidence indicates that each functional group contains roughly the same number of species. All of these observations would be consistent with soil having fractal structure across the size-scale perceived by protozoa. The method described will be useful for comparing the effects on the soil protozoan community of different soil treatments (e.g. liming and biocides).

In vitro cultivation and developmental cycle in culture of a parasitic dinoflagellate (Hematodinium sp.) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters.

Dinoflagellates are common and often important parasites of aquatic organisms, but their developmental cycles are poorly known and have not been established in in vitro culture. The parasitic dinoflagellate (Hematodinium sp.) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters has been cultivated in vitro in 10% foetal calf serum in a balanced Nephrops saline. In culture the parasite undergoes a characteristic cycle of development. Circulating sporoblasts from the host's haemolymph in vitro generate 2 kinds of flagellated uninucleate dinospore, macrospores and microspores, either of which will, after 5 weeks in fresh medium, germinate to produce multinucleate unattached filamentous trophonts. These trophonts multiply by fragmentation and growth and may be serially subcultured in this form, at 2 week intervals, indefinitely. If not subcultured, the filamentous trophonts give rise to colonies of radiating filaments ('gorgonlocks') which subsequently attach to the substratum to form flattened web-like 'arachnoid' multinucleate trophonts. Arachnoid trophonts become arachnoid sporonts when they synthesize trichocysts and flagellar hairs and may give rise to secondary arachnoid sporonts or to dinospores which initiate a new cycle.

Presence of apicomplexan-type micropores in a parasitic dinoflagellate, Hematodinium sp.

Structures resembling apicomplexan micropores were found by transmission electron microscopy in in vitro-cultured and in in vivo forms of the parasitic dinoflagellate Hematodinium sp. from crustacean hosts. Uptake of colloidal gold indicated a cytostomal function for the micropores.

The evolutionary expansion of the trypanosomatid flagellates.

The trypanosomatids combine a relatively uniform morphology with ability to parasitise a very diverse range of hosts including animals, plants and other protists. Along with their sister family, the biflagellate bodonids, they are set apart from other eukaryotes by distinctive organisational features, such as the kinetoplast-mitochondrion and RNA editing, isolation of glycolysis enzymes in the glycosome, use of the flagellar pocket for molecular traffic into and out of the cell, a unique method of generating cortical microtubules, and bizarre nuclear organisation. These features testify to the antiquity and isolation of the kinetoplast-bearing flagellates (Kinetoplastida). Molecular sequencing techniques (especially small subunit ribosomal RNA gene sequencing) are now radically reshaping previous ideas on the phylogeny of these organisms. The idea that the monogenetic (MG) trypanosomatids gave rise to the digenetic (DG) genera is losing ground to a view that, after the bodonids, the African trypanosomes (DG) represent the most ancient lineage, followed by Trypanosoma cruzi (DG), then Blastocrithidia (MG), Herpetomonas (MG) and Phytomonas (DG), with Leptomonas (MG), Crithidia (MG), Leishmania (DG) and Endotrypanum (DG) forming the crown of the evolutionary tree. Vast genetic distances (12% divergence) separate T. brucei and T. cruzi, while the Leishmania species are separated by very short distances (less than 1% divergence). These phylogenetic conclusions are supported by studies on RNA editing and on the nature of the parasite surface. The trypanosomatids seem to be able to adapt with ease their energy metabolism to the availability of substrates and oxygen, and this may give them the ability to institute new life cycles if host behaviour patterns allow. Sexual processes, though present in at least some trypanosomatids, may have played only a minor part in generating diversity during trypanosomatid evolution. On the other hand, the development of altruistic behaviour on the part of some life cycle stages may be a hitherto unconsidered way of maximising fitness in this group. It is concluded that, owing to organisational constraints, the trypanosomatids can undergo substantial molecular variation while registering very little in the way of morphological change.

Comparison of the effects of immune killing mechanisms on Trypanosoma brucei parasites of slender and stumpy morphology.

Trypanosoma brucei slender forms predominate over stumpy forms as the parasite population grows but at the peak of a parasitaemic wave and during remission of infection stumpy forms predominate. To determine whether this change in predominance might be caused by selective killing of slender forms, the fates of slender and stumpy form trypanosomes in two in vitro assays of immune-mediated killing were compared. Parasite populations in which > 90% of cells were of slender morphology were observed to be killed by antibody-dependent complement-mediated lysis approximately five times faster than populations in which < 15% of cells were slender and most were of intermediate or stumpy morphology. Quantification of the relationship between the proportion of slender forms in the population and the rate of lysis indicated that slender forms were killed approximately 7.3 times faster than other forms. In an opsonization assay, no differences were observed between slender and stumpy forms in the extent to which they attached to macrophages in an antibody-dependent manner. These results suggest that the change in proportions of slender and stumpy forms at the peak of a parasitaemic wave results from slender forms being more susceptible to complement-mediated killing as the antibody response develops.

Isolation and ultrastructural features of a new strain of Dimastigella trypaniformis Sandon 1928 (Bodonina, Kinetoplastida) and comparison with a previously isolated strain.

A strain of the free-living flagellate Dimastigella trypaniformis Sandon was isolated from the gut contents of the Australian termite Mastotermes darwiniensis Froggatt. The fine structure of the isolate (Ulm strain) was compared with a strain isolated from soil in Scotland (Glasgow strain). The biflagellated trophozoites possess great morphological similarities with the bodonids and cryptobiids such as a polykinetoplastic mitochondrial complex, a flagellar paraxial rod, and a cytopharyngeal complex. However, characteristic features are also found such as the two opposed basal bodies and the two flagella, one anteriorly and the other recurrently directed, the latter adhering to a ventral furrow, a rostrum as well as a cytostome and a cytopharynx with rostral structures. These organisms have a typical motion with a highly flexible cell body. They are able to form resistant cysts in the culture. The two strains are distinguishable by small differences in their growth characteristics in different media and qualitative ultrastructural features.

The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3-D reconstruction.

Computer-aided 3-D reconstruction of trypanosomes from 0.35-micron-thick sections imaged on the Zeiss 902 electron microscope are being used to study the dynamics of cell organization. Segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. Direct counts of glycosomes from Trypanosoma evansi indicate 30-40 per cell and for the closely related T. brucei, 65 per cell. These figures contrast with the estimates of others who have used model-based morphometric methods to obtain a value of 230 per cell.

Multinuclear forms in a dyskinetoplastic strain of Trypanosoma evansi in mice.

The production of short stumpy and multinuclear trypanosomes in a Chinese strain of dyskinetoplastic Trypanosoma evansi maintained in rabbits and mice is described. Production of multinuclear trypanosomes was increased following passage through a reptile (gecko), in which the trypanosomes did not multiply, and transfer back to mice. The multinuclear trypanosomes showed more nuclei than flagella indicating that disruption of the normal cell cycle had taken place and not simply inhibition of cleavage. A Chinese kinetoplastic T. evansi treated similarly rarely produced stumpy or multinuclear forms.

A comparison of multiplication rates in primary and challenge infections of Trypanosoma brucei bloodstream forms.

The hypothesis that division of Trypanosoma brucei slender bloodstream forms is dependent upon the availability of a host-derived growth factor has been tested by superimposing challenge doses of slender-form trypanosomes onto preexisting infections at a time during the primary infection when stumpy forms predominated. The challenge populations grew in the doubly-infected mice indicating that depletion of a putative growth factor by the expanding population of the primary infection had not prevented division of the trypanosomes although slight reductions in multiplication rates were observed. This effect was independent of the variable antigen type (VAT) of the trypanosomes and of their stock of origin.

Trypanosome sociology and antigenic variation.

Survival of the trypanosome (Trypanosoma brucei) population in the mammalian body depends upon paced stimulation of the host's humoral immune response by different antigenic variants and serial sacrifice of the dominant variant (homotype) so that minority variants (heterotypes) can continue the infection and each become a homotype in its turn. New variants are generated by a spontaneous switch in gene expression so that the trypanosome puts on a surface coat of a glycoprotein differing in antigenic specificity from its predecessor. Homotypes appear in a characteristic order for a given trypanosome clone but what determines this order and the pacing of homotype generation so that the trypanosome does not quickly exhaust its repertoire of variable antigens, is not clear. The tendency of some genes to be expressed more frequently than others may reflect the location within the genome and mode of expression of the genes concerned and may influence homotype succession. Differences in the doubling time of different variants or in the rate at which trypanosomes belonging to a particular variant differentiate into non-dividing (vector infective) stumpy forms have also been invoked to explain how a heterotype's growth characteristics may determine when it becomes a homotype. Recent estimations of the frequency of variable antigen switching in trypanosome populations after transmission through the tsetse fly vector, however, suggest a much higher figure (0.97-2.2 x 10(-3) switches per cell per generation) than that obtained for syringe-passed infections (10(-5)-10(-7) switches per cell per generation) and it seems probable that most of the variable antigen genes are expressed as minority variable antigen types very early in the infection. Instability of expression is a feature of trypanosome clones derived from infective tsetse salivary gland (metacyclic) trypanosomes and it is suggested that high switching rates in tsetse-transmitted infections may delay the growth of certain variants to homotype status until later in the infection.

An estimate of the size of the metacyclic variable antigen repertoire of Trypanosoma brucei rhodesiense.

A group of 27 variable antigen type (VAT)-specific monoclonal antibodies (McAbs) have been made against metacyclic forms of a cloned stock of Trypanosoma brucei rhodesiense. In combination, these labelled in immunofluorescence 99.3% of trypanosomes in salivary probes from tsetse flies. The 0.7% of unlabelled trypanosomes were believed to be uncoated forms. The ability of a mixture of antibodies to kill metacyclics in vitro by complement-mediated lysis, thus neutralizing their infectivity for mice, was tested. The antibody mixture consisted of 24 McAbs plus 3 VAT-specific rabbit antisera. In 12 replicate experiments this mixture of antibodies prevented infection of mice. Parallel controls showed that neutralization was probably antibody-mediated and VAT specific. However, we have not been able to repeat these results on a long-term basis; this may be due to a loss of neutralizing activity by one of the McAbs. The successful neutralization experiments indicate that the number of VATs in the metacyclic repertoire of one stock of T. b. rhodesiense is limited to at most 27.

Biology of African trypanosomes in the tsetse fly.

African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian host's immune response; and a unique flagellum-to-host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.

The requirement for epimastigote attachment during division and metacyclogenesis in Trypanosoma congolense.

In cultures of tsetse proboscis stages during the development of Trypanosoma congolense, attached epimastigote forms multiply and give rise to free nondividing metacyclic trypanosomes. Prevention of attachment by shaking the cultures or by providing a polypropylene substratum does not inhibit epimastigote division but does prevent the differentiation of metacyclics. We conclude that epimastigote attachment forms a necessary part of the program of metacyclic development.

Loss of variable antigen during transformation of Trypanosoma brucei rhodesiense from bloodstream to procyclic forms in the tsetse fly.

A pleomorphic line of Trypanosoma brucei rhodesiense expressing a single variable antigen was used to quantify the rate of loss of the surface coat from bloodstream forms transforming to procyclics in the tsetse fly, Glossina morsitans, and in in vitro culture. Loss of variable antigen occurred at similar rates in the crop and anterior portion of the midgut of tsetse flies and in in vitro culture, but in the posterior portion of the fly midgut it occurred 2-3 times faster. The posterior portion of the midgut is the most important site for transformation of bloodstream-form trypanosomes to procyclics, and the dynamics of at least one component of this process are therefore not accurately paralleled in vitro.

Onset of expression of the variant surface glycoproteins of Trypanosoma brucei in the tsetse fly studied using immunoelectron microscopy.

The acquisition of the variant surface glycoprotein (variable antigen) coat by metacyclic stage Trypanosoma brucei in the salivary glands of the tsetse fly, Glossina morsitans, has been studied in situ by transmission and scanning electron microscopy using monoclonal antibodies raised against metacyclic variable antigen types and complexed with horseradish peroxidase or colloidal gold. The coat is acquired after binary fission has ceased but while the parasite is still attached to the gland epithelium, i.e. before the mature metacyclic is released into the gland lumen. The variable antigen type heterogeneity previously observed in discharged mature metacyclics is here demonstrated in the nascent (attached) metacyclic population. The variant surface glycoprotein genes are thus not expressed in a fixed sequence since different metacyclic variable antigen types are present ab initio. The distribution of immunogold-marked nascent metacyclics of a particular variable antigen type, as shown by quadrat analysis of a scanning electron micrograph montage of the infected salivary gland epithelium, conforms to a Poisson series. This provides evidence that individual variant surface glycoprotein genes are stochastically activated and suggests that selective activation occurs after trypanosome division has ceased.